
Proceedings of the AACR

Volume 57 | April 2016

**Part** **B: Abstracts 2697-5293**

TABLE OF CONTENTS

BIOINFORMATICS AND SYSTEMS BIOLOGY:

Bioinformatic Tools for Analysis and Mathematical Modeling of Clinical Samples

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Cycle Control and Checkpoints

DNA Double-Strand Break Repair Defects in Cancer: Therapeutic Strategies and Molecular Basis

DNA Methylation 1

Hypoxia and Oxidative Stress

Pharmacological Inhibitors of Cyclin-dependent Kinases

Senescence, Cell Death, and Unfolded Protein Response

Transcriptional Regulation and Gene Expression in Human Malignancies

Translational and Therapeutic Relevance of Perturbations of Gene Regulation in Malignancy

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Mechanisms of Drug Resistance 3

Monoclonal Antibodies and Antibody-Drug Conjugates

New Mechanisms of Anticancer Drug Action

Novel Targets and Pathways

Preclinical Radiotherapeutics

Small Molecule Inhibitors

CANCER CHEMISTRY:

Structural and Chemical Biology

CLINICAL RESEARCH:

Biomarkers for Gastrointestinal, Hematologic, and Uncommon Cancers

Circulating Biomarkers 2

Molecular Classification and Genomic Applications

Novel Approaches to Pediatric Cancers

IMMUNOLOGY:

Immune Checkpoints 2

Innate Immune System, Myeloid Cells, and Tumorigenesis

TUMOR BIOLOGY:

Antiangiogenic Therapy: Inhibitors and Resistance

Hematological Microenvironment

Microbiome in Cancer

Stemness Properties of Breast and Ovarian Cancer

Stemness Properties of Leukemias and Carcinomas

Tumor Angiogenesis: Host Interactions and the Tumor Microenvironment

Tumor Angiogenesis: Mediators and Mechanisms

EPIDEMIOLOGY:

Epidemiology of Cancer Prognosis and Survival

Exogenous Exposures and Cancer Risk

PREVENTION RESEARCH:

Behavioral and Social Science Studies across the Cancer Prevention Continuum

ENDOCRINOLOGY:

Clinical Endocrinology

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Death 1

Cell Death 2

Cell Death 3

Genetic Instability in Cancer: Molecular Basis and Tools

Genomic Technologies

Genomic Technologies and Analyses

Tumor Suppressor Genes and Pathways

Tumor Suppressors: TP53 Pathway

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Cell Death Pathways and DNA Repair

Gene and Vector-Based Therapy

Novel Antitumor DNA-Reactive Agents

Novel Targets

Oncogenes and Tumor Suppressor Genes as Therapeutic Targets

Targeting Protein Kinases, Death Pathways, and the Tumor Microenvironment

CANCER CHEMISTRY:

Proteomics and Mass Spectrometry

Therapeutics

CLINICAL RESEARCH:

Biomarkers for Breast Cancer

Circulating Biomarkers 3 / Immune Biomarkers

Novel Molecular Diagnostics and Imaging

IMMUNOLOGY:

Immune Modulation from Non-Immunotherapy: Preclinical

Mechanisms and Applications of Immune-based Therapies

TUMOR BIOLOGY:

Chemical and Viral Carcinogenesis

Host-Tumor Interactions

Imaging and Therapeutics of Metastasis

Immune Cell Activity

Mechanisms of Tumorigenesis in Animal Models of Cancer 2

Molecular and Cellular Imaging of Cancer 1

Molecular and Cellular Imaging of Cancer 2

New Cell Lines and 3D Models

EPIDEMIOLOGY:

Biomarkers of Endogenous and Exogenous Exposures

PREVENTION RESEARCH:

Diet and Cancer

CANCER CHEMISTRY:

Drug Design and Delivery

CLINICAL RESEARCH:

Clinical Qualification of Impactful NGS-based Biomarkers

EPIDEMIOLOGY:

Endogenous and Exogenous Factors in Cancer Epidemiology throughout the Life Course

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Identifying Targets and Combinations through Novel Approaches

IMMUNOLOGY:

Potentiating Immunotherapy Responses with Next Generation Agents and Combinatorial Partners

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Genomic Alterations and Their Functional Consequences

Oncogenic Cell Signaling: Mechanisms and Translational Insight

MULTIDISCIPLINARY:

New "Cool Tools" for Cancer Discovery

TUMOR BIOLOGY:

Therapeutic Studies in Cell Culture and Mouse Models

Tumor-supporting Microenvironment

Advocates Poster Session 2

MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

Cell Signaling

DNA Methylation 2

Epigenetic Biomarkers and Therapies

Germline/Somatic Genomics and Personalized Medicine

Histone Modifications and Chromatin Dynamics

Protein Modification and Transcriptional Regulation

Receptors

Wnt, AKT, and Cell Survival Pathways

EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

Cellular Responses to Anticancer Drugs

Combination Therapies and Approaches to Sensitizing Cancer Cells to Drugs

Epigenetic Agents

HDAC, Methyltransferase Inhibitors, and Novel Anticancer Agents

Molecular Characterizations of Tumors and Translational Studies

Novel Chemotherapies

Targeted Therapy

CANCER CHEMISTRY:

Drug Design

IMMUNOLOGY:

Immune Modulating Agents 2

Immune Response Monitoring: Preclinical

CLINICAL RESEARCH:

Biomarkers for Gastrointestinal Cancer

Biomarkers for Genitourinary Cancers: Prostate

Immune Modulation from Non-Immunotherapy and Antibodies: Clinical

Predictive and Prognostic Biomarkers

TUMOR BIOLOGY:

Cell Adhesion and Tumor Treatment

Cellular and Molecular Dynamics of Cancer Migration and Invasion

Extracellular Microenvironment

Immunomodulation and Immunotherapy

Molecular Carcinogenesis

Therapeutic Studies in Patient-derived Xenografts

EPIDEMIOLOGY:

Description of Cancer Trends and Next-Generation Sequencing in Epidemiology

PREVENTION RESEARCH:

Chemoprevention Studies

BIOINFORMATICS AND SYSTEMS BIOLOGY:

New Bioinformatic Tools, Databases, Portals, and Data Resources

#

#  Tuesday, April 19, 2016

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### Bioinformatic Tools for Analysis and Mathematical Modeling of Clinical Samples

#2697

Drug combinations for breast cancer based on synthetic lethality screens in yeast.

Michael Schwarz,1 Erwin Tomasich,1 Maximilian Marhold,1 Andreas Heinzel,2 Paul Perco,2 Peter Horak,3 Bernd Mayer,2 Michael Krainer1. 1 _Medical University of Vienna, Department for Internal Medicine I - Oncology, Vienna, Austria;_ 2 _emergentec biodevelopment GmBH, Vienna, Austria;_ 3 _SMZ Ost, Department for Medicine II - Oncology, Vienna, Austria_.

Synthetic lethality describes an interdependent relationship between two genes, where the loss of either one alone can be compensated, while the simultaneous loss of both genes causes a non-viable phenotype. In recent years, first therapeutics based on this concept entered the clinic, most notably the PARP inhibitors for BRCA1/2-mutated cancers.

In the present study, we analyzed synthetic lethal interactions in yeast to identify new and potentially synergistic drug combinations for breast cancer therapy. We were able to confirm significantly enhanced cytotoxicity for predicted drug pairs in breast cancer cell lines in vitro.

First, a predictor built from publicly available yeast genetic interactions in the Data Repository of Yeast Genetic INteraction (DRYGIN) was used to predict potential synthetic lethal genetic interactions in human. Independently, a data set containing all pharmacological approaches, targeted or cytostatic, in breast cancer therapy was created. These drug combinations and their respective targets were then analyzed regarding their coverage of predicted synthetic lethal interactions. New drug combinations, previously unused in breast cancer therapy, were identified in silico by combining drugs already in use for breast cancer therapy (individually or in other combinations) in such a manner that their combination covers one or more potential synthetic lethal gene pairs. From this set of new drug combination and their synthetic lethal gene pairs we further pursued the predicted interdependencies between farnesyl diphosphate synthase (FDPS) and tubulin, beta 1 (TUBB1) and between FDPS and phosphoinositide-dependent kinase-1 (PDPK1) as well as prostaglandin-endoperoxide synthase 2 (PTGS2). Drugs targeting these genes are celecoxib (PDPK1, PTGS2), an anti-inflammatory drug, and zoledronic acid (FDPS), a bone degradation inhibitor, as well as the cytotoxic agent paclitaxel (TUBB1).

We performed cell viability and cytotoxicity assays to determine therapeutic effects of celecoxib, zoledronic acid and paclitaxel alone and in combination on selected breast cancer cell lines.

Our results showed statistically significant decreases in cell viability for the combinational treatment with zoledronic acid and paclitaxel as well as with the per se non-cytotoxic combination of zoledronic acid and celecoxib when compared to single agent treatment.

In conclusion, we present a bioinformatics approach to predict potentially synergistic gene interactions based on synthetic lethality found in yeast and a strategy for utilizing these interactions for identifying new potentially synergistic drug combinations.

#2698

Transcriptional profiling of human TAMs highlights differences in breast and endometrial tumour microenvironments.

Matina Fragkogianni,1 Luca Cassetta,1 Agnieszka Swierczak,1 Lisa Wiechmann,2 Harriet O. Smith,3 Andrew H. Sims,4 Hui Zhang,5 Lisa M. Coussens,6 J. W. Pollard1. 1 _MRC Centre for Reproductive Health, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom;_ 2 _Northeast Medical Group, Yale New Haven Health, CT;_ 3 _Department of Obstetrics & Gynecology and Women's Health, Albert Einstein College of Medicine, NY; _4 _CRUK Edinburgh Cancer Centre, Edinburgh, United Kingdom;_ 5 _Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, NY;_ 6 _Oregon Health & Science University, OR_.

Background: Breast and endometrial cancers are two of the most frequently diagnosed reproductive cancers in women in the western world. Previously, research has focused on cancer cells, however increasing evidence shows that the tumour microenvironment plays an important role in cancer progression. Macrophages are almost entirely derived from circulating monocytes and are recruited into tumour sites. In murine studies, tumour-associated macrophages (TAMs) have been implicated in promoting angiogenesis, immune suppression and resistance to treatment. Their density has been associated with poor prognosis in breast cancer and decreased survival in endometrial cancer. However, little is known about the function of TAMs in humans. The aim of this study is to examine the transcriptional profiles of human TAMs in order to investigate their biological relevance and potential for therapeutic intervention.

Methods: RNA-sequencing was performed on purified normal macrophages and TAMs from breast tissue (4 breast cancer and 4 healthy breast) and endometrium tissue (5 endometrial cancer and 9 healthy endometrium). Statistical analysis using Limma was used to identify significantly differentially expressed genes (FDR< 0.05) with a minimum log2 fold change of 1.5. Gene set enrichment (GSEA) analysis and gene ontologies (GO) were employed for functional analysis and identification of important biological pathways.

Results: Transcriptome profiling revealed a significantly altered gene expression profile of TAMs when compared to normal macrophages. Furthermore, comparison of TAMs between breast and endometrial cancer also revealed differences suggesting that different tumour microenvironments induce different gene expression profiles in TAMs. Interestingly, comparison of normal macrophages between breast and endometrial tissue also revealed differences in gene expression suggesting tissue specificity for macrophages. Functional analysis of significant genes revealed similar biological pathways to those of murine studies suggesting that TAMs in humans may have similar functions. We were able to extract TAM-specific genes by comparing with publicly available datasets that could serve as markers for their identification. Finally, we identified a list of transmembrane receptors that could act as potential targets for targeted killing of TAMs.

Conclusion: This is the first study to carry out genome-wide profiling of TAMs in human breast and endometrial cancer. Expression profiles differed between TAMs and healthy patients, as well as between breast and endometrial cancer suggesting cancer specificity for TAMs and revealing not only potential TAM-specific markers, but also identifying possible cancer- and TAM-specific therapeutic targets.

#2699

A cancer treatment based on synergy between anti-angiogenic and immune cell therapies.

Luis F. Soto-Ortiz. _East Los Angeles College, Monterey Park, CA_.

A mathematical model integrating tumor angiogenesis and tumor-targeted cytotoxicity by immune cells was developed to identify the therapeutic window of two distinct modes to treat cancer: 1) an anti-angiogenesis treatment based on the monoclonal antibody bevacizumab that targets tumor vasculature, and 2) immunotherapy involving the injection of unlicensed dendritic cells to boost the anti-tumor adaptive response. The angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) contributes to the immunosuppressive tumor microenvironment, which is responsible for the short-lived therapeutic effect of cancer-targeted immunotherapy. The effect of immunosuppression on the width of the therapeutic window of each treatment was quantified. Experimental evidence has shown that neutralizing immunosuppressive cytokines results in an enhanced immune response against infections and chronic diseases. The model was used to determine treatment protocols involving the combination of anti-VEGF and unlicensed dendritic cell injections that enhance tumor regression. The model simulations predicted that the most effective method to treat tumors involves administering a series of biweekly anti-VEGF injections to disrupt angiogenic processes and limit tumor growth. The simulations also verified the hypothesis that reducing the concentration of the immunosuppressive factor VEGF prior to an injection of unlicensed dendritic cells enhances the cytotoxicity of CD8+ T cells and results in complete tumor elimination. Feasible treatment protocols for tumors that are diagnosed late and have grown to a relatively large size were identified.

#2700

Transcriptome analyses reveal fusion transcripts and transcript variants that are recurrent across sample series of testicular germ cell tumors.

Andreas M. Hoff, Sen Zhao, Bjarne Johannessen, Rolf I. Skotheim. _Department of Molecular Oncology, Institute for Cancer Research, Oslo University Hospital-Norwegian Radium Hospital, Oslo, Norway_.

Testicular germ cell tumors (TGCT) are the most frequently diagnosed solid tumors in young men. Embryonal carcinoma (EC) comprises a histological subgroup of TGCT that exhibits pluripotent characteristics similar to embryonic stem (ES) cells. The genetic drivers underlying malignant transformation of TGCT, including ECs, are unknown. To elucidate the abnormal genetic events potentially contributing to TGCT malignancy, such as the formation of fusion genes or expression of aberrant transcript variants, we have previously performed RNA sequencing of EC cell lines and their non-malignant ES cell line counterparts. From this dataset, we identified and characterized eight novel fusion transcripts and one gene with alternative promoter usage, ETV6. These were the first fusion genes to be shown in TGCT (Hoff et al., Cancer Research, in press).

To search for recurrence of the originally nominated TGCT specific fusion transcripts and other transcript variants in an external sample series, we have here parsed raw RNA sequencing data from 102 primary TGCTs obtained from The Cancer Genome Atlas (TCGA; unpublished). In this process, we have evaluated multiple fusion finder algorithms and approaches to determine the optimal method, on the basis of sensitivity and specificity, to query specific transcript variants and breakpoints in RNA sequencing datasets.

We were able to detect the recurrent fusion transcripts; RCC1-ABHD12B, RCC1-HENMT1, CLEC6A-CLEC4D and the transcript variant of ETV6, which was previously detected from our in-house RNA sequencing dataset from EC cell lines. We also demonstrate that expression of several fusion transcripts correlate significantly with the histological subtype. In conclusion, the novel fusion transcripts and ETV6 transcript variant are expressed from a high fraction of primary TGCTs with low differentiation grade, as detected across independent sample series.

#2701

Combining whole-exome and RNA-Seq data improves the quality of PDX mutation profiles.

Manuel Landesfeind, Bruno Zeitouni, Anne-Lise Peille, Vincent Vuaroqueaux. _Oncotest GmbH, Freiburg, Germany_.

Patient-derived xenograft tumor models (PDX) are of increasing interest for anti-cancer agent testing due to their close resemblance to patient tumors. An accurate molecular characterization of the models is essential 1) to select the PDX that best fit the genetic requirements for a successful cancer therapy investigation and 2) to identify potential predictive biomarkers of response. In this study, we evaluated the quality of mutation profiles from whole-exome sequencing (WES) in terms of concordance with previously acquired mutation data in a large collection of PDX. Further, we analyzed the persistence of disclosed mutations at the transcript level with RNA-Seq.

From 339 PDX, DNA was extracted and enriched in exonic regions with Agilent SureSelect kits before Illumina HiSeq 2000 sequencing with a minimum expected average-of-coverage of 100X. Raw paired-end reads were analyzed by a PDX-specific bioinformatics pipeline to identify the human mutation profile. Sequenom Oncocarta and Sanger sequencing data acquired for 29 cancer genes in 272 PDX was used to evaluate the WES mutation profiles. In parallel, 92 PDX were profiled with RNA-Seq (100M sequencing reads required) and we investigated the expressed mutation profiles by comparing with mutations from WES data.

Among 502 point mutations found with classical methods, 95% were retrieved by WES analyses, revealing the very high sensitivity of the PDX-specific bioinformatics pipeline. 5% of mutations were missed because of a low coverage, particularly in the STK11 gene and in the KRAS gene of pancreatic models, possibly due to poor gene enrichment and high mouse stroma content, respectively. Deeper sequencing could potentially overcome this lack of coverage. Additionally, the WES analysis pipeline displays a high specificity, reporting only 1 additional mutation at gene positions covered with the classical methods. Finally, 507 mutations were detected by WES at positions not interrogated by classical methods emphasizing the necessity for next-generation sequencing (NGS) to obtain a comprehensive mutational spectrum. The number of mutations found using RNA-Seq data was on average two times lower and covered 15% of the mutations detected in WES. This was mainly due to the non-expression of genes or isoforms (40%), the mono-allelic expression of genes (30%), and low coverage data (15%). RNA-Seq analysis restricted to expressed genes represents a substantial complement to WES mutation data and enhances understanding of actual gene alterations in cancer cells.

This study demonstrated the high quality of mutation profiles obtained by WES and highlights the importance of integrating expression data to accurately predict the impact of a mutation at the protein level. An accurate molecular characterization of models is crucial for the selection of PDX with a specific genetic background for the evaluation of anticancer agents.

#2704

Radiotherapy and immunotherapy in cancer: A mathematical model.

Raphael Serre, Xavier Muracciole, Joseph Ciccolini, Sebastien Benzekry, Dominique Barbolosi. _SMARTc Unit, Aix Marseille Université, INSERM, CRO2 UMR_S 911, Marseille, France_.

The rise of immunotherapy is a major breakthrough in oncology. Recently, the combination of radiotherapy with the blockade of immune checkpoint inhibitors such as the PD1-PDL1 axis or the CTLA4 pathway has shown a synergistic potential: in addition to the direct cell kill induced by irradiation, radiotherapy unleashes neoantigens that can further induce an anti-tumoral immune response. However, this immune response can be inhibited by the immunosuppressive nature of the tumor micro-environment (TME). Hence, radiotherapy combined with immune check-point inhibitors is a promising solution and is the subject of preclinical and clinical research. However, defining the most efficient scheduling between radiotherapy and immunotherapy is a crucial issue that cannot be properly addressed solely by empirical trial-and-error practices. Consequently, developing mathematical models that can describe the synergy between immune checkpoint inhibitors and radiotherapy is critical. Hence, we have built a pharmocodynamic model of the combination of radiotherapy with inhibitors of the PD1-PDL1 axis and/or the CTLA4 pathway. We describe a mathematical representation of how a growing tumor first elicits and then inhibits an anti-tumoral immune response. This anti-tumoral immune response is described by a primary and a secondary response. The primary immune response appears first and is down-regulated by the PD1-PDL1 axis, while the secondary immune response happens next and is down-regulated by the CTLA4 pathway. We describe the effects of irradiation by a slightly modified version of the Linear-Quadratic model. In particular, we explain the biphasic relationship between the size of a tumor and its immunogenicity, as measured by the abscopal immune response. The ability of the model to forecast pharmacodynamic endpoints was retrospectively validated by reproducing results from experimental studies investigating radiotherapy and immune checkpoint inhibitors. This model clarifies the issue of synchronisation of immunotherapy with radiotherapy and it also explains why the CTLA4 blockade often occurs with a delay. The model also explains why a sustained response to immunotherapy may or may not happen after treatment discontinuation. We believe that this mathematical model could be further used as a simulation tool to help decision-makers determine the optimal scheduling between radiotherapy and immunotherapy and could be a building block for the in-silico design of optimized multimodal strategies.

#2705

A computational algorithm to predict tumor growth and cancer stem cell proportion in-vitro and in-vivo from single-cell observations.

Alexander T. Pearson, Patrick Ingram, Shoumei Bai, Euisik Yoon, Trachette Jackson, Ronald J. Buckanovich. _University of Michigan, Ann Arbor, MI_.

We have developed a new mathematical algorithm and corresponding computer software that uses observations of single cell behavior to predict the growth of the cancer stem-like cell proportion in larger cancer cell groups. Cancer stem-like cells (CSCs) have been implicated in ovarian cancer tumor growth, chemotherapy resistance, and disease recurrence. Aldehyde dehydrogenase (ALDH) is a primary discriminator of CSCs in ovarian cancer as well as in other cancer types. Unfortunately, the rarity and ability of CSCs to rapidly differentiate makes them difficult to study in-vitro and in-vivo. Microfluidic capture devices now allow us to grow and evaluate single ovarian cancer cells in isolated culture. We deployed microfluidic devices to evaluate the different self-renewal and asymmetric division patterns of ALDH+ and ALDH(-) ovarian CSCs. We analyzed data gathered from arrays of single cell chamber observations and found that purified ALDH+ cells were more proliferative than ALDH(-) cells in both cell-line (n = 112, p < 0.001) and primary (n = 41, p = 0.008) ovarian cancer specimens. Importantly, ALDH+ cells could produce both ALDH+ and ALDH(-) cells, whereas ALDH- cells were only able to produce ALDH- cells. Based on this hierarchy-defining data, we developed an easy to use computer algorithm on the freely-available software R to predict cancer cell population growth in-vitro and in-vivo. In our algorithm, size changes of cell populations are simulated by drawing from observed events. We compared the predictions from our hybrid microfluidics chip and computational algorithm with validation experiments in-vivo and in-vitro for both cell line and primary ovarian cancer cells. We found a superb correlation between observed and predicted in-vitro total and CSC counts for both cell line and primary ovarian cancer cells (correlation r = 0.98, p < 0.0001). Furthermore, this approach appropriately predicted changes in cell growth in the presence of the CSC-promoting growth factor EGFL6 both in vitro and in vivo, over time frames of up to 28 days. The single cell division based sampling algorithm developed here allows for rapid and inexpensive means to predict in-vivo ovarian tumor growth based on microfluidic chip culture and can easily be adapted to evaluate new therapeutic options across other cancer subtypes.

#2706

Thermodynamic and molecular orbital analysis of the effects caused by incorporation of novel anti-tumor agent Trifluridine to DNA.

Jun Koseki, Kenta Tsunekuni, Masamitsu Konno, Naohiro Nishida, Koichi Kawamoto, Yuichiro Doki, Masaki Mori, Hideshi Ishii. _Osaka University, Suita, Japan_.

Trifluridine (FTD), known as main component of TAS-102, is beginning to be applied as anti-tumor agent due to the highly efficacious antitumor potency although there is little to distinguish FTD structure from thymidine structure. TAS-102 has first been used in Japan in clinical therapy as an oral agent. Some experimental results hypothesized that FTD pharmacological antitumor effect can be arisen by inhibition of thymidylate synthase and incorporation of FTD itself into DNA. One of them is the thermal denaturation experiments for DNA duplexes of containing some FTD or not, performed by J. C. Markley et al. Their experiments have shown that the DNA duplexes containing FTD are slightly less stable, with a melting temperature about three degrees lower than not containing duplexes. However, the change of thermodynamic structure and inter-nucleobase interaction changes are not yet known.

In this presentation, we have performed molecular dynamics (MD) simulations and ab initio molecular orbital (MO) calculations to analyze the differences of thermodynamics structure and inter-nucleobase interaction between containing and not containing FTD. From these results, we sought to qualitatively understand the effects of FTD. At the first step, we have estimated the stabilization structures of same two DNA duplexes (containing FTD and not) used by J. C. Markley, with energy minimization. Then, a large differences were shown between these structures. The hydrogen bond breaking is observed between FTD and complementary Adenine base while the not containing DNA duplex keeps having a stabilized structure. The behavior means that the essential difference of intra-DNA interactions was arisen. At the second step, MD simulations were performed to analyze the distribution of distances between FTD and complementary base (i.e. thermally available conformations). With some conformations from MD sampling, the MO calculations were performed to discuss the difference of intra-base interactions.

From our results, we found that thermodynamic conformational change of DNA duplex containing FTD occurs even if it is in internal body temperature, and the break hydrogen bonding between FTD and paired base was observed more frequently. In addition, strong interactions between FTD and nearby π-conjugate base were observed. These behaviors might be one of the factor of inducing unstabilization in DNA duplexes.

#2707

Quantitative analysis of the role of androgen receptor in radiation treatment for prostate cancer.

Mengdi Qian,1 Alexandru Almasan,2 Evren Gurkan-Cavusoglu1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

The purpose of this study is to quantitatively investigate the mechanism of androgen receptor (AR) and non-homologous end joining (NHEJ) interaction after and ionizing radiation (IR) and androgen deprivation therapy (ADT) treatment. In most clinical treatment approaches, a combination of ADT and IR is used in order to improve prostate patients' overall survival probability. It is found that AR interacts with NHEJ pathway, a major double strand break (DSB) repair pathway. We have developed a mathematical model of the NHEJ pathway that is composed of a series of ordinary differential equations. The kinetic rate constants constitute the unknown parameters of the model and we have estimated these parameters using least square estimation. The data sets used in the parameter estimation were obtained from the literature and they were from both in vitro experiments as well as clinical data from prostate cancer patients. Both sets of data demonstrated the effects of AR on DSB repair by NHEJ. The in vitro data suggest that AR enhances DSB repair by inducing expression/activity of DNA-PKcs, which is a key protein in NHEJ. The clinical data show that the effect is through the first NHEJ protein complex that is recruited to DSB, Ku70/80. We have developed the models for IR treatment alone or in combination with ADT to determine which of these mechanisms can explain the behavior in both data sets simultaneously. We have carried out sensitivity analysis and identifiability tests on all the parameters from both models in order to determine the parameters that are reliably estimated. We have shown that when the rate constant for the initiation protein Ku70/80 is decreased to half in the IR+ADT case compared to the IR only case, the model outputs captured the observations in both experimental and clinical data. The rate constant for Ku70/80 is proved to be both sensitive and identifiable, indicating that the two-fold difference in the rate constants of IR only and IR+ADT cases is biologically relevant. Therefore, we conclude that when ADT is applied, AR significantly slows down the kinetic activity of Ku70/80, thus the DSB repair by NHEJ. The suppression of AR activity through ADT has the potential to enhance the IR treatment outcomes for the prostate cancer patients that are resistant to IR-only treatment.

#2708

Development of an individualized 3D transport model of topotecan for a patient-derived orthotopic xenograft model of pediatric neuroblastoma.

Abbas Shirinifard, Suresh Thiagarajan, Yogesh T. Patel, Abigail D. Davis, Megan O. Jacus, Stacy L. Throm, Jessica Roberts, Vinay Daryani, Clinton F. Stewart, András Sablauer. _St. Jude Children's Research Hospital, Memphis, TN_.

Resistance to chemotherapeutics and targeted therapies in pediatric solid tumors including neuroblastoma is a common cause of poor clinical outcome. These failures in part stem from shortcomings in understanding inter- and intra-tumor heterogeneities of drug penetration due to heterogeneities in blood perfusion. Herein we propose to develop an individualized 3D transport model of topotecan (TPT) for a patient-derived orthotopic xenograft model of pediatric NB5 neuroblastoma to account for inter- and intra-tumor heterogeneities in blood perfusion. The transport model uses a 3D reaction-diffusion equation to simulate diffusion of TPT from blood vessels into the tumor tissue and its flux in and out of intracellular space. Our transport model takes three types of inputs to predict TPT exposure maps defined over the volume of an individual tumor: a) plasma concentration-time profiles from an individualized physiologically-based pharmacokinetic (PBPK) model of TPT (separate cohort), b) 3D blood perfusion map of the individual tumor from contrast enhanced ultrasound (CEUS) using VisualSonics VEVO 2100 imaging system, and c) in vitro TPT cellular uptake and efflux kinetics from two-photon imaging. We use in vitro pharmacodynamics (PD) experiments with NB5 cells exposed to TPT to derive probabilistic PD-rules for drug effects (e.g., γ-H2AX response). Based on these rules and the exposure maps, we then compute probabilities of effects for the entire tumor volume. We will validate the predicted drug effect maps by comparing them to the observed effects measured by immunohistochemistry marker for γ-H2AX from the same tumor (location matched) using spatial correlation techniques.

#2709

Mathematical modeling for prediction of secondary BRCA1 and BRCA2 mutations in ovarian cancers with deleterious germline BRCA1 and BRCA2 mutations.

Dana-Adriana Botesteanu,1 Doron Levy,1 Jung-Min Lee2. 1 _Department of Mathematics and Center for Scientific Computation and Mathematical Modeling (CSCAMM), University of Maryland, College Park, MD;_ 2 _Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD_.

Background:

DNA damaging agents such as cisplatin or carboplatin and poly(ADP-ribose) polymerase inhibitors (PARPi) have clinical activity in germline BRCA1 and BRCA2 mutation-associated ovarian cancers. However, BRCA1 or BRCA2-mutated tumors often develop resistance to these drugs. Restoration of BRCA function due to secondary BRCA mutations has been recognized as one of the clinical resistance mechanisms. The aim of this mathematical analysis is to study the emergence and frequency of secondary BRCA mutations in a cohort of BRCA-deficient in silico ovarian cancer-positive cases.

Methods:

We develop a 2D stochastic cell-cycle specific mathematical model of BRCA-deficient ovarian cancer growth and progression prior to and post-treatment, which includes debulking surgery, platinum-based chemotherapy or PARPi. This is a continuum model based on a combination of ordinary differential equations using Gompertzian growth kinetics subject to random discrete-time jump processes. This approach enables us to reflect the inter-individual heterogeneity in disease progression and drug administration reported in published data. Calculations are performed for in silico cancer-positive growth curves in which the inception of the first ovarian malignant cell is assumed to have already occurred.

Results:

Our modeling approach generates an empirical cumulative distribution of disease-free survival times prior to recurrence or disease progression, given the heterogeneous cancer progression illustrated by our in silico cohort. Our preliminary computational results indicate that an initially rare resistant cell clone carrying a secondary BRCA mutation pre-exists to treatment in circa 90% of the simulated progression curves. For the remaining 10% of the simulated curves, it is more likely that the secondary mutations are acquired during chemotherapy or PARPi via increased mutation rates, possibly conferred by the increasing amount of damaged DNA lesions caused by the cancer treatment.

Conclusions:

Our mathematical model provides some insight on the dynamics of secondary BRCA mutations that cannot be accessed through existing genomic analysis methods, as current DNA sequencing depth thresholds cannot detect the presence of rare tumor cell clones carrying such mutations. We predict that a large proportion of initially resistant cell clones with restored BRCA function more likely originate prior to rather than post-treatment. If such clones get selected for by subsequent platinum-based chemotherapy or PARPi in a Darwinian fashion, our model generates an empirical distribution of disease-free survival times prior to recurrence. This may have direct implications on the possible duration of subsequent treatment response in our simulated ovarian cancer-positive population.

#2710

Model evolution technique as a novel concept for characterization of tumor heterogeneity in dynamic contrast enhanced MRI studies.

Hassan Bagher-Ebadian,1 Azimeh NV Dehkordi,2 Rasha Alamgharibi,3 David Nathanson,1 Hamid Soltanian-Zadeh,1 Arbab S. Ali,4 Stephen Brown,1 Meser M. Ali,1 Tom Mikkelsen,1 James R. Ewing1. 1 _Henry Ford Hospital, Detroit, MI;_ 2 _Shahid Beheshti University, Tehran, Islamic Republic of Iran;_ 3 _Oakland University, Rochester, MI;_ 4 _Georgia Regents University, Augusta, GA_.

Introduction: Many studies have shown that tumor vascular network and the assortment of tumorous cells inside and on the periphery of solid tumors are spatially heterogeneous. Variation in cell packing density (VCPD), hypoxia, acidosis, and elevated interstitial fluid pressure (IFP) are main characteristic features of solid tumors. Elevated IFP and VCPD in solid tumors can be generally relevant to the pathological structures at the cellular level that is fundamental to understanding the chance of response to treatment and recurrence. Therefore, non-invasive quantification of tumor heterogeneity for the same types of tumors can play an important role in diagnosis and treatment planning.

Hypothesis: In this pilot study, using Nested Model (NM) selection technique, Model Evolution (ME) concept is framed and introduced to quantify the evolutions of 3 different physiologically NM that are derived from standard Tofts model, throughout the course of Dynamic Contrast Enhanced (DCE) Magnetic Resonance Imaging (MRI) experiment. Using ME technique for pharmacokinetic (PK) modeling and DCE-MRI data analysis, a heterogeneity measure is formulated and introduced based on the evolutionary profile of the estimated extra-cellular extra-vascular (ve) volume. We hypothesized that the ME profiles in the course of DCE-MRI experiment, highly depend on the inward diffusion and outward convection of contrast agent concentration and contain abundant information for describing the compartmentalization and heterogeneity levels of solid tumors.

Material and Methods: 24 athymic Nude rats with U251n rat tumor model of cerebral tumor were studied. Look-Locker T1 mapping and DCE-MRI experiments (Dual Gradient Echo, 150 image sets at 4.0 sec intervals over 10 min: matrix = 128x64, five 2.0 mm slices, NE= 2, NA=1, TE/TE/TR = 2.0/4.0/40ms with bolus intravenous injection of the Magnevist at 0.25 mmol/kg) acquired at 7T field strengths. In each animal, in-vivo measurement of tumor IFP was done right after the DCE-MRI experiment using a wick-in-needle technique. The ME technique was applied on DCE-MR data of 24 U251n rat tumors to characterize the heterogeneity of each tumor and then the results were compared to their known in vivo measure of IFPs.

Results and Conclusions: Results of this pilot study clearly attest that the evolutionary profile of ve can be used to characterize the heterogeneity level of solid tumors. The ME results imply that as the slop of the evolutionary profile increases, the IFP of tumor increases. Also, the latency of the profile during the course of MR experiment can reliably explain the tumor compartmentalization and their elevated IFP. This pilot study confirms that the ME concept can make a paradigm shift in non-invasive quantification of tumor heterogeneity from DCE-MRI studies

#2711

Spatiotemporal progression patterns in metastatic lung cancer treated with bevacizumab.

Jeremy M. Mason, Paul K. Newton, Gino K. In, Sonia Lin, Peter Kuhn, Jorge J. Nieva. _University of Southern California, Los Angeles, CA_.

We describe a Markov chain mathematical model of lung cancer progression based on a longitudinal dataset of 722 lung cancer patients. Specifically, we investigate the patterns of metastatic spread in lung cancer and how the VEGF inhibitor, bevacizumab, alters these metastatic patterns. Stochastic models were used to simulate metastatic spread by means of random walk processes on directed graphs. We created spatiotemporal diagrams of cancer progression to analyze the differences between patients treated and not treated with bevacizumab. Patients with squamous lung cancer or brain metastases at baseline were ineligible for bevacizumab and excluded from analysis. Our results demonstrated that not only does bevacizumab extend survival, but it also alters patterns of metastatic spread. Patients treated with bevacizumab were characterized by greater heterogeneity in their pathways of metastatic progression, compared to those patients not treated with bevacizumab, which showed more homogeneity in their pathways. Furthermore, we quantify this spread and characterize metastatic sites as 'spreaders' and 'sponges' based on their probability of spreading the disease.

#2712

Joint somatic mutation and germline variant identification and scoring from tumor molecular profiling and ct-DNA monitoring of cancer patients by high-throughput sequencing.

Francisco M. De La Vega,1 Ryan T. Koehler,1 Yannick Pouliot,1 Yosr Bouhlal,1 Austin So,1 Federico Goodsaid,1 Sean Irvine,2 Len Trigg,2 Lincoln Nadauld3. 1 _TOMA Biosciences, Foster City, CA;_ 2 _Real Time Genomics, Hamilton, New Zealand;_ 3 _InterMountain Healthcare, Saint George, UT_.

Cancer tumor profiling by targeted resequencing of actionable cancer genes is rapidly becoming the standard approach for selecting targeted therapies and clinical trials in refractory cancer patients. In this clinical scenario, a tumor sample is obtained from an FFPE block and sequenced by targeted next-generation sequencing (NGS) to uncover actionable somatic mutations in relevant cancer genes. Some of the challenges that arise in analyzing tumor-derived NGS data include distinguishing between somatic and germline variants in the absence of normal tissue data, recognizing pathogenic germline variants, and identifying sequencing errors (which occur at about 0.5% rate). Additional challenges arise when considering other clinical applications of NGS such as sequencing cell-free tumor DNA (cf-DNA) from plasma samples to monitor disease response or disease recurrence. Here we present a principled approach to identify both single-nucleotide and small insertion/deletion somatic mutations and germline variants from NGS data of tumor tissue that leverages the allelic fraction patterns in tumors and prior information from external databases through the use of a Bayesian Network algorithm. Our approach allows us to score each putative mutation or variant with respect to its probability of belonging to each variant class, versus classification as a sequencing error. The method enables the joint calling of related samples form the same patient, such as cases where a cf-DNA sample and primary tumor sample are both profiled improving sensitivity and specificity. We validated our method by analyzing data obtained with the TOMA OS-Seq targeted sequencing RUO assay for 98 cancer genes from a mixture of well-known genomes, patient case triads (where normal, tumor and cf-DNA are available), and a retrospective analysis of tumor patient data that underwent clinical tumor profiling for therapy selection.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Cycle Control and Checkpoints

#2713

Combination of PLK1 and PI3K inhibitors shows strong synergy in anaplastic thyroid cancer.

Emrullah Yilmaz,1 Arturo Orlacchio,2 Antonio Di Cristofano2. 1 _Montefiore Medical Center, Bronx, NY;_ 2 _Albert Einstein College of Medicine, Bronx, NY_.

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer with median of 6 months of survival from diagnosis. Molecular alterations are well defined recently. p53 mutations, and activation of PI3K, RAS and BRAF are among the most common alterations. PLK1 overexpression has also been identified in ATC.

We have previously shown that PLK1 inhibitors are effective in ATC cell lines. However PLK1 inhibition in cell lines with PI3K activation resulted in escaping growth arrest and subsequent mitotic catastrophe through mitotic slippage. Since the PI3K activation is common in ATC, there is risk of generating polyploid, genetically unstable cell populations by targeting these tumors with a PLK1 inhibitor. So we tested the effect of combining PLK1 and PI3K inhibitors in ATC cell lines.

We combined PLK1 inhibitor BI6727 and PI3K inhibitor BKM120 and measured the cell viability using Alamar Blue. Combination of these two drugs resulted in significant synergy in mouse derived PTEN deleted ATC cell lines. We observed the same synergy in the human ATC cell lines with PIK3CA mutations.

BI6727 showed significant G2/M arrest in the cell lines. The combination two drugs enhanced the G2/M arrest. In a low dose that the single drugs showed no inhibition in cell cycle, combination resulted in G2/M arrest.

We treated the cells with a dual PI3K and PLK1 inhibitor Rigosertib. Although we have seen some sensitivity of the cells with PIK3CA mutation, the cell lines with PTEN were strongly resistant to Rigosertib.

Our results show that combination of PLK1 and PI3K inhibitors is an effective treatment for ATC cells with PI3K activation. This combination results in cell cycle arrest suggesting the role of using combination therapies in this aggressive cancer.

#2714

Inhibition of human telomerase by new purine analogs.

Wilnelly M. Hernandez-Sanchez,1 Diane Baus,1 Anthony J. Berdis,2 Derek J. Taylor1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Cleveland State University, Cleveland, OH_.

Telomeres are the end caps of linear chromosomes that help to ensure genomic stability. Because of the inability of DNA polymerases to synthesize the extreme ends of the lagging strand, telomeres get slightly shorter each time a cell divides. When telomeres get extremely short, a state of senescence is triggered which halts cell division. Some cells that require an extended lifespan (e.g. embryonic cells, germ lines and stem cells) use an enzyme called telomerase to maintain telomeres at a length that is sufficient to escape senescence. Whereas telomerase is undetectable, in adult somatic tissue, it is upregulated in 85-90% of all metastatic tumors; enabling cancer cells to divide indefinitely. Due to this unique property of cancer cells, targeting telomerase is a viable chemotherapeutic approach. Nucleotide analogs have been shown to inhibit telomerase activity (e.g. azidothymidine or AZT), albeit modestly, to promote telomere shortening in cancer cell lines. Also, most of these nucleotide analogs are limited by requirement of high dosage (e.g. The IC50 of AZT varies from 0.25 to 1.35 mM). Therefore, while promising, there is a need to develop other nucleotide analogs that are more potent in inhibiting telomerase activity. We have screened a series of new purines analogs using a direct in vitro telomerase assay. These nucleotide analogs were developed to have different chemical modifications around the indole group of the nucleobase. We have identified two purine analogs (5-MeCITP and 6-NITP) that inhibit telomerase activity in vitro. 5-MeCITP was measured to have an inhibition constant (ki) of ~45μM, which is significantly efficient. Future efforts will include evaluation of the lead nucleoside analogs in telomerase positive cancer cell lines to determine their effect on telomere length and cell survival.

#2715

Cell cycle regulated mitophagy: a key step at the G1/S metabolic checkpoint.

Daniela Annibali. _KU Leuven, Leuven, Belgium_.

How tumor cells coordinate their high demand for biosynthetic processes with nutrient availability, proliferation rate and oxidative stress tolerance is still not completely understood. We recently showed that highly proliferating cells, whether normal or tumorigenic, display different metabolic requirements throughout the cell cycle, in term of glucose and glutamine utilization. The role of autophagy in cancer cells is still debated, but recent evidence pointed to its involvement in the regulation of cellular bioenergetics and nutrients utilization. To investigate autophagy regulation during cancer cells proliferation and to characterize in detail the metabolic behaviour associated with tumor cell proliferation in the different phases of the cell cycle, we used synchronized HeLaS3, a human epithelial carcinoma cell line. Treatment of the cells with a double thymidine block (DTB) results in an arrest at the G1/S boundary and the possibility to study their progression through a complete division cycle. Here we show that in the highly proliferating HeLaS3 cancer cells basal autophagy is a cell cycle regulated phenomenon, responsible for mitochondria quality control and that it plays an essential role at the G1/S checkpoint. Both general autophagy genetic inhibition and mitophagy pharmacological inhibition with Mdivi-1 during cell cycle progression lead the cells to enter faster in S phase. However, probably due to the high levels of oxidative stress dictated by their proliferation rate, autophagy-incompetent cells start to accumulate insulted and less functional mitochondria. Autophagy is likely to be involved in the induction of resistance mechanisms to treatments in different types cancers. Because of this, there is an increased interest in the possibility to use inhibitors of the autophagic pathways as adjuvants for current anti-tumor therapies. Further investigations of the mechanisms here described will be of importance to elucidate whether drugs which inhibit autophagy may be a viable and effective approach in combination with chemotherapy for the treatment of highly proliferating cancers.

#2716

Lipid mediated "nutrients sensing" checkpoint in G1 phase of the cell cycle.

Deven Patel,1 David Foster2. 1 _The Graduate Center of CUNY, New York, NY;_ 2 _The Hunter College of CUNY, New York, NY_.

One of the hallmarks of cancers is dysregulation of G1 phase cell cycle progression- where cells decide their fate whether they should divide, arrest or undergo apoptosis. There is a growth factor-dependent Restriction point (R), where cells commit to replicate its genome or to enter a state of quiescence. Unicellular organisms only respond to the nutrients, a point known as START. In mammalian system, we have shown equivalent to START, "Cell Growth" checkpoints, where it senses Essential Amino Acids (EAAs), a "Conditionally" EAA glutamine (Q), and at last a checkpoint regulated by mTOR, which we think it senses nutrients and is the final arbitrator of the G1-S phase progression. mTOR- mammalian/mechanistic target of rapamycin integrates the growth factor signaling with nutrient sensing signaling and known to be involved in protein translation and proliferation. Another key nutrient needed for cell growth is lipids which is required as building blocks for plasma membrane synthesis and signaling molecules. Phosphatidic acid has been shown to be required for the activity of mTORC1 and mTORC2. In this study, we investigated the involvement of lipids in the cell cycle progression and how the signals are integrated. Upon lipid deprivation, normal fibroblasts cells arrest in G1-phase of the cell cycle. There is a distinct and distinguishable lipid sensing checkpoint from EAAs and Q. Temporally it is different from other nutrient sensing checkpoints in the late G1-phase of the cell cycle. In summary, there is a series of "nutrient sensing" checkpoints late in the G1-phase followed by mTOR "Growth" checkpoint. Dysregulation of these checkpoints in cancer cells may present better opportunity to target them with different therapeutic approach.

#2717

Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma.

Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Ogan Abaan, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sean R. Davis, Sven Bilke, Paul S. Meltzer. _NCI-CCR, Bethesda, MD_.

The unlimited proliferative capacity of cancer cells is closely linked to maintenance of their telomeres, which shorten with each cell division in normal cells. Cancer cells are able to maintain telomere length by multiple mechanisms, including activation of telomerase and the recombination based alternative lengthening of telomeres (ALT) pathway. ALT is prevalent in osteosarcoma, with approximately 50% of osteosarcoma cases using ALT for telomere maintenance. Mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 correlate with activation of the ALT pathway in several tumor systems. While loss of ATRX is a frequent event in osteosarcoma tumors, alterations of DAXX have not been reported. We characterized the telomere maintenance mechanisms utilized by 11 osteosarcoma cell lines. Of these, 45% (5/11) were ALT positive and 45% (5/11) were telomerase positive. One cell line possessed features of both telomere maintenance mechanisms. Among ALT positive osteosarcoma cell lines, we observed frequent loss of ATRX expression (4/5) and a previously unreported translocation resulting in disruption of DAXX. The translocation abolishes recruitment of DAXX to nuclear PML bodies and prevents normal DAXX function. By reintroducing full length DAXX, we were able to suppress telomere maintenance by ALT as evidenced by multiple assays including loss of C-circles and ALT-associated PML bodies, thus demonstrating that continued DAXX deficiency is necessary for maintenance of the ALT mechanism. Suppression of ALT by DAXX reintroduction did not result in compensatory activation of telomerase. This first demonstration of ALT suppression by DAXX supports a mechanistic connection between loss of the ATRX/DAXX chromatin remodeling complex and telomere maintenance by ALT. Understanding this relationship may uncover vulnerabilities specific to ALT tumors that could potentially lead to the development of targeted therapies for diverse cancers that depend on the ALT pathway.

#2718

Synthesis and characterization of novel benzylpyrazole-based BUB1 kinase inhibitors with anti-tumor activity.

Marion Hitchcock, Gerhard Siemeister, Hans Briem, Amaury Ernest Fernandez-Montalvan, Simon Holton, Anne Mengel, Ursula Mönning, Michael Brands, Karl Ziegelbauer, Dominik Mumberg, Franz von Nussbaum. _Bayer Pharma AG, Berlin, Germany_.

BUB1 (budding uninhibited by benzimidazoles 1) is a serine/threonine protein kinase. The protein is bound to kinetochores and plays a key role in the establishment of the mitotic spindle checkpoint and chromosome congression prior to anaphase.

Inhibition of BUB1 kinase represents a novel approach for cancer treatment: whereas cell cycle arrest is the predominant mode of action of a number of antimitotic cancer drugs (e.g. taxanes and vinca alkaloids), BUB1 inhibition results in aneuploidy and cell death by driving cells through mitosis irrespective of DNA damage and misattached chromosomes.

Here, we report the characterization of a novel benzylpyrazole lead-structure series inhibiting BUB1 exemplified by BAY-320, a novel, first-in-class small molecule inhibitor of BUB1 kinase. This structure class was initially discovered as a single hit in a high-throughput screen, and resulted in a lead compound by chemical optimization. Benzylpyrazole BAY-320 is highly selective for BUB1 with single digit nanomolar biochemical potency and single-digit micromolar cellular potency (HeLa proliferation assay). Synergistic effects can be observed when BUB1 inhibitor BAY-320 is combined with low doses of paclitaxel affecting chromosome segregation and cell proliferation. X-ray data of benzylpyrazoles allowed for a better understanding the binding mode for rational property design. Further data on structure-activity relationship including pharmacokinetic, drug metabolism and the synthesis of BAY-320 and analogues will be presented.

These results validate the benzylpyrazoles as novel selective BUB1 inhibitors and BUB1 as a promising approach for cancer treatment.

#2719

ADA3, a cell cycle regulator, regulates chromosome segregation.

Shashank Srivastava, Shakur Mohibi, June Wang-France, Sameer Mirza, Xiangshan Zhao, Hamid Band, Vimla Bnad. _University of Nebraska Medical Center, Omaha, NE_.

The RNA polymerase II mediated transcription requires the higher degree structure of chromatin to be relieved so that general transcription machinery can access the DNA. The acetylation of DNA bound histones at specific loci is the major epigenetic modification by which opening of chromatin is achieved. HATs (Histone Acetyl Transferase) are the enzymes that catalyze the acetylation of histones and require association with mediator proteins for their function. One such mediator protein is ADA3 (Alteration/Deficiency in Activation 3), which was initially discovered as a component of multi-protein complex that contains either GCN5 (General Control Non-repressed 5) or PCAF (p300/CBP Associated Factor) as HAT and subsequent studies showed that ADA3 associates with another HAT, p300. As a HAT interacting protein, ADA3 enhances the acetylation of histones as well as non-histone proteins, such as p53.

We have recently shown that ADA3 is a cell cycle regulatory protein and is important for both G1 to S phase transition as well as mitosis. Conditional deletion of Ada3 from Ada3FL/FL MEFs (Mouse Embryonic Fibroblasts) causes cell cycle arrest and severe mitotic defects. To further explore the mechanism of ADA3 mediated mitosis, we performed ChIP-seq analyses and found that ADA3 bound to higher order repeat region of the centromere across most of the chromosomes. Further studies showed ADA3 interaction with centromere was mediated by a centromeric protein CENP-B. More importantly, siRNA mediated knockdown of ADA3 decreases the occupancy of CENP-B at centromere and causes chromosomal segregation defects during mitosis. These studies demonstrate a novel function of ADA3 in cell cycle regulation. Our current studies are focused on defining the exact mechanism of recruitment of ADA3 complex to CENP-B to regulate mitosis and its effect on genomic stability.

#2721

Discovery and development of orally available subnanomolar potent checkpoint kinase 1 inhibitors as potential anticancer therapies.

Alex C. Vo,1 Janelle Taylor,1 Robert Rosler,1 Dina Leviten,1 Teresa Sierra,1 Richard Boyce,2 Robert Boyle,2 Scott Peterson,1 Kevin Klucher1. 1 _Oncothyreon, Seattle, WA;_ 2 _Sentinel Oncology, Cambridge, United Kingdom_.

Checkpoint kinase 1 (Chk1) is a serine/threonine protein kinase that regulates cell division by arresting progression in the S & G2 phases of the cell cycle in response to genotoxic stress. Pharmacological inhibition of Chk1 selectively uncouples the completion of DNA replication from G2/M phase transition in tumor cells lacking parallel cell cycle checkpoint controls, resulting in mitotic catastrophe and cell death. These properties make Chk1 inhibition a unique therapeutic approach as a single agent or as a means to enhance the efficacy of DNA-targeted chemotherapeutic drugs.

In this report, we describe our progress in the development of orally bioavailable, potent and selective Chk1 inhibitors derived from an aminopyrazole chemical scaffold. Starting with the lead compound, ONT-2409 (IC50 of <0.1 nM against the Chk1 enzyme), a series of Chk1 inhibitors have been developed. Lead candidates have been generated with potent biochemical IC50s against Chk1 (IC50s =0.1-0.3 nM). In a panel of 125 protein kinases the top candidates were shown to be highly selective for Chk1, with little appreciable activity against Chk2 or other kinases involved in cell cycle control/DNA damage response. The lead candidates have potent single agent activity against a panel of diverse cancer cell lines (EC50s=0.03-0.4 µM) and show synergistic enhancement in the activity of clinically relevant DNA targeted chemotherapeutics. Mechanistically, the lead candidates abrogate gemcitabine-induced cell cycle arrest and induce apoptotic cell death. Biomarker studies show the lead candidates inhibit gemcitabine-induced auto-phosphorylation of Chk1 at S296 and reduce phosphorylation of CDK1 at Y15. Compared with ONT-2409, the optimized candidates display improved cell permeability/efflux ratios, superior intrinsic microsomal and hepatic half-lives, and have excellent oral bioavailability in mice and rats. Lead candidate Chk1 inhibitors have also demonstrated potent potentiation of DNA targeted chemotherapies and single agent activity in xenograft tumor models

#2722

Reviving kinesin-5 inhibitors: Evidence for a powerful combinatorial strategy.

Emma G. Sturgill, Ryoma Ohi. _Vanderbilt University, Nashville, TN_.

Mitosis serves as a powerful opportunity for therapeutic intervention in the treatment of neoplastic diseases like cancer. Anti-mitotic pharmacological agents tame the proliferative capacity of tumor cells by interfering with proper function of the mitotic spindle. Several next generation anti-mitotics target kinesins involved in assembling the mitotic spindle. Of these mitotic kinesins, the kinesin-5 Eg5 (also known as KSP) is perhaps the most compelling because of its primacy during spindle assembly in animal cells. Indeed, Eg5-inhibitors (K5Is) induce a potent and lethal mitotic arrest in cell culture and murine xenografts; however, K5Is failed to induce tumor regression during Phase II trials. This devastating clinical performance has halted further development of K5I monotherapies and motivated investigation into the underlying reason(s) for K5I inefficacy.

We previously showed that tumor cells in culture are capable of acquiring K5I resistance by employing parallel spindle assembly pathways. By studying a limited number of K5I-resistant cell lines, we showed that resistance involved the kinesin-12 Kif15; however, the generality of this finding was unclear. In this study we capitalize on the latest methodologies in targeted genome editing to study mechanisms of K5I resistance on a broader cell population level. We find that cells require Kif15 in all cases of K5I resistance, and that rescue is sensitive to the timing and extent of Kif15 expression. We propose that K5Is steer cells into an evolutionary bottleneck that requires Kif15, and that adaptive changes occur during passage through the bottleneck that enable populations to recover and diversify. These findings prompt a search for pharmacological inhibitors of Kif15 to test in combination with K5Is as an anti-mitotic chemotherapeutic regimen.

#2723

The synergistic efficacy of Chk1/Chk2 inhibitors and doxorubicin in the treatment of acute lymphoblastic leukemia.

Andrea Ghelli Luserna di Rora, Ilaria Iacobucci, Enrica Imbrogno, Enrico Derenzini, Anna Ferrari, Valentina Robustelli, Viviana Guadagnuolo, Cristina Papayannidis, Maria Chiara Abbenante, Sandro Grilli, Giovanni Martinelli. _University of Bologna, Bologna, Italy_.

Different Checkpoint kinase inhibitors (Chk-i) have been developed to increase the cytotoxic effect of genotoxic agents inhibiting the key elements of the DNA damage response (DDR) pathways. Our group has already showed the efficacy of this class of compounds in single agent in different in vitro/ex vivo/in vivo studies for the treatment of acute lymphoblastic leukemia (ALL). The aim of the study was to evaluate the efficacy of a Chk1/Chk2 inhibitor in combination with the topoisomerase II inhibitor doxorubicin for the treatment of ALL. Firstly we evaluate the efficacy of doxorubicin on human B (NALM-6, NALM-19 and REH) and T (MOLT-4, RPMI-8402 and CEM) ALL cell lines in term of reduction of the cell viability, modification of cell cycle profile and activation of the DDR pathways. Cells were treated with doxorubicin (0.25-2.5 uM) for 24 and 48 hours and the reduction of the cell viability was quantified using WST-1 reagents. In all the cell lines treated the cytotoxic effect of doxorubicin was time and dose dependent. The induction of the apoptosis (Pi/Annexin V) and the effect on cell cycle profile (Pi staining) was also evaluated in all the cell lines. Due to the inhibitory effect of the compound on the topoisomerase II enzyme and due to the activation of the cell cycle checkpoint, cells were arrested in G2/M phase. Then the effectiveness of the Chk-i as a chemo-sensitizer agent was evaluated. Different cell lines were treated with doxorubicin (5, 10, 25 and 50 nM for the more sensitive cell lines; 50, 100, 250 and 500 nM for the less sensitive cell lines) in combination with the Chk-i (2, 5 and 10 nM) for 24 and 48 hours. The combination showed a synergistic effect in term of reduction of the cell viability and induction of apoptosis. The effect of the combination was also analyzed using western blot looking for specific marker of activation of the DDR pathway showing the same synergistic effect. Moreover the effect of the combination on cell cycle profile was evaluated using a double staining Pi/Anti-phospho-Histone H3 ser10 (marker of mitosis). Cell lines were pre-treated for 18 hours with doxorubicin and then with the Chk-i for different time points (1, 2, 3, 6 and 9 hours). The treatment with Chk-i removed the G2/M arrest induced by the pre-treatment with doxorubicin, progressively reducing the number of cells in G2/M phase, increasing the percentage of cells positive for the mitotic marker p-HH3 (ser10) and increasing the percentage of cells in sub-G1 phase. In our opinion the combination between the Chk1 inhibitor,LY2606368, and the topoisomerase II inhibitor, doxorubicin, could be a promising strategy for the treatment of B/T-ALL.

Supported by ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project.

#2724

Collaborative regulation of cell cycle progression by Chk1 and E2F1 in small cell lung cancer cells.

Wei-Hsun Hsu, In-Gyu Kim, Guanhua Rao, Justine McCutcheon, Shuo-Tse Hsu, Yu-Wen Zhang, Giuseppe Giaccone. _Georgetown University, Washington, DC_.

Introduction

Small cell lung cancer (SCLC) harbors very frequent mutations in p53 and Rb, key cell cycle regulators in normal cells. In absence of such tumor suppressors, SCLC cells rely on Chk1 for cell cycle arrest in the event of DNA damage. High expression of E2F1 and Chk1 have been found in SCLCs, which is associated with poor prognosis in various solid tumors. In human SCLC cell lines, Chk1 inhibition sensitizes chemotherapy agents. The interaction between Chk1 and E2F1 is understudied. Here, we report a collaborative role of Chk1 and E2F1 in the cell cycle regulation in SCLC cells.

Methods

Three Rb-mutated human SCLC cell lines (GLC4, NCI-H128, and NCI-H209) were used. GLC4 carries mutant p53; H209 and H128 are wild type. Chk1 inhibition was achieved by either siRNA knockdown or LY2940930; E2F1 was knocked down by siRNA. Ectopic overexpression of Chk1 or E2F1 was achieved using Lonza electroporation kit. Cell viability was measured by Cell-Titer Glo assay. Cell cycle analysis was assayed by PI staining and FACS. Western blotting was used to evaluate caspase activation and other signaling proteins.

Results

Chk1 inhibition by siRNA knockdown or LY2940930 treatment enhanced cisplatin cytotoxicity in SCLC cell lines, regardless of p53 status. Also, a significant decrease of E2F1 protein was observed following Chk1 inhibition in these cells. Knockdown of E2F1 by siRNA enhanced the cytotoxic activity of cisplatin in the GLC4 and H209 cells. In GLC4 cells, Chk1 siRNA combined with cisplatin treatment caused activation of caspase-2 and caspase-3 as well as the cleavage of BID. Interestingly, knocking down caspase-2 reversed caspase-3 activation resulting from the combination of Chk1 siRNA and cisplatin, and restored the level of E2F1 protein. In contrast, ectopic overexpression of Chk1 resulted in an increase of E2F1, while increase of Chk1 expression was also noticeable upon E2F1 overexpression. It has been shown that Chk1 inhibition can abrogate cell cycle arrest and increase phospho-histone H3 expression (mitotic marker). We found that E2F1-overexpressing GLC4 and H128 cells displayed higher levels of phospho-histone H3 when treated with LY2940930, compared to that of Mock-transfected controls. Furthermore, exogenous E2F1 overexpression significantly reduced the IC50 of cisplatin (1331nM vs. 3698nM; p=0.0028 by paired t-test) in GLC4 cells, but not that of LY2940930 (14nM vs. 19nM; p=0.25).

Conclusions

Either downregulation of Chk1 or overexpression of E2F1 leads to cell cycle progression, resulting in more cell death when combined with DNA damaging agents. Decrease of E2F1 by Chk1 inhibition might represent a mechanism of self-protection to avoid excessive mitosis after DNA damage, and this regulation is likely involving caspase-2. Further exploration of Chk1 and E2F1 regulatory mechanisms is warranted in SCLC.

#2725

Probing mitotic functions of BUB1 kinase using the small molecule inhibitors BAY-320 and BAY-524.

Anna P. Baron,1 Conrad von Schubert,1 Fabien Cubizolles,1 Gerhard Siemeister,2 Marion Hitchcock,2 Anne Mengel,2 Jens Schröder,2 Amaury Fernández-Montalván,2 Martin Lange,2 Franz von Nussbaum,2 Dominik Mumberg,2 Erich Nigg1. 1 _Biozentrum, University of Basel, Basel, Switzerland;_ 2 _Bayer Pharma AG, Berlin, Germany_.

The maintenance of correct chromosome number (euploidy) during cell division is ensured by a highly conserved surveillance mechanism termed 'spindle assembly checkpoint' which safeguards correct chromosome segregation by delaying anaphase onset until all chromosomes are properly bi-oriented on the spindle apparatus. The mitotic kinase BUB1 (budding uninhibited by benzimidazoles 1) was reported to contribute to both chromosome congression and checkpoint function, yet the role of BUB1 catalytic activity in these processes remains a matter of debate.

To differentiate between catalytic and non-catalytic functions of BUB1 we compared phenotypes provoked by BUB1 protein depletion with specific BUB1 kinase inhibition using two novel small molecule inhibitors of BUB1, termed BAY-320 and BAY-524.

BAY-320 and BAY-524 were highly potent and selective ATP-competitive inhibitors of BUB1 kinase activity with IC50 values in the single digit nanomolar range (at 10 micromolar ATP concentration). By monitoring phosphorylation of Thr120 in histone H2A, we showed that both compounds acted as potent BUB1 kinase inhibitors both biochemically and in human cells. We found that BUB1 inhibition substantially altered the chromosomal association of Shugoshin and the chromosomal passenger complex without major effects on global Aurora B function. Consequently, inhibition of BUB1 kinase clearly impaired chromosome arm resolution but, in stark contrast to depletion of BUB1 protein, only had a minor effect on cell cycle and SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of paclitaxel, synergistically affecting chromosome segregation and cell proliferation.

These findings are highly relevant to both our understanding of BUB1 kinase function during mitosis and the prospects of BUB1 as a target of anti-cancer therapies. In this regard, BAY-320 and BAY-524 are first-in-class inhibitors of BUB1 kinase and their potential utility as anti-cancer agents is being explored.

#2726

WWP2 is required for normal cell cycle progression.

Byeong Hyeok Choi,1 Xun Che,1 Changyan Chen,2 Luo Lu,3 Wei Dai1. 1 _Department of Environmental Medicine, New York University Langone Medical Center, Tuxedo Park, NY;_ 2 _Center for Drug Discovery, Northeastern University, Boston, MA;_ 3 _Division of Molecular Medicine, Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Torrance, CA_.

WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of AktS473, a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression.

#2727

LOX is a novel mitotic spindle-associated protein essential for mitosis.

Myriem Boufraqech, Darmood Wei, Urbain Weyemi, Lisa Zhang, Martha Quezado, Petr Kalab, Electron Kebebew. _National Cancer Institute, Bethesda, MD_.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices. LOX is highly expressed in aggressive cancers; has been associated with a shorter survival and regulates cancer progression in a variety of human malignancies. Here, we report a new function of LOX in mitosis in anaplastic thyroid cancer cell line (THJ-16T), breast cancer cell line (MDA-MB231) and HeLa cells. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression suggesting that LOX binds to stabilized microtubules only. Functional studies of LOX depletion resulted in decreased cellular proliferation, G2/M cell cycle arrest, and formation of gigantic nuclei. Further, LOX knockdown led to reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B expression. Lastly, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules and this effect was synergistic. Our results show identify a novel function of LOX in cancer cell mitosis, and that loss of LOX expression in cancer cells can enhanced effects of anti-microtubules agents used in cancer therapy.

#2728

In silico identification of a MAD2-interacting motif in MAD2 spliced isoforms suggest a functional interaction with the spindle assemble checkpoint in cancer.

Miguel Ramirez-Otero, Alejandro Lopez-Saavedra, Marco Andonegui, Jose Diaz-Chavez, Luis Alonso Herrera. _Inst. Nacional de Cancerología, Mexico, Mexico_.

Abnormal chromosome segregation plays a key role in cancer development. MAD2 is a component of the spindle assembly checkpoint (SAC), a cell cycle control mechanism that ensures an accurate segregation of chromosomes during mitosis. Changes in MAD2 expression have been associated with chemo-resistance both to spindle inhibitors and to DNA damaging agents. Also, a previous study has shown that the exogenous expression of MAD2β, a splicing variant of MAD2, was associated with resistance to Adriamycin and Vincristine in gastric cell lines. Additionally, we have previously identified that exogenous overexpression of MAD2γ upon paclitaxel-induced SAC activation in the colorectal cancer cell HCT116, reduces drug-induced mitotic arrest. These findings suggested a possible structural interaction of MAD2 isoforms with SAC components. To determine possible structural interactions between MAD2 isoforms and key SAC components (i.e. MAD1 and CDC20), we performed an in silico analysis of MAD2 isoforms, Interestingly, we found that alternative splicing of MAD2 generates a premature stop codon and a frameshift in exon 4 in MAD2γ and MAD2β. This change generates a new C-terminal region in MAD2γ and MAD2β isoforms that comprise 16 amino acids, which are not present in the major isoform (MAD2α). We aligned this region with the amino acid sequence of CDC20 from various species and identified a MAD2-interacting motif (MIM). This finding suggests that MAD2 isoforms may interact with the active conformation of MAD2 (C-MAD2). Since MAD2 isoforms and CDC20 may compete for the same region in MAD2, we propose a new model whereby MAD2 isoforms inhibits SAC by interfering with C-MAD2/CDC20 formation. This model helps to explain previous results where MAD2 isoforms over expression seem to have an opposite role in SAC signaling.

#2729

Estrogen receptor-negative breast cancer cell lines exhibit hypersensitivity to the CHK1 inhibitor LY2606368.

Jean-Bernard Lazaro, Kalindi Parmar, Geoffrey I. Shapiro, Alan D. D'Andrea. _Dana-Farber Cancer Inst., Boston, MA_.

Checkpoint kinase 1 (CHK1) inhibitors are currently under investigation as chemopotentiating agents due to the role of CHK1 in establishing DNA damage checkpoints in the cell cycle. A novel CHK1/CHK2 inhibitor, LY2606368, as a single agent causes replication catastrophe, DNA double strand breaks and apoptosis (King C et al. Mol Cancer Ther. 2015). Accordingly, LY2606368 is currently in clinical development as a single agent and in combination with both cytotoxic and targeted agents. As subsets of breast cancers exhibit genomic instability and DNA repair deficiencies, we assessed the effect of LY2606368 as a single agent on breast cancer cell lines. We characterized the IC50's for growth inhibition by LY2606368 of a large panel of cell lines assembled on the basis of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression status. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is pathologically negative for expression of ER/PR and HER2 protein. As previously reported for other CHK1 inhibitors (Bryant C et al. BMC Cancer. 2014; Albiges L et al. Breast. 2014), TNBC cell lines exhibited high sensitivity to LY2606368. Interestingly, hypersensitivity to LY2606368 was observed in ER-negative cells, regardless of HER2 status. In addition, ER-positive cells were comparatively resistant suggesting that high sensitivity to LY2606368 occurs in the absence of ER and is not restricted to TNBC. No correlation was found between TP53 mutational status and sensitivity to LY2606368. Consistent with the observed hypersensitivity, ER-negative cell lines exposed to LY2606368 exhibited high levels of γH2AX and phospho-Ser1981-ATM demonstrating appearance of DNA double strand breaks. The concomitant appearance of phospho-Ser345-Chk1 marked aborted checkpoint activation by upstream detector/effector kinases (e.g. ATR, ATM). ER-positive cells did not engage in initiation of the DNA damage response or significant checkpoint activation when exposed to comparable doses of LY2606368. Collectively, these results suggest that the CHK1 inhibitor LY2606368 is likely to be more cytotoxic in ER-negative breast cancer types. Homologous recombination and other DNA repair deficiencies can explain the need for CHK1-dependent checkpoint activation during each replication cycle. On the other hand, ER-positive breast cancers might be less sensitive to monotherapy because of the absence of unresolved DNA damaging events that create checkpoint dependency.

In summary, triple negative status does not directly determine the sensitivity of breast cancer cell lines to the CHK1 inhibitor LY2606368, although TNBC cells are in general hypersensitive. The absence of ER might be a more promising marker of sensitivity to LY2606368 as a monotherapy in breast cancer.

#2730

**Association of** TERT **promoter mutations with telomerase expression in melanoma.**

Seungjae Lee,1 Patricia Opresko,2 Alberto Pappo,1 John Kirkwood,3 Armita Bahrami1. 1 _St. Jude Children's Research Hospital, Memphis, TN;_ 2 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 3 _University of Pittsburgh Medical Center, Pittsburgh, PA_.

Telomerase reverse transcriptase (TERT) promoter mutations occur at a high frequency in melanoma, but the functional consequences of these mutations in melanoma remain to be clarified. In a large study of melanoma samples by The Cancer Genome Atlas network, mRNA expression analysis using RNA sequencing data showed that only TERT promoter mutations at C228T were associated with high TERT expression levels. The effect of hotspot C250 or C242T/C243T tandem mutations on telomerase expression has not yet been determined. To assess telomerase expression levels in melanomas harboring C250 or C242T/C243T mutations, we used real-time quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in a sample set of pediatric melanocytic tumors for which the methylation status and the mutational profile of the TERT promoter were known. We previously showed that in a subset of melanoma, TERT expression is mediated epigenetically by promoter hypermethylation rather than by promoter point mutations. Formalin-fixed paraffin-embedded (FFPE) tissues from 10 metastatic melanomas, of which 8 harbored a hotspot TERT promoter mutation (5 C250T; 2 C228; 1 C242T/C243T) and 2 carried a hypermethylated wild-type TERT promoter were examined. For controls, 9 atypical Spitz tumors with a wild-type unmethylated TERT promoter were also studied. Total RNA was isolated from FFPE tumor tissues and converted to cDNA. TERT expression levels were determined by RT-qPCR by using the TaqMan® Gene Expression Assay and normalized with GAPDH as the endogenous control. In the atypical Spitz tumor samples, TERT mRNA expression was undetectable or negligible. In contrast, TERT expression in all melanoma samples was elevated, ranging from 3- to 71-fold (median, 25-fold) when compared to an atypical Spitz tumor as a reference. Our data support that in melanoma, TERT promoter hotspot C250 and C242T/C243T mutations, similar to C228T mutations, correlate with TERT overexpression and that C250 and C242T/C243T mutations likely contribute biologically to tumorigenesis in melanoma.

#2731

Impact of hypomethylating agents on hTERT expression and synergistic effect in combination with imetelstat, a telomerase inhibitor, in AML cell lines.

Joshua Rusbuldt, Jacqueline Bussolari, Aleksandra Rizo, Fei Huang. _Janssen Research & Development, LLC, Spring House, PA_.

The catalytic subunit of human telomerase (hTERT) is highly expressed in acute myeloid leukemia (AML). Transcriptional factors and epigenetic modifications regulate this expression. Studies suggest hypermethylation promotes hTERT overexpression. Imetelstat is a competitive telomerase inhibitor. Decitabine (DAC) and azacitidine (AZA) are DNA methyltransferase inhibitors (DNMTIs) used in the treatment of AML. Combining imetelstat and DNMTI may be an improved treatment option for AML. We investigated effects of DAC and AZA on hTERT expression and cell viability in AML cell lines OCI-AML3 and OCI-AML5, and evaluated growth inhibition when combined with imetelstat. OCI-AML3 had higher baseline expression of hTERT compared with OCI-AML5. Dose-dependent downregulation of hTERT expression was observed with DAC and AZA treatments. OCI-AML5 was more sensitive to DAC (1 μM, 46% viability) than OCI-AML3 (1 μM, 95% viability) whereas sensitivity to AZA was similar in both cell lines (1 μM, 29% vs 33% viability). After removal of the drugs, growth inhibition was not sustained and cell proliferation rebounded. Single-agent imetelstat reduced both hTERT expression and telomerase activity but resulted in only limited growth inhibition in these cell lines. It was hypothesized that combining DNMTI with imetelstat may exert more potent growth inhibition. To test this, both cell lines were treated with DAC or AZA for 72 hours followed by different concentrations of imetelstat for 2 to 4 weeks. Dose-dependent synergistic activity was demonstrated for DAC followed by imetelstat: OCI-AML3 cell viability changed from 95% with DAC to 61% and 10% after 2 and 4 weeks of imetelstat, respectively; OCI-AML5 cell viability changed from 46% with DAC to 6% and 1% after 2 and 4 weeks of imetelstat, respectively. In contrast, no enhanced activity was observed in either cell line with AZA followed by imetelstat, but imetelstat was able to slow the growth rebound after AZA removal. Additional studies are ongoing to further understand the mechanism of action of this drug combination in AML and to evaluate different combination sequences of DNMTI with imetelstat for optimal anticancer activity. In summary, DAC and AZA reduced hTERT expression and differentially inhibited cell viability in AML cell lines. Although at the same concentration AZA was more active than DAC in OCI-AML3 and OCI-AML5 lines, the sustainability of growth inhibition after washout differed. Growth rebounded after removal of AZA or DAC treatment. Dose-dependent synergistic activity demonstrated by DAC followed by imetelstat was not seen with AZA followed by imetelstat. Distinctly different mechanisms of action of DAC and AZA may underlie their differential activity when combined with imetelstat, warranting further investigation.

#2732

Myelosuppression in patients treated with the telomerase inhibitor imetelstat is not mediated through activation of toll-like receptors.

Gabriela M. Baerlocher,1 Joshua Rusbuldt,2 Fei Huang,2 Jacqueline Bussolari2. 1 _Inselspital/University Hospital of Bern, CH-3010 Bern, Switzerland;_ 2 _Janssen Research & Development, LLC, Spring House, PA_.

Imetelstat is a covalently lipidated 13-mer N3′-P5′ thio-phosphoramidate (NPS) oligonucleotide (5′-TAGGGTTAGACAA-3′) that is complementary to the RNA template region (hTR; 3′-AUCCCAACCUGUU-5′) of telomerase. Imetelstat is believed to act as a direct competitive inhibitor of telomerase enzyme activity and to not have an antisense mechanism of action (MOA) typical of other oligonucleotides. The purpose of this study was to assess whether the thrombocytopenia observed in some patients with myeloproliferative neoplasm treated with imetelstat can be attributed to activation of toll-like receptors (TLRs) by imetelstat. Imetelstat and related control oligonucleotides (Mis-match and Sense) were tested using 7 different HEK-Blue™ TLR cell lines, each stably coexpressing a human TLR gene (TLR2, 3, 4, 5, 7, 8, and 9) and a NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. After 20 hours' incubation, the cells were measured for absorbance at 650 nm for SEAP activity. Each TLR experiment was performed twice, with 3 replicates per experiment. The results showed that the positive control for each individual TLR was recognized by the corresponding TLR, leading to a strong SEAP activity, whereas imetelstat at a clinically relevant concentration range (10 µM-60 µM) and the control oligonucleotides related to imetelstat yielded no induction of SEAP activity (similar level to untreated). These data demonstrate that imetelstat has no stimulatory effect on the majority of the tested TLRs. Imetelstat yielded slightly higher induction on TLR8 than the untreated control, but significantly lower than the positive control. Although thrombocytopenia has been observed in patients with myeloproliferative neoplasms treated with imetelstat, these data show that it cannot result from imetelstat acting as a ligand for TLRs. TLR9, associated with thrombocytopenia, was not activated by imetelstat and the slight activity of imetelstat on TLR8 is likely irrelevant as no link between TLR8 activation and thrombocytopenia has been reported. These findings are consistent with 13-mer sequence of imetelstat, differing from oligonucleotides known to stimulate TLR9, which comprise 2 CpG islands separated by 6-10 nucleotides and are longer than 21 nucleotides. Ex vivo studies have provided evidence that imetelstat may induce thrombocytopenia in patients with myeloproliferative neoplasms by blocking the terminal maturation of normal megakaryocyte precursors: megakaryocytic differentiation requires upregulation of telomerase activity which is inhibited by imetelstat. Correct understanding of the MOA of imetelstat is vital to guide its clinical use and strategies to mitigate thrombocytopenia in patients with myeloproliferative neoplasms.

#2733

Novel function of the RAI2 protein in genomic integrity of breast cancer cells.

Stefan Werner, Kerstin Borgmann, Klaus Pantel, Harriet Wikman. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

We have recently identified the RAI2 protein as putative metastasis-suppressor related to dedifferentiation, early occurring bone metastasis formation, and survival of hormone dependent breast carcinomas. Nevertheless, low RAI2 expression is also predictive for poor patient outcome in hormone independent tumors and is furthermore associated with mutation of p53 in primary breast tumors. Thus, besides sustaining luminal differentiation, RAI2 might possess a general function as tumor suppressor that is, as indicated by association with p53 status, possibly related to maintenance of genomic stability.

In order to characterize its potential role in genomic integrity, we screened lysates from RAI2 depleted MCF-7 and KPL-1 breast cancer cell lines with phosphorylation-specific antibody arrays. Additionally, we analyzed changes in expression of cell cycle related genes and proteins by qPCR and Western Blot analysis. We also conducted cytogenetic assays to investigate whether RAI2 depletion affects chromosomal stability and mitotic progression. Finally, we evaluated a possible association of RAI2 expression with aneuploidy in published expression data sets from breast cancer patients.

As revealed by antibody array analysis, RAI2 depletion causes activation of p53 protein in MCF-7 and KPL-1 breast cancer cells, which was accompanied by a significant increase of senescent cells in the corresponding cell populations. Furthermore RAI2 depletion causes downregulation of several key factors of G2/M transition, like Aurora kinases A and B as well as Cyclins A2, B1 and B2. Concomitantly, we found in RAI2-depleted cell cultures reduction of mitotic index, increase of unattached chromosomes in metaphase and lagging chromosomes and during anaphase. This finding was further approved by an increase of numerical aberrations in RAI2 knockdown cells. Finally, we could show an association between low RAI2 expression and higher level of chromosomal instability in primary tumors from breast cancer patients.

In conclusion, we found that loss of RAI2 function is associated with decreased mitotic fidelity. Thus, besides sustaining differentiation in hormone dependent breast tumor, RAI2 also acts as general tumor suppressor that maintains genomic integrity.

Reference: Werner S et al., Cancer Discovery. 2015 May;5(5):506-19

#2734

Click colorimetric EdU proliferation and TUNEL assay: Conversion of click chemistry from fluorescent to colorimetric for use with bright field microscopy.

Scott T. Clarke,1 Carmen Finnessy,1 Fernada M. Bosada,2 Ben Armstrong,2 Michael O'Grady1. 1 _Thermo Fisher Scientific., Eugene, OR;_ 2 _University of Oregon, Eugene, OR_.

We present here the conversion of click chemistry fluorescence-based EdU proliferation assay and TUNEL apoptosis assay to use with bright field microscopy. Advantages of having a colorimetric signal are the retention of the click chemistry work flow while allowing for compatibility with standard H&E staining and protocols.

Proliferation using copper-catalyzed labeling of the thymidine analog EdU (ethynyl deoxyuridine) was introduced in 2007. This click-based assay has advantages over the traditional antibody based BrdU assay. The small molecule detection is simple, rapid and gives a bright signal useful for multiplexing. Additionally, recent improvements to the chemistry allow for GFP and R-PE compatibly with the EdU and TUNEL assays.

Several approaches for creating a colorimetric signal for EdU proliferation were attempted. The method presented here had the shortest protocol and uses a biotin-based click reaction followed by streptavidin -horseradish peroxidase (HRP) step. This two-step reaction creates an essentially covalent link between the EdU incorporated in the DNA and the enzyme reporter. Then standard HRP chromogens such as DAB were used to produce a signal marking the proliferating cells. Examples of proliferation using colorimetric EdU labeling in adult Zebrafish caudal fin, mouse cardiac, and rat intestine, mammary and uterine FFPE treated tissue are presented. Multiplexing with alkaline phosphatase for two color detection are also presented. Two color detection of EdU with BrdU or EdU with progesterone receptor (PR) are shown. Most HIER protocols are compatible with EdU detection. For EdU alone no HCl treatment is required. For EdU/BrdU double labeling the BrdU protocol for DNA revelation is used. Anti-BrdU mouse antibody clone MoBU-1 is used because of its lack of cross-reactivity with EdU.

Additionally, we present the click chemistry-based apoptotic TUNEL assay colorimetric detection using the same approach. Clickable biotin adapters are used to couple HRP to the ethynyl tails created by TdT enzyme extension at 3ʹ end of double stranded breaks. We provide examples of this late stage indicator of apoptosis visualized with DAB substrate and stained with standard H&E and other histological stains.

#2735

Clinico-pathological and molecular features associated with TP53 mutation in 3457 molecularly-profiled colorectal cancers (CRCs).

Joanne Xiu,1 Thierry Soussi,2 Ryan Bender,1 Sandeep Reddy,1 Wafik S. El-Deiry3. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden;_ 3 _Fox Chase Cancer Center, Philadelphia, PA_.

Deregulation of the p53 tumor suppressor gene (TP53) is a key event contributing to transformation and aggressive metastatic features of CRC. Patients with TP53 mutation are often resistant to therapy and carry a poor prognosis. We investigated TP53 mutation in a cohort of 3457 CRCs to identify molecular features specific to TP53-mutated CRC tumors. The 3457 CRC clinical samples were evaluated for tumor profiling (Caris Life Sciences, Phoenix, AZ). Tests included Sanger or next generation sequencing (NGS), protein expression by immunohistochemistry (IHC) and gene amplification by in situ hybridization (ISH). TP53 mutation was observed in 2106 or 61% of CRCs analyzed. 2018 or 96% of these mutant TP53 tumors carried one TP53 mutation, 83 (4%) carried 2 mutations, 4 carried 3 and 1 tumor carried 4 mutations. Among the ~2200 mutations found in TP53, 37% were found at one of the six hotspots within the DNA binding domain (R175, G245, R248, R249, R273 and R282). Overall, 1554 (71%) were missense mutations, 367 (17%) nonsense, 209 (9.5%) frameshift, 45 (2%) small in-frame in-dels, and 25 (1.1%) mutations that affect splicing. In this cohort, TP53 mutation was more prevalent in male patients (64% vs. 57%, P<0.0001) and was more likely to occur in tumors that originated from the left colon (69%) as compared to the right colon (45%, p<0.0001). TP53 mutation rate was not correlated with patient age, histology or whether the tumor sample was taken from the primary or metastatic sites. When the molecular features of TP53-mutated tumors were compared to those of wild-type TP53, mutated tumors carried significantly higher Her2 IHC expression (2.5% vs. 1.0%, p=0.0039) and gene amplification (3.7% vs. 1.4%, p=0.0002), as well as higher MGMT (61% vs. 53%, p<0.0001) and TOPO2A expression (92% vs. 81%, p<0.0001). On the other hand, lower EGFR expression (57.4% vs. 70%, p<0.0001), PTEN expression (47.9% vs. 61%, p<0.0001), microsatellite instability (2.5% vs. 11.5%, p<0.0001), ERCC1 (18% vs. 24%, p<0.0001) and TS expression (31% vs. 38%, p<0.0001) were associated with TP53-mutated tumors. TP53-mutated CRCs carried higher rates of APC mutation (63% vs. 53%, p<0.0001), but lower rates of KRAS (46% vs. 54%, p<0.0001), PIK3CA (11.6% vs. 22%, p<0.0001), PTEN (2% vs. 5.2%, p<0.0001) , GNAS (1% vs. 8.3%, p<0.0001) and AKT1 (0.6% vs. 1.7%, p=0.0016) mutation. In this cohort of 3457 molecularly profiled CRCs, TP53 mutation was more prevalent in males and tumors that originated from the left colon. Distinct molecular features associated with TP53 mutation in CRC included lower frequency of PI3K/Akt/mTor pathway activation and were more likely to be microsatellite stable. . Our findings suggest differential presence of therapeutic targets in CRC tumors based on TP53 mutation status.

#2736

Regulation of cytokinesis by polo-like kinase 4.

Michael F. Press,1 Bin Xie,1 Simon Davenport,1 Yu Zhou,1 Neil O'Brien,2 Michael Palazzolo,2 Tak Mak,3 Joan Brugge,4 Dennis J. Slamon2. 1 _University of Southern California, Los Angeles, CA;_ 2 _Geffen School of Medicine at UCLA, Los Angeles, CA;_ 3 _University of Toronto, Toronto, Ontario, Canada;_ 4 _Harvard Medical School, Boston, MA_.

Introduction. The serine/threonine mitotic kinase, polo-like kinase 4 (PLK4), is known to play a critical role in centrosome duplication in preparation for cell division. Based on our preliminary observations with a PLK4 kinase inhibitor, we investigated the possibility that PLK4 may also play a role in regulation of cytokinesis.

Experimental Procedures. Immunofluorescence was used to localize PLK4 and phospho-PLK4 in cultured human breast, ovarian, and colorectal cancer cell lines throughout the cell cycle without and with CFI-400945 PLK4 inhibitor and MG-115 protease inhibitor. Flow cytometry and videomicroscopy were used to analyze the consequences of PLK4 inhibition on cytokinesis.

Results. Using immunofluorescence, PLK4 was localized to centrosomes; however, we also found that phospho-PLK4 was cleaved and distributed to kinetochores (metaphase and anaphase), cleavage furrow (telophase), and middle body (cytokinesis) during cell division in colorectal, ovarian, and breast cancer cells. Distribution of phospho-PLK4 to the cleavage furrow and middle body raised the possibility that this kinase plays a functional role in cytokinesis. Using either CFI-400945 PLK4 kinase inhibitor or the MG-115 protease inhibitor, we found that PLK4 accumulated in centrosomes with inhibition of translocation of PLK4 to the middle body. This change in subcellular distribution of PLK4 was associated with generation of large, multi-nucleated tumor cells or tumor cells with polyploidy. Videomicroscopy confirmed that treatment with CFI-400945 PLK4 inhibitor was associated with prevention of cellular abscission in treated cells.

Conclusions. These observations demonstrate a role for phospho-PLK4 in facilitation of cytokinesis. A regulatory role for PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.

#2737

Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.

Sarine Markossian,1 Alexei Arnaoutov,1 Nakhle S. Saba,2 Vladimir Larionov,3 Mary Dasso1. 1 _Division of Molecular and Cellular Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD;_ 2 _Section of Hematology and Medical Oncology, Department of Medicine, Tulane University, New Orleans, LA;_ 3 _Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). CIN is associated with poor prognosis in many cancer types, and targeting of CIN is an attractive strategy for anti-cancer therapeutics. The mechanisms causing CIN and its contributions to tumor initiation and growth are not well defined, partly because there is no straightforward, quantitative assays for CIN in human cells. To address this problem, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in mammalian cells, with which we can easily score the rates of CIN within one cell division under different experimental conditions.

We have constructed a HAC encoding copies of enhanced green fluorescent protein (eGFP) fused to the destruction box (DB) of hSecurin, a substrate of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, which becomes active during anaphase to catalyze the proteolysis of critical mitotic target proteins. This HAC also contains tet operator (tetO) arrays and sequences encoding the tetracycline repressor fused to monomeric cherry fluorescent protein (tetR-mCherry). We have produced human U2OS cells (U2OS-Phoenix) carrying this HAC, in which we monitor HAC segregation in two ways: First, APC/C degrades the DB-eGFP fusion expressed from the HAC at anaphase onset, and DB-eGFP re-accumulates in the daughter cells after G1 phase, when APC/C becomes inactive. Daughter cells that do not obtain a copy of the HAC will thus be GFP negative in the subsequent interphase. Second, because tetR-mCherry binds to the tetO arrays, the HAC itself could be followed by live imaging. Following the HAC by live cell imaging experiments, we show that U2OS-Phoenix cells have low inherent levels of CIN, but HAC missegregation is markedly increased by treatment with Reversine, an inhibitor of Mps1, and microtubule agents Nocodazole and Paclitaxel.

In summary, we have developed new assays to score CIN levels in human cells and have shown that CIN levels increase upon chemical disruption of mitotic progression, which makes our assays ideal for chemical screens.

#2738

**Establishing an** in vitro **cell culture model for studying the function of FAM190A deletions.**

Eric S. Calhoun. _Alma College, Alma, MI_.

A growing number of studies have reported a variety of genetic alterations to FAM190A (CCSER1) in cancer. Interestingly, a large percentage of these are focused on deleting downstream exons producing in-frame arrangements for this protein. An analysis of coding sequences reveals that regions of FAM190A are highly conserved throughout the N-terminus, however, with no recognizable protein domains. Published immunofluorescence and shRNA knockdown studies have since suggested that FAM190A is localized to the midbody during cytokinesis and may function in maintaining genome stability. Unfortunately, attempts to overexpress FAM190A were not successful by these authors. The aim of our current study was to try to generate an in vitro model that would allow us to examine the effect of a C-terminal deletion on cellular division and FAM190A's ability to associate with the spindle and/or midbody structures. To accomplish this, full length FAM190A and a deletion mutant construct lacking exons 8-11 were separately cloned into mammalian expression vectors that include a N-terminal HaloTag Fusion marker and a modified CMV promoter. Current studies are focused on introducing these constructs into the pancreatic cancer cell line, PL-5, to generate FAM190A stably expressing cell lines.

#2739

**Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine** in vitro **and** in vivo **.**

Kun-I Lin,1 Chih-Chien Lin,2 Ming-Feng Chen,3 Chang-Han Chen,4 Li-Yen Shiu3. 1 _Departments of Obstetrics & Gynecology, Chang Bing Show Chwan Memorial Hospital, Changhua County, Taiwan; _2 _Department of Cosmetic Science, Providence University, Taichung City, Taiwan;_ 3 _Cell Therapy and Research Center, E-Da Hospital, Kaohsiung City, Taiwan;_ 4 _Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung City, Taiwan_.

Carnosic acid (CA), one of the purified components of rosemary, has shown anti-inflammatory, chemo-preventive, and anticancer activities. In this study, we treated melanoma cell line B16F10 with CA and detected the anticancer effects. After 24-hour treatment, CA decreased cell proliferation rate, wound healing activity, and inhibited colony formation of B16F10. Additionally, CA induced G0/G1 cell cycle arrest, up-regulated p21 expression, and down-regulate cyclin E, cyclin D and CDK4 expression. Carmustine (BCNU) and Lomustine (CCNU) are used in clinical treatment of melanoma. To assay the combinational effects of CA and BCNU or CCNU, B16F10 cells were treated with CA, BCNU, CCNU alone, or CA combined with BCNU or CCNU. Our results showed, BCNU and CCNU arrested cell cycle at G2/M phase after 24-hour treatment. Dramatically, CA enhenced BCNU- and CCNU-induced cell cycle arrest at G2/M phase, and decreased cyclin B1 and p-CDK1 expresion. In the in vivo xenograft model, B16F10 cells (5×105) were inoculated subcutaneously in C57BL/6 mice, and the tumor grew for one week. The mice were divided into six groups and provided different treated strategies: control, CA (50 mg/kg) alone, BCNU (50 mg/kg) alone, CCNU (25 mg/kg) alone, CA combined with BCNU, and CA combined with CCNU. After two-week treatment, compared with control group, CA had better treated effects than BCNU or CCNU. Compared with CA, BCNU or CCNU treatment alone, the combined strategies inhibited the tumor growth in melanoma mice model more effectively. Furthermore, BCNU and CCNU treatment in melanoma mice caused high values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). However, CA significantly decreased AST and ALT values caused by BCNU and CCNU in combined trestment group. Therefore, CA is more safe and effective than BCNU and CCNU, and the combined treatment strategies of CA and BCNU or CCNU result in an increased antitumor efficacy. CA may be suitable for translation to future clinic.

#2740

Mechanistic basis of Palbociclib combinatorial activity in ER+ breast cancer and non-breast indications.

Stephen Dann,1 Jing Yuan,1 John Chionis,1 Chaoting Liu,1 Tao Xie,1 Nathan V. Lee,1 Enhong Chen,1 Ping Wei,1 Paul A. Rejto,1 David J. Shields,2 Todd VanArsdale1. 1 _Pfizer, La Jolla, CA;_ 2 _Pfizer, Pearl River, NY_.

Phosphorylation of the retinoblastoma protein (Rb) by cyclin-dependent kinases 4 and 6 (CDK4/6) is a critical checkpoint for G1/S cell cycle progression and commitment to cellular proliferation. Human malignancies often subvert these control mechanisms through a range of genetic and biochemical adaptations. Accordingly, tumors that depend on CDK4/6 activity for proliferation and survival are particularly sensitive to inhibition of this pathway by palbociclib (IbranceTM), a highly selective inhibitor of CDK4/6 kinase activities. Treatment regimen of palbociclib with letrozole significantly improved progression-free survival in a randomized phase 2 study of women with advanced estrogen receptor-positive (ER+), HER2-negative breast cancer. Likewise, in ER+ breast cancer models palbociclib and estrogen antagonists combine for greater anti-proliferative activity, increased hallmarks of cellular senescence and prolonged durability of response following drug removal. Dual inhibition of CDK4/6 and ER signaling demonstrated robust anti-tumor activity in xenograft studies. The addition of Palbociclib to other targeted therapeutics elicits improved activity in pre-clinical models of several non-breast indications and these effects also manifest through modulation of cellular proliferation, senescence and growth arrest. Data will be presented on the molecular basis of combination benefit with Palbociclib in ER+ breast and other oncology indications.

#2741

A novel branched DNA-based multiplex method for telomere analysis at the single-cell level by flow cytometry.

Steve Lai, Leslie Sias, Castle Funatake. _Affymetrix, Inc., Santa Clara, CA_.

Telomeres are protective structures at the ends of chromosomes, containing multiple repeats of the repetitive sequence (TTAGGG) in vertebrates. It undergoes progressive shortening during normal aging. Abnormalities in the regulation of telomere length are implicated in various diseases including cancers. Current methods for telomere analysis are tedious and lack the flexibility to allow identification of unique cell subsets in a heterogeneous sample. Here we describe a novel flow cytometric method for analyzing relative telomere length at the single-cell level based on the branched DNA technology. This method, named PrimeFlow Assay can be combined with antibody staining and mRNA detection enabling simultaneous analysis of telomere length as well as protein and RNA for a comprehensive view in any specific cell subset. With this method, we investigated various cell lines and primary cells of human and mouse origin. Our results indicate that transformed cell lines possess longer telomeres than primary cells. In human peripheral blood mononuclear cells, we observed that lymphocytes had longer telomeres compared to monocytes, consistent with previously published results by another flow cytometric method. These data demonstrate that the PrimeFlow assay is a valuable method for high-throughput analysis of relative telomere length that may be combined with the analysis of protein and gene targets and, thus, provides data at the single-cell level for a more complete understanding of sample heterogeneity.

#2742

USP37 promote oncogenesis by stabilizing protein involved in DNA damage repair pathway and provides a novel way to modulate Chk1 activity.

mayank singh,1 Amy C. Burrows,2 Andrew Dickson,2 Matthew Summers1. 1 _Ohio State University, Columbus, OH;_ 2 _lerner research institute, Cleveland, OH_.

Dysregulation of the Ubiquitin proteasome system (UPS), which comprises nearly 1000 different components is associated with many aspects of oncogenesis and has emerged as a novel therapeutic target. We have previously shown that USP37 (Ubiquitin specific peptidase 37) antagonizes the tumor suppressor APCCDH1and it promotes S phase entry while data from other groups have shown that it stabilizes oncoproteins, such as c-Myc. We have found USP37 protein levels to be high in many cancerous cell lines as compared to untransformed cells and it appears to help tumor survive the high level of replication stress associated with oncogene expression. It was found that USP37 over expression confers survival advantage to cancer cells while its depletion enhances chemo sensitization in response to variety of replication stresses. USP37 over expressing cells were able to resolve γ-H2AX foci much more effectively then the wild type or cells in which USP37 was depleted. Our data indicate that USP37 binds and stabilizes the active form of CHK1 as evident by its protein levels when USP37 over expressing or depleted cells were exposed to replication stress. Mechanistically, our analyses indicate that USP37 stabilizes CHK1 by its deubiquitination, preventing its degradation. As CHK1 has been pursued as a therapeutic target with limited success our work defines a novel way to modulate activity of CHK1 while simultaneously targeting multiple oncogenes and thereby providing a rationale to evaluate USP37 inhibitor over CHK1 inhibitor as a potential therapeutic target in different cancers.

### DNA Double-Strand Break Repair Defects in Cancer: Therapeutic Strategies and Molecular Basis

#2743

Finding determinants of PARP inhibitor sensitivity using genome-wide and focused CRISPR screens.

Stephen Pettitt,1 Dragomir B. Krastev,1 Feifei Song,1 Alan Ashworth,2 Christopher J. Lord1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA_.

We are using genomic approaches to study the mechanism of action of targeted cancer drugs, particularly poly(ADP-ribose) polymerase (PARP) inhibitors. One of these drugs, olaparib was recently approved for use in ovarian cancers with germline BRCA1/2 mutations and many other clinical trials with this and other PARP inhibitors are underway. Experimental study of potential resistance mechanisms can inform the ongoing clinical development of these drugs.

We previously carried out a loss of function mutagenesis screen in haploid cells, which showed that PARP1 was required for cellular toxicity of olaparib and other PARP inhibitors. This supported the hypothesis that PARP inhibitor toxicity occurs via a poisoning mechanism where inhibited PARP is tightly bound to DNA, forming a toxic lesion.

Recently we carried out a similar screen using genome-wide lentiviral CRISPR-Cas9 mutagenesis in diploid cells. Many PARP inhibitor resistant clones had loss of function Parp1 mutations as expected. However, we also isolated a point mutation affecting a single amino acid in the Parp1 DNA binding domain. This mutant encodes a stable Parp1 protein that cannot bind DNA and does not become trapped in the presence of inhibitors. Thus the CRISPR screen implicated PARP1 DNA binding directly in determination of PARP inhibitor toxicity and was extremely informative about the mechanism of action compared to conventional loss-of-function mutagenesis.

We extended this approach by synthesising a high-density focused sgRNA library targeting only PARP1. We developed a reporter cell line that allows us to selectively isolate in-frame mutations that preserve PARP1 protein expression. By deep sequencing mutagenised and appropriately selected cells we identified a series of subtle mutations in PARP1 that result in PARP inhibitor resistance, giving us a detailed insight into structure-function relationships in PARP1. The approach is applicable to other drug targets, providing a framework for understanding drug action and predicting clinical resistance.

#2744

Synthetic lethal killing of RAD54B-deficient cancer cells by PARP1 silencing and inhibition.

Erin N. McAndrew, Chloe C. Lepage, Kirk J. McManus. _University of Manitoba, Winnipeg, Manitoba, Canada_.

Colorectal Cancer (CRC) is the second leading cause of cancer-related deaths in North America. Currently, many chemotherapeutics are non-specific and preferentially kill cancer cells based on their high proliferation rate. However, due to the lack of tumor specificity, many treatments have unwanted side effects. Accordingly, identifying novel therapeutic strategies and drug targets that better target and combat cancer are needed. To address this need, synthetic lethal (SL) approaches are now being explored in many cancer contexts. Synthetic lethality refers to the lethal combination of two independently viable mutations and functions by exploiting the aberrant genetics contained within cancer cells. RAD54B is an excellent candidate to exploit using a SL paradigm as it normally functions in homologous recombination repair (HRR) and hypomorphic expression and/or function are implicated in tumorigenesis in a wide array of cancers. We predict that RAD54B and PARP1 are SL, as BRCA1 and BRCA2, which also encode functions within HRR, are SL with PARP1. To identify PARP1 as a novel SL interactor of RAD54B, we employed an established RNAi-based screening approach along with a RAD54B isogenic CRC model. Conceptually, following PARP1 silencing, a SL interaction will result in fewer cells within the RAD54B-deficient cells relative to RAD54B-proficient controls. Accordingly, we sought to determine whether PARP1 silencing or inhibition (Olaparib or BMN673) would induce selective killing within RAD54B-deficient HCT116 cells relative to controls. As predicted, PARP1 silencing with either individual or pooled siRNA duplexes and inhibition with both Olaparib and BMN673 was associated with statistically significant decreases in the number of RAD54B-deficient cells relative to controls. To confirm the SL interaction did not occur due to de novo mutations within the RAD54B-deficient cells, dual silencing of RAD54B and PARP1 was performed within the parental line and confirmed the above findings. Finally, real-time cellular analyses were conducted and revealed the decrease in cell numbers was due to cellular cytotoxicity rather than cell cycle arrest. Collectively, these data show that RAD54B and PARP1 are SL, and identify PARP1 is a candidate lead target in CRCs harboring RAD54B defects.

#2745

A genetic screen to identify tumor suppressive DDR genes in breast cancer.

Katerina D. Fagan-Solis, Gaorav P. Gupta. _University of North Carolina Chapel Hill, Chapel Hill, NC_.

To maintain genomic integrity, cells utilize a DNA damage response (DDR) mechanism that 1) functions to repair damaged DNA efficiently and 2) commits cells to apoptosis if damage is irreparable. Failure of this mechanism results in genomic instability and cancer predisposition. Defects in DDR have been implicated in both sporadic and familial breast and ovarian cancer predisposition syndromes (e.g. BRCA1, BRCA2, and PALB2), although the specific pathway components that are disrupted have not been elucidated. In particular, an impaired DDR may contribute to the pathophysiology of triple-negative (ER-/PR-/HER2-) breast cancer (TNBC) due to the extent and complexity of the genomic instability that is observed. We identified the DNA damage sensing Mre11- Rad50-Nbs1 (MRN) complex as an essential mediator of the oncogene-induced DDR. Disrupting the MRN complex in mouse models promotes the development of genomically unstable, metastatic breast cancer. The clinical relevance of this pathway is highlighted by the observation that components of the Mre11 complex are perturbed in a significant fraction of human TNBCs, which correlates with clinical outcomes. However, lack of MRN suppression in a majority of TNBCs indicates that alternative DDR components may also be inactivated to promote breast cancer development. Our goal is to identify additional DDR genes that, when perturbed, promote the development of genomically unstable breast cancers. In order to accurately distinguish driver genes from bystander genes we have developed a platform to identify DDR genes that mediate oncogene-induced senescence in mammary epithelial cells using both shRNA and CRISPR pooled libraries. Analysis of newly identified tumor suppressor genes, compared to what is already known about Mre11 and p53 (positive controls), will enable us to determine causality of specific DDR mutations in breast cancer development and importantly, the genomic instability phenotype that is typical for TNBC. The insights gleaned from this study may ultimately provide the means to determine the optimal treatment regimen for each patient with TNBC, and to prevent over-treatment of patients where certain chemotherapies may not be effective.

#2746

Combined inhibition of Wee1 and Poly(ADP Ribose)Polymerase 1/2 synergistically inhibits acute leukemia cells by enhancing DNA damage and inducing apoptosis in vitro and in vivo.

Tamara B. Garcia,1 Jonathan C. Snedeker,2 Christopher C. Porter1. 1 _University of Colorado School of Medicine, Aurora, CO;_ 2 _Johns Hopkins University, Baltimore, MD_.

Introduction: The goal of this study was to test the hypothesis that pharmacologic inhibition of Wee1 can sensitize acute leukemia cells to Parp1/2 inhibition by inducing defects in homologous recombination.

Methods: Human cell lines MV4;11 and Molm-13 (AML), Jurkat (T-ALL), and Reh (B-ALL) were treated with various concentrations of a Wee1 inhibitor (AZD1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry In some experiments, cells were split into fresh media to recover for an additional 72 hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots examining γH2AX as well as comet assays were used to measure DNA damage induction. Inhibitory phosphorylation of BRCA2, a critical protein in the homologous recombination pathway, was examined by Western blot. Finally, wild type Bl6 mice were injected with a murine AML cell line (Mll-Enl+FLT3-ITD+) and treated daily with olaparib and/or AZD1775. Leukemia progression was determined by measuring luciferase activity biweekly via In Vivo Imaging Systems (IVIS).

Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 4 cell lines tested, with CI values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Comet assays and western blots for γH2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Western blots demonstrated increased inhibitory phosphorylation of BRCA2 in cells treated with AZD1775 suggesting impaired homologous recombination upon Wee1 inhibition. In preliminary in vivo studies, mice with AML treated with olaparib and AZD1775 displayed prolonged survival compared to mice treated with single agents.

Conclusion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML, T-ALL, and B-ALL cell proliferation, DNA damage and apoptosis than either drug alone. Inhibition of Wee1 resulted in increased BRCA2 inhibitory phosphorylation, suggesting that this increased sensitivity to PARP1/2 inhibition could be due to impairment of the homologous recombination pathway. Preliminary studies indicate that combined inhibition of Wee1 and Parp1/2 enhances survival in a murine AML model. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in patients for whom conventional chemotherapeutics may not be well tolerated.

#2747

PARP inhibitors act synergistically with cisplatin and doxorubicin in BRCA competent cells by compromising chemotherapy-induced replication arrest.

Lea Schaaf,1 Matthias Schwab,2 Simon Heine,1 Walter E. Aulitzky,3 Heiko van der Kuip1. 1 _Dr. Margarete Fischer-Bosch Institute and University of Tuebingen, Stuttgart, Germany;_ 2 _Dr. Margarete Fischer-Bosch Institute and Department of Clinical Pharmacology, University Hospital Tuebingen, Stuttgart and Tuebingen, Germany;_ 3 _Robert Bosch Hospital, Stuttgart, Germany_.

PARP inhibitors as monotherapy have emerged as promising antitumor drugs in particular for malignancies with nonfunctional BRCA1/2 proteins. Furthermore, preclinical data as well as first results from clinical studies suggest that PARP inhibition can potentiate cytotoxic effects of conventional chemotherapy independently of the BRCA status. Mechanisms underlying these combinations have predominantly been attributed to an enhancement of DNA damage through interference with DNA repair. Apart from its central function in DNA damage repair processes the enzymatic function of PARP1/2, namely Poly(ADPribosy)lation (PARylation), PARP has also been implicated in stabilizing replication fork arrest upon Topoisomerase I (TopoI) poisoning. We aimed to determine if the function of PARP1/2 at the stalled replication fork might be important for synergistic effects of PARP inhibitors with widely used chemotherapeutics other than TopoI inhibitors. For this, the BRCA competent ovarian and colon cancer cell lines OVCAR8, OVCAR3, HCT116, and LOVO as well as primary tissue samples from ovarian carcinoma patients were treated with cisplatin, doxorubicin, etoposide, 5-FU, and paclitaxel for one hour in presence or absence of the three commonly used PARP1/2 inhibitors Rucaparib, PJ34, and A966492. DNA fiber spreading analyses, quantification of PARylation and 53BP1/γH2AX spot formation, as well as analysis of DNA strand break repair capacity were performed within the first hours after treatment. Furthermore, cell lines were analyzed for long term survival using colony forming assays. Importantly, PARylation was only observed at early time points after treatment with cisplatin and doxorubicin, but not upon 5-FU, etoposide and paclitaxel. Consequently, PARP inhibition had only synergistic effects on long term survival together with cisplatin and doxorubicin but not with the other tested chemotherapeutics. Inhibition of PARylation caused a significant delay of cisplatin adduct removal and strand break repair upon doxorubicin. More importantly, combination of PARP inhibitors with both cisplatin and doxorubicin led to double strand break formation selectively in S/G2 phase cells as detected by 53BP1/γH2AX spots in Geminin positive cells. This was caused by a complete circumvention of chemotherapy-induced fork slowing in cell lines and primary tumor cells. The inability of initiating replication fork arrest upon chemotherapy in cells with blocked PARP1/2 activity was confirmed by the presence of new origin firing and significantly reduced replication stops.

In conclusion our work implicates that PARP inhibitors have significant effects not only in BRCA deficient cells but also in proliferating BRCA competent cells when combined with adduct-forming, replication stress-inducing chemotherapeutics.

#2748

Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10.

Audrey L. Turchick, Peter M. Glazer. _Yale University, New Haven, CT_.

There is growing evidence that many solid tumors have defective DNA repair pathways and cell cycle checkpoints. This has spurred development of therapeutics to specifically target cancer cells with defective DNA repair, but few have been successful outright due to problems such as general toxicity. One novel anti-cancer agent of interest to us is 3E10, a unique cell-penetrating, anti-DNA autoantibody. This molecule is non-toxic to normal cells on its own but has deleterious effects on cells with DNA repair deficiencies. Specifically, 3E10 has been shown to be synthetically lethal in BRCA2-deficient human cancer cells, to be synergistic with radiation and other commonly used DNA damaging therapies, and to reduce the efficiency of Rad51 mediated strand exchange and thus homologous recombination (HR). The goal of this project is to better understand how 3E10 can be used as a new paradigm for cancer treatment. New evidence suggests that 3E10 inhibits Exo1 exonuclease activity. Additionally, preliminary data shows that 3E10 physically interacts with Rad51, ultimately inhibiting Rad51 nuclear localization and foci formation. Additionally, a point mutation in 3E10 that confers increased DNA binding mediates increased cell death in BRCA2-deficient human cancer cells. Furthermore, studies investigating the potential therapeutic effect of 3E10 in patient derived primary melanoma cell lines show that 3E10 is synthetically lethal with PTEN deficiency. This data suggests that 3E10 inhibits HR by binding to DNA at double strand breaks and occupying sites normally processed by core repair machinery, as well as by physically interacting with Rad51. Overall, 3E10 holds great promise as a novel therapeutic agent for various cancers, including PTEN deficient melanomas.

Reference:

Hansen, J. et al. Targeting cancer with a lupus autoantibody. Science Translational Medicine 4, (2012)

#2749

A novel small molecule inhibitor targets the DNA double strand break repair protein Ku70/80.

Daruka Mahadevan,1 Alfred C. Gallegos,2 Lauren N. Dominick,2 Laurence S. Cooke,3 Trace N. Bartels,4 Josef Vagner,5 Terry O. Matsunaga,6 Eric Weterings2. 1 _The West Clinic, Memphis, TN;_ 2 _University of Arizona, Dept. of Radiation Oncology, Tucson, AZ;_ 3 _University of Tennessee, Memphis, TN;_ 4 _University of Arizona, Dept. Of Radiation Oncology, Tucson, AZ;_ 5 _University of Arizona, Bio5 Institute, Tucson, AZ;_ 6 _University of Arizona, Dept. of Medical Imaging, Tucson, AZ_.

Purpose: Non-Homologous End-Joining (NHEJ) is the primary pathway for the repair of DNA double strand breaks (DSBs) in human and mammalian cells. NHEJ is frequently upregulated in diverse solid malignancies and is a determining factor in the process of carcinogenesis. In addition, upregulation of the NHEJ pathway can reduce the efficiency of anti-cancer therapies including radiation or chemotherapy. Inhibition of NHEJ by siRNA reduces the growth of NHEJ-upregulated tumors and alleviates radiation-refractory disease. The Ku70/80 heterodimer is the central scaffolding protein of the NHEJ pathway, which recruits and activates all other key NHEJ factors. Therefore, Ku70/80 protein is a logical target for disruption of the NHEJ process.

Experimental Procedures: We report the identification of a prospective binding pocket in the Ku70/80 crystal structure, located in close proximity to the DNA-binding domain and the Ku70/80 heterodimer interface. Based on this pocket, an idealized virtual ligand ('protomol') was created and utilized to screen 'in silico' an extensive database of small molecule compounds for potential ligands. Nine of the highest ranking hits were chosen for biological evaluation.

Results: We identified one compound (designated 'Compound L'), which proved capable of disrupting the in vitro binding of Ku70/80 to a DNA substrate in a dose-dependent manner with an IC50 of 3.5 µM. In addition, Compound L showed dose-dependent inhibition of DNA-Dependent Protein Kinase Catalytic Subunit (DNA-PKCS) activity, which requires Ku70/80 as a co-factor with an IC50 of 2.5 µM. Finally, we showed that Compound L synergistically sensitizes a glioblastoma cell line to ionizing radiation, with a marked sensitization at ~20-25 µM. This latter observation clearly demonstrates the ability of Compound L to disrupt NHEJ-mediated repair of radiation-induced DSBs.

Conclusions: Compound L is an active inhibitor of the Ku70/80 heterodimer. To the best of our knowledge, no such compound has been reported in the peer-reviewed literature. The structure of Compound L will be utilized as a scaffold for a 'structure-activity relationship' (SAR) based hit-to-lead drug development aimed at designing a novel class of anti-cancer agents targeting the Ku70/80 heterodimer. We predict Ku70/80 inhibitors would have applicability as single-modality drugs, following a synthetic lethality approach, or as sensitizing agents for concurrent radiation or chemotherapy in a variety of solid malignancies.

Support: P30 CA023074 .

#2750

Mutations on the homologous recombination pathway in 13 cancer types.

Joanne Xiu,1 Ryan Bender,1 Brian Abbott,1 Zoran Gatalica,1 Sandeep Reddy,1 Mohamed Salem,2 Shelly Seward3. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC;_ 3 _Karmanos Cancer Institute, Detroit, MI_.

Homologous recombination (HR) is important in DNA double-strand break repair. HR defects promote carcinogenesis and are associated with selective sensitivity to PARP-inhibitors and DNA-damaging agents. We collected 1029 tumor samples in 13 cancer types and used next-generation sequencing (NGS) to survey genes in the HR pathway. NGS on 591 genes was performed using formalin-fixed paraffin-embedded samples on the Illumina NextSeq platform (Caris Life Sciences, AZ). Mutations in as low as 5% of cells can be detected with > 99 % confidence. Deletions larger than 27bp may not be detected by this method. Pathogenic or presumed pathogenic variants are counted as mutations. The table summarizes mutation rates of 7 key genes—ATM, BRCA1, BRCA2, CHEK1, CHEK2, PALB2 and PTEN—included in this pilot study. Another 17 HR genes—ATR, ATRX, BARD1, BLM, BRIP1, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, MRE11A, NBN, RAD50, RAD51, and RAD51B—were also analyzed. PTEN mutations were seen in 6.3% of tumors, ATM in 5%, BRCA1 in 2%, BRCA2 in 2%, PALB2 in 1%, and CHEK2 in 1%. No CHEK1 mutations were observed. Overall, 15% of tumors carry at least one mutation in any of the 7 key genes. The highest mutation rates were seen in endometrial (43%), glioblastoma (34%), and gastric cancers (23%). The highest rates of ATM (9.7%), BRCA2 (6.5%), and PALB2 (6.5%) were seen in gastric cancer, while the highest CHEK2 (5.6%), BRCA1 (7.3%) and PTEN (44%) mutations were seen in cholangiocarcinoma, ovarian and endometrial tumors, respectively. One 53-year old pt with metastatic poorly-differentiated gastric adenocarcinoma experienced ongoing radiographic partial response and dramatic symptom relief following 4 cycles of FOLFOX without surgery; tumor analysis revealed a nonsense PALB2 (S326*) gene mutation, while the other 23 HR genes were wild type. ERCC1 showed intact expression by IHC.

Mutation rates of 7 genes on the HR pathway in 13 cancer types

---

Endometrial | ATM | BRCA1 | BRCA2 | CHEK1 | CHEK2 | PALB2 | PTEN | Any of the 7 genes

Endometrial (N=35) | 0 | 0 | 0 | 0 | 2.9% | 3.0% | 44.1% | 42.9%

GBM (N=47) | 2.1% | 2.1% | 0 | 0 | 0 | 0 | 30.4% | 34.0%

Gastric (N=31) | 9.7% | 0 | 6.5% | 0 | 0 | 6.5% | 0 | 22.6%

Bladder (N=38) | 2.6% | 0 | 5.4% | 0 | 0 | 0 | 10.8% | 18.4%

Kidney (N=41) | 2.5% | 0 | 0 | 0 | 5.0% | 0 | 10.0% | 17.1%

Ovarian (N=82) | 3.7% | 7.3% | 1.2% | 0 | 1.2% | 0 | 1.3% | 14.6%

Breast (N=108) | 4.6% | 2.8% | 1.9% | 0 | 0.9% | 1.0% | 3.8% | 13.9%

Cholangiocarcinoma (N=36) | 2.8% | 0 | 2.8% | 0 | 5.6% | 0 | 2.9% | 13.9%

CRC(N=254) | 6.3% | 2.0% | 1.6% | 0 | 0.4% | 0 | 4.0% | 13.0%

Pancreatic (N=62) | 4.8% | 1.6% | 3.2% | 0 | 0 | 1.7% | 3.3% | 12.9%

NSCLC(N=234) | 6.5% | 0 | 0.9% | 0 | 0 | 1.4% | 2.6% | 11.1%

Neuroendocrine(N=35) | 2.9% | 0 | 0 | 0 | 0 | 0 | 5.7% | 8.6%

Esophageal (N=26) | 3.8% | 0 | 0 | 0 | 0 | 0 | 4.0% | 7.7%

Overall(N=1029) | 5.0% | 1.6% | 1.6% | 0 | 0.8% | 0.8% | 6.3% | 15.2%

Thus, mutation rates of at least 8 to 43% in the HR pathway are reported from 13 cancer types. This method can potentially identify responders to DNA-damaging agents including platinum.

#2751

Frequent BRCA2 somatic mutations in colorectal cancer patients with microsatellite instability (MSI).

Safoora Deihimi,1 Avital Lev,1 Elena Shagisultanova,2 Joanne Xiu,3 Michael Slifker,4 Qifang Xu,5 David T. Dicker,1 Eric A. Ross,4 Roland Dunbrack,5 Wafik S. El-Deiry1. 1 _Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Division of Medical Oncology, Anschutz Cancer Center, University of Colorado, Denver, CO;_ 3 _Caris Life Sciences, Phoenix, AZ;_ 4 _Biostatistics and Bioinformatics Facility, Fox Chase Cancer Center, Philadelphia, PA;_ 5 _Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA_.

Microsatellite instability (MSI) is a hallmark of mismatch repair (MMR)-deficient cancers including colorectal cancer (CRC). MSI represents 15% of CRCs as a result of either epigenetic silencing of MLH1 or mutations in one of the MMR genes: MLH1, MSH2, MSH6 and PMS2. MSI tumors have a better prognosis than microsatellite stable (MSS) tumors while responding differently to treatments, including relative resistance to 5-FU and high clinical benefit from immune checkpoint therapy. Deficient MMR can lead to deficient DNA double-strand-break repair. We recently reported on 26 MSI-High and 558 non-MSI-High CRCs profiled by Caris Life Sciences. MSI-High CRCs had a high mutation rate (50%) in BRCA2 (Shagisultanova et al., 2015 ASCO Annual Meeting Abstract No. e14684 "Association of increase in BRCA2 gene mutations in microsatellite instable (MSI-H) colorectal cancer (CRC) with increased c-MET expression"). We hypothesized there might be a pattern with specific BRCA2 mutations in MSI-H CRCs targeting the coding microsatellites in BRCA2. BRCA2 mutations could be potential targets in clinical therapy. We further investigated functional mutation patterns in BRCA2 in both MSI-H and MSS groups. Of 1104 profiled CRCs in the COSMIC v73 database, somatic BRCA2 mutations were mapped for 101 MSI-High versus 916 MSS CRCs. MSI-High CRCs showed a significantly higher mutation rate in BRCA2 as compared to MSS (38% vs 6%, P<0.0000001). A higher rate of damaging mutations (42% vs. 2%, P< 0.0001) and a relatively distinct pattern of protein mutation distribution could be clearly mapped in MSI-High CRCs versus the MSS group. We found that specific mutations in coding microsatellites of BRCA2 can be impacted by MMR defects. We found 72 unique BRCA2 mutations in MSI-H CRCs not previously seen in either breast cancer or pancreatic cancer as reported in COSMIC v73. However, using the BIC database (http://research.nhgri.nih.gov/bic/) we detected 5 BRCA2 deleterious mutations that have been reported as germline mutations in breast cancer. We used the consensus result from five predictors and available 3-D structural information to predict deleterious properties of mutations including damaging BRCA2 protein structure and disruption of interactions with partner proteins including DSS1 and RAD51. Targeting BRCA2 mutations in MSI tumors and using the concept of synthetic lethality might be effective in BRCA-deficient CRCs with MSI.

#2752

Characterization of the ATM-SPOP pathway in prostate cancer tumorigenesis.

Joshua Fried,1 Rebecca Boohaker,2 Qinghua Zeng,2 Wei Zhang,2 Bo Xu2. 1 _University of Alabama Birmingham, Birmingham, AL;_ 2 _Southern Research Institute, Birmingham, AL_.

The Speckle type Poz Protein (SPOP), an E3 ubiquitin ligase adaptor, has recently been identified as the gene that has the most common somatic point mutations in prostate cancer. SPOP mutations are associated with genomic alterations, indicating a role for SPOP in the maintenance of genome stability. We, and others, have recently demonstrated a critical role of SPOP in the DNA damage response (DDR), suggesting SPOP mutants may represent a subgroup of patients that have hyper sensitivity to DNA damaging therapies. However, how SPOP mutations might impact its function and their roles in the progress of prostate tumorigenesis remain to be extensively studied. Genomic studies have found that SPOP mutations are clustered within its substrate binding pocket. Of a particular interest, Serine119 is found to be frequently mutated to Asparagine (S119N). Using computational modeling, we found that Ser119 resides in the SBC-MATH binding interface and is in close contact with the non-polar residue of the SPOP-binding consensus motif. Therefore we hypothesized that phosphorylation of Serine 119 might directly affect substrate binding. First we characterized prostate cancer cells expressing this mutation and we found that they displayed a suboptimal DNA damage response with prolonged DNA repair. Using an in situ proximity ligation assay, we demonstrate that Serine 119 is essential for SPOP ionizing irradiation induced interaction with the Ataxia Telangiectasia Mutated kinase (ATM), a major hub protein in the DDR. With the evidence of SPOP being a phospho-protein, we further provide in vitro evidence that ATM phosphorylates SPOP on Serine 119 in response to DNA damage. Characterization of the functional significance of ATM-mediated SPOP phosphorylation indicates a wide range of downstream targets regulating cell cycle progression and DNA repair. Taken together, our data reveal a critical pathway linking ATM and SPOP in regulation of prostate cancer initiation and therapeutic responses to DNA damage. This also provides the first evidence of a pathophysiological relevant mutation linked to ATM phosphorylation in the DDR.

#2753

Landscape of somatic mutations in DNA repair genes in prostate cancer.

Santosh Yadav,1 Muralidharan Anbalagan,2 Melody Baddoo,3 Erik Flemington,4 Krzysztof Moroz,4 Kathleen Hering-Smith,1 Nick Makridakis5. 1 _Department of Medicine, Tulane University, New Orleans, LA;_ 2 _Deartment of Structural and Cellular Biology, Tulane University, New Orleans, LA;_ 3 _Louisiana Cancer Research Center, New Orleans, LA;_ 4 _Department of Pathology, Tulane University, New Orleans, LA;_ 5 _1Globe Health Institute, Boston, MA_.

Prostate Cancer (PCa) is a genetic disease that is characterized by multiple genomic alterations. Studies have shown that there is increasing associations between DNA repair genes and PCa. Previously we have reported somatic mutations in Polb and Polk enzymes that are critical for base excision repair and translesion DNA synthesis respectively. In the present study we identified frequently mutated genes that are involved in DNA repair pathway by sequencing all exons of DNA repair pathway genes (129). A total of 57 tumors with matched non tumor tissue was used. Tumors include 24 Caucasians, 21 African Americans (AA). PCa tumors that were ≥6 gleason score and ≥50% of viable tumor cells were only included. All the PCa were procured from Tulane University (TU) Hospital, New Orleans and ethical approvals were obtained from the TU Local Research Ethical Committee. Genomic DNA from tumors was using agilent SureSelect kit was customized to enrich the target that contained all exons of 129 genes of DNA repair pathways. Samples were sent for sequencing by Illumina HiSeq 2000. We validated experimentally somatic mutations in genes that were either affected by somatic non-synonymous or synonymous mutations with a frequency significantly greater than background rates (P<0.05). We considered a somatic mutation to be validated if the targeted sequencing confirmed the presence of the mutation in tumor and it was not found in the matched normal sample. Overall we identified 875 somatic mutations in AA, in which 414 were non-synonymous, 427 synonymous, 14 were stop-gain and 1 was frameshift mutation. AA demonstrated 52 somatic mutations/tumor and Caucasians exhibited 46 somatic mutations/tumor. In terms of Nucleotide excision repair (NER), Mismatch repair (MMR) and Base excision repair (BER) we identified variation in NER, MMR and BER pathways in AA [highest mutation in NER (mean-66) followed by MMR (mean-47) and then BER pathways (mean-37)] whereas our report shows that MMR, BER and NER equally mutated in Caucasians. Furthermore analysis of distribution of mutations between low grade (6) and high-grade Gleason scores (8 or >8) in AA and Caucasians revealed that 12 mutations per tumor for Gleason score 6, versus 22.3 mutations for Gleason 8, 9 or 10. Due to the limitation of samples with gleason score 6 in both races we could not draw a trend individually. Analysis of mutation spectra across the DNA repair genes revealed that C> T transitions (27%) markedly higher in AA PCa followed by T > C transitions (24%). Interestingly in Caucasians PCa C > T transitions (26.8%) were higher including marked increase of C > A (17%) and T > G (17%) transversions. Understanding the mutational changes at the individual patient level could enable personalized treatment and patient selection based on tumor mutational profile for clinical drug testing might be critical for development of new treatments strategies. Support PC094628, and NIH grant 8 P20 GM103518

#2754

**Antibody-based tools for** in vitro **and live cell analysis of endogenous PARP1, an essential human DNA repair enzyme.**

Andrea Buchfellner,1 Larisa Yurlova,1 Stephanie Dennison,1 Benjamin Ruf,1 Ulrich Rothbauer,2 Tina Romer1. 1 _ChromoTek GmbH, Martinsried, Germany;_ 2 _NMI Tübingen, Reutlingen, Germany_.

DNA damage caused by normal cell activity or exogenous genotoxic agents is a constant threat to the genome. The nuclear enzyme Poly(ADP-ribose) polymerase 1 (PARP1) is rapidly activated by DNA lesions such as single-strand breaks and signals their presence by attaching ADP-ribose units to chromatin-associated proteins. To improve our understanding of PARP1 within a disease context, there is an ongoing need to develop novel PARP1 detection systems.

Here we describe the development of a VHH directed against human PARP1. The variable domain of single chain antibodies (VHH or nanobody) is a versatile research tool for a variety of applications. The high binding affinity of VHHs, their small size (15 kDa) and their robust expression in various cellular systems make them preferable to conventional antibodies. Moreover, VHH domains can be selected to recognize and bind their target structures within living cells.

This highly specific PARP1 VHH was selected by phage display using a VHH library from an immunized alpaca. We characterized its affinity, selectivity and activity both in vitro and in live cells.

To validate the in vitro interactions of this PARP1-specific VHH, we developed a PARP1 immunoprecipitation reagent by conjugating the PARP1-specific VHH to an immobilizing matrix (termed PARP1-Trap). This PARP1-Trap was shown to bind with high specificity to human PARP1 and not to other members of the PARP family. Importantly, the binding of the PARP1-Trap to PARP1 leaves the enzymatic activity of PARP1 unaffected. The epitope of the PARP1-Trap was localized to the N-terminal domain of PARP1 and consists of the three-dimensional motif of zinc fingers 2 and 3 together.

To determine whether the PARP1 VHH recognizes its target structure also in a cellular environment, the VHH was genetically fused to a fluorescent protein and expressed in living cells (termed Chromobody). The interaction of the PARP1 Chromobody with PARP1 was visualized using a protein-protein interaction assay called fluorescent two-hybrid (F2H). The F2H principle is based on a tethering strategy: a GFP-tagged protein (here GFP-PARP1) is enriched at a protein interaction platform engineered into F2H-BHK cells and serves as bait, whereas the RFP-tagged PARP1 VHH serves as a prey. Using the F2H assay, we could show that the PARP1 Chromobody is functional within living cells and specifically recognizes its antigen in a cellular environment. Moreover, by monitoring the PARP1 Chromobody signal after microirradiation, we were for the first time able to follow the recruitment of endogenous PARP1 to sites of DNA damage in living cells.

In summary, we developed a novel PARP1 VHH for both biochemical and live cell analysis of human PARP1. We anticipate that PARP1-VHH based reagents will provide new insights into the PARP1 enzyme. For example, the use of the PARP1-Trap coupled with mass spectrometry analysis may lead to the identification of hitherto unknown PARP1 interaction partners.

#2755

Regulation of 53BP1 by the structural nuclear protein NuMA.

Naike Salvador Moreno. _Wake Forest Baptist Medical Center, Winston Salem, NC_.

DNA double-strand breaks (DSBs) can cause genomic instability and cancer through point mutations and genomic translocations if they are not properly repaired. DSBs are repaired by two major pathways: homologous recombination (HR) or nonhomologous end joining (NHEJ). The cellular choice between these two pathways is critical to cell survival and is altered in cancers. 53BP1, which promotes NHEJ by blocking CtIP-dependent DNA resection, is a key protein mediating repair pathway choice. Here, we present a new negative regulation mechanism of 53BP1 that relies on the structural nuclear protein NuMA. Apart from its role in mitotic spindle assembly, NuMA influences chromatin organization during the interphase and modulates the chromatin response to DNA damage. We identified an interaction between NuMA and 53BP1 by proteomics analysis of a 53BP1 pull-down and confirmed this interaction by NuMA immunoprecipitation. We measured greater than 50% decreased 53BP1-NuMA binding in response to DSB, which suggest that, in the absence of DNA damage, NuMA may restrain 53BP1 diffusion. This hypothesis is supported by fluorescence correlation spectroscopy (FCS) measurements in cells expressing GFP-tagged 53BP1, as well as by microirradiation experiments in which NuMA overexpression or silencing inhibited or promoted 53BP1 recruitment at DNA damage sites, respectively. 53BP1 has an essential role in B cell class switch recombination (CSR) and mediates PARPi sensitivity in BRCA1-null cells. We will present how modulating NuMA expression affects both aspects of 53BP1 function. The results from our work will help to understand how 53BP1 is controlled, and shed light on the mechanisms regulating this modulator of PARPi sensitivity in the presence or absence of DNA damage.

#2756

SMARCAD1 depletion enhances hyperthermia-mediated radiosensitization by decreasing resection and enhancing stalled replication forks.

Sharmistha Chakraborty, Clayton R. Hunt, Shashank Hambarde, Kalpana Mujoo, Nobuo Horikoshi, Raj K. Pandita, Abid Mattoo, Bin Teh, Tej K. Pandita. _THMRI, Houston, TX_.

Hyperthermia is the most effective chemo- and radio-sensitizer in use clinically for containing tumor growth. Hyperthermia is proposed to induce degradation of BRCA2, the protein involved in homologous recombination (HR) repair of DNA double strand breaks and, thus sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition. We and other laboratories have reported that hyperthermia inhibits DNA damage repair. Since HR includes single-strand annealing, gene conversion and break-induced replication, it is critical for DNA replication during recovery of stalled replication forks, but it is not yet clear how hyperthermia affects these important steps in HR.

Here we report that SMARCAD1 depletion increases hyperthermia induced cell death and chromosome damage in irradiated S-phase cells. Combined SMARCAD1 depletion and hyperthermia treatment decreased repair and recruitment of repairosome proteins to ISce-1 induced DSBs as well as decreased IR-induced repairosome foci formation. This effect was limited to S-phase cells, where homologous recombination is up regulated and the predominant pathway of DSB repair. Hyperthermia decreased the quantity of 5` single stranded DNA intermediates at endonuclease-generated DNA break sites, an effect that was further decreased by SMARCAD1 depletion. Consistent with these results, simultaneous SMARCAD1 depletion and hyperthermia increased replication fork stalling and affected firing of new replication origins. The results presented here support a mechanism by which SMARCAD1 depletion and hyperthermia mediate radiosensitization through inhibition of the DNA resection step that precedes strand homology search during HR repair of IR-induced DSBs.

#2757

Mcl-1 dictates DNA double-strand break repair pathway choice.

Gou Chen, Ke Xu, Maohua Xie, Taofeek K. Owonikoko, Suresh S. Ramalingam, Paul W. Doetsch, Xingming Deng. _Emory University, Atlanta, GA_.

DNA double-strand breaks (DSBs) are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways. The choice of pathway is a critical aspect of DSB repair that is poorly understood. We show that Mcl-1 acts as a functional switch in pathway choice between HR and NHEJ. Mcl-1 is cell cycle-regulated, with expression peaking in S/G2 phase via reduction of its ubiquitination when HR occurs. Mcl-1 depletion reduces HR and enhances NHEJ. Mcl-1 overexpression results in a net increase in HR over NHEJ. Mcl-1 promotes HR-dependent DSB repair following DNA replication stress, which contributes to maintenance of genomic integrity. Mcl-1 directly interacts with Ku via its BH1 and BH3 domains. This interaction is required for Mcl-1 to inhibit Ku-mediated NHEJ, and promote Mre11 complex-mediated DNA resection and HR-dependent DSB repair. Thus, Mcl-1, in addition to its antiapoptotic function, plays an unexpected role in directing DSB repair pathway choice by skewing the balance toward HR during cell cycle progression.

#2758

FANCF, a Fanconi anemia core complex protein involved in monoubiquitination of FANCD2, also has a role with nuclear alpha spectrin in DNA interstrand crosslink repair.

Muriel W. Lambert,1 Deepa Sridharan,2 Pan Zhang3. 1 _Rutgers New Jersey Medical School, Newark, NJ;_ 2 _Lawrence Berkeley National Laboratory, Berkeley, CA;_ 3 _Weill Cornell Medical College, Cornell University, New York City, NY_.

Nonerythroid nuclear Α spectrin (ΑSpII) is critical for repair of DNA interstrand crosslinks (ICLs) and for genomic stability. We have previously shown that there is a deficiency in ΑSpII in the inherited chromosomal instability disorder, Fanconi anemia (FA), which has a defect in ability to repair DNA ICLs and a predisposition to cancer. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex essential for monoubiquitination of FANCD2 (FANCD2-Ub), a process which is critical for ICL repair. However, whether any of these FA core proteins play additional roles in ICL repair is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in steps in the ICL repair process in which ΑSpII also plays a role. Immunofluorescence microscopy was used to determine whether FANCF co-localizes with ΑSpII in nuclear foci in normal human cells after they are damaged with an ICL agent, 8-methoxypsoralen plus UVA light (8-MOP). Time course measurements showed that FANCF co-localized in nuclear foci with ΑSpII and followed a similar time course for formation. This time course was similar to that of the ICL repair protein, XPF, which produces incisions at sites of ICLs and acts downstream of FANCD2-Ub. FANCF foci, like ΑSpII and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. This association of FANCF with ΑSpII was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ΑSpII after ICL damage. These studies indicate that FANCF associates with ΑSpII and is involved with ΑSpII in the repair process. Since we have demonstrated that ΑSpII, like XPF, acts downstream of FANCD2-Ub and that FANCF co-localizes with ΑSpII after ICL damage, this suggests that FANCF interacts with ΑSpII in repair events downstream of FANCD2-Ub. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ΑSpII levels to normal and to formation of FANCF nuclear foci, which co-localized with ΑSpII foci. This indicates that ΑSpII is needed in localization of FANCF to sites of damage. These studies support a model we have proposed in which ΑSpII acts as a scaffold in the recruitment of proteins involved in the repair process to sites of ICL damage. They also show that FANCF has an additional function in ICL repair besides monoubiquitination of FANCD2. We propose that after DNA damage and monoubiquitination of FANCD2, ΑSpII and FANCF act downstream of FANCD2, along with XPF, and that the interaction between these proteins at sites of damage is critical for the repair process and maintenance of genomic stability.

#2759

USP21 stabilizes the BRCA2/Rad51 complex to ensure cancer genome maintenance.

Jinping Liu, Alex J. Kruswick, Hien Dang, Andy D. Tran, Xin W. Wang, Philipp Philipp. _NIH, Rockville, MD_.

Cancer is a disease of defective genome maintenance. Paradoxically, however, tumor growth relies on efficient DNA repair to mitigate the detrimental impact of replication-associated DNA double-strand breaks (DSBs). Deleterious mutations in BRCA2, a tumor suppressor that promotes homologous recombination (HR), consequently result in increased susceptibility to genotoxic stress in rapidly dividing tumor cells. While these findings underscore the importance of tightly controlled BRCA2 function for tumor growth, little is known about the factors that regulate this process. Here, we establish the ubiquitin-specific protease USP21 as a positive regulator of BRCA2 stability. USP21 interacts with, deubiquitinates and stabilizes the BRCA2/Rad51 complex and promotes efficient Rad51 loading to DSBs. As a result, depletion of USP21 decreases HR efficiency, causes an increase in DNA damage load and impairs tumor cell survival. Importantly, BRCA2 overexpression partially restores the USP21-associated HR defect. Moreover, we show that USP21 is overexpressed in hepatocellular carcinoma (HCC), where it promotes BRCA2 stability and inversely correlates with patient survival. These findings demonstrate a novel deubiquitination mechanism to control BRCA2 levels and point to USP21 as a potential therapeutic target in BRCA2-proficient tumors.

#2760

Does a short open reading frame (sORF)-encoded protein participate in DNA repair.

Les Hanakahi, Matthew Summerlin, Yi-Chia Tang, Jinho Heo. _University of Illinois College of Pharmacy, Rockford, IL_.

Non-homologous end joining (NHEJ) is one of the major mechanisms for rejoining of DNA double-strand breaks. Knowledge of the molecular mechanism of NHEJ will help us to understand how this process preserves genome integrity, and how NHEJ errors cause chromosomal rearrangements that lead to cancer. Several critical participants in the NHEJ pathway have been identified and some mechanistic steps have been characterized. At present, a significant gap in our understanding of NHEJ is how NHEJ factors assemble at DSBs. It is plausible that additional, as yet unidentified, factors contribute to NHEJ assembly. We have biochemical and biological data implicating a short open reading frame (sORF)-encoded protein in NHEJ. sORFs are found in all genomes. Ribosome profiling, mass spectroscopy and RNA sequencing have predicted the existence of numerous sORF-encoded proteins of <100 amino acids in size. While the functional significance of the majority of these small proteins is not clear, we recently discovered that a 69 amino acid protein encoded by chromosome 7 open-reading frame 49 (C7orf49) interacts with critical NHEJ factors in human cells, and stimulates NHEJ in cell-free extracts. Treatment of cells with the DSB-inducing agent etoposide increased C7orf49 expression and nuclear localization, and knockdown of C7orf49 sensitized cells to etoposide. C7orf49-deficient cells exhibited a mild proliferation defect and increased sensitivity to etoposide. Our data suggest that this small protein may play an important role in double-strand beak repair by NHEJ and highlights the potential biological relevance of sORF-encoded proteins in the DNA damage response and in DNA repair.

#2761

Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation.

Selma M. Cuya, Kellie M. Regal, Robert C.A.M. Van Waardenburg. _University of Alabama at Birmingham, Birmingham, AL_.

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3' and 5' phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 utilizes a two-step catalytic cycle that centers on the formation of an obligatory Tdp1-DNA covalent complex (Tdp1-cc) through its nucleophilic histidine (Hisnuc), resulting in dissociation of the adduct, while its general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg (H493R) mutant stabilizes the Tdp1-cc and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab or substitutions of the Hisnuc transforms yeast Tdp1 into a potent toxin via stabilization of Tdp1-cc. We propose that stabilization of this Tdp1-DNA covalent complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two catalytic Hisgab (H493R or H493N) mutants and one Hisnuc (H263A) mutant of hTdp1 in HEK293 cells. Doxycycline-induced expression of Tdp1H263A, Tdp1H493R, and Tdp1H493N mutant enzymes induced Tdp1-dependent cytotoxicity without additional genotoxic stress. Utilizing two different immuno-assays, we validated that the observed Tdp1-dependent toxicity correlates with stabilization of their enzyme-DNA covalent complex. Moreover, all of these Tdp1 catalytic mutants show reduced catalytic activity compared to wild type hTdp1, but they do not all show a stabilized Tdp1-cc in this in vitro assay. This indicates a significant difference between in vitro Tdp1 activity and cellular Tdp1 activity, which is most likely due to the difference in substrate; a small oligonucleotide with a 3'phospho-tyrosyl modification versus covalent complex of full length Topo1 with genomic DNA. However, these results confirm our previous yeast studies: Stabilization of the Tdp1-cc converts a DNA repair enzyme into a cellular toxin, which constitutes a potential novel therapeutic strategy to treat cancer. In addition, we are comparing schedule dependent 'drug'-combinations of our toxic Tdp1 mutant expression with topotecan, etoposide (targets Topo2-cc) and cisplatin to evaluate the potential therapeutic value of this novel Tdp1 targeted strategy.

This work is in part supported by the ADDA, UAB ACS-IRG, and DOD OCRP WX81WH-15-1-0198. 

### DNA Methylation 1

#2762

Common DNA methylation patterns in cancer and placental cells involved in migration and invasion, immune escape, and angiogenesis induction.

Akpeli V. Nordor,1 Sophie Richon,2 Thierry Fournier,3 Dominique Bellet,4 Virginie Dangles-Marie,5 Martin J. Aryee6. 1 _Institut Curie, PSL Research University, Département de Recherche Translationnelle, Laboratoire d'Investigation Préclinique; Université Paris Descartes, Sorbonne Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé, Paris, France;_ 2 _Institut Curie, PSL Research University, CNRS, UMR 144, Paris, France;_ 3 _Université Paris Descartes, Sorbonne Paris Cité, INSERM, UMR-S 1139; PremUP Foundation, Paris, France;_ 4 _Institut Curie, PSL Research University, Département de Biopathologie, Laboratoire d'Oncobiologie; Université Paris Descartes, Sorbonne Paris Cité, Unité de Technologies Chimiques et Biologiques pour la Santé, Paris, France;_ 5 _Institut Curie, PSL Research University, Département de Recherche Translationnelle, Laboratoire d'Investigation Préclinique; Université Paris Descartes, Sorbonne Paris Cité, Faculté de Pharmacie, Paris, France;_ 6 _Massachusetts General Hospital and Harvard Medical School, Department of Pathology and Center for Cancer Research, Charlestown, MA_.

Identifying common patterns of regulation in cancer and placental cells might shed new light on cellular programs allowing aggressive tumor development. Indeed, as it has been described for the first time more than a century ago, cancer and placental cells (trophoblasts) share astonishingly similar phenotypes : migration and invasion, immune escape, and angiogenesis induction. These common phenotypes, relying on common genomic sequences and transcriptomic profiles, may result from a shared epigenomic regulation program. This might be especially true for tumor cells leading eventually to metastasis and cytotrophoblasts (a trophoblasts subset) during placental implantation at the beginning of pregnancy.

In order to investigate such common epigenomic patterns, we carried out comparisons of DNA methylation in cancer and placental cell genomes. This study involved: cancer samples (primary tumor vs. normal tissue) across various tissues (including breast, colon, liver, lung, prostate, thyroid, uterus), on one hand; and placenta samples (early vs. late term) either heterogeneous (chorionic villi) or homogenous (ex vivo cytotrophoblasts), on the other hand. Cancer data were data downloaded from The Cancer Genome Atlas web portal. Placenta data were downloaded from the Gene Expression Omnibus web portal. In addition, our group generated original data from ex vivo cytotrophoblasts samples. All data were generated on the Illumina Infinium 450K array. Data analysis was carried out thanks to computational methods for epigenomics recently described.

This first direct comparison of cancer and placental cells epigenomes, leveraging both published data and original data, led to the identification of large hypomethylated blocks common to cancer and placental cells. Such common patterns have recently been described as a universal defining epigenetic alteration in human solid tumors. These megabase-scale DNA methylation marks differentiate primary tumors from normal tissues. Likewise, they differentiate early term placentas from late term placentas. Moreover, genes belonging to genomic regions displaying common large hypomethylated blocks overlapping in cancers and placentas are enriched for pathways involved in migration and invasion, immune escape, and angiogenesis induction.

Common DNA methylation patterns in cancer and placental cells identified in this pilot study might contribute to the epigenomic regulation of cellular programs allowing aggressive tumor development. Further analyses of these common patterns, as well as analyses of differences in cancer and placental cell epigenomes, could eventually lead to the identification of critical epigenomic switches that prevent healthy placentas to degenerate into tumors, while they allow aggressive tumors to develop. Ultimately, such epigenomic switches could also represent innovative targets in oncology.

#2763

DNA methylation profiling unveils TGF-β hyperresponse in tumor associated fibroblasts from lung cancer patients.

Miguel Vizoso,1 Marta Puig,2 F. Javier Carmona,1 María Maqueda,3 Antonio Gomez,1 Anna Labernardie,4 Marta Gabasa,2 Saioa Mendizuri,2 Rafael Ikemori,2 Xavier Trepat,4 Sebastian Moran,5 Enrique Vidal,5 Noemí Reguart,6 Alexandre Perera,3 Manel Esteller,1 Jordi Alcaraz2. 1 _Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain;_ 2 _University of Barcelona, Barcelona, Spain;_ 3 _Technical University of Catalonia (UPC), Barcelona, Spain;_ 4 _Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain;_ 5 _Bellvitge Biomedical Research Institute-IDIBELL, L'Hospitalet de Llobregat, Spain;_ 6 _Hospital Clínic de Barcelona, Barcelona, Spain_.

There is growing interest in defining the aberrant molecular differences between normal and tumor-associated fibroblasts (TAFs) that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer (NSCLC) patients, and reported a widespread hypomethylation concomitantly with focal gain of DNA methylation; in addition, we found evidence that a fraction of lung TAFs are fibrocytes in origin. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-β pathway. To get insights on the functional implications of the latter impact, we analyzed the response of lung TAFs to exogenous TGF-β1 in terms of activation and contractility. We found a larger expression of a panel of activation markers including α-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-β1 elicited a larger contractility in TAFs than in CFs as assessed by traction force microscopy. These findings reveal that lung TAFs are hyperresponsive to TGF-β1, which may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer.

#2764

Detection of early gastric cancer with Sox17 DNA methylation analysis of gastric washes.

Yoshiyuki Watanabe, Ritsuko Oikawa, Hiroyuki Yamamoto, Fumio Itoh. _St. Marianna Univ. School of Medicine, Kawasaki, Japan_.

Aim: Although minimal invasive treatment is widely accepted for early stage of gastric cancer (EGC), we still do not have any appropriate risk markers to detect residual neoplasia and some potential of recurrence. We previously reported that aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia (Gastroenterology 2009, Tumor Biology 2011). Our goal is to verify the potency of our three selected candidate genes (Sox17, MINT25, miR34) as a treatment marker for EGC. Method: We conducted a multicenter, prospective study using selected three candidate genes (Sox17, MINT25 and miR34) by Methylated CpG Island Amplification Microarray (MACAM) based analysis and analyzed the DNA methylation levels before and after endoscopic resection (ER) for EGC using gastric washes. We statistically analyzed sensitivity, specificity and AUC with DNA methylation levels of before / after ER and information of recurrent cases in 2 years. Results: 352 EGC cases were registered and endoscopically removed. 182 cases could followed endoscopically for 2 years (51.7%). Sensitivity, specificity and AUC were as follows. Sox17 (70.0%, 48.1%, 0.646), MINT25 (70.0%, 35.2% 0.555), miR34 (65.0%, 49.4%, 0.578). Conclusion: We have developed a new methodology for gastric cancer detection by DNA methylation in gastric washes. It is easy and useful for early detection of recurrence after endoscopic treatment in GCa patients.

#2765

Asbestos exposure-related DNA methylation markers: a validation study in non-small cell lung tumors.

Eeva Kettunen,1 Kristina Stuopelytė,2 Hector Hernandez-Vargas,3 Henrik Wolff,1 Sisko L. Anttila,4 Zdenko Herceg,3 Sonata Jarmalaite,2 Kirsti Husgafvel-Pursiainen1. 1 _Health and Work Ability, Finnish Inst. of Occupational Health, Helsinki, Finland;_ 2 _Division of Human Genome Research Centre, Faculty of Natural Sciences, Vilnius University, Vilnius, Lithuania;_ 3 _Epigenetics Group, International Agency for Research on Cancer, Lyon, France;_ 4 _Dept of Pathology, HUSLAB, Helsinki University Central Hospital & Health and Work Ability, Finnish Inst. of Occupational Health, Helsinki, Finland_.

Introduction. We have earlier explored the epigenome-wide DNA methylation in lung tumors. The study subjects, mostly smokers, consisted of lung cancer patients highly exposed to asbestos and those without exposure. We found methylation markers that associated either with sample type or exposure (asbestos or tobacco smoke) (Kettunen et al submitted). In this validation study, we focused on estimating the prevalence of the distinct methylation markers in a larger material of lung tumor patients with detailed exposure data available.

Study design, subjects, and methods. We investigated the DNA methylation of selected markers in a validation set of 69 non-small cell lung cancer cases, tumor (T) and normal lung (N) from each. The patients, given a written informed consent, had been interviewed for detailed data on work and smoking history (as tobacco smoking pack-years, PY), and the pulmonary asbestos fiber counts quantified. Smoking-related methylation was studied in lung cancer groups of heavy-smokers (PYs higher than 36) vs lighter-smokers (PYs 36 or less) whereas methylation in asbestos-exposed lung tumor patients were compared with that in non-exposed lung tumor patients. DNA methylation was studied in tumor (T) and matched peripheral normal (N) lung tissue using pyrosequencing.

Results. Numerous CpG positions in T vs N and eleven sites in asbestos-exposed tumors had indicated significant methylation variation (MVPs). Twenty-six differentially methylated positions (DMPs) having mean delta beta of at least 10% had been revealed in T vs N. Tobacco smoking- and asbestos exposure-related methylation sites were distinct, involving different CpG sites. We chose to validate five MVPs and four DMPs, both hypo- and hypermethylated CpG sites that were investigated using two instrumentation PSQ96MA and Pyromark Q24. The validation study set indicated good performance in pyrosequencing and we could demonstrate methylation of selected sites in relation to malignancy and exposure in this larger material.

Conclusions. The central observations of the methylation showing distinct changes in relation to sample types and patients' exposure status could be validated in a larger material. Significant methylation variation was observed in lung tumors.

#2766

Genome-wide miRNA methylation analyses in non-small cell lung cancer patients.

Gerwin Heller,1 Corinna Altenberger,1 Thais Topakian,1 Barbara Ziegler,1 György Lang,2 Adelheid End-Pfützenreuter,2 Irene Steiner,3 Sonja Zehetmayer,3 Balazs Döme,2 Walter Klepetko,2 Martin Posch,3 Christoph C. Zielinski,1 Sabine Zöchbauer-Müller1. 1 _Medical Univ. of Vienna, Department of Medicine I, Vienna, Austria;_ 2 _Medical Univ. of Vienna, Department of Thoracic Surgery, Vienna, Austria;_ 3 _Medical Univ. of Vienna, Section for Medical Statistics, Vienna, Austria_.

Deregulated DNA methylation (referred to as methylation) leading to transcriptional inactivation of certain genes occurs frequently in non-small cell lung cancers (NSCLC). Besides protein encoding genes also microRNA (miRNA) encoding genes were found to be targets for methylation in NSCLCs, however, the number of known methylated miRNA genes is still small. Thus, we investigated genome-wide methylation of miRNA genes in primary tumors (TU) and corresponding non-malignant lung tissue samples (NL) of 50 NSCLC patients using methylated DNA immunoprecipitation followed by custom designed tiling microarray analyses (MeDIP-chip). Differentially methylated miRNA genes between TU and NL samples were identified using paired t-statistics with permutation adjusted p-values for step down multiple testing. Using this approach, 201 differentially methylated probes between TU and NL samples were found. These probes were annotated resulting in the identification of 39 tumor-specifically methylated miRNA genes. 79% of them are associated with a 5´ CpG island. While some of these miRNA genes were already known to be methylated in NSCLCs (e.g. miR-9-3, miR-124) methylation of the vast majority of them was unknown so far.

We selected 6 miRNA genes (miR-10b, miR-1179, miR-137, miR-572, miR-3150b and miR-129-2) for gene-specific methylation analyses in TU and corresponding NL samples of 108 NSCLC patients and observed statistically significant tumor-specific methylation of these miRNA genes which confirmed our MeDIP-chip data (p<0.0001, respectively). Using miRWalk2.0 software, we searched for predicted targets of the 6 miRNAs and identified several oncogenic/cell proliferation promoting factors. Examples of them are TFAP2C and HOXA1 (predicted miR-10b targets), HMGB3, CCNE1 and FGF11 (predicted miR-1179 targets), HOXD11 and ONECUT1 (predicted miR-572 targets). By analysing RNA-seq data of > 1.000 NSCLC patients from The Cancer Genome Atlas database, we found that many of the predicted miRNA targets are upregulated in TU samples (e.g. CCNE1, HMGB3, HOXD11, TFAP2A, ZIC2). Based on these findings, we investigated if miR-1179 indeed targets CCNE1 in vitro. Thus, we transfected miR-1179 mimics into CCNE1 expressing NSCLC cell lines (HTB182 and NCI-H1650). Using RT-PCR, we found downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls. In addition, we observed downregulated CCNE1 expression in cells transfected with miR-1179 mimics compared to cells transfected with controls by Western blot analyses. Additional functional analyses of miR-1179 targets as well as of other miRNA targets are ongoing.

In conclusion, we identified a large number of tumor-specifically methylated miRNA genes in NSCLC patients and found that some of these miRNAs target certain oncogenes in NSCLC cells. Overall, our findings emphasize the impact of miRNA gene methylation on the pathogenesis of NSCLCs.

#2767

Expression of miR-1252 and its target PRR4 gene in laryngeal carcinoma.

NUR BUYRU, SEDA EKIZOGLU, JALAL GULIYEV, EMIN KARAMAN, TURGUT ULUTIN. _Istanbul University, Istanbul, Turkey_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide and includes neoplasias of the paranasal sinuses, the oral cavity, the trachea, the pharynx and the larynx. The most important risk factors are tobacco use, alcohol consumption and infection with human papillomavirus (HPV). Besides the exogenous risk factors, genetic factors may play a key role in the development of the HNSCC. Cancer of the larynx accounts for 25% of all head and neck malignancies.

The PRR4 (Prolin Rich Protein 4) gene is localized on human chromosome 12p13 and encodes a protein which is secreted from the acinar cells of the lacrimal gland, the submandibular gland, the parotis and the sublingual glands. Recent studies have demonstrated that PRR4 may play an important role as an antimicrobial protein to protect the ocular surface and the oral cavity. Additionally, it has been shown that PRR4 is highly expressed in the human submucosal glands.

microRNAs are small non-coding RNAs which regulate gene expression at the post-transcriptional level. They bind to their target RNA with their seed sequence complementary to the 3'UTR region. This interaction results in either up- or down-regulation of the target messenger RNA.

To understand the role of the PRR4 gene in laryngeal carcinogenesis, we investigated its mRNA expression levels in laryngeal tumor tissue and adjacent non-cancerous tissue samples from 90 patients by quantitative real-time PCR. As a complementary region between the seed sequence of miR-1252 and 3'UTR region of PRR4 gene exists, we also analyzed whether miR-1252 binds to this region and regulates the expression of PRR4.

We found that PRR4 gene expression was decreased in 65 (72.2%) and increased in 24 (26.7%) of 90 tumor tissues when compared to normal tissue. Statistically significant downregulation of the PRR4 mRNA was observed in laryngeal tumors (p<0.001). No significant correlation was found between PRR4 gene expression and clinicopathological parameters including age, sex, stage, histological grade and tumor location (p>0.05). We also analyzed miR-1252 expression levels in a subset of tumor samples. miR-1252 upregulation was observed only in 24% of tumor tissues compared to non-cancerous tissue. The miR-1252 level was same in the 62.5% of the remaining samples. We did not observe any correlation between the PRR4 and miR-1252 expression levels. Our results indicate that the PRR4 gene may have an impact in the progression of laryngeal carcinoma.

#2768

Global DNA hypomethylation is an important feature of melanoma.

Goran Micevic,1 Nicholas Theodosakis,1 Janis M. Taube,2 Marcus W. Bosenberg,1 Nemanja Rodic1. 1 _Yale School of Medicine, New Haven, CT;_ 2 _The Johns Hopkins Hospital, Baltimore, MD_.

Aberrant DNA methylation occurs nearly universally in cancer. Paradoxically, changes in DNA methylation are bi-directional: there is a focal increase in DNA methylation (hypermethylation), and a global overall decrease in DNA methylation (hypomethylation). Focal hypermethylation plays well described roles in silencing tumor suppressor genes in many malignancies, including melanoma. However, the functional role of global hypomethylation in tumorigenesis is relatively poorly understood. Melanoma is the deadliest form of skin cancer, accounting for approximately 74,000 new cases and 10,000 deaths in the United States in 2015. Changes in DNA methylation are also arguably one of the most common features of melanoma. Similar to other malignancies, most studies have focused on focal hypermethylation, identifying genomic loci differentially methylated between melanocytes and melanoma cells. However, global changes in DNA methylation have not been well described thus far. In this study, we describe global changes in DNA methylation in two melanoma clinical cohorts (JHH cohort, n=13 nevi and n=39 melanomas; NCI cohort n=31 nevi and n=97 melanomas) relative to benign nevi using a semi-quantitative immunohistochemistry based assay. We report a global decrease in DNA methylation levels in melanoma relative to benign melanocytic nevi consistent between both cohorts (p < 0.001, Student's t-test). Furthermore, we studied the functional effect of increasing the level of global DNA methylation in a panel of five early passage melanoma cell lines. Increases in global DNA methylation suppressed proliferation of melanoma cells (p <0.05, Student's t-test), and increased the proportion of apoptotic cells (p <0.01, Student's t-test). Global DNA hypomethylation is thus an important epigenetic feature of melanoma that may regulate proliferative capacity, and merits further investigation.

#2769

Response to 5FU therapy in colorectal cancer is affected by EZH2-mediated BclX splicing.

Natalya Benderska,1 Maria Gazouli,2 Gerasimos Aravantinos,2 Arndt Hartmann,1 Regine Schneider-Stock1. 1 _FAU Erlangen-Nürnberg, Erlangen, Germany;_ 2 _University of Athens, Erlangen, Greece_.

Background and Aims: The chromatin modifier EZH2 is remarkably up-regulated in many cancer types including colon cancer. High levels of EZH2 have been correlated with tumor metastasis and regulation of tumor-initiating cell subpopulation. Since, DZNep, an EZH2 inhibitor, can inhibit the growth capability of colon cancer by inducing apoptosis; down-regulation of EZH2 seems to be a promising therapeutic approach. Our preliminary data showed that EZH2 levels were remarkably increased in blood samples from metastatic colorectal cancer patients who did not respond to the combination therapy with bevacizumab-irinotecan-5FU in comparison to patients who were responders and developed a stable disease. Many apoptosis-related proteins possess a dual function (pro- and anti-apoptotic) due to alternative splicing. Therefore we aimed to find possible apoptosis-associated EZH2 targets to explain these interesting findings.

Methods: HCT116 colon cancer cells were used for DZNep and 5FU treatment and its efficiency was verified by Western blot analysis. In vivo splicing assay was performed using BclX minigene construct. The mRNA expression of EZH2, caspase 2, Death-associated protein kinase (DAPK) and BclX isoforms was assessed by quantitative real-time PCR in cells (BclX also in blood of patients) and normalized to the GAPDH. Cell viability was examined by Crystal Violet staining.

Results: Western Blot for caspase 3 cleavage indicated increased apoptotic levels after DZNep treatment. Combined DZNep and 5FU treatment of cells led to a synergistic effect in reducing cell viability. DZNep treatment specifically promoted pro-apoptotic BclXs isoform, but did not affect the shift of pro-apoptotic isoforms of caspase 2 and DAPK. Using a BclX minigene transfection we confirmed this effect exogenously. The ratio BclXs/Xl was significantly altered by overexpression of different splicing factors such as ASF/SF2, Sam68, after DZnep treatment. Moreover, patients who did not respond to the combination therapy had the highest EZH2 and BclXl isoform levels.

Conclusion: Our data indicate a new interesting link between epigenetic regulation and the splicing machinery. Detailed investigation of EZH2-mediated regulation of BclXs/Xl ratio could have the potential for prediction of 5FU-based therapy response in colon cancer patients.

#2770

DERL3 hypermethylation alters rhabdomyosarcoma cell metabolism.

Pere Llinas, Paula Lopez, Manel Esteller. _Idibell, Barcelona, Spain_.

Physiological cellular activity requires a precise control of the proteome. However, cancer cells have a distorted protein profile. It is likely that intrinsic defects in protein homeostasis participate in human tumorigenesis, such as alterations in de novo protein synthesis, or defects in protein degradation. In this last case, the ubiquitin (Ub) proteasome system (UPS) targets a variety of proteins, including functional proteins that are no longer needed. Without appropriate protein homeostasis maintained by the UPS, healthy cells can undergo malignant transformation, and this observation has been therapeutically exploited by the development of proteasome inhibitors as anticancer agents. Many of the cytoplasm UPS-degraded proteins are retrotranslocated from the endoplasmic reticulum (ER). In this regard, ER possesses a quality control mechanism, termed the ER-associated degradation mechanism (ERAD), that is induced in response to ER stress by a transcriptional program, known as the unfolded protein response (UPR), which leads to the accelerated degradation of unfolded proteins. Within the ERAD pathway, the Derlin proteins play a critical role. It has been proposed that Derlins form an export channel in the membrane of the ER through which the ERAD substrates pass to reach the proteasome.

A previous study in our lab has shown that DERL3 silencing by DNA methylation deregulate SLC2A1 (GLUT1) degradation, promoting the Warburg effect. SLC2A1 is a key glucose receptor up-regulated in most solid tumors, allowing cancer cells to get the glucose they need for tumor progression.

In this study we use an embryonic rhabdomyosarcoma cell line as a model to find out the role of DERL3 in these tumors, as the percentage of promoter hypermethylation is these tumors is approximately 60%. Rhabdomyosarcoma (RMS) is one of the leading causes of cancer related deaths among children, being the histological variant of embryonic rhabdomyosarcoma the most common soft tissue sarcoma of childhood and adolescence, with 350 cases per year in USA.

The restoration of DERL3 expression in vitro promotes GLUT1 degradation and GLUT4 up-regulation, becoming cells sensitive to insulin. Moreover, it has been shown that DERL3 confers sensitivity against an ERAD related drug, Eeyarestatin I, opening a new approach for RMS treatment. All together, these initial findings highlight the role of DERL3 in RMS metabolism.

#2771

Epigenetic regulation of the cocaine and amphetamine regulated transcript (CART) in cancer.

Kate Connor, Sudipto Das, Bruce Moran, Laoighse Mulrane, William M. Gallagher, Darran O'Connor. _University College Dublin, Dublin, Ireland_.

The cocaine and amphetamine regulated transcript (CART) is well documented in the central nervous system for its role in appetite regulation and reward pathways. In recent studies CART has been proven to be a poor prognostic marker in lymph node-negative, estrogen receptor (ER)-positive breast cancer patients, and has also been shown to be associated with worse survival in small bowel carcinoid tumours. The regulation of CART in these different cancer types however remains largely unknown. The presence of a large CpG island in the putative promoter region and the lack of expression of CART in many cancer cell lines suggest a possible epigenetic mechanism of suppression.

A panel of various cancer cell lines (breast, colorectal, endometrial and ovarian) were assessed for endogenous CART expression via western blot and RT-PCR. Cells were then treated with forskolin and leptin; agents which have been previously shown to induce CART expression in the CNS. Cells were also treated with CART conditioned media, which has been previously shown to be the only factor capable of inducing CART expression. Treatment was carried out with varying doses of two DNA methyltransferase inhibitors, 5-Aza-2′-deoxycytidine and RG108, followed by examination of CART expression post treatment. Bisulfite sequencing of the promoter region of CART was performed in all cell lines and a small selection of formalin fixed paraffin embedded (FFPE) tissue samples. Whole methylome sequencing was carried out to confirm this promoter region of CART and study the global effects of treatment with epigenetic modifiers and CART peptide.

Assessment of a collection of cell lines via western blot has shown CART expression to be negative across the panel. Moreover, treatment with forskolin and leptin did not result in re-expression of CART. Treatment with 5-Aza-2′-deoxycytidine has however resulted in increased CART mRNA levels, and subsequent bisulfite sequencing of the promoter region of CART has revealed dense methylation at the CpG island. Treatment with CART conditioned media may also induce promoter demethylation and subsequent re-expression of CART. Collectively, this data suggests the potential involvement of DNA methylation in the regulation of CART.

#2772

SPAG6 and L1TD1 are transcriptionally regulated by DNA methylation in non-small cell lung cancers.

Corinna Altenberger,1 Gerwin Heller,1 Barbara Ziegler,1 Erwin Tomasich,1 Maximilian Marhold,1 Leonhard Müllauer,2 György Lang,3 Adelheid End-Pfützenreuter,3 Balazs Döme,3 Britt-Madeleine Arns,4 Walter Klepetko,3 Christoph C. Zielinski,1 Sabine Zöchbauer-Müller1. 1 _Department of Medicine I, Medical University of Vienna, Austria;_ 2 _Department of Pathology, Medical University of Vienna, Austria;_ 3 _Department of Thoracic Surgery, Medical University of Vienna, Austria;_ 4 _Landesklinikum Thermenregion Hochegg, Grimmenstein, Austria_.

DNA methylation is one of the major epigenetic mechanisms regulating transcriptional activity and is involved in the pathogenesis of non-small cell lung cancers (NSCLC). In a recent study we identified a large number of tumor-specifically methylated genes in NSCLCs (Heller et al., Carcinogenesis 2013). For further detailed analyses we selected the genes SPAG6 (Sperm Associated Antigen 6) and L1TD1 (LINE-1 Type Transposase Domain Containing 1). By analysing publically available IlluminaHiSeq RNA-seq data we observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. In addition, we investigated SPAG6 and L1TD1 mRNA expression in 5 NSCLC cell lines and found SPAG6 as well as L1TD1 mRNA expression frequently downregulated in all of these cell lines compared to normal human bronchial epithelial cells (NHBECs). Subsequently, we treated cells of NSCLC cell lines which did not express SPAG6 or L1TD1 with the epigenetically active drugs 5-aza-2´-deoxycytidine and Trichostatin A and observed re-expression of both genes suggesting that transcriptional regulation of SPAG6 and L1TD1 is mediated by DNA methylation in NSCLCs. Bisulfite genomic sequencing of parts of the 5´ region of SPAG6 and L1TD1 revealed that the vast majority of CpG sites indeed are methylated in NSCLC cells in contrast to NHBECs. Moreover, we analysed SPAG6 and L1TD1 methylation in TU and NL samples of 147 stage I-III NSCLC patients using the gene-specific approach methylation-sensitive high resolution melt analysis (MS-HRM). Differences in SPAG6 as well as in L1TD1 methylation between TU and NL samples were statistically significant for both genes and confirmed that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs. Additionally, we investigated SPAG6 and L1TD1 protein expression in TU and NL samples of 35 NSCLC patients by immunohistochemistry. In the vast majority of SPAG6 or L1TD1 methylated TU samples, protein expression of these genes was downregulated in tumor cells. Moreover, we performed cell proliferation, cell viability and colony formation assays in vitro and observed that ectopic expression of L1TD1 but not of SPAG6 reduced tumor cell proliferation, viability and the ability of NSCLC cells to form colonies. To further investigate the role of L1TD1 in the pathogenesis of NSCLCs, in vivo studies are being carried out. Overall, our results demonstrate that DNA methylation is the major mechanism for frequent downregulation of SPAG6 and L1TD1 expression in NSCLCs.

#2773

Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation.

Michelle P. Vaz,1 Stephen Y. Hwang,1 Ashwini Patil,2 Jillian Phallen,1 Lauren Murphy,1 Cynthia A. Zahnow,1 Victor E. Velculescu,1 Hariharan Easwaran,1 Stephen B. Baylin1. 1 _Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, MD;_ 2 _Krieger School of Arts and Sciences, Advanced Academic Programs, Baltimore, MD_.

Herein, we define a model for how cigarette smoke and chronic inflammation induce human lung cancer via evolution of a co-ordinated pattern of progressive cancer-associated epigenetic abnormalities which prime cells for addiction to a single key genetic alteration, KRAS mutation. Non-clonogenic, non-tumorigenic, epigenetically stable human bronchial epithelial cells (HBEC) were exposed to cigarette smoke condensate (CSC) for 15 months. Earlier studies have established a requirement for the simultaneous disruption of three oncogenes to fully transform these cells. Genome-wide DNA methylation, expression, chromatin changes, as well as binding to chromatin of key epigenetic regulators and cell phenotypic features were examined over time in exposed versus non-exposed cells. CSC exposure acutely causes, within 10 days, a change we have previously associated with DNA damage, tightening of DNA methyltransferase 1 (DNMT1) and EZH2 to chromatin. While the EZH2 binding decreases with prolonged exposure, DNMT1 remains tightly bound to chromatin. Chronic exposure causes progressive, but stochastically variable, global DNA methylation changes which begin by six months and progress over the time course of the study to include hypermethylation of gene promoters which are frequent in human lung cancer. ChIP-seq analyses reveal, preceding the above methylation changes, that promoters of such methylated genes have an initial recruitment of EZH2, which begins at 10 days and then decreases with time. In contrast, recruitment of EZH2 increases with time and remains dominant for these same genes in the non-exposed controls. Following 10 months of exposure, CSC treated cells begin to clone in soft agar, often a feature of transformation, yet do not form tumors in immunodeficient mice. At this time point, gene expression studies show the top signaling pathway change is strong activation of MAP-kinase and KRAS pathways. Yet genome-wide, exome sequencing reveals no known lung cancer driver gene mutations. Remarkably, overexpression of mutant KRAS alone now markedly enlarges the soft agar colonies, and the cells are now fully transformed and form tumors in mice. Our study reveals, in a chronic cigarette exposure model relevant to the time course for evolution of KRAS mutant human lung adenocarcinoma, a key initial role for smoking induced epigenetic changes which facilitate addiction to the oncogene.

#2774

High levels of 5-hydroxymethylcytosine (5hmC) predict biochemical recurrence after prostatectomy in ERG negative prostate cancer.

Siri H. Strand, Soren Hoyer, Anne-Sofie Lynnerup, Christa Haldrup, Tine M. Storebjerg, Michael Borre, Torben F. Orntoft, Karina D. Sorensen. _Aarhus University Hospital, Aarhus, Denmark_.

Background: Prostate cancer (PC) can be stratified into distinct molecular subtypes based on TMPRSS2-ERG gene fusion status, but its potential prognostic value remains controversial. Likewise, routine clinicopathological features cannot clearly distinguish aggressive from indolent tumors at the time of diagnosis, thus new prognostic biomarkers are urgently needed. The DNA methylation variant 5-hydroxymethylcytosine (5hmC, an oxidized derivative of 5-methylcytosine) has recently emerged as a new diagnostic and/or prognostic biomarker candidate for several human malignancies. However, this remains to be systematically investigated for PC. In this study, we determined 5hmC levels in 311 PC (stratified by ERG status) and 228 adjacent non-malignant (NM) prostate tissue specimens by immunohistochemical analysis of a tissue microarray, representing a large radical prostatectomy (RP) cohort with long clinical follow-up. We investigated possible correlations between 5hmC and routine clinicopathological variables and assessed the prognostic potential of 5hmC by Kaplan-Meier, uni- and multivariate Cox regression analyses in ERG+ (n=178) vs. ERG- (n=133) PCs using biochemical recurrence (BCR) as endpoint.

Results: We observed a borderline significant (p=0.06) reduction in 5hmC levels in PC compared to NM tissue samples, which was explained by a highly significant (p<0.001) loss of 5hmC in ERG- PCs. ERG status was not predictive of BCR in this cohort (p=0.73) and no significant association was found between BCR and 5hmC levels in ERG+ PCs (p=0.98). In contrast, high 5hmC immunoreactivity was a significant adverse predictor of BCR after RP in ERG- PCs, independent of Gleason score, pathological tumor stage, surgical margin status, and pre-operative PSA level (HR (95% CI): 1.62 (1.15-2.28), p=0.006).

Conclusions: This is the first study to demonstrate a prognostic potential for 5hmC in PC. Our findings highlight the importance of ERG stratification in PC biomarker studies, and suggest that epigenetic mechanisms involving 5hmC are important for the development and/or progression of ERG- PC.

#2775

Characterization of transcriptional and epigenetic signatures of benign thyroid adenomas: Can we improve preoperative diagnosis of thyroid nodules.

Audrey H. Choi, Ryan Lew, Michael O'Leary, Yuman Fong, John H. Yim, Maria A. Hahn. _City of Hope, Duarte, CA_.

Introduction: It has been estimated that as many as 50,000 patients in the United States receive unnecessary thyroidectomies due to the inability to distinguish benign thyroid nodules from malignant ones. Up to 30% of fine needle aspirates performed for thyroid nodules diagnosis are indeterminate due to overlapping cytological features between benign and malignant nodules. At present, commercially-available diagnostic tests for thyroid nodules are based on the molecular differences observed between thyroid cancer compared to normal thyroid tissue. However, the molecular signature of benign thyroid nodules is neither well-characterized nor included in currently available diagnostic panels. The objective of this study was to determine whether thyroid adenomas (TA) are distinct from normal thyroid tissue and papillary thyroid cancer (PTC).

Methods: Whole transcriptome analysis was used to assess differences in transcriptional activity in 9 TA and 12 PTC tissue pairs (with matched normal adjacent thyroid tissue from the same patients). In order to evaluate epigenetic alterations associated with TA development, we used reduced representation bisulfite sequencing (RRBS) in 114 thyroid specimens (40 PTC, 28 TA and 46 matching adjacent thyroid tissues). This approach provides single base resolution of DNA methylation genome wide.

Results: According to our data, transcriptome activity divided analyzed specimens into three separate groups: PTC, TA and adjacent thyroid tissues. TA demonstrated a unique transcriptional pattern, distinct from both normal adjacent thyroid tissue and PTC. Similar results were obtained by analysis of epigenetic alterations in these tissues. According to the clustering analysis of DNA methylation patterns, the majority (18 of 28) of benign nodules forms a separate cluster, distinct from adjacent normal thyroid and PTC tissue clusters. Within this group, 14 of 28 TA demonstrated a distinct DNA methylation signature associated with TA-specific hypermethylation. In fact, only 4 of 28 TA demonstrated a DNA methylation signature similar to normal thyroid tissue, suggesting that the majority of TA is not equivalent to normal thyroid.

Conclusion: According to whole transcriptome analysis and genome wide analysis of DNA methylation, TA is frequently associated with specific transcriptional and DNA methylation signatures compared to normal thyroid tissue and PTC. These data indicate that the majority of thyroid adenomas are associated with a unique molecular pathway that is distinct from PTC development. Use of the adenoma-specific molecular signature can be an essential factor in the improvement of PTC diagnostic panels, helping to reduce unnecessary thyroidectomies.

#2776

The relationship between UHRF1 overexpression and LINE-1 hypomethylation in esophageal squamous cell carcinoma.

Kenichi Nakamura, Yoshifumi Baba, Keisuke Kosumi, Kazuto Harada, Hironobu Shigaki, Keisuke Miyake, Junji Kurashige, Takatsugu Ishimoto, Masaaki Iwatsuki, Naoya Yoshida, Hideo Baba. _Academia, Kumamoto, Japan_.

Background: Global DNA hypomethylation contributes to oncogenesis through multiple mechanism. The level of long interspersed nucleotide element-1 (LINE-1) methylation is regarded as a surrogate marker of global DNA methylation, and is drawing interest as a good indicator for predicting cancer prognosis. However, the mechanism by which LINE-1 (global DNA) methylation is controlled in cancer cells remains to be fully explored. Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) plays an essential role in DNA methylation. Previous studies have demonstrated that UHRF1 overexpression might be a mechanism of underlying DNA hypomethylation in hepatocellular carcinoma, and contribute to poor prognosis. Nonetheless, the epigenetic role of UHRF1 in esophageal squamous cell carcinoma (ESCC) remains unclear.

Methods: Using 16 frozen tissues (matched pairs of cancer and normal epithelium), and 160 curatively resected esophageal cancer specimens, we analyzed the relationship between UHRF1 expression, LINE-1 methylation levels and patient outcome. We measured UHRF1 expression by real-time PCR and immunohistochemistry, and LINE-1 methylation levels by bisulfite- pyrosequencing technology. In addition, to investigate the relationships between UHRF1 expression and LINE-1 methylation level (i.e., global DNA methylation level) using UHRF1 overexpression vector and siRNA for UHRF1 with ESCC tissues and ESCC cell lines.

Results: In ESCC tissues, UHRF1 expression were significantly associated with LINE-1 methylation levels, and UHRF1 overexpression correlated with poor prognosis in our cohort of 160 ESCC patients. Furthermore, UHRF1 vector-mediated overexpression caused LINE-1 (global DNA) hypomethylation, and conversely, UHRF1 knockdown using siRNA increased DNA methylation in ESCC cell lines.

Conclusion: These results may suggest that UHRF1 is an epigenetic key regulator of DNA methylation, moreover, can be applicable to attractive cancer therapies.

#2777

One-carbon metabolism genetic variant and genome-wide DNA methylation in breast tissues from healthy women.

Min-Ae Song,1 Theodore M. Brasky,1 Catalin Marian,1 Daniel Y. Weng,1 Cenny Taslim,1 Adana A. Llanos,2 Ramona G. Dumitrescu,3 Zhenhua Liu,4 Joel B. Mason,5 Bhaskar V. Kallakury,6 Jo L. Freudenheim,7 Peter G. Shields1. 1 _The Ohio State University, Columbus, OH;_ 2 _Rutgers School of Public Health and Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 3 _Distilled Spirits Council of the United States, Washington, DC;_ 4 _University of Massachusetts, Amherst, MA;_ 5 _Tufts University, Medford, MA;_ 6 _Georgetown University, Washington, DC;_ 7 _University at Buffalo, Buffalo, NJ_.

Altered DNA methylation is an early event in carcinogenesis. Little is known about the mechanism of altered methylation in breast tissue; possible factors include diet such as alcohol and folate intake, and genetic variation for enzymes in one carbon metabolism. Examination of the association of these factors with methylation in breast tissues from healthy women provides insight into these changes. Blood and glandular breast tissues from 81 women with no history of cancer and who underwent reduction mammoplasty were assayed. The 96-plex Illumina BeadXpress® or TaqMan® SNP Genotyping assays assessed SNPs, genome-wide DNA methylation profiling was performed using the Illumina Infinium HumanMethylation450 BeadChip.The Affymetrix GeneChip Human Trascriptome Array 2.0 was used to compare gene expression level with methylation change in fresh frozen breast tissues. Biological networks of differentially-methylated (DM) genes were assigned using the Ingenuity Pathway Analysis (IPA). Fifty-seven CpG sites were DM in comparisons of genotype for eight SNPs in FTHFD, MTHFD1, MTHFR, MTR, MTRR, and TYMS (P<5.0 x 10-5 for each). SNPs in FTHFD were associated with 56% of the DM CpGs. SNPs in FTHFD and MTR were associated with DM CpG sites in their own genes. Six methylation and gene expression pairs were modestly to weakly correlated (P<0.05), five positively correlated (HCN4, FRMD4A, FTHFD, SLC39A7, and LOC63930) and one negatively correlated (ADAMTS14). Four DM CpGs identified by SNPs in MTRR, MTHFR, and FTHFD were significantly associated with alcohol consumption and/or breast folate. Forty-five DM genes were available in the IPA database. IPA revealed enrichment for genes (91%) involved in cancers. The top-scoring network was "Energy Production, Molecular Transportation, Nucleic Acid Metabolism" (score=32). The top molecular and cellular functions were Amino Acid Metabolism (ALDH1L1, MTR, and PTPRN2), Cell-to-Cell Signaling (DLG3, GRN, HLA-DQB1, PTPRN2, and SLC6A3), Cellular Function and Maintenance (ADAMTS14, ADMTS2, CRIPT, DCL1, DLG3, GRN, KAG2, PTPRN2, and RXRB). High concordance of methylation levels for all DM loci analyzed was found between HM450 and pyrosequencing on 75 technically validated samples (Spearman correlation r=0.98, P<1.0x10-47). This is the first comprehensive study of the association between variation in one-carbon metabolism genes and genome-wide DNA methylation in histologically normal breast tissues. These SNPs, particularly FTHFD, as well as alcohol intake and folate exposure appear to affect DNA methylation in the breast of healthy women. The finding that SNPs in FTHFD and MTR are associated with their own methylation is also novel and highlights a role for these SNPs as methylation quantitative trait loci. Understanding of the role of one carbon metabolism in altered DNA methylation could provide insight into prevention of breast tumors.

#2778

Interaction of Microsatellite Instability (MSI) and tumor for Differential DNA Methylation in Colorectal carcinoma.

Muhammad G. Kibriya,1 Mohammed Kamal,2 Mustafizur Rahman,2 Shantanu Roy,1 Maruf Raza,2 Rupash Paul,2 Habibul Ahsan,1 Zahidul Haq,2 Farzana Jasmine1. 1 _University of Chicago, Chicago, IL;_ 2 _Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh_.

Introduction: Colorectal Cancer (CRC) is one of the most common malignancies worldwide. The role of MSI and KRAS somatic mutation in tumor tissue is well known in CRC. In genome-wide scale, we explored whether differential methylation is associated with MSI status.

Methods: We carried out a genome-wide methylation assay (Illumina 450K) for a total of 250 paired samples from 125 CRC patients (m=72, f=53) at different stages (stage1:25, stage II: 33 and stageIII: 67). Of them 101 had left-sided (descending colon to rectum) CRC and 30 had MSI, and 34 had somatic mutation in KRAS (rs112445441).

Results: MSI was more frequent in the right-sided tumor (54% vs. 17%, p=0.003). Frequency of KRAS mutation was not different between right and left-sided CRC (29% vs. 27%). Among the patients with microsatellite stable (MSS) CRC (n=95), paired comparison of methylation data between tumor and corresponding normal tissue revealed a total of 1641 tumor-specific differentially methylated loci (DML) covering 686 genes that were significant at FDR 0.001 and the magnitude of difference (delta beta) was at least 20%; Similar analysis in patients with MSI (n=30) revealed 6209 tumor-specific DML covering 2316 genes. This suggested that MSI is associated with methylation change in much larger number of genes.

We could not find any methylation signature from normal colon tissue that could predict MSI or KRAS mutation in the corresponding tumor tissue. However, we identified 413 genes to be differentially methylated in tumor tissue compared to corresponding normal tissue only in presence of MSI, irrespective of KRAS mutation status, tumor staging, and location of tumor. These are "MSI-associated tumor-specific genes". Among these, 19 DML covering17 genes showed delta-beta >30%. The list was enriched in genes associated with biologically relevant GO-terms like, "negative regulation of cell proliferation", "regulation of MAP kinase activity", "regulation of biological process" etc.

We also identified 240 genes differentially methylated in tumor tissue compared to corresponding normal tissue irrespective of MSI status, KRAS mutation, tumor staging, and location of tumor. These are "general tumor-specific genes". Among these loci, 87 DML (49 genes) showed delta-beta >30%.

Conclusions: Our study shows evidence of association between MSI and DNA methylation in the pathogenesis of CRC.

#2779

A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

Andrew D. Kelly,1 Heike Kroeger,2 Jumpei Yamazaki,1 Rodolphe Taby,2 Frank Neumann,2 Sijia Yu,1 Justin T. Lee,1 Rong He,2 Shoudan Liang,2 Yue Lu,2 Matteo Cesaroni,1 Sherry A. Pierce,2 Steven M. Kornblau,2 Carlos E. Bueso-Ramos,2 Farhad Ravandi,2 Hagop M. Kantarjain,2 Jaroslav Jelinek,1 Jean-Pierre J. Issa1. 1 _Temple University School of Medicine, Philadelphia, PA;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Acute myeloid leukemia (AML) causes the most leukemia-related deaths in the United States, and has frequent epigenetic aberrations, including a CpG island methylator phenotype (CIMP). CIMP defines unique molecular subtypes of other cancers and has been linked to mutations in IDH1/2, however the clinical consequences of CIMP and the role of IDH1/2 mutations in AML remain unclear.

Methods: To measure genome-wide CpG methylation we used Digital Restriction Enzyme Analysis of Methylation (DREAM) on AML bone marrow samples and normal peripheral blood controls. For validation we used methylation data from patient samples from The Cancer Genome Atlas (TCGA) on the Illumina Infinium HumanMethylation450 platform. We also used RNA-seq data from TCGA, and microarray data from GEO (GSE6891). Statistical analysis was done using R.

Results: Genome-wide analysis of variably methylated CpG sites in 96 AML bone marrow samples using DREAM revealed two distinct CpG island methylator phenotypes by hierarchical clustering: IDH-CIMP (I-CIMP) in which 7/10 cases had oncogenic IDH1/2 mutations, and AML-CIMP (A-CIMP), which lacked any mutations in IDH1/2. At median follow-up of 6.16 years, A-CIMP cases, but not I-CIMP cases were associated with longer overall survival (median OS, years: A-CIMP = Not reached, P=0.08; I-CIMP =3.35, P=0.50; CIMP-negative =1.17). We validated and extended these findings using TCGA data. In this cohort A-CIMP cases also had significantly longer OS compared to CIMP-negative (median OS, years: A-CIMP =2.34, P=0.01; I-CIMP =1.25, P=0.89; CIMP-negative =1.00). Aberrant hypermethylation in A-CIMP occurred preferentially at CpG islands by a factor greater than 3, while I-CIMP cases demonstrated a slight preference for hypermethylation at sites outside CpG islands. Interestingly, A-CIMP was enriched in CEBPA (19%) and WT1 mutations (14%), but inversely correlated with IDH, TET2, and NPM1 mutations. Functional pathway analysis revealed that genes hypermethylated in A-CIMP are associated with pluripotency maintenance - including PAX6, GBX2, and HOXA9 - and RNA-seq data largely, but not entirely, recapitulated methylation-based patterns. There was a strong correlation between promoter CpG island methylation and gene expression for many A-CIMP genes. Finally, the transcriptional program associated with A-CIMP was found to correlate with outcome and genetic backgrounds in both TCGA and additional independent datasets.

Conclusions: Taken together, our data suggest that CIMP in AML is complex, multifactorial and cannot be explained solely by coding gene mutations (e.g. IDH1/2, TET2). There is an association between A-CIMP and curability in multiple AML datasets that cannot be recapitulated by mutational data alone and that may be worth validating in prospective studies.

#2780

Genome-wide methylation analysis reveals methylator subtypes of Barrett's esophagus and esophageal adenocarcinoma.

Ming Yu,1 Sean Maden,1 Andrew Kaz,2 Tai Heinzerling,1 Rachele O'Leary,1 Matthew Stachler,3 Adam Bass,4 Amitabh Chak,5 Joseph Willis,6 Sanford Markowitz,7 William Grady8. 1 _Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Clinical Research Division, Fred Hutchinson Cancer Research Center; Department of Medicine, University of Washington School of Medicine; Gastroenterology Service, VA Puget Sound Health Care System, Seattle, WA;_ 3 _Department of Pathology, Brigham and Women's Hospital and Harvard Medical School; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA;_ 4 _Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA;_ 5 _Division of Gastroenterology, Division of Oncology, University Hospitals Case Medical Center, Cleveland, OH;_ 6 _Department of Pathology, University Hospitals Case Medical Center, Cleveland, OH;_ 7 _Department of Medicine, University Hospitals Case Medical Center; Case Comprehensive Cancer Center, Case Western Reserve University and Case Medical Center, Cleveland, OH;_ 8 _Clinical Research Division, Fred Hutchinson Cancer Research Center; Department of Medicine, University of Washington School of Medicine, Seattle, WA_.

Esophageal adenocarcinoma (EAC) arises from Barrett's Esophagus (BE) through a progression sequence driven by the accumulation of epi/genetic alterations. Genetic alterations and aberrantly methylated genes/loci, the best-characterized epigenetic alteration, vary among individual BE and EAC cases. To better understand the molecular heterogeneity in BE/ EAC, we analyzed genome-wide DNA methylation patterns in BE (N=98), EAC (N=23), and normal esophagus and stomach (N=78) collected via Esophagus Translational Research Network (BETRNet) using Human Methylation 450K (HM450K) arrays. We identified methylation-based subtypes using a model-based recursively partitioned mixture model and unsupervised resampling-based hierarchical cluster analysis with the most variable probes based on standard deviation. All analyses were performed in R. Four methylator subtypes were identified in the BE/EAC dataset using our clustering approach: high (HM); intermediate (IM); low (LM); and minimal (MM). The clusters showed widespread methylation differences in CpGs primarily located in CpG islands (72% of 414 total probes). Each methylation subtype showed distinct genetic and clinical features suggesting underlying biological differences (Table 1). HM exhibited methylation patterns reminiscent of a CpG Island Methylator Phenotype (CIMP). Among BE/EAC cases with known TP53 mutation status, the majority of mutants were among HM and IM clusters (15/19, 78.9%), Finally, identification of methylation subtypes in an independent HM450K dataset of EAC samples (N=89) from the Cancer Genome Atlas validated the existence of the BE/EAC methylation subtypes. In summary, our data suggest genome wide alterations in DNA methylation occur in the BE stage of the BE-to-EAC progression. We also identified four distinct methylation subtypes of BE/EAC, including a CIMP-like HM subtype, and found more TP53 mutants in the HM and IM subtypes, revealing molecular heterogeneity in different methylation subtypes.

Table 1. Characteristics of primary BE/EAC dataset and subtypes from cluster analyses.

---

Cluster Characteristic | |  | Subtypes  | |  | |

|

|  | |

High N (%) | Intermediate N (%) | Low N (%) | Minimal N (%) | Total N (%)

|

|

Case Counts | Total  | 31 (26%) | 44 (36%) | 28 (23%) | 18 (23%) | 121 (100%)

|

|  | BE  | 23 (23%) | 34 (35%) | 24 (24%) | 17 (17%) | 98 (100%)

|

|  | EAC  | 8 (35%) | 10 (43%) | 4 (17%) | 1 (4%) | 23 (100%)

|

Demographic Variables | Gender | M | 28 (28%) | 35 (35%) | 23 (23%) | 14 (14%) | 100 (100%)

|

|

Gender | F | 3 (14%) | 9 (43%) | 5 (24%) | 4 (19%) | 21 (100%)

|

|  | Age (mean ± sd)  | 68 ± 11 | 63 ± 13 | 61 ± 14 | 61 ± 10 | 64 ± 13

|

TP53 Mutations*** | |

Mutation*  | 6 (32%) | 9 (47%) | 4 (21%) | 0 (0%) | 19 (100%)

|

|  | WT  | 6 (30%) | 8 (40%) | 5 (25%) | 1 (5%) | 20 (100%)

|

|  | Unknown  | 19 | 27 | 19 | 17 | 82

|

*** Mutation categories included: Frame Shift Deletion; In Frame Insertion; In Frame Deletion; Missense Mutation; Nonsense Mutation; and Splice Site  | |  | |  | |  | |

#2781

Novel putative diagnostic methylation biomarkers for prostate cancer identified by whole genome DNA methylation profiling.

Arup Ratan Chakraborty,1 Erica Hlavin Bell,1 Simon Kriste,2 Vanessa Drendel,2 Joseph McElRoy,1 Martin Werner,2 Anca Grosu,2 Arnab Chakravarti1. 1 _Ohio State University, Columbus, OH;_ 2 _University Medical Center Freiburg, Freiburg, Germany_.

BACKGROUND

Aberrant patterns of DNA methylation is a typical hallmark of cancer. Several studies have reported DNA hypermethylation of a significant number of genes in various malignancies. This suggests a role of hypermethylation in the initiation and progression of cancer. In this study, we investigated genome-wide alterations in DNA methylation in prostate cancer (PCa). We compared alterations between tumor (Tu) and adjacent epithelial (Ep) tissues in order to identify hypermethylated genes in promoter regions associated with initiation and progression of prostate cancer.

METHODS

The study included 33 tumor samples including 21 paired Tu and Ep samples from 21 radical prostatectomy patients. Genomic DNA was isolated from FFPE tissue blocks. Genome-wide DNA methylation profiling was performed using the Infinium Illumina 450K array. A mixed linear model, with a random effect for patient, main effect of tissue, and covariate of array batch, was employed to identify probes that differed in methylation between tissue types. Multiple comparison adjustment was performed using the FDR (false discovery rate) method.

RESULTS

In total, 8791 hypermethylated CpG sites in the promoter regions were identified in Tu versus Ep tissue with FDR adjusted P <0.05. Hypermethylation status of our top-ranked hypermethylated genes (with an estimated mean absolute difference 0.3 or greater) between tumor and epithelial tissue was also observed in publicly available methylation data in PCa. To find out if promoter hypermethylation was associated with the regulation of gene expression, we analyzed gene expression of the 20 top-ranked hypermethylated genes using publicly available mRNA expression data in PCa. Several genes exhibited reduced expression in PCa samples. Similar observation was also noted in in vitro studies as significant (P <0.05) increase in gene expression following 5-Azacytidine treatment was detected only in PCa cell lines but not in normal prostate cell line.

CONCLUSIONS

This study identified promoter hypermethylation of several genes in PCa. These newly identified hypermethylated genes are potential biomarker candidates for PCa diagnosis. Currently we are evaluating the functional significance of these hypermethylated genes in prostate cancer.

This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC).

#2782

The effect of LINE-1 methylation level on survival in pancreatic cancer.

Kensuke Yamamura,1 Yoshifumi Baba,1 Lei Zhou,2 Xiaobo Zhang,2 Keisuke Kosumi,1 Katunobu Taki,1 Kazuto Harada,1 Takatsugu Ishimoto,1 Katsunori Imai,1 Daisuke Hashimoto,1 Akira Chikamoto,1 Toru Beppu,1 Tan Xiaodong,2 Hideo Baba1. 1 _Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan;_ 2 _China Medical University Shengjing Hospital, Shenyang, China_.

Background: Genome-wide DNA hypomethylation plays an important role in genomic instability and carcinogenesis. DNA methylation in the long interspersed nucleotide element-1 (LINE-1) is a good indicator of the global DNA methylation level. In previous studies, we demonstrated that LINE-1 hypomethylation was strongly associated with a poor prognosis in some types of cancers including esophageal cancer, gastric cancer, and hepatocellular carcinoma, supporting its potential role as a prognostic marker. However, the relationship between LINE-1 hypomethylation and clinical outcome in pancreatic cancer remains unclear.

Methods: LINE-1 methylation level in 126 samples resected pancreatic cancers was measured by pyrosequencing assay, and the prognostic value of LINE-1 methylation level in pancreatic cancer was examined.

Results: Pancreatic cancers showed significantly lower LINE-1 methylation levels compared to matched non tumorous parenchyma (P=0.039; N=36). Tumoral LINE-1 methylation range was 41.3-92.8 (N=126; mean: 77.7; median: 78.5; standard deviation: 5.7). However, LINE-1 hypomethylation was not significantly associated with overall survival, cancer specific survival, disease-free survival [log-rank p=0.30, p=0.18 and p=0.50, respectively].

Conclusion: The present result suggested that LINE-1 hypomethylation in pancreatic cancer is not associated with poor prognosis. In this study, our cohort was small, therefore further accumulation of data may be needed in order to confirm the effect of LINE-1 methylation level on survival in pancreatic cancer.

#2783

Integrated genomics and epigenomics analysis reveals genome-wide effect of the DNA methyltransferase inhibitor 5-azacitidine (Aza) on regulation of transcription.

Yuchen Bai,1 Emanuele Palescandolo,2 John Whitaker,3 Vinod Krishna,1 Vipul Bhargava,1 Satya Saxena,1 Xiang Yao,3 David Pocalyko,1 Kurtis Bachman1. 1 _Johnson & Johnson, Spring House, PA; _2 _Johnson & Johnson, Beerse, Belgium; _3 _Johnson & Johnson, La Jolla, CA_.

DNA methyltransferase inhibitors (DNMTi) such as 5-azacytidine(Aza) are clinically used in treating hematologic malignancies. Promising effects were also observed in solid tumors, although the exact mechanism underlining the efficacy was unclear. There has been accumulating evidence suggesting an enhanced immune response to the tumor cells following such epigenetic therapies. Cancer immunotherapies targeting checkpoint inhibition in combination with epigenetic agents such as a DNMTi could hold promise for those patients that do not respond to immunotherapies alone. Aza was also found to stimulate an interferon response in tumor cells via inducing expression of endogenous retroviruses. In this study, we were interested to further explore the mechanisms of AZA on transcription regulation across the whole regulome.

HCT-116 is a colon cancer cell line genetically defined by microsatellite instability (MSI) and mutations in KRAS(G13D) and PIK3CA(H1047R). In this study, HCT-116 cells were treated with DMSO (control) or 1μM Aza for 42 and 96 hours. RNA-seq, Mass spectrometry proteomics, RRBS, Chip-Seq, ATAC-Seq and Hi-C data was collected. Data was analyzed by integrative computational methods. Treatment with Aza resulted in global demethylation at CpG islands(CGI), which was moderately correlated with increased expression of the corresponding genes. DNA methylation, DNA accessibility, H3K4me3 (active promoter histone mark), H3K27me3 (repressive histone mark) and RNA expression revealed and cross-verified different states of genomic regions. Pathway and gene set enrichment analysis found activation of anti-viral defense and interferon pathways, as well as increased expression of cancer-testis antigens. IRF7 expression was increased by Aza, and IRF binding motifs were the most enriched TF motifs at the immediate promoter regions of the up-regulated genes. The data also suggested that Aza may remove gene repression by disrupting the scaffolding function of DNMTs. The effects of Aza on promoter-enhancer interactions were also examined by Hi-C. In conclusion, we found that DNMTi such as Aza regulates gene transcription through multiple modes of actions.

#2784

Mitochondrial genomic backgrounds affect nuclear DNA methylation and gene expression.

Carolyn J. Vivian,1 Amanda E. Brinker,1 Gerald C. Gooden,2 Christophe Legendre,2 Samuel Turpin,1 Devin C. Koestler,1 Bodour Salhia,2 Danny R. Welch1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Translational Genomics Research Institute, Phoenix, AZ_.

Increasing evidence shows mitochondrial DNA (mtDNA) genetics contribute to complex diseases like cancer, but the mechanisms responsible are mostly unknown. A new genetically engineered mouse model was created that contains nuclear DNA from one mouse strain and mtDNA from different mouse strain(s), designated Mitochondrial Nuclear Exchange (MNX). We have demonstrated that specific mtDNA-nDNA combinations could modify metastasis efficiency. The underlying mechanism is hypothesized to be due to mtDNA-driven changes in nDNA expression, but how mtDNA regulates the coordinated gene expression is not yet known. Since DNA methylation is an important epigenetic modification that occurs in all vertebrate genomes and retrograde and anterograde cross-talk exists between the nuclear and mitochondrial genomes, we hypothesized that the nuclear epigenome, specifically methylation, changes as a result of mitochondrial haplotype. To test this hypothesis, we performed Meth-Seq using the Agilent Mouse SureSelectXT, RNA-Seq using Illumina HiSeq 2500®, and Affymetrix GeneChip® Mouse Transcriptome Array. Initial studies were performed using four male mouse brains (8 wk) that were pooled from different litters and cages of wild-type and MNX mice. Significant and selective differential DNA methylation and gene expression patterns were observed and indicate that there are some global, some subtle, but mostly reproducible, differences between these strains. Pathway analysis show clustering of changes involving cell adhesion, ion channels, cell surface receptors, metabolism and molecules involved in transport. Together these observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to cancer pathogenesis. Support: Susan G. Komen for the Cure (SAC11037), Natl Fndn Cancer Res, Steiner Family Fund for Metastasis Research, Kansas Bioscience Authority CA134981, P30-CA168524

#2785

The role of heat shock factors for cancer stem cell regulation.

Ankita Thakkar, Bin Wang, Tan A. Ince. _University of Miami, Miller School of Medicine, Miami, FL_.

Introduction: Heat shock factor family is a group of transcription factors that can bind specifically to heat shock elements (HSE) within the promoter region of heat shock protein (HSP). The HSF pathway activation helps cells survive under stress by preventing protein misfolding and aggregation. Three heat shock factors (HSF1, HSF2 and HSF4) have been identified in humans, among them, HSF1, which is believed to be the master regulator of cellular response to stress, is by far the best-characterized member of the HSF family.

We first identified that high HSF1 is associated with high tumor grade and poor survival in multiple cancer models including breast, ovarian, lung and colon cancers. In addition, our recent data indicate that HSF1 drives a distinct transcription program that is unrelated to heat shock response in malignant tumors, and this HSF1 "non-heat shock related transcription program" correlates with poor outcome in breast cancer patients. Moreover, we confirmed that HSF1 drives the Cancer Stem Cell (CSC) phenotype in multiple breast cancer cell lines and facilitate chemo-treatment resistance by increasing CSC frequency in breast cancer cells.

Previous studies have shown that HSF2 and HSF4 may play a role in cancer progression either on their own or through interaction with HSF1. HSF2 forms hetero-trimers with HSF1 and facilitate its function in response to heat shock. Inactivation of HSF4 in p53 null mouse models causes cell senescence and suppression of tumorigenesis, with great similarities to HSF1-/- p53 null mice.

Results: In an attempt to investigate how HSF1 is activated to regulate CSC phenotype, we constructed phospho HSF1 at three different sites. We observed that there is no correlation of phospho activation of HSF1 with CSC phenotype. In follow-up work to the studies above, we found that both HSF2 and HSF4 are also significantly up-regulated in CSCs, suggesting that they may cooperate with HSF1 in regulating CSCs. Moreover, we discovered that HSF4 is over-expressed in breast cancers compared to normal mammary gland, and over-expression of HSF4 induces tumorsphere formation in breast cancer cells.

Conclusion: These observations indicated that in order to gain a complete understanding of the role of the HSFs in cancer it is now necessary to study all three HSFs together.

#2786

Differences in gene-specific DNA methylation in blood DNA as a biomarker for early detection of hepatocellular carcinoma in populations at risk.

Katarzyna Lubecka,1 Lucinda Kurzava,1 Kirsty Flower,2 Hannah Buvala,1 Samer Gawrieh,3 Suthat Liangpunsakul,3 Naga Chalasani,3 James M. Flanagan,2 Barbara Stefanska1. 1 _Purdue University, West Lafayette, IN;_ 2 _Imperial College London, London, United Kingdom;_ 3 _Indiana University School of Medicine, Indianapolis, IN_.

Hepatocellular carcinoma (HCC), the most prevalent type of primary liver cancer, is the second leading cause of cancer death worldwide. It is estimated that early HCC detection would increase the cure rate from 5% to 80%. Approximately 85% of individuals with HCC have underlying liver cirrhosis which is the main risk factor for developing HCC. Every year up to 5% of cirrhotic patients are diagnosed with HCC. The biggest challenge that physicians face today is to distinguish cirrhotic patients that will develop cancer from those that will not. Thus, early detection biomarkers that can be implemented as screening tools in populations at risk are of high interest.

In our previous genome-wide study, we demonstrated that blood DNA has different DNA methylation patterns specific to HCC samples collected before diagnosis (pre-diagnostic) compared to healthy individuals without cancer. We established 10 differentially methylated probes corresponding to 10 genes that distinguish pre-diagnostic HCC blood samples from healthy controls. In the current study using pyrosequencing, we tested the diagnostic potential of these probes in blood samples from a prospective cohort of cirrhotic patients without HCC at the time of blood collection. Twenty eight of these patients developed HCC within 4 years of follow-up. These 28 cases were matched with 23 cirrhotic controls on gender, age, ethnicity, hepatitis C, and diabetes.

Five out of the 10 tested probes distinguished cirrhotic patients who subsequently developed HCC (cases) from those who stayed cancer free (controls) (p˂0.05, Mann-Whitney U test). The highest differences were observed at CpG sites located within BARD1 whose methylation was consistently lower in cases vs. controls. Using this probe, 22 cases were correctly identified as cases (79% sensitivity), whereas only 6 controls were falsely included into cases (74% specificity). Analysis of the panel of the 5 probes increased sensitivity to 86% with a threshold of 2 positive probes, while maintaining 74% specificity. Receiver Operating Characteristics (ROC) curve analysis confirmed high predictive value for the 5 probes in the cirrhotic population [Area Under the Curve (AUC) = 0.80]. The established DNA methylation biomarkers have better values of performance compared to current recommended methods for HCC surveillance, such as serum alpha-fetoprotein (AFP) that can miss up to 40% of patients.

Our results establish the possible diagnostic value of gene-specific DNA methylation in blood DNA for HCC early detection in populations at risk such as individuals with liver cirrhosis. This study was supported by Showalter Trust and PCCR Awards granted to BS.

#2787

Expression of Olfactomedin 4 is regulated by methylation in the benign prostate epithelial cell-RWPE1 and PC-3 prostate cancer cells.

Hongzhen Li. _NIH-NHLBI, Bethesda, MD_.

Expression of Olfactomedin 4 gene (OLFM4) plays a critical tumor suppressor role in human and murine prostate cancer. We have previously reported that loss or reduced OLFM4 expression is associated with the progression of human prostate cancer. We and others have further determined that deletions within the OLFM4 gene are present in about 10 to 25% of human prostate cancer samples. However, we have observed that loss of expression of OLFM4 were found in more than 58% of the samples obtained from advanced prostate cancer patients. Therefore, we sought to study the role of epigenetic factors in the regulation of OLFM4 gene expression in normal and prostate cancer cell lines. Methylation of CpG sites in the promoter region of key genes is an important epigenetic mechanism in cancer development. We examined the methylation status of CpG sites in the OLFM4 promoter region in the benign immortalized prostate epithelial cell line (RWPE1), androgen-responsive cancer cells (LNCaP, VCaP), and androgen-independent cancer cells (PC-3 and DU145).

There are eight CpG sites (-681, -666, -562, -486, -91, +4, +34) in the promoter regions of OLFM4 gene. The pattern of methylation status was detected and compared between RWPE1 cell and cancer cell lines LNCaP, VCaP, PC-3 and Du145. To test whether hypermethylation of CpG sites of OLFM4 gene can block the expression of OLFM4 gene, RWPE1 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 µM) from 24h to 96h and PC-3 cells were treated with 5-azadeoxycytidine (5-Aza.) (5 µM) for 3 days and 7 days. The methylation status of CpG sites was compared in the presence or absence of 5-Aza. at different time points. The addition of 5-Aza. consistently reduced the percent methylation of the CpG sites. Correspondingly, the expression of the OLFM4 gene was induced after treatment with 5-Aza. in both RWPE1 and PC-3 cells. These results suggest that expression of OLFM4 is regulated by the methylation status of OLFM4 gene in the RWPE1 and PC-3 prostatic cell lines.

#2788

Inhibition of DNA methylation suppresses intestinal tumor organoids by inducing an anti-viral response.

Yoshimasa Saito, Kasumi Sakai, Toshihide Muramatsu, Toshiaki Nakaoka, Masaki Kimura, Hidetsugu Saito. _Keio University Faculty of Pharmacy, Tokyo, Japan_.

Background and Aim

It is believed that the anti-tumor effect of DNA methylation inhibitors is mediated by re-activation of epigenetically silenced tumor suppressor genes in cancer cells. However, recent studies have proposed that the major anti-tumor effect of DNA methylation inhibitors is induction of interferon-related genes via dsRNAs-containing endogenous retroviruses. These studies used conventional cancer cell lines, which are maintained in 2D culture condition with serum-containing medium and are considered to be relatively artificial models of cancer cells. Recently, 3D culture system for stem cells known as organoid culture has been developed. Lgr5-positive stem cells form organoids that closely recapitulate the properties of original tissues. To investigate the effect of DNA demethylation on tumor organoids, we have established intestinal tumor organoids from tumors of ApcMin/+ (Min) mice and subjected them to 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment and Dnmt1 knockdown.

Methods

Min mice were treated with 5-Aza-CdR (1 μg/body weight, n=12) or PBS (n=11) by subcutaneous injection weekly from 6 weeks of age. At 21 weeks of age, mice were dissected and number of intestinal polyps was counted. Stem cells were isolated from intestinal tumors of Min mice and maintained by organoid culture. Treatment with 5-Aza-CdR and lentivirus-mediated knockdown of Dnmt1 were performed in organoids derived from intestinal tumors. Expression profiles of genes after treatment with 5-Aza-CdR and Dnmt1-knockdown were analyzed.

Results

Treatment of Min mice with 5-Aza-CdR significantly reduced the average number of intestinal adenomas from 66 to 44 (male) and from 65 to 47 (female). The average number of large adenomas (≧3mm) in Min mice treated with 5-Aza-CdR was significantly decreased from 24 to11, whereas there was no significant difference in the average number of small adenomas (< 3mm). We successfully established organoids derived from intestinal tumors of Min mice by 3D culture with serum-free medium including epidermal growth factor (EGF) and Noggin. DNA demethylation induced by 5-Aza-CdR treatment and Dnmt1 knockdown significantly reduced the cell proliferation of the tumor organoids. Microarray analyses of the tumor organoids after 5-Aza-CdR treatment and Dnmt1 knockdown revealed that interferon-related genes including interferon regulatory factor 7 (Irf7) were activated by DNA demethylation. Gene ontology and pathway analyses clearly demonstrated that these genes activated by DNA demethylation are involved in the anti-viral response.

Conclusions

DNA demethylation suppresses the proliferation of intestinal tumor organoids by inducing an anti-viral response including activation of interferon-related genes. These findings suggest that treatment of colon cancers with DNA methylation inhibitors such as 5-Aza-CdR may be an effective therapeutic approach inhibiting cancer stem cells by immune response.

#2789

Investigating the role of DNA methylation in pediatric choroid plexus tumors.

Malgorzata Pienkowska,1 Sanaa Choufani,1 Andrei Turinsky,1 Diana Merino,1 Ana Novokmet,1 Michael Brudno,1 Rosanna Weksberg,1 Adam Shlien,1 Cynthia Hawkins,1 Eric Bouffet,1 Uri Tabori,1 Richard Gilbertson,2 David Malkin1. 1 _The Hospital for Sick Children, Toronto, Ontario, Canada;_ 2 _Cambridge Cancer Center, Cambridge, United Kingdom_.

Choroid plexus tumors (CPTs) are rare neoplasms of the central nervous system most commonly found in the pediatric population. CPTs represent 1- 4% of all childhood brain tumors, with 10- 20% occurring during the first year of life. Within this family of tumors, choroid plexus carcinoma (CPC) is the malignant neoplasm which is categorized as a grade III tumor by the WHO. Choroid plexus papilloma (CPP) is a benign form classified as a grade I tumor, and atypical choroid plexus papilloma (aCPP) as a grade II tumor. Distinction between these tumor subtypes is essential for treatment stratification.

Previous studies performed in our laboratory suggest that CPTs are highly unstable and harbor unique patterns of chromosome-wide gains and losses. To better understand the complexities of tumor biology of CPTs as well as to identify better molecular biomarkers to distinguish between aggressive and benign forms of CPTs we performed a genome-wide DNA methylation study using Illumina Human Methylation450 BeadChip. We analyzed genome-wide DNA methylation profiles from 34 CPT (14 CPCs, 5 aCPPs and 15 CPPs) samples. Differential DNA methylation analysis did not identify significant differences between aCPPs and CPPs, therefore we explored CPC-specific DNA methylation signature in comparison to CPPs. Using a median beta value difference of 0.3 or greater and an FDR adjusted p-value<0.05, we identified 3361 CpGs that showed significant difference in methylation between CPCs and CPPs or aCPPs. Two-way clustering performed using Pearson's correlation and average linkage for both the sample tree and the gene tree revealed segregation between the majority of CPCs and CPPs or aCPPs. Two main clusters were discovered within CPCs that were due to differences in TP53 mutation status. Pathway analysis on a 1328 gene set overlapping the 3361 CpGs using the IPA software revealed nine canonical pathways with GABA receptor on top of the list and several biofunction categories associated with cellular growth and proliferation that were significantly enriched in CPCs in comparison with CPPs or aCPPs. To identify minimal CPC specific signature, we applied a difference in DNA methylation of 40% and a p-value of 0.001. This increased statistical stringency led to the identification of 59 CpG sites encompassing 33 candidate genes. Of them, 3 genes were validated by pyrosequencing in the initial CPT cohort (n=34) and a new replication cohort of n=23 CPT samples. Next, we tested the sensitivity of the CPC specific DNA methylation signature against DNA methylation profiles of several brain tumor datasets extracted from GEO database and found that CPC-DNA methylation signature was highly specific. Our data suggest that dysregulation of epigenetic mechanisms contribute to the molecular events leading to tumor development and progression in CPC and that our DNA methylation based biomarker signature can have prognostic value for this disease and enable better treatment strategies.

### Hypoxia and Oxidative Stress

#2790

Sensitizing the hypoxic tumor microenvironment with OMX, a breakthrough oxygen delivery protein: From protein engineering to clinical trial.

Ana Krtolica,1 Natacha Le Moan,1 Philberta Leung,1 Youngho Seo,2 Jonathan Winger,1 Henry Van Brocklin,2 Michael Kent,3 Stephen Cary1. 1 _Omniox Inc, San Carlos, CA;_ 2 _University of California San Francisco, San Francisco, CA;_ 3 _University of California at Davis, Davis, CA_.

Hypoxia, or lack of oxygen, is a key modulator of the tumor microenvironment and is associated with immunosuppression, invasiveness, angiogenesis, radiotherapy resistance and poor patient outcomes. Methods to increase oxygen in the hypoxic tumor niche have failed due to the long distance oxygen must diffuse from dysfunctional vasculature to the hypoxic cells. To address this problem, we engineered OMX oxygen carriers that bind oxygen with high affinity and can specifically deliver oxygen to severely hypoxic tumor regions without afecting normoxic tissues. Here, we show that OMX extravasates through leaky tumor vasculature, penetrates into and delivers oxygen to hypoxic tumor tissue. This reduces hypoxia in the tumor microenvironment and sensitizes tumors to therapy. Specifically, OMX accumulation in tumors leads to an increase in tumor oxygenation in mouse human xenograft and immunocompetent rat orthotopic models, as assessed by direct oxygen measurement (oxygen probe), by18F-FMISO PET in vivo imaging and by IHC and ELISA assessment of hypoxia markers (pimonidazole, CAIX, CCI, HIF-1). Moreover, OMX-mediated tumor oxygenation increases efficacy of radiation therapy as demonstrated by ex-vivo clonogenic assay and tumor growth delay. Importantly, while radiation alone only moderately delays tumor growth, combination of OMX and radiation therapy leads to tumor eradication in >50% of tumors. In addition to the enhancement of radiation therapy with OMX, we are currently exploring the capacity of OMX to ameliorate immunosuppressive microenvironment caused by hypoxia.

Results from a Phase 0 clinical trial in canine cancer patients indicate that OMX is well tolerated and safe to use in combination with standard of care radiation therapy even in aged and fragile canine patient populations. Similar to rodent tumor models, OMX penetrates the tumor tissue in spontaneous canine brain and melanoma tumors. Furthermore, OMX accumulation correlates with reduced hypoxia in the tumor microenvironment as assessed by multiple hypoxia markers using quantitative IHC and ELISA. Taken together, these preclinical safety and efficacy data in both rodents and canines strongly support our IND submission to initiate human clinical trials in newly diagnosed glioblastoma patients.

Results from completed and ongoing studies support the potential of OMX as modulator of hypoxic tumor microenvironment that may significantly impact the effectiveness of radiotherapy, chemotherapy and immunotherapy in a variety of solid tumors where hypoxia is major contributor to therapeutic resistance.

#2791

Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines.

Justin Montoya,1 Kieran Finch,2 Jan Simper,3 Robert J. Arceci,1 Eiman A. Aleem1. 1 _Phoenix Children's Hospital & UA College of Medicine-Phoenix, Phoenix, AZ; _2 _UA College of Medicine-Phoenix, Phoenix, AZ;_ 3 _Arizona State University, Phoenix, AZ_.

Background

Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients' response to therapy.

The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including acute myeloid leukemia (AML), Ewings sarcoma (ES), osteosarcoma (OS), and neuroblastoma (NB), under normoxia and hypoxia, and (2) to determine the potential mechanisms of drug resistance under hypoxia.

Methods and Results

In the present study the IC50 values were determined for the following targeting agents: linsitinib, picropodophylin (insulin-like growth factor-1 receptor), YM155 (survivin), AZD 8055 (mTOR), dovitinib (FLT3, c-kit, FGF4), and Palbociclib (CDK4/CDK6) in a panel of 15 AML, 4 ES, 2 OS and 2 NB cell lines using Cell Titer Glo assay. The effect of hypoxia was differential. It depended on the cell line and the compound used. However, hypoxia induced universal resistance to YM155 in all cell lines tested. YM155 had significant cytotoxicity in AML cell lines (IC50 between 2.5-1775 nM) in normoxia, however, the IC50 values in hypoxia were in the micromolar range, with an increase of > 100 fold. The effect of hypoxia on YM155 response in ES cell lines was less pronounced with (IC50s between 1-10 nM) in normoxia compared to (IC50 between 6 and 350 nM) in hypoxia. We studied the effect of hypoxia on the cell cycle using propidium iodide and flow cytometry in MV4-11 AML cells. Treatment with YM155 for 24 h in normoxia induced a pronounced decrease in the percentage of cells in S phase and G2/M phase and an increase in the sub-G1 phase. However, no change in cell cycle phases was detected under hypoxia. Ongoing studies are focusing on determining the mechanisms of hypoxia-induced YM155 resistance through studying the expression of proteins regulating hypoxia, cell cycle, and apoptosis using western blot.

Conclusion

These data demonstrate that hypoxia induced different effects in a panel of pediatric cancer cell lines. However, it conferred pronounced resistance to YM155 in AML and ES cell lines. Hypoxia is likely a key factor in programming treatment resistance and, thus, relapse and poor outcomes.

#2792

Impact of hypoxia on tyrosine kinase activation in cancer cells.

Yao Dai, Dietmar Siemann. _University of Florida, Gainesville, FL_.

Purpose: Metastasis is the major cause of therapeutic failure and high mortality in cancer patients. Growing evidence has demonstrated that dysregulated intracellular tyrosine kinase (TK) activity can play an important role in promoting disease onset and progression, while low oxygen tensions (hypoxia) are a typical external driving force that facilitates tumor cell dissemination. However, whether hypoxia-induced metastasis is mediated by TK-signaling is largely unknown. In this study, we have identified potential TK(s) that can mediate hypoxia-induced cell functions associated with metastasis, and further determine the therapeutic potential of small molecule TK inhibitors in intervening these functional behaviors.

Methods: Cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h), with or without treatment of desired molecular agents. To test cellular phenotypes, cells were seeded into appropriate culture vehicles and cell migration and invasion will be examined by scratch and transwell-based assays. At the molecular level, pan-TKs phosphorylation in malignant versus normal cells, as well as in cancer cells with or without hypoxic exposure were detected by Western blotting. To further investigate the association of p-Src and HIF-1α in patient tissues, tissue microarray (TMA) with prostate cancer patients was conducted by immunohistochemical staining followed by independent quantification on the H-score of the two proteins.

Results: TK phosphorylation in cancer cells is generally stronger than in normal cells. Similar observation is found in patient tumor tissues versus adjacent normal counterparts. When exposing cells to hypoxia, short term hypoxic exposure (2-6 h) generally induces greater cell migration and invasion than prolonged hypoxic exposure (24 h). Such functional behaviors tend to be associated with elevated tyrosine kinome activation, although the exact TKs in different cell lines may vary. Subsequent kinome screening reveals that the well-known oncoprotein c-Src is activated under particularly acute hypoxia. Further, small molecule inhibitors targeting these TKs have shown stronger inhibition on cell migration and invasion under hypoxia than normoxia. Finally, TMA analysis suggests that p-Src expression is significantly associated with HIF-1α particularly in prostate cancer patients with advanced disease.

Conclusion: The data reveal that short term hypoxia is able to enhance metastasis-associated phenotypes by activating a group of TKs such as p-Src. In addition, Src inhibitors are showing higher potential in impeding hypoxia-induced functional behaviors. These findings suggest that at least in some cancers over-activated TKs can be used as indirect markers for hypoxia and more importantly, molecular agents targeting hypoxia-activated TKs may hold therapeutic potential to prevent metastatic spread of cancer cells that have experienced particularly short-term oxygen depletion.

#2793

ADP- dependent glucokinase enhances hypoxia- inducible factor-α target gene transactivation through modulation of ROS levels in hypoxic human cancer cells.

Sergio Rey, Luana Schito, Marianne Koritzinsky, Bradly G. Wouters. _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

ADP- dependent glucokinase (ADPGK) is a glycolytic enzyme catalyzing the reaction of glucose→glucose 6-P by using ADP instead of ATP. ADPGK is remarkably conserved across various phyla from archaea to humans. Despite an important role priming glycolysis in conditions of metabolic stress in extremophile organisms, human ADPGK does not contribute to glycolysis. Recent work shows that ADPGK is essential for ROS generation during T-cell activation. Intriguingly, ADPGK protein levels are upregulated across a panel of cancer cell lines and primary tumors. Hereby we hypothesized that the increase of cellular ROS in response to mild (~1% O2) hypoxia is dependent on ADPGK therefore contributing to the stabilization of hypoxia- inducible factor (HIF)-1α, a central regulator of the transcriptional response of cancer cells to hypoxia.

We achieved ADPGK loss-of-function (LOF) utilizing lentiviral shRNA- mediated knockdown (>75%) in HCT-116 and HT-29 (colon); or H460 (lung) cancer cell lines. Intracellular ROS levels under hypoxia (1% O2) were analyzed by CM-H2DCFDA fluorescence and flow cytometry. Protein levels of HIF-1α (and -2α), and selected HIF-α targets were assessed by immunoblot assays. HIF-1α (and -2α) transcriptional activity was measured through a RT-qPCR array developed in house targeting 84 hypoxia- responsive transcripts in HCT-116 cells exposed to 20, 10, 5, 1, .2 and <.02% O2 for 24 h. Baseline non-hypoxic oxygen consumption and glycolytic activities were measured in real-time with an automated bioanalyzer (Seahorse Biosciences) whereas 3D spheroid growth assays and HCT-116 xenografts were used to assess the effect of ADPGK LOF on tumor mass.

ADPGK LOF decreased ROS and HIF-1α (-and 2α) protein levels in a cell type- and O2- dependent manner in HCT-116, HT-29 and H460 cells. Results from our RT-qPCR array show a blunted transcriptional response to hypoxia with maximal inhibition at .2% O2 (~15 mmHg). Immunoblots confirmed a striking impairment of CA9 hypoxic induction in addition to a significant decrease of the HIF-α targets IGFBP3, ERO-1α and DDIT4. 3D spheroid assays showed a significant growth rate increase after ADPGK knockdown. Addition of the antioxidant N-Ac-Cysteine during the spheroid formation phase mimicked the effect of ADPGK LOF in control cells whereas it further enhanced cell growth in ADPGK knockdown. However, baseline O2 consumption and glycolysis were not affected. Consistently with results of 3D spheroid assays, we observed enhanced tumor growth of HCT-116 xenografts bearing ADPGK LOF.

Our work uncovers a hitherto unknown function of ADPGK in cancer cells whereby it enhances ROS- dependent HIF-1α (and -2α) hypoxic stabilization and transactivation therefore limiting tumor growth. Further work will be focused on the mitochondrial origin of ADPGK- dependent hypoxic ROS generation as suggested by previous studies in primary T-lymphocytes.

#2794

Six-transmembrane epithelial antigen of prostate 4 (STEAP4): an important link between hypoxia, inflammation, nutrition and tumorigenesis in the intestine.

Xiang Xue, Yatrik Shah. _University of Michigan, Ann Arbor, MI_.

Hypoxia occurred in the inflammatory foci can further increase the pro-inflammatory response. Moreover, inflammatory hypoxia is critical for altering tissue metabolism and nutritional response, which can further sustain the inflammation and drive tumorigenesis. However, the mechanisms that link hypoxia, nutrition, inflammation and tumorigenesis are not clear. Through genome-wide expression analysis we identified six-transmembrane epithelial antigen of prostate 4 (STEAP4) as a gene highly induced by hypoxia. STEAP4 is an oxidoreductase that regulates inflammatory stress, fatty acid, glucose and iron metabolism. Here we demonstrate that the increase in STEAP4 is dependent on epithelial HIF-2α in mouse models of colitis. Luciferase reporter assay and ChIP assay confirmed that STEAP4 is a HIF-2α target gene. Compared to normal healthy controls, the gene expression of STEAP4 was highly induced in tissues from colitis patients. Transgenic mice overexpressing GFP-tagged STEAP4 specifically in intestinal epithelial cells demonstrated that STEAP4 was expressed primarily on the plasma and mitochondrial membrane. Interestingly, these mice had no change in serum and tissue minerals except for a slight increase in intestinal iron, specifically mitochondrial iron, but no change in systemic iron levels. STEAP4 transgenic mice were highly susceptible to acute experimental colitis model. Moreover, STEAP4 was increased in human colorectal cancer (CRC) and predicts poor prognosis. Consistently, tumor numbers in the STEAP4 transgenic overexpression mice were increased compared to wild-type littermates in a colitis-associated CRC tumor model. Mechanistically, overexpression of STEAP4 increased oxidative stress and DNA damage in the intestine. In conclusion, we have identified a novel gene induced in colitis and CRC that could link hypoxia-induced metabolic changes, inflammation and cancer.

#2795

MARVELD1 regulates hypoxia-induced oxidative stress response and tumor growth.

Ming Shi, Yuanfei Yao, Yu Li. _Harbin Institute of Technology, Harbin, China_.

Nuclear factor MARVELD1 (MARVEL domain-containing 1) is a potential tumor suppressor, which negatively regulates tumor cell proliferation. It is widely expressed in human normal tissues, but down-regulated in multiple primary carcinomas. In this study, we show that MARVELD1 acts as tumor suppressor via its ability to inhibit reative oxygen species (ROS) in response to hypoxic condition. Data showed that overexpression of MARVELD1 in uterine cancer cells significantly suppressed tumor growth, while MARVELD1 silencing promoted colony formation of tumor cells in hypoxic environment. Furthermore, we found that ROS production in hypoxia-treated cancer cells was elevated by MARVELD1 silencing. Mitochondria function assay showed that compared to RL952/vector cells, a decrease in oxygen consumption and derived mitochondrial ATP production was observed in hypoxia-treated RL952/MARVELD1 cells. Moreover, considering that ROS that induced by hypoxia promoted tumor cell proliferation, our data further indicated ROS generation that inhibited by MARVELD1 alleviated the growth of uterine tumor cells. Taken together, MARVELD1 regulated hypoxia-induced oxidative stress response and cell growth through inhibiting ROS generation in uterine cancer, suggesting that targeting MARVELD1 might be a novel strategy to cancer therapy.

Keywords: MARVELD1, Hypoxia, Oxidative stress, ROS

#2796

Regulation of HIF-1α by hnRNP A18 contributes to tumor promotion under hypoxic conditions.

Elizabeth T. Chang, Palak Parekh, Qingyuan Yang, France Carrier. _University of Maryland School of Medicine, Department of Radiation Oncology and Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD_.

The RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) is a new protein translation regulator found to be elevated in many cancers. Previous work from our lab has shown that hnRNP A18 is able to bind and regulate the translation of a group of mRNAs under stress conditions, including hypoxia. Hypoxia has largely been recognized as an innate characteristic of solid tumors and plays numerous cellular and physiologic roles such as cell proliferation, invasion, migration, metastasis, and angiogenesis. Given its extensive function in solid tumors, combined with computational results showing a possible hnRNP A18 signature motif in the 3'UTR of HIF-1α, we sought to determine if hnRNP A18 binds HIF-1α mRNA and how this affects tumor promotion and growth. Results: Our data indicates that hnRNP A18 protein is able to bind HIF-1α mRNA. Under hypoxic conditions, down-regulation of hnRNP A18 significantly decreases HIF-1α mRNA stability, leading to reduced amounts of HIF-1α protein levels in melanoma cells. Treatment with CoCl2 increases hnRNP A18 protein levels in melanoma cells, but not in normal melanocytes. Additionally, colony survival assay demonstrates that down-regulation of hnRNP A18 decreases cells' survival rates under a hypoxic environment. The functional significance of this effect was observed in a mouse xenograft model where down-regulation of hnRNP A18 significantly reduced melanoma and breast tumor growth. Immunohistochemistry staining of xenograft specimens showed overlapping localization of hnRNP A18 to the tumor hypoxic regions. Lastly, proteome profiling of the xenograft tumors showed a significant decrease of key angiogenic-related factors (e.g., angiogenin, endoglin, VEGF) when hnRNP A18 is reduced. Conclusion: Heterogenous ribonucleoprotein A18 is able to regulate HIF-1α mRNA and modulate its protein levels under hypoxic conditions. The oxygen-deprived regions generated in the central core of a solid tumor leads to up-regulation of hnRNP A18 and consequently HIF-1α, resulting in tumor growth and promotion. New therapeutics targeting hnRNP A18 would therefore be a logical next-step in developing a novel mechanism-based therapy for cancer treatment.

#2797

Role of the ST6Gal-I glycosyltransferase in protecting tumor cells against hypoxia.

Robert B. Jones. _University of Alabama at Birmingham, Birmingham, AL_.

A change in surface glycosylation was one of the first identified hallmarks of a cancer cell, however an understanding of how altered glycosylation affects cancer cell biology remains unclear. Our group has shown that ST6Gal-I, a glycosyltransferase that adds negatively charged sialic acids to surface receptors in a α2-6-linked manner, is upregulated in many cancer types. Through modulating the activity of various receptors, ST6Gal-I can act as a pro-survival factor in a variety of settings, including resistance to some chemotherapeutics. Tumor microenvironment plays a crucial role in cancer progression. One key component of the tumor microenvironment is the availability of oxygen, and in solid tumors there can be areas of hypoxia. Hypoxia leads to the induction of hypoxia inducible factors (HIFs), which are transcriptional regulators of the cellular response to low oxygen tension. HIFs such as HIF-1a, regulate the transcription of many proteins necessary for cells to survive when challenged by hypoxia. In this study we demonstrated that forced overexpression of ST6Gal-I in both ovarian and pancreatic cancer cell lines increased the stabilization of HIF-1a after treatment with the hypoxia mimetics desferrioxamine and DMOG, and when grown in a hypoxic environment. Correspondingly, knockdown of ST6Gal-I inhibited the stabilization of HIF-1a in these same conditions. Additionally, ST6Gal-I activity stimulates pro-survival signaling through phosphorylated NFκB and AMPK in these same models. These signaling pathways have been shown to be important for cellular survival in hypoxic environments. Markers of HIF-1a-induced transcription were investigated through qRT-PCR, and it was revealed that the ST6Gal-I-mediated stabilization of HIF-1a increased the levels of PDHK1and GLUT1 mRNA. Both GLUT1 and PDHK1 promote the shift in glucose metabolism when cells are in a low oxygen environment. Taken together, these data indicate that ST6Gal-I acts as a pro-survival factor by eliciting a more robust response by HIF-1a. The finding that a glycosyltransferase regulates the cellular response to hypoxia represents a novel paradigm in hypoxia signaling.

#2798

Enhanced HIF1α inhibition through dual inhibition of CDK and HSP90.

Shuai Zhao, David T. Dicker, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

A prevalent characteristic of solid tumors is the presence of hypoxic areas. Intratumoral hypoxia plays a well-known role in chemotherapy and radiotherapy resistance and is associated with poor prognosis as well as enhanced metastasis. Hypoxia-inducible factor 1α (HIF1α) is a major mediator of the cellular response to hypoxia, which promotes malignant proliferation and progression in cancers. HIF-1α expression is increased in a variety of tumors but this is not restricted to hypoxic regions. Understanding and targeting the mechanism of cancer-related HIF1α stabilization may help to improve current cancer treatments. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., Cell Cycle, 2013). Another previously acknowledged VHL-independent HIF1α stabilizer is the heat shock protein 90 (HSP90) that has been correlated with adverse prognosis and used as a therapeutic target in cancer. We investigated potential crosstalk between CDK1-mediated and HSP90-mediated HIF1α stabilization and the potential for therapeutic targeting. We found that under hypoxia, the interaction between HSP90 and HIF1α proteins is impaired by CDK1 inhibition in HCT116 colon cancer cells. Moreover, the level of HIF1α protein is increased by heat shock (40°C) treatment (as compared to 37°C), which can be reversed by the treatment with the HSP90 inhibitor, geldanamycin. Interestingly, administration of the CDK1 inhibitor, Ro-3306, is also able to decrease heat shock-induced HIF1α. Our results suggest that CDK1 activity may contribute to HSP90-mediated HIF1α stabilization. On the other hand, HIF1α level is decreased by HSP90 inhibition under hypoxia, which can be further reduced by the combination of HSP90 inhibition and CDK1 inhibition or knockdown. The joint inhibitory effect of CDK1 and HSP90 is observed in other cancer cell lines. In addition, we evaluated the combinational treatment of CDK4/HSP90 inhibition and found it robustly suppresses HIF1α expression in glioblastoma cell lines (i.e. T98G and SNB19). Ongoing studies are examining the biological function of CDK/HSP90 dual inhibition on cancer cell invasiveness as well as effects on cell viability. Our findings provide a rationale for targeting HIF1α through a novel combination of CDK and HSP90 inhibitors, a therapeutic strategy with clinical implications.

#2799

Exosomes from hypoxic pancreatic cancer cells confer resistance to subsequent hypoxia insult.

Mary C. Patton, Haseeb Zubair, Girijesh K. Patel, Seema Singh, Ajay P. Singh. _Mitchell Cancer Institute, Mobile, AL_.

Pancreatic cancer (PC) remains one of the leading causes of cancer-associated deaths in the US due to its highly aggressive nature and lack of effective treatments. Pancreatic tumors are characterized by a dense, desmoplastic reaction and poor vasculature leading to reduced oxygen levels within the tumor microenvironment. It is believed that this encounter of pancreatic tumor cells to intratumoral hypoxic condition is a major cause of their persistent survival under a variety of stresses, increased aggressiveness and therapeutic-resistance. However, underlying molecular mechanisms responsible for PC's adaptation to hypoxia (along with enhanced growth and aggressiveness) remain elusive. In this study, we examined the role of exosomes, which are nanoscale biological vesicles with a lipid bilayer membrane, in imparting survival benefit to PC cells. For this, we first examined the effect of conditioned media (CM) from PC cells cultured under hypoxic (0.1% O2, H-CM) or normoxic (21% O2, N-CM) conditions on the growth of fresh PC cells cultured under hypoxic (0.1% O2) conditions. We found that H-CM conferred significant survival benefit to cancer cells under hypoxia as compared to N-CM . Thereafter, we isolated exosomes from N-CM (N-exo) or H-CM (H-exo) and used them to treat pancreatic cancer cells under hypoxic culture conditions. We observed that H-exo imparted a significantly higher survival advantage to cancer cells under hypoxia as compared to N-exo. Interestingly, protein quantification indicated the presence of higher levels of exosomes from cells cultured under hypoxic conditions as compared to those grown in normoxic environment. Western blot analysis with anti-CD9 and anti-CD63 antibodies confirmed enrichment of exosomes. Moreover, Dynamic Light Scattering-based size distribution suggested that H-exo were smaller in size compared to N-exo. Further studies are underway to identify the molecular determinants in exosomes responsible for inducing growth-promoting effects under hypoxia. Overall, our data provides the first evidence that hypoxic PC cells secrete exosomes that enhance survivability under hypoxic conditions.

#2800

Target RNA sequencing analysis of hypoxia induced expression of stem cell genes in primary cultures of prostate cancer cells.

Hong Yin, Adam H. Greer, Glenn Mills. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Hypoxia plays important regulatory roles in expanding the tumor stem cell population. In prostate cancer, hypoxia is a feature of poor prognosis. The association of prostate cancer stem cells with hypoxia has been investigated. The altered stem cell gene expression in prostate cancer cells under the hypoxic condition has been documented. The majority of the investigations were performed with established prostate cancer cell lines. However, little is known about the expression of stem cell genes in primary cultures of prostate cancer. In this study, we examined the hypoxia-regulated expression of stem cell genes with primary cultures of human prostate cancer. Cells were isolated from three patient's prostate cancer samples. Isolated cells were cultured in prostate epithelium culture medium CnT-52. After 48 hours of exposure to 1% oxygen, RNA was extracted and quantified. The library was prepared with Illumina's TruSeq Targeted RNA Expression Stem Cell Panel. The sequencing was performed on Miseq platform with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter and analyzed further by Truseg Target RNA v1.0(Illumina), Cufflinks Assembly &DE( Illumina), and Biomedical Genomics Workstation(Qiagen).The results showed that of the 100 tested stem cell genes, seven genes were significantly upregulated(FDR p-value<0.05). They are BMP1, BMP2, FZD7, JUND, NOTCH1, NOTCH3, and PPARD. Three genes, PSEN2, SDAD1, and SOX2, were significantly down-regulated. To validate sequencing results, we examined NOTCH1 and NOTCH3 expression with RT-PCR. Compared to cells under normoxic condition, hypoxia induced expression of NOTCH1 increased by 3.7, 3.9 and 3.1 fold, respectively among three hypoxic cultures, while the expression of NOTCH3 increased by 8.1, 5.9, and 7.5 fold, respectively. However, the investigation of hypoxia affected expression of stem cell genes in LNCaP, an established prostate cell line, showed that the significant decreased expression of BMP1, FZD7, PPARD, and NOTCH3 was seen under hypoxic condition(FDR p-value <0.05). RT-PCR analysis showed that expression of NOTCH3 in hypoxic LNCaP, PC3, and DU145 cells was decreased by 7.7, 6.1, and 1.2 fold, respectively. The expression profile of stem cell genes in response to hypoxia was different between primary cultures of prostate cancer cells and established prostate cancer cell lines. The activation of BMP-NOTCH signaling could be one character of stemness of prostate cancer in response to hypoxia.

#2801

Arginylation as a novel regulator of prostate cancer progression.

Michael Birnbaum, Akhilesh Kumar, Fangliang Zhang. _University of Miami, Miami, FL_.

Post-translational arginylation, mediated by Arginyltransferase (Ate1), is an important protein modification involved in stress response yet remains poorly studied. Our lab recently reported that a loss of Ate1 is sufficient to induce tumorigenesis and loss of contact inhibition in fibroblasts. However, the role of Ate1 in cancer initiation and progression is not clear. This study examines the effects of Ate1 loss in prostate cancer models of tumorigenesis and progression. We first show that Ate1 is required for normal cellular responses to several types of stress present in cancer. Preliminary data in fibroblasts shows that Ate1 is essential for cell death following oxidative stress, hypoxia, UV and gamma radiation, and apoptosis-inducing drugs. These results inspired further tests to examine if cancer progression may be directly stimulated by a loss of Ate1 through increased cell survival and invasiveness. To study the effects of lost Ate1, we characterize established prostate cancer cell lines with Ate1 stably knocked down. In PC-3 prostate cancer cells, a reduction of Ate1 corresponded with increased invasiveness in the Boyden Chamber invasion assay, and reduced H2O2-induced cell death. In LnCaP prostate cancer cells, reduced Ate1 induced significantly higher anchorage-independent growth during a soft-agar growth assay. In conclusion, our data suggests that the loss of Ate1 in prostate cancer serves to stimulate cancer progression, and that Ate1 protein expression levels may predict malignancy.

#2802

Exogenous pyruvate supports oxygen-independent tumor cell proliferation by serving as an oxygen surrogate to maintain homeostasis of NAD+/NADH.

Chengqian Yin, Dan He, Shuyang Chen, Nianli Sang. _Drexel University, Philadelphia, PA_.

Hypoxia is usually associated with solid tumors and ischemic lesions. Because oxygen is the common final electron acceptor in a variety of metabolic reactions, hypoxia leads to cell injury and death in normal tissues but promotes tumor progression and metastasis. Hypoxic cells use pyruvate from glycolysis as electron acceptor to support ATP production; anaerobic glycolysis, in which the glycolytic product pyruvate is converted to lactate, has been well established as an adaptive energy strategy for hypoxic cells. In tumor cells, pyruvate may also be used as a substrate to support an active biosynthesis, as well as to maintain the homeostasis of NAD+/NAD. However, whether exogenous pyruvate promotes hypoxic tumor cell proliferation by serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxia and ρ0 cell with defective mitochondrial electron transfer chain, we show that exogenous pyruvate sustains the proliferation of tumor cells that cannot use oxygen as the electron acceptor. Pyruvate-derived metabolites including acetyl-coA, α-ketoglutarate, succinate, alanine, and aspartate were not able to substitute pyruvate to support oxygen-independent proliferation. Knockdown of metabolic enzymes including pyruvate carboxylase, pyruvate dehydrogenase and citrate synthetase showed no negative effects on pyruvate-facilitated hypoxic cell proliferation, whereas knockdown of lactate dehydrogenase significantly inhibited proliferation, indicating that the crucial role of exogenous pyruvate is to serve as the electron acceptor other than a substrate for proliferating biosynthesis. Consisting with this notion, we show that exogenous pyruvate significantly increased lactate generation and increased the ratio of NAD+/NADH. We also show that in our model cells, hypoxia did not cause accumulation of reactive oxygen species, suggesting that pyruvate-facilitated proliferation of hypoxic cells is not achieved by alleviating oxidative stress. Finally, we show that well-oxygenated cells release pyruvate, providing an in vivo source of pyruvate exogenous to the hypoxic cells. Taken together, our data support a novel model that well-oxygenated cells release pyruvate, which is utilized by hypoxic cells as an electron acceptor to facilitate proliferation. This model provides a potential new therapeutic target to suppress tumor progression.

#2803

Distinct roles of intracellular ROS in cisplatin and evofosfamide cytotoxicity.

Fanying Meng, Deepthi Bhupathi, Geraldine Chan, Charles P. Hart. _Threshold Pharmaceuticals, Inc., South San Francisco, CA_.

Cisplatin is one of the most widely used anticancer agents for the treatment of an array of malignancies. Although multiple mechanisms are involved in cisplatin efficacy, the most prominent and best understood modes of action are the generation of DNA lesions and induction of apoptosis and mitochondrial reactive oxygen species (ROS)-dependent effects on mitochondrial integrity. However, intrinsic or acquired cisplatin resistance can become a limiting factor in cisplatin-based therapy. Evofosfamide is a hypoxia-activated prodrug that is reduced at its nitroimidazole group and preferentially under hypoxic conditions releases Br-IPM, a DNA cross-linker. Evofosfamide is currently being investigated in multiple clinical trials for the treatment of cancer including indications and settings where resistance to cisplatin is observed (e.g. second-line non-small cell lung cancer; NCT01403610). We have previously demonstrated both shared and distinct DNA repair pathways involved with evofosfamide and cisplatin DNA damage. In the current study, we have conducted comparative studies to identify any distinctions in the relative role of ROS underlying cell sensitivity to cisplatin and evofosfamide to help evaluate the potential of evofosfamide to treat cisplatin-resistant tumors. The cisplatin-sensitive cervical cancer cell line ME180, the intermediate cisplatin-resistant human ovarian cancer cell line SK-OV-3, and cisplatin-resistant human lung cancer cell line A549 were investigated with respect to their chemosensitivity to evofosfamide. Unlike cisplatin, evofosfamide exhibited similar potency across all three cell lines. Addition of the ROS scavenger N-acetyl-L-cysteine (NAC) reduced the potency of cisplatin- but not evofosfamide-mediated cytotoxicity, indicating ROS plays a role in cytotoxicity mediated by cisplatin, but not evofosfamide. ROS levels were investigated using the dye H2DCFDA. Cisplatin induced a concentration-dependent and NAC-inhibitable increase of cellular ROS in cisplatin-sensitive ME-180 cells but not in cisplatin-resistant A549 cells. In contrast, ROS levels were not affected by the addition of evofosfamide under normoxia and hypoxia in both ME180 and A549 cells. In order to ascertain whether mitochondrial pathway-mediated apoptosis was involved in cytotoxicity mediated by evofosfamide and cisplatin, mitochondrial membrane depolarization was investigated with the dye JC-1. Cisplatin induced a concentration-dependent JC-1 polarization and these effects were abolished by NAC. In contrast, evofosfamide did not affect mitochondrial membrane potential. In conclusion, this data demonstrates that ROS is an essential factor in cisplatin-mediated cytotoxicity, but not in evofosfamide-mediated cytotoxicity.

#2804

Disrupted integrity in IκB kinase alpha promotes lung adenocarcinoma progression via an oxidative stress-inflammation circuitry.

Na-Young Song, Jami Willette-Brown, Mahesh Dalta, Yinling Hu. _NCI-Frederick, Frederick, MD_.

Recent advanced research has highlighted that multiple drivers and somatic aberrations drive intratumor heterogeneity. Multiple hits in different nucleotides of a single critical gene and diverse deregulatory mechanisms in one tumor have challenged to understand how these various forms of a single gene cooperate with other drivers in cancer development. IκB kinase alpha (IKKα) down-or-up-regulation and mutations have been detected in many human cancers; however, the function of IKKα in lung adenocarcinoma (ADC), a major type of human lung cancers, has not been explored. To study how the different mutant forms of IKKα regulate KrasG12D-induced lung ADC development, we crossed KrasG12D mice with Ikkα floxed mice and two Ikkα knock-in mice and found that a lung specific IKKα deletion (Ikkα∆Lung) and two different IKKα point mutants (IkkαKA/KA: K→A at amino acid 44 and IkkαAA/AA: S→A at amino acids 176 and 180) promote the progression of KrasG12D-induced lung ADCs. The IKKα mutants integrate into the KrasG12D-mediated diverse oncogenic pathways; additionally the mutants increase levels of pulmonary macrophage infiltration, ROS, and DNA damage compared to KrasG12D-induced lung ADCs. IKKα deficiency results in decreased levels of Nrf2, a transcription factor that regulates expression of genes encoding many anti-oxidants, and increased levels of NADPH oxidase 2 (Nox2), which form an oxidative stress-inflammation circuitry partially regulated by a new identified pathway of IKKα, aryl hydrocarbon receptor and Nox2. Treatment with a Nox inhibitor (apocynin) or a ROS scavenger (N-acetyl cysteine), and/or Nox2 null bone marrow transplant inhibits the pathogenesis of ADCs. Also, we observed various aberrations of IKKα in human lung ADCs. The study not only demonstrates that IKKα serves as a gatekeeper to suppress oncogenic oxidative stress in lung ADC development, but also provides an advanced model that explains how the different mutant forms of a signal gene integrate into distinct signalings to enhance the crucial driver KrasG12D-induced lung cancer development.

#2805

Protein oxidation in pancreatic neoplasia and cancer.

Michelle Schultz,1 Andrew Diaz,1 Sharon Smite,1 Brian DeCant,1 David Bentrem,2 Paul Grippo1. 1 _University of Illinois-Chicago, Chicago, IL;_ 2 _Northwestern University Feinburg School of Medicine, Chicago, IL_.

Rationale- Peroxiredoxin-1 (Prdx1) is overexpressed in pancreatic cancer patient serum and correlates with worse prognosis. Prdx1 is an important antioxidant protein whose deletion causes the formation of malignant lesions in mice. Prdx1's redox function depends on its reduction by two other Thioredoxin system members, Thioredoxin (Txn) and Sulfiredoxin (Srxn). Prdx1 may also contribute to tumorigenesis by oligomerizing and becoming a nuclear chaperone when it's overoxidized and its redox function is inhibited.

Objective- Our objective was to investigate disruptions in the Txn system during pancreatic neoplasia and to determine how that affects phosphorylation of Kras effectors.

Findings- Prdx1 expression was elevated and Srxn expression was reduced in pancreatic lesions of El-Kras, Pdx1-Cre/LSL-Kras (KC-Pdx1), and P48-Cre/LSL-Kras (KC-P48) mice. Txn was absent in lesions of KC-Pdx1 and KC-P48 mice but present and differentially located in EL-Kras lesions. In primary pancreatic acinar cells, oxidation of the Txn system with auranofin increased ERK and AKT phosphorylation in EL-Kras mice, but not in KC-P48 mice, indicating differential regulation of the Txn system in KC-P48 mice. This could be due to the presence of a higher molecular weight Prdx1 being present in KC-p48 mice and not in EL-Kras mice. This suggests that the effect of mutant Kras on the thioredoxin system in these two models may be different. Prdx1 and (p)ERK also interacted in vitro and inhibition of ERK and AKT phosphorylation modified the expression of Txn and Srxn in pancreatic cell lines. We also found that in human pancreatic cancer patient tissue, Prdx1 was overexpressed in pancreatic cancer as compared to adjacent normal tissue.

Conclusions- The Txn system is disrupted during pancreatic neoplasia and may be associated with modified ERK and AKT regulation in Kras mutant pancreatic tissue.

#2806

Oxidative stress limits metastasis of human melanoma cells.

Elena Piskounova, Michail Agathocleous, Malea Murphy, Zeping Hu, Ralph DeBerardinis, Sean Morrison. _Children's Research Institute at UTSW Medical Center, Dallas, TX_.

Solid cancer cells commonly enter the blood and disseminate systemically, but are highly inefficient at forming distant metastases for poorly understood reasons. We studied the complex and poorly understood process of metastasis by using a clinically relevant model of melanoma metastasis: patient-derived xenografts (PDX) that differ in their capacity to metastasize in NOD-SCID-Il2rg−/− (NSG) mice, mirroring their metastasis histories in the patients from which they were derived. We systematically dissected distinct steps of the metastatic cascade in vivo to show that melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficiently metastasizing melanomas. Using Liquid Chromatography-Mass Spectrometry (LC-MS) and flow cytometry, we discovered that melanoma cells in the blood and visceral organs experienced high levels of oxidative stress not observed in subcutaneous tumours. This work demonstrates that successfully metastasizing melanomas undergo reversible functional and metabolic changes during metastasis that increases their capacity to withstand oxidative stress through an increased dependence on NADPH-generating enzymes in the folate pathway. Chronic antioxidant treatment promotes circulating melanoma cell survival and distant metastases in NSG mice. Folate pathway inhibition, using low-dose methotrexate or depletion of several NADPH-regenerating enzymes, inhibits distant metastases without significantly affecting the growth of subcutaneous tumours in the same mice. We are currently investigating the role of other NADPH-regenerating pathways such as the pentose phosphate pathway in metastasis of human melanoma cells. Finally, we hypothesize that other stress types may also play a role in limiting metastasis in vivo and we are looking for additional metabolic adaptations that may increase stress-resistance of metastasizing melanoma cells and represent novel therapeutic targets in metastatic disease.

#2807

Coordinated upregulation of peroxiredoxins in breast cancer.

Jillian Muhlbauer,1 Shelley A. Phelan2. 1 _Harvard University, Cambridge, MA;_ 2 _Fairfield University, Fairfield, CT_.

Peroxiredoxins (Prdxs) are a group of thiol-specific antioxidant proteins that are involved in many cellular processes, including growth, proliferation, differentiation, and the stress response. The family includes six proteins in humans, which show differences in substrate specificity, tissue expression, and cellar localization and distribution. Cancer cells commonly exhibit aberrantly regulated expression of antioxidants, which can support cancer cell survival in the face of elevated levels of reactive oxygen species. Breast cancers exhibit significantly elevated Prdx levels of Prdx. We previously reported overexpression of many Prdx proteins in the MCF-7 breast cancer line, and further demonstrated their importance in cell survival and doxorubicin-resistance in this line. To further investigate the misregulation of these proteins in human breast cancer, we compared Prdx levels between breast tumor and adjacent normal breast tissue from 20 breast cancer patients. We showed that most patients exhibited elevated levels of several Prdx proteins in the tumor tissue. We have gone on to analyze the promoters of these genes in an effort to identify potential mechanisms of coordinate regulation, as well as downstream targets that may mediate their role in cell survival. Together, we feel that this family of proteins may be useful biomarkers, and also targets, in breast cancer biology.

#2808

Oxidative status of mouse azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer model and the effects of COX-2 inhibitor.

Gorkem Kismali,1 Aykut Gokturk Uner,2 Ogunc Meral,1 Merve Alpay,3 Dilek Ulker Cakir,4 Funda Kosova,5 Tevhide Sel1. 1 _Ankara University, Ankara, Turkey;_ 2 _Harvard University, Boston, MA;_ 3 _Duzce University, Duzce, Turkey;_ 4 _Canakkale Onsekiz Mart University, Canakkale, Turkey;_ 5 _Celal Bayar University, Manisa, Turkey_.

Background: Colorectal cancer (CRC) is becoming increasingly common in Asian and European countries and still remains the second leading cause of cancer deaths in the United States. The prevention of carcinogenesis by anti-inflammatory agents including nonsteroidal anti-inflammatory drugs (NSAIDs), selective cyclooxygenase-2 (COX-2) inhibitors, and natural products is an area of considerable interest and research. Numerous anti-inflammatory agents have been identified as potential CRC chemopreventive agents but vary in their mechanism of action.The objective of this study is to explore the oxidative status of the mouse model of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colitis-associated cancer, and effects of COX-2 inhibitor in these animals.

Materials and Methods: Totally 40 mice were randomized divided to four groups All animals except control and Cox-2 inhibitor alone group received AOM/DSS to establish colitis-associated cancer model as reported elsewhere. Control group animals were fed with conventional mice diet. COX-2 preferential inhibitor meloxicam was used to minimize side effects such as gastrointestinal hemorrhage Meloxicam were used i.p. 5mg/kg tree times in a week for meloxicam alone and AOM/DSS + meloxicam group. Oxidative stress markers Superoxide dismutase (SOD), Glutation peroxidase (GPx), Malondialdehyde (MDA) and Advanced Oxidation Protein Products (AOPP) were measured by spectrophotometrically in sera.

Results: The combination treatment of Meloxicam and AOM/DSS significantly increased (p<0,05) SOD activities. GPx activities were found significantly increased (p<0,001) in mice treatment with Meloxicam and AOM/DSS combinations or alone. There were no differences between the control and treatment groups of MDA levels. Advanced Oxidation Protein Products levels of Meloxicam and AOM/DSS combination group were found higher than the other groups.

Conclusion: Meloxicam and /or AOM/DSS treatment not caused lipid peroxidation, but increased the antioxidant enzymes and Advanced Oxidation Protein Products levels.

#2809

ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors.

Smitha Antony,1 Yongzhong Wu,1 Diana C. Haines,2 Guojian Jiang,1 Jennifer L. Meitzler,1 Agnes Juhasz,1 Jiamo Lu,1 Krishnendu K. Roy,1 James H. Doroshow1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Pathology/Histotechnology Laboratory, Leidos Biomedical Research, Inc. Frederick National Laboratory for Cancer Research, Frederick, MD_.

Reactive oxygen species (ROS) produced by the NADPH oxidase (Noxs) homologs participate in signaling cascades regulating proliferation. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms modulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 and WM-852 melanoma cells by generating Nox5-overexpressing and knockdown cells. Stable overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased protein tyrosine phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Likewise, in human WM-852 melanoma cells, transient overexpression of Nox5 decreased p27Kip1 expression and knockdown of endogenous Nox5 resulted in increased p27Kip1 and gamma-H2AX expression with decreased cell growth. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in UACC-257 Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing UACC-257 cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, UACC-257 cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that ROS signaling by Nox5 in human melanoma could modulate multiple signaling networks that regulate Hif-1α and p27Kip1 expression, contributing in part, to cell proliferation and the ability to grow in the absence of serum or growth factors.

#2810

Pterostilbene, a natural phytoalexin, weakens the antioxidant defenses of aggressive cancer cells in vivo: a pituitary gland- and Nrf2-dependent mechanism.

Maria Benlloch,1 Soraya L. Valles,2 Maria L. Rodriguez,2 J. Antoni Sirerol,2 Javier Alcacer,3 Jose Pellicer,2 Rosario Salvador,2 Concha Cerda,4 Guillermo T. Saez,5 Jose M. Estrela2. 1 _San Vicente Martir Catholic University, Valencia, Spain;_ 2 _Univ. of Valencia, Valencia, Spain;_ 3 _Pathology Laboratory, Quiron Hospital, Valencia, Spain;_ 4 _Service of Clinical Analysis-CDB, General University Hospital, University of Valencia, Valencia, Spain;_ 5 _Univ. of Valencia and Dr. Peset University Hospital, Valencia, Spain_.

Polyphenolic phytochemicals have anticancer properties. However, in mechanistic studies lack of correlation to the bioavailable concentrations is a critical issue. We studied the underlying mechanisms using different human melanomas (A2058, MeWo and MelJuso) and pancreatic cancers (AsPC-1 and BxPC-3) (with genetic backgrounds correlating with most tumors in patients), growing in nude mice as xenografts, and pterostilbene (Pter, 3',5'-dimethoxy-4-stilbenol; abundant in e.g. blueberries and a natural dimethoxylated analog of resveratrol). RESULTS: Intravenous administration of Pter decreased human melanoma and pancreatic cancer growth (an effect associated with lower rates of tumor cell proliferation and increased apoptosis) in vivo. However Pter, at levels measured within the tumors, did not affect cancer growth or viability in vitro. Pter inhibited pituitary production of the adrenocorticotropin hormone (ACTH), decreased plasma levels of corticosterone and, thereby, down regulated the glucocorticoid receptor- and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent antioxidant defense system in growing cancers (i.e. the glutathione and thioredoxin systems, superoxide dismutases, catalase, and NADPH supplying dehydrogenases). Exogenous corticosterone or genetically-induced Nrf2 overexpression in the cancer cells prevented the inhibition of tumor growth and antioxidant defenses in these malignant cells. Glutathione depletion (selected as a potential anti-cancer strategy) facilitated the complete elimination by chemotherapy of cancer cells isolated from mice treated with Pter. CONCLUSIONS: This report shows a novel link between a neuroendocrine system- and stress response-dependent mechanism and the regulation of cancer growth in vivo. Natural polyphenols can interfere with the growth and defense of cancer cells by down-regulating the pituitary gland-dependent ACTH synthesis. Lower levels of plasma ACTH cause a decrease in the suprarenal glands-dependent glucocorticoid production, thus decreasing the glucocorticoid receptor and Nrf2-dependent signaling/transcription and the antioxidant protection of melanoma and pancreatic cancer cells. Hence facilitating identification of molecular targets to sensitize aggressive cancers to oncotherapy.

#2811

Antioxidant compounds and their effects on ECM-detached ovarian cancer cells.

Calli A. Davison-Versagli. _Saint Mary's College, Notre Dame, IN_.

Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynecological cancers. Approximately 70% of all EOC patients are diagnosed at advanced stages of the disease. In these patients, ovarian cancer cells shed from the primary tumor and accumulate in ascites in the peritoneal cavity. These shed cells have shown the ability to attach and form secondary tumors throughout the peritoneal cavity, making EOC more difficult to treat. A better understanding of the molecular mechanisms involved in the survival of ovarian cancer cells in ascites could lead to the development of improved chemotherapeutics aimed at eradicating this nonadhesive subset of cells and thus resulting in better survival rates of EOC patients. While non-tumorigenic epithelial cells require attachment to the extracellular matrix (ECM) for survival, ovarian cancer cells surviving in ascites gain the ability to survive in anchorage independence. Striking increases of reactive oxygen species (ROS) mediated by ECM-detachment has been shown to inhibit energy production and inhibit cell viability in non-tumorigenic mammary epithelial cells. Consequently, neutralization of ROS with antioxidant compounds enhance ATP production and promote survival in anchorage independence. Furthermore, previous publications from our laboratory suggest that antioxidant enzymes are critical for metabolic maintenance and survival of ECM-detached breast cancer cells. While these recent studies reveal a novel role for antioxidant activity in metastatic breast cancer, antioxidant activity and its effects on ECM-detached ovarian cancer cells has yet to be unveiled. Currently, we have found that neutralization of ROS through treatment with a variety of antioxidant compounds increases metabolic efficacy in ECM-detached ovarian cancer cells. More specifically, ECM-detached SKOV3 ovarian cancer cells treated with antioxidant compounds Trolox (a vitamin E analog), Vitamin C, and Epigallocatechin gallate (EGCG) results in large increases in ATP levels. Given these data, we are further working to understand the mechanisms involved in this metabolic increase. In addition, we are working to understand the relationship between this increase in metabolism and survival in anchorage independence. Future studies include looking at this phenomenon in other ovarian cancer cell lines as well as using mouse xenograft studies to examine the effects of antioxidant compounds on survival of metastatic ovarian cancer cells.

#2812

Targeting reactive oxygen species in human cancer cell growth and metastasis in nude mouse.

Haruhiko Inufusa. _Gifu University, Gifu, Japan_.

[Purposes of the study] Reactive oxygen species (ROS) are associated with clinical condition, and closely involved in cancer growth and metastasis. Twendee X is a composition consisting of Vitamin C, Glutamine, Cystine, Riboflavin, Succinic acid, Fumaric acid, Coenzyme Q10, and Niacin. Twendee X strongly reduces oxygen radicals or ROS in vitro and in vivo (World Intellectual Property Organization WO/2013/072441 COMPOSITION FOR PROTECTION AGAINST CELL-DAMAGING EFFECTS). Effects of Twendee X on human colon cancer cell lines growth and metastasis in nude mouse was examined.

[Experimental procedures] Human colon cancer cell line RPMI4788 human gastric cancer cell line MKN45 was used in this study. Ten nude mice (BALB/C nu-nu, 4wks, Male) per each group of experiments were used, and repeated twice. Two cell lines were inoculated in subcutaneous of nude mouse, and tumor growth monitored for days. Lung metastasis was counted weeks after injection RPMI4788 cells into the tale vein of nude mouse. Twendee X was added to the free drink water around 37mg/kg/day by calculation. Serum of tumor bearing nude mouse was sampled just before sacrifice. Free Calpe Diem (Diacron International, Italy) was used to measure ROS related marker d-ROMs test (detect hydrogen peroxide). Natural Killer cell (NK) activity also measured in tumor bearing nude mouse.

[Data] Tumor growth of RPMI4788 and MKN45 was reduced 25% and 40% respectively by Twendee X administration. Metastatic nodule of RPMI4788 was reduced to 1/7 by Twendee X administration. Increase value of d-ROMs test reduced to 60% compare to the control by Twendee X administration. NK activity of RPMI4788 tumor bearing nude mouse was reduced to 28% compare to the normal mouse, and increased to 56% by Twendee X administration.

[Conclusion] Twendee X has significant effects of reducing tumor growth and inhibition metastasis. Hydrogen peroxide level (d-ROMs test) of tumor bearing nude mouse in serum was significantly reduced. ROS effects on immune cell activity, and Twendee X recovered suppressed NK activity. Thus, Twendee X effects on ROS inhibition and suppressed RPMI 4788 tumor growth and metastasis to the lung of nude mouse. Metastatic nodules receiving larger blood flow compare to the tumor in the back of mouse, thus means anti-tumor immune cells in blood may easier attack cancer cells.

#2813

The synergistic anticancer activity of 1'-acetoxychavicol acetate and sodium butyrate in human hepatocellular carcinoma cells.

Isao Matsui-Yuasa, Akiko Kojima-Yuasa. _Grad Sch Human Life Science, Osaka City Univ., Osaka, Japan_.

Introduction: It has been suggested that the combined effect of natural products may improve the effect of treatment against the proliferation of cancer cells. We evaluated the combination of 1'-acetoxychavicol acetate (ACA), obtained from Alpinia galangal, and sodium butyrate (NaB), a major short chain fatty acid, on the growth of HepG2 cells. ACA exhibits chemopreventive effects on chemically induced tumors in mouse skin as well as on rat oral, colonic, esophangeal, and pancreatic tumors. ACA also exerts antitumor activity by inducing apoptosis in various tumor cells. On the other hand, sodium butyrate has multiple effects on tumor cells cultured in vitro, including, most notably, inhibition of cell proliferation and induction of apoptosis, as well as initiating the differentiation of various carcinoma cells. The aim of our study was to elucidate the synergistic interaction of ACA and NaB on cell viability in the human hepatocellular carcinoma cell line HepG2 and to examine the mechanism of the anti-cancer effect of combined ACA and NaB treatment.

Methods: HepG2 cells or HT-29 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS.

Results: The number of HepG2 cells was synergistically decreased via apoptosis induction with the combined treatment of ACA and NaB. In ACA- and NaB-treated cells, intracellular reactive oxygen species (ROS) levels and NADPH oxidase activities were increased significantly. The decrease in cell number after combined treatment of ACA and NaB was improved with the treatment of catalase. These results suggest that an increase in intracellular ROS levels is involved in cancer cell death. AMP-activated protein kinase (AMPK) plays an essential role in controlling processes related to tumor development. In ACA- and NaB-treated cells, AMPK phosphorylation was induced significantly, and this induction improved when cells were pretreated with catalase. These results suggest that the increase in intracellular ROS is involved in the increase of AMPK phosphorylation.

Conclusion: These findings are consistent with the hypothesis that combination treatments can sensitize cancer cells more effectively than individual treatments. We reported that combination treatment of ACA and NaB synergistically induced apoptotic cell death via an increase in intracellular ROS and an increase in pAMPK levels. Our findings may provide new insight into the development of novel combination therapies against hepatocellular carcinoma.

#2814

Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro.

Garret B. Nelson,1 Sheau-Fung Y. Thai,1 Carlton P. Jones,1 Audrey Barbee,2 Micaela Killius,2 Jeffrey A. Ross1. 1 _US Environmental Protection Agency, Research Triangle Park, NC;_ 2 _Contractor to US Environmental Protection Agency, Research Triangle Park, NC_.

A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at several concentrations for 72 hours. Only limited cytotoxicity was observed at concentrations up to 300 µg/ml for all of the nanomaterials. Small increases in 8-oxo-deoxyguanosine were induced by some of the nanomaterials, but did not achieve statistical significance. No increases in ethenoadenosine or ethenocytidine were detected by ELISA assays for any of the tested nanomaterials. Several of the nanomaterials exhibited concentration related increases in levels of apurinic/apyrimidinic sites, endogenous DNA adducts measured by ³²P-postlabeling, lipid peroxidation, and ROS. Consistent with these findings, several of the nanomaterials also affected expression of genes involved in p53, ATM, and mismatch repair pathways. Integrin signaling pathways were also altered by a majority of the nanomaterials tested. There was general agreement between activity in DNA damage assays and extent of pathway transcriptional alteration. One out of the cerium oxide nanomaterials tested did not induce a high enough incidence of differentially expressed genes relative to controls to allow analysis at the pathway level, and also elicited the lowest response in multiple DNA damage assays. Taken together, these data are consistent with the contribution of DNA damage induced by reactive oxygen species as mediators of potentially adverse biological effects following exposure to engineered titanium and cerium oxide nanomaterials, and suggests the utility of short term in vitro tests to predict relative potencies of these particles.

This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

### Pharmacological Inhibitors of Cyclin-dependent Kinases

#2815

Antitumor activity of CDK4/6 inhibition in combination with radiation therapy on glioblastoma stem cells.

Christopher Nguyen,1 Yuji Piao,2 Juan Emmanuel Martinez-Ledesma,2 Jianwen Dong,2 Soon Yeung Park,2 Roel Verhaak,2 Erik Sulman,2 John F. De Groot2. 1 _Rice University, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX_.

Almost 80% of glioblastoma tumors have alterations in the CNKN2A-CDK-Rb pathway. Cyclin dependent kinases 4 and 6 (CDK4/6) act as cell cycle regulators and have been recognized as therapeutic targets for glioblastoma (GBM). In the current study, we determined the antitumor activity of CDK4/6 inhibition (palbociclib and abemaciclib) alone and in combination with radiation on a panel of glioblastoma stem cells (GSCs). The GSCs were molecularly characterized using multiple 'omimcs' techniques and Western blots identified specific phospho- and total protein levels. Approximately 50% of cell lines have homozygous deletion of CDKN2A although CDK4 and CDK6 protein expression was significantly elevated in most of the GSCs. RB protein was constitutively phosphorylated in only a subset (45%): GSC11, GSC262, GSC231, GSC304 and GSC7-2. In our panel of GSCs, there were total of three cell lines with CDKN2A loss, RB phosphorylation and CDK4/6 overexpression (GSC262, GSC11 and GSC231). Our data showed that GSC262, GSC11, GSC231 (CDKN2A deletion, RB intact), GSC7-2 and GSC300 (CDKN2A WT, RB intact) were sensitive to CDK4/6 treatment (IC50 ≤ 1µM). CDK4/6 inhibitor treated GSCs showed G1 cell cycle arrest and decreased phospho-RB levels in GSC262 and GSC7-2 cells. We next determined whether CDK4/6 inhibition combined with radiation has additive or synergistic antitumor effect. A clonogenic assay demonstrated that neurosphere formation was significantly decreased by CDK4/6 inhibition in combination with radiation compared to either treatment in GSC262 (resistant to radiation treatment) or GSC7-2 (sensitive to radiation treatment) cell lines. The radiation resistant GSC (GSC262) treated with radiation and CDK4/6 inhibitor became sensitive to radiation therapy whereas radiation sensitive GSC (GSC7-2) had a significant decrease in colony formation compared to radiation alone. CDK4/6 combined with radiation increased γ-H2AX protein expression levels and decreased cyclin D1 protein expression in a time dependent manner compared to single drug treatment. These data indicate synergistic antitumor effects of CDK4/6 inhibition combined with radiation therapy in GSCs in vitro. Further investigation is ongoing to evaluate this combination therapy.

#2816

Evaluation of Cdk5 inhibitors in neural crest tumors.

Robin E. Norris,1 John J. Pink,2 Tej Pareek,2 John L. Letterio1. 1 _Rainbow Babies & Children's Hospital, Cleveland, OH; _2 _Case Comprehensive Cancer Center, Cleveland, OH_.

Cyclin-dependent kinase 5 (Cdk5) is a ubiquitously expressed proline-directed serine threonine kinase active mainly in post-mitotic neurons due to abundant expression of its obligate activating partners p35 and/or p39. Cdk5 has been viewed narrowly as a neuron-specific kinase and as an essential regulator of neuronal function and migration.

Recently, Cdk5 activity has been implicated in mediating tumor invasion, progression, and metastasis. We have found invariably high expression and activity of Cdk5/p35 in a series of pediatric tumor cell lines including neuroblastoma.

To evaluate the potential role of Cdk5 inhibitors in the treatment of neural crest tumors, we evaluated the in vitro kinase activity and growth inhibition of 4 Cdk5 inhibitors (AT7519, dinaciclib, flavopiridol, and seliciclib). We compared the in vitro activity against Cdk2/cyclin E, Cdk5/p25, Cdk5/p35, Cdk7/cyclin H, and Cdk9/cyclin K. Using a DNA-based proliferation assay, we assessed the in vitro growth inhibition in a panel of 10 neural crest-derived tumors (melanoma, neuroblastoma, Ewing sarcoma).

All Cdk5 inhibitors demonstrated enzyme and growth inhibition at clinically achievable concentrations. Dinaciclib was the most potent inhibitor of Cdk5/p35 with an enzyme IC50 of 0.0002 μM, compared to 0.003 μM for AT7519, 0.021 μM for flavopiridol, and 0.097 μM for seliciclib. Dinaciclib was also the most potent inhibitor of Cdk2. However, flavopiridol and AT7519 were the most potent inhibitors of Cdk7 and Cdk9, respectively.

Across all cell lines, dinaciclib was the most potent inhibitor of growth. The median (range) growth inhibition IC50 for dinaciclib was 0.006 μM (0.004 - 0.14), compared to 0.079 μM (0.045-0.16) for flavopiridol, 0.52 μM (0.14 - >15) for AT7519, and 13.2 μM (7.8 - 22.1) for seliciclib. Interestingly, within the Ewing sarcoma cell lines, there was disparate sensitivity to the 2 most potent Cdk5 inhibitors, dinaciclib and AT7519. Based on the activity of these Cdk inhibitors in neural-crest tumors, we expanded our evaluation of AT7519 in 6 pediatric tumor cell lines (medulloblastoma, rhabdomyosarcoma, osteosarcoma) and found similar in vitro potency with a median (range) IC50 of 0.43 μM (0.20 - 1.26).

AT7519, dinaciclib, flavopiridol, and seliciclib demonstrated in vitro growth inhibition in a panel of neural crest and pediatric solid tumor cell lines at clinically achievable concentrations. Further in vivo evaluations and in vitro combination studies including this novel class of agents in models of neural crest and select pediatric cancers are warranted.

#2817

An inhibitor to the WEE1 kinase has pre-clinical activity in mutant KRAS/LKB1-deficient non-small cell lung cancer.

Amanda L. Richer,1 Jacqueline M. Cala,1 Kelley O'Brien,1 Vashti M. Carson,2 Timothy G. Whitsett,2 Landon J. Inge1. 1 _St. Joseph's Hospital and Medical Center/Norton Thoracic Insitute, Phoenix, AZ;_ 2 _The Translational Genomics Research Institute, Phoenix, AZ_.

Within eukaryotic cells, the G1/S and G2/M cell cycle checkpoints activate in response to intracellular or extracellular stress to prevent defects in DNA synthesis and mitosis. Deregulation of the cell cycle is a fundamental characteristic of cancer, with loss of the G1/S checkpoint playing an important role in carcinogenesis. Phenotypically, loss of the G1/S checkpoint within tumor cells results in increased reliance upon the G2/M checkpoint for adaptation to stress and DNA damage repair. This dependence has led to interest towards inhibition of the G2/M checkpoint for therapeutic treatment. Efforts have produced several clinically relevant compounds that target the regulatory proteins of the G2/M checkpoint. One of these compounds the WEE1 kinase inhibitor, AZD1775 displays favorable activity in a variety of pre-clinical tumor models in combination with DNA damaging agents and in particular, AZD1775 shows enhanced activity in tumor cells harboring inactivating mutations to p53. However, whether AZD1775 or other G2 checkpoint inhibitors have improved activity in the context of other mutations remains poorly understood. The tumor suppressor, LKB1 (STK11) is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly co-mutated with oncogenic mutations to KRAS (mtKRAS). Functional studies have revealed that mtKRAS/LKB1-deficient (LKB1-) NSCLC cells display increased activation of the G2/M checkpoint and this characteristic of mtKRAS/LKB1- NSCLC has been proposed as a point for therapeutic intervention. Based upon the reported response of tumor cells to AZD1775, we investigated the pre-clinical effects of AZD1775 in mtKRAS/LKB1- NSCLC. Employing NSCLC cell lines, we find that AZD1775 has increased activity in mtKRAS/LKB1- cells in vitro. mtKRAS/LKB1- NSCLC cells display increased protein levels of markers of DNA damage (phosphorylated γ-H2AX) and apoptosis (cleaved PARP) and reduced cell viability with AZD1775 treatment, compared to mtKRAS NSCLC with functional LKB1. The effects of AZD1775 in mtKRAS/LKB1- NSCLC cells were enhanced in the presence of DNA damaging agents (radiation, cisplatin). Using a genetically-engineered mouse model of mtKRAS/LKB1- NSCLC, combined treatment of AZD1775 and cisplatin were found to increase overall survival, compared to cisplatin mono-therapy in vivo. Collectively, these findings suggest that application of AZD1775 with DNA damaging agents may have therapeutic efficacy in mtKRAS/LKB1- NSCLC, a genomic subgroup of lung cancer with no effective therapeutic regimens.

#2818

An unbiased tumor cell panel profiling method to identify drug-drug interactions reveals synergy between the CDK4 and CDK 6 inhibitor abemaciclib and the Raf dimer and pan-Raf inhibitor LY3009120 in Ras mutant cancers.

Xueqian Gong,1 Wenjuan Wu,1 Li-Chun Chio,1 Susan Pratt,1 Constance King,1 Yue Webster,1 Maria Jose Lallena,2 Karsten Boehnke,3 Raquel Torres,3 Philip Iversen,1 Christoph Reinhard,1 Shih-Hsun Chen,1 Richard Bechmann,1 Sheng-Bin Peng,1 Sean Buchanan1. 1 _Eli Lilly and Company, Indianapolis, IN;_ 2 _Eli Lilly and Company, Spain;_ 3 _Eli Lilly and Company, Alcobendas, Spain_.

Drug sensitivity profiling across genomically characterized panels of tumor cells can identify the molecular determinants of drug response. By testing compound combinations in an unbiased format, the same methodology can be used to identify the genomic context of drug-drug synergy. Based on this principle, we developed an unbiased combination screening protocol to identify synergistic interactions with LY3009120, a novel Raf dimer inhibitor that inhibits all three Raf isoforms (Peng et al. 2015, Cancer Cell 28:384-98). Inhibitors of the Ras-MAPK pathway have proven very effective in the treatment of BRAF-mutant melanoma but are, in general, only partially effective in the treatment of BRAF-mutant colorectal cancer and Ras mutant cancers. LY3009120 combined with various compounds was screened across panels of genomically characterized tumor cells. These screens identified a strong synergy with abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6). Statistical analysis of effects in over 500 cancer cell lines showed that mutations in BRAF or Ras family genes were strongly associated with sensitivity to this combination. Strong synergy was observed in skin, colorectal, lung and pancreatic cancers with Ras/Raf mutations, but was also observed in various cancer cells wild type for Ras pathway genes. This included tumor types sensitive to single agent abemaciclib, such as mantle cell lymphoma, ER+ breast cancers, certain leukemias, squamous non-small cell lung cancer, and/or lung cancer with receptor tyrosine kinase activation. In vitro and in vivo analyses of the effects of the combination treatment on signaling pathways in KRAS mutant cancers led to potential mechanistic explanations for the differing efficacy of the combination, which manifests as regression of tumor xenografts in rodent models.

#2819

Characterization of the mechanisms of early and later stages of resistance to the selective CDK4/6 inhibitor palbociclib.

Katia Bouchekioua-Bouzaghou, Catherine Lenihan, Alice Shia, Edmund Wilkes, Pedro Casado-Izquierdo, Pedro Cutillas, Peter Schmid. _Barts Cancer Institute, London, United Kingdom_.

Background: Dysregulation of the cyclin D-CDK4/6-Rb axis occurs in a substantial proportion of ER-positive (ER+) breast cancers promoting proliferation and resistance. Adding the CDK4/6 inhibitor palbociclib to endocrine treatment has led to a substantial improvement of the outcome of patients with ER+ metastatic breast cancer. However, with the expected clinical implementation of CDK4/6 inhibitors, acquired resistance is emerging as a major clinical challenge. Using long-term culture with palbociclib, we have established MCF7 and T47D cell lines with 8-10 fold increased IC50 values as a model of acquired resistance. Characterization of resistant cell lines using phosphoproteomic analysis and RNAseq revealed increased activity in the PI3K/AKT/mTOR pathway which can be targeted therapeutically by co-treatment with CDK4/6 inhibitors and PI3K/AKT/mTOR inhibitors (Lenihan C, et al, San Antonio Breast Cancer Symposium 2015). The present work addresses whether palbociclib still has target effects and/or residual activity in resistant cells, to characterize cell cycle pathways in resistant cells, to establish the role of ER-signaling in resistant cells, and to study if palbociclib resistance might be associated with a more aggressive phenotype.

Results: Western blot analysis of newly generated resistant clones demonstrated significant reduction of phospho RB (ser780), in keeping with maintained target inhibition with palbociclib. Although total RB decreased in some clones, the protein remained detectable in all resistant clones. Cumulative population doubling confirmed palbociclib still has an effect on cell proliferation in resistant clones. Subsequent long-term culture in the presence of palbociclib altered the effect of the drug; these cells gradually lose the inhibition of pRB, as well as the residual anti-proliferative effect of palbociclib, suggesting potential loss of palbociclib target effects. Mutational analysis of RB is ongoing. Cell cycle analysis of resistant clones demonstrated an increase in cyclin E expression suggesting up-regulation of alternative cell cycle pathways. Morphological changes of resistant clones and multiple phosphorylation changes in the Rho/Rac pathway showed by our phosphoproteomic analysis suggested that resistant clones might exhibit a more aggressive phenotype. Tumor spheroid-based invasion assays and transwell migration assays confirmed increased migration and invasion of resistant clones compared to sensitive cells.

Conclusions: Whilst Palbociclib has sustained target effects and anti-proliferative activity during early stages of resistance, these effects gradually diminish during later stages of acquired resistance. Palbociclib resistant breast cancer cells also exhibit a more aggressive phenotype. In vivo investigation is ongoing to better characterize the escape mechanisms involved in CDK4/6 inhibitors resistance.

#2820

Effects of a CDK 4/6 inhibitor, G1T38, in androgen receptor sensitive and resistant models of castrate resistant prostate cancer.

James P. Stice,1 Hannah S. White,2 Suzanne E. Wardell,1 Alex Y. Yllanes,1 Scott A. Lawrence,1 Holly Alley,1 Donald P. McDonnell,1 Jay C. Strum2. 1 _Duke University, Durham, NC, NC;_ 2 _G1 Therapeutics, Durham, NC, NC_.

Introduction: Resistance to endocrine therapies, via expression of mutations or variants of the androgen receptor (AR), remains an impediment to enduring therapeutic responses in advanced castration resistant prostate cancer (CRPC). AR-dependent upregulation of cyclins leads to activation of the Cyclin D1-cyclin dependent kinase 4/6 (CDK 4/6) complex and cell proliferation, suggesting that targeting of this axis may be effective in CRPC. We have developed a series of potent and selective CDK 4/6 inhibitors, of which G1T38 has an IC50 in the low nanomolar range, and >3000 fold selectivity for CDK4/6 over CDK 2/cyclin A/E complexes. The efficacy of G1T38 was evaluated using models of CRPC; the results of which have significant clinical implications.

Methods: For proliferation assays, cells were treated for 5-10 days with G1T38 or standard of care comparators prior to quantitation of cell number/viability. Cell cycle progression and apoptosis were assayed by flow cytometry using propidium iodide or Annexin V/Sytox red staining, respectively. Orchiectomized Nu/Nu or NOD SCID Gamma mice bearing 22rV1 or LnCAP-AR-F876L xenograft tumors, respectively, were treated with G1T38 or clinically relevant comparators.

Results: G1T38 inhibited the growth of prostate cancer cell lines expressing wild type AR (LnCAP and VCAP), resistance associated AR variant AR v7 (22rV1 and LnCAP95), and LnCAP cells overexpressing the MDV3100 resistant AR mutation F876L. Corresponding decreases in cell cycle G0/G1 progression and in Rb phosphorylation (S807/811) were observed in all cell lines tested, whereas no changes were observed in Cyclin D1, E or Cdk2, 4, or 6 expression. Growth inhibition by G1T38 was dependent on Rb, and not AR, status as DU145 (AR-/Rb-) cells were not growth inhibited by G1T38, while PC3 (AR-/Rb+) were growth arrested. Treatment of G1T38 in combination with the anti-androgen MDV3100 increased the sensitivity of VCAP and 22RV1 cells to growth inhibition. Interestingly, MDV3100 in combination with G1T38 at high doses produced a synergistic apoptotic effect in LnCAP, VCAP, and 22rV1 cells, which could be attenuated by the caspase inhibitor Q-VD-OPH. The growth of 22rV1 cells and LnCAP AR F876L cells were assessed when propagated as xenografts. In 22RV1 cells, pharmacologic CDK 4/6 inhibition, significantly resulted in tumor regression compared to vehicle or docetaxel. In the LnCAP-AR-F876L xenograft model, tumor growth and doubling time was significantly decreased by low and high dose G1T38 treatment compared to control and MDV3100.

Conclusions: The CDK 4/6 inhibitor G1T38 exerts anti-proliferative effects in relevant models of CRPC when used as a stand-alone agent or when tested in combination with MDV3100. G1T38 inhibited xenograft tumor growth to a greater extent than other available therapies, highlighting the utility of CDK 4/6 inhibition in prostate cancer and a potential new paradigm in CRPC treatment.

#2821

Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes.

Daniel Mueller, Frank Totzke, Thomas Weber, Christian Beisenherz-Huss, Diane Kraemer, Carolin Heidemann-Dinger, Constance Ketterer, Chris Eckert, Michael H.G. Kubbutat. _ProQinase GmbH, Freiburg, Germany_.

Members of the family of cyclin dependent kinases (CDKs) have been recognized as pivotal regulators of cell cycle progression for more than 20 years. Concordant to their central role in the control of cell division they have been in the focus of research of proliferation associated diseases ever since, most prominently amongst these cancer.

Although initial results obtained from first and second generation, low specificity CDK inhibitors (e.g. Flavopyridol, Roscovitine, Dinaciclib, AT7519, R547) have been sobering the recent approval of the first CDK-inhibitor Palbociclib for the treatment of certain forms of breast cancer clearly demonstrates the suitability of cell cycle CDKs as targets in oncology.

Furthermore, in addition to cell cycle CDKs a second group of CDKs have been shown to have important roles in the regulation of gene transcription, and several of the "transcriptional" CDKs have become interesting targets in oncology.

Recent results underline the notion that for being effective in the treatment of cancer, CDK inhibition requires very high specificity towards the respective target CDK(s). For example CDK1 knockdown or CDK9 inhibition have been shown to be synthetically lethal in combination with MYC overexpression.

Selectivity of compounds within the family of CDKs could so far only be tested using a quite limited number of CDK-Cyclin complexes expressed in human cells. To date there are 20 CDK genes and at least 17 different Cyclin genes described, many of which give rise to different variants, e.g. there are 3 D-type cyclins, two A- and E-type cyclins etc.. Experimental data indicates that at least 50-60 different, biologically relevant CDK-Cyclin complexes may exist, but only a limited number of these are available for biochemical testing of drug candidates so far.

We have recombinantly expressed and purified 28 different CDK-Cyclin complexes, covering a significant part of the CDK family, and established in-vitro kinase-activity assays for these recombinant enzymes.

The resulting CDK panel represents the most comprehensive array for biochemical testing of this enzyme group currently available. We characterized the specificity of several CDK inhibitors that have been or are currently in preclinical or clinical development with this CDK collection. Results will be presented showing the specificity of these inhibitors not only for CDKs but also for CDKs complexed to different Cyclins. In several cases we could detect signifcant differences in the inhibition of the same CDK complexed to different Cyclins, e.g. a 10fold difference was seen for CDK6 complexes with Cyclin D1-3. A >100 fold difference was detected for CDK3 complexed to either Cyclin E1 or Cyclin C.

This screening panel allows generating data on compound selectivity early in development, diminishing the risk of designing a compound with suboptimal target specificity.

#2822

TPX2 overexpression is essential for the survival of MYC-driven triple negative breast cancer.

Julia Rohrberg, Alexandra Corella, Sanjeev Balakrishnan, Andrei Goga. _University of California, San Francisco, San Francisco, CA_.

Triple-negative breast cancer (TNBC) presents the most challenging subtype with the poorest clinical outcome and no targeted therapy. Our lab previously showed that oncogenic MYC pathways are deregulated in human TNBC tumors and predict patients' poor prognosis. However, clinical development of small molecule inhibitors for MYC has not been successful yet. We identified a synthetic-lethal interaction in which inhibition of cyclin-dependent kinase 1 (CDK1) resulted in the selective killing of MYC overexpressing cancer cells. CDK1 is a mitotic kinase that regulates a large number of substrates and its inhibition causes proliferation arrest in all cells, which may be associated with toxicity. We hypothesize that the MYC-CDK1 synthetic lethality can be attributed to the loss of function of only one or a handful of CDK1 substrates. To identify those substrates, we combined bioinformatics analysis of gene expression data from patient samples and cell based screening approaches. We identified TPX2 depletion to efficiently kill MYC overexpressing cells, while sparing normal cells. Using analog-sensitive CDK1 we confirmed that TPX2 is a direct substrate of CDK1. TPX2 is highly overexpressed in TNBC and predicts patients' poor prognosis. Its loss of function efficiently kills TNBC cells in a MYC dependent manner.

We hypothesize that TPX2 is a novel synthetic lethal interaction partner of MYC. Our studies will help elucidate the biology of MYC-driven cancer such as TNBC, which will eventually lead to the development of less toxic and more efficacious targeted therapies.

#2823

Identification of highly selective inhibitors of cyclin-dependent kinase 12.

Axel Choidas,1 Carsten Schultz-Fademrecht,1 Carsten Degeenhardt,1 Peter Habenberger,1 Uwe Koch,1 Jan E. Eickhoff,1 Ann Kathrin Greifenberg,2 Peter Nussbaumer,1 Matthias Geyer,2 Bert M. Klebl1. 1 _Lead Discovery Center GmbH, Dortmund, Germany;_ 2 _Research Center Caesar & University of Bonn, Bonn, Germany_.

CDK12 and CDK13 belong to the cyclin-dependent kinase (CDK) family, which includes at least 20 different human CDKs and CDK-like enzymes. One subclass of CDKs (e.g. CDK7, CDK8, CDK9, CDK12 and CDK13) preferentially regulates transcription by phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII), whereas another subgroup of CDKs plays a pivotal role in controlling cell cycle progression (e.g. CDK1, CDK2, CDK4, CDK6 and CDK7). Both groups - cell cycle as well as transcriptional CDKs - have been reported to be involved in cancer development and progression. Recent reports indicated CDK12 to be involved in DNA damage repair mechanisms and linked its mutation to high-grade serous ovarian carcinoma rendering CDK12 a promising target for drug development. However, the pivotal role of individual CDKs and their complex regulation requests for highly selective inhibitors to avoid unwanted adverse effects. Non-specific first-generation CDK-inhibitors cause toxicity in vivo issues. They showed little to no therapeutic window in clinical trials and thus shifted the focus to the optimization of selective small molecule CDK inhibitors.

To identify selective small molecular weight inhibitors of CDK12 and CDK13 we employed a rationale drug discovery approach based on a focused kinase library screen. Screening of slightly over 16.000 compounds identified several hit compound classes. Medicinal chemistry based optimization yielded nanomolar CDK12 inhibitors. The selectivity of these improved CDK12 inhibitors is excellent, when tested against 12 CDK family members. Such selective CDK12 inhibitors have also shown promising physicochemical properties, indicating lead- and drug-likeness. Although, potency on the target still needs to be improved, the frontrunners from our selective CDK12 inhibitor family have shown cellular inhibition of CDK12 substrate phosphorylation such as those of RNA polymerase II. Currently, potency of these CDK12 inhibitors is being improved by using structure-guided medicinal chemistry-based optimization. The resulting lead candidates will then be tested in in vivo cancer models.

#2824

G1T38, a novel, oral, potent and selective CDK4/6 inhibitor for the treatment of Rb competent tumors.

Jessica A. Sorrentino, John E. Bisi, Patrick J. Roberts, Jay C. Strum. _G1 Therapeutics, Inc, Research Triangle Park, NC_.

The development of therapeutically effective inhibitors of the cyclin dependent kinase (CDK) family has been challenging due to a poor understanding of target and structural biology leading to the development of drugs that are toxic with limited efficacy. Recently, the first highly selective CDK4/6 inhibitor, palbociclib, was approved by the FDA for use in combination with letrozole as a first line treatment in patients with ER+/ HER2- metastatic breast cancer. While this first approved CDK4/6 inhibitor is highly efficacious, it causes severe myelosuppression during daily treatment resulting in at least a 7 day treatment holiday in every 28 day cycle to allow recovery of neutrophil counts. This leads to an increased risk of febrile neutropenia, potential tumor growth during the treatment holiday, and emergence of drug resistance. CDK4/6-induced myelosuppression is the result of on-target inhibition of hematopoietic stem and progenitor cell proliferation causing a narrow therapeutic window between tumor efficacy and neutropenia. Thus, a next generation CDK4/6 inhibitor will need to produce a potent, selective G1 arrest in Rb competent tumors while minimizing the effect on the bone marrow.

With a new understanding of CDK4/6 biology, we have generated compounds with unique properties that maintain tumor efficacy while minimizing inhibition of bone marrow proliferation. Here we describe G1T38, a novel, oral, potent, and selective CDK4/6 inhibitor. Biochemical profiling demonstrates G1T38 is a competitive, nanomolar inhibitor of CDK4/6 with highly selectivity for CDK4-cyclin D1 and CDK6-cyclin D3. Kinome profiling exhibits on-target selectivity across 468 independent kinases at 100 nM, with no significant activity against other cell cycle or mitotic kinases. G1T38 elicits a precise G1 arrest profile along with loss of Rb phosphorylation in Rb competent cells with no impact in Rb deficient cells up to three orders of magnitude above the biochemical IC50. In cell proliferation assays, G1T38 exhibits a low EC50 (<100 nM) in Rb competent cell lines compared to >3 μM in Rb null cells. In vivo, daily oral treatment with G1T38 causes significant, durable growth inhibition of tumors in a HER2/neu GEMM and in MCF7 xenograft breast cancer models. G1T38 is cleared from the plasma but significantly accumulates in tumors. The level of G1T38 in the tumor correlates with significant reductions in Rb phosphorylation and tumor cell proliferation. Additionally, daily oral dosing of G1T38 in mice, rats and dogs for up to 28 days has shown a dose dependent decrease in neutrophils without severe neutropenia.

These data demonstrate that the unique pharmacokinetic and pharmacodynamic properties of G1T38 allow it to be highly efficacious against tumors while having a mild effect on the bone marrow, thus making it optimal for use as a daily oral antineoplastic agent.

#2825

Identification of CDK4/6-response biomarkers using estrogen receptor-positive breast cancer patient-derived xenografts (PDX).

Violeta Serra,1 Marta Palafox,1 Maria-Teresa Herrera,2 Martin A Rivas,1 Marta Guzmán,1 Olga Rodriguez,1 Judit Grueso,1 Meritxell Bellet,1 Mafalda Oliveira,1 Cristina Saura,1 Emmanuelle di Tomaso,3 Giordi Camponigro,3 Nicholas C. Turner,2 Javier Cortés,1 José Baselga4. 1 _Vall d'Hebron Inst. of Oncology, Barcelona, Spain;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Novartis Pharmaceutical Corporation, Cambridge, MA;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Endocrine resistance is a clinical challenge for the treatment of estrogen receptor positive (ER+) breast cancer (BC). CDK4/6 blockade in combination with endocrine therapy has shown clinical activity in metastatic ER+ BC refractory to anti-hormonal treatment. However, there is a need for biomarkers that can predict the response to this treatment and improve patient stratification. We aimed to address this issue using xenograft models established from samples of ER+ BC patients.

Six ER+ PDXs were treated with continuous doses of a CDK4/6 inhibitor (LEE011, 75mg/kg, 6IW) and a PI3K-alpha inhibitor (BYL719, 35mg/kg, 6IW) as single agents and in combination, and intrinsic sensitivity to these agents was evaluated. The models were then genomically characterized using a capture-based sequencing panel and by digital PCR.

One PDX model was intrinsically sensitive to single-agent CDK4/6 inhibition and experienced tumor regression, but all individual tumors eventually escaped therapy after 50 days of treatment. This particular model harbored an ESR1-mutation and concomitant losses of CDKN2A/B. At relapse, we identified the acquisition of an RB1 frameshift mutation. Interestingly, upfront combined treatment with a PI3K-alpha inhibitor delayed the onset of tumor progression. Two out of the remaining five CDK4/6-resistant PDXs harbored either a frameshift mutation in RB1 (plus loss of heterozygosity) or had low pRb protein expression. Two other resistant models harbored CCND1 and MYC amplifications. The remaining one harbored a TSC1 loss. In all the CDK4/6-resistant PDX, however, the combination of CDK4/6 and PI3K-alpha inhibition resulted in tumor regression.

From our results, we conclude that loss of G1-cell cycle checkpoint control, such as mutation/loss of RB1 and CCND1-amplification, is associated with lack of response to CDK4/6 blockade in ER+ BC PDX. The addition of a PI3K-alpha inhibitor results in improvement of disease control in all experimental models tested.

#2826

Sustained melanoma regression is achieved with continuous palbociclib and PLX4720 treatment but not with intermittent or sequential dosing.

Karen E. Sheppard,1 Carleen Cullinane,1 Claire Martin,1 Laura Kirby,1 Nicole Haynes,1 Kelly Waldeck,1 Richard Young,1 Todd VanArsdale,2 Grant McArthur1. 1 _Peter MacCallum Cancer Ctr., East Melbourne, Australia;_ 2 _Pfizer Oncology, San Diego, CA_.

In melanoma the development of resistance to BRAF inhibitors limits clinical responses. Thus the search for novel single agent and combination therapies as well as sequencing strategies that overcome or delay the emergence of resistance is needed. The p16-CDK4- cyclinD-RB1 pathway (CDK4 pathway) is deregulated in approximately 90% of melanomas and most melanoma cell lines are sensitive to palbociclib a specific CDK4/6 inhibitor. We hypothesized that dual targeting the MAPK/ERK and CDK4 pathways would lead to robust inhibition of the CDK4/Cyclin D complex and consequently induce greater tumor regression than single agent treatment. Proliferation and colony formation assays demonstrated that sequential and intermittent treatment with PLX4720 and palbociclib was not as effective as continuous combinational dosing. Only the combination of PLX4720 and palbociclib overcame the development of resistance leading to sustained inhibition of proliferation, cell death and induction of senescence. In A375 and HT144 human tumor xenograft models, palbociclib and PLX4720 initially induced tumor regression and tumor stasis, respectively, however resistance eventually developed to both agents. In contrast, the combination treatment induced rapid and sustained tumor regression. Biomarker studies indicate resistance to single agent PLX4720 was due to reactivation MAPK/ERK pathway and resistance to palbociclib to partial restoration of phosphorylated RB1. Tumor regression in the combination treatment was associated with the infiltration of leukocytes including activated natural killer cells indicating that these cells were involved in tumor clearance. These data demonstrate that dual targeting CDK4/6 and mutant BRAF evades resistance to single agents and leads to sustained tumor regression

#2827

**PD0332991-induced stromal senescence promotes melanoma growth** in vivo **.**

Xiangnan Guan, Kyle Lapak, Rebecca Hennessey, Christin Burd. _The Ohio State University, Columbus, OH_.

Cellular senescence, a process in which cells permanently exit the cell cycle, has pleiotropic biological effects. Several lines of evidence suggest that senescent cells within the tumor microenvironment can elicit a pro-inflammatory secretome that promotes cancer initiation and progression. This proinflammatory phenotype differs amongst senescent cells triggered by distinct stimuli; therefore, multiple forms of senescent cells likely exist within the tumor stroma during cancer evolution and treatment. Here, we sought to determine how stromal senescence initiated by clinically relevant treatments would influence melanoma growth. To generate senescent stromal populations, we treated presenescent MEFs (p4) with ultraviolet light (UV), Mitomycin C (MMC), or a CDK4/6 inhibitor (CDK4/6i). In contrast to p4 fibroblasts, cells treated with UV, MMC or CDK4/6i exhibited cell cycle arrest, increased SA-β-gal positivity and elevated p16INK4a expression. Pronounced 53BP1 and γH2AX foci were only observed in the UV and MMC treated cells, suggesting the CDK4/6i treatment did not induce DNA damage. Profiling of gene expression using a TaqMan mouse inflammation expression panel revealed distinctions amongst the 4 fibroblast lines. Of the senescent cell populations, CDK4/6i fibroblasts showed the highest number of elevated pro-inflammatory transcripts. To define the in vitro paracrine growth effects induced by stromal senescence, melanoma cell lines harboring either Braf600E, Ras, or unknown (wild-type) driver were co-cultured with each fibroblast population. In general, melanoma cells grew similarly on senescent and non-senescent fibroblasts; however, tumor cell growth was genotype-dependent. Notably, CDK4/6i fibroblasts never stimulated additional tumor cell growth in vitro. In contrast, syngeneic transplantations of fibroblasts and tumor cells into immune competent mice revealed that all senescent fibroblasts stimulated tumor growth in vivo. However, whether senescent and non-senescent fibroblasts promoted tumor growth at the same rate was genotype-dependent. Since this is the first study to examine the paracrine effects of senescent stromal cells using an immune competent mouse, we performed IHC for CD45 to assess whether immune cell recruitment differs in the presence of senescent stromal fibroblasts. No increase in CD45 positive infiltrates was observed in the melanoma lines co-injected with CDK4/6i fibroblasts. Together, this work reveals that melanoma growth in response to senescent stromal fibroblasts is genotype dependent, and that this stromal promotion of cancer growth can be accurately assessed only in the context of an immune competent, syngeneic host. Moreover, these data suggest that stromal senescence induced by emerging CDK4/6 inhibitor therapies and/or regimens combining chemotherapy/radiation with immune checkpoint blockade may also promote tumor growth through the paracrine effects of senescent bystanders.

#2828

Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer.

Neil A. O'Brien,1 Dylan Conklin,1 Tong Luo,1 Ondrej Kalous,1 Erika von Euw,1 Sara A. Hurvitz,1 Richard P. Beckmann,2 Colleen Mockbee,2 Dennis J. Slamon1. 1 _UCLA, Los Angeles, CA;_ 2 _Oncology Discovery Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN_.

The cyclin D:CDK-4/6:Rb axis is dysregulated in a number of different cancers and is implicated in resistance to hormonal therapy in breast cancer. Pharmacologically targeting cyclin dependent kinase 4 and 6 (CDK4 and 6) has proven to be a successful therapeutic approach in ER+ breast cancer (BC). This study aimed to identify the molecular subtypes of BC that are sensitive to the novel CDK4 and 6 inhibitor, abemaciclib, and identify the best combination strategies for the clinical development.

Growth inhibition activity of abemaciclib was assessed in a panel of 46 BC cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. In vivo activity of abemaciclib was assessed in cell line xenograft models of ER+ and HER2+/ER+ BCs. For ER+ BC, mice were treated daily with clinically achievable doses of abemaciclib (50-75 mg/kg) as a single agent or in combination with tamoxifen or fulvestrant. Combinations with trastuzumab, docetaxel and tamoxifen were assessed in the HER2+/ER+ xenografts.

Sensitivity to abemaciclib was observed predominately in multiple luminal BC cell lines and a small subset of triple negative cell lines that had intact Rb signaling. Activating mutations in PIK3CA also marked for abemaciclib sensitivity. Abemaciclib potentiated the anti-proliferative effects of cytotoxic/anti-mitotic agents when given simultaneously or 48 hours prior to treatment in vitro. Significant tumor growth inhibition (TGI) was observed with single agent abemaciclib in the ER+ BC cell line xenografts. In ZR751 xenografts, the addition of either tamoxifen or fulvestrant to abemaciclib induced complete inhibition of tumor growth for the 12 weeks of treatment. In the MCF7 model, treatment was withdrawn after five weeks, which triggered tumor regrowth in each of the single agent arms. However, complete responses were maintained in the combination arms for a further six weeks post drug withdrawal. In HER2 amplified xenografts, abemaciclib single agent treatment induced significant TGI in trastuzumab sensitive and resistant xenografts, and combination with trastuzumab further increased this anti-tumor effect. The addition of tamoxifen to this combination induced a further increment in TGI. Consistent with the in vitro findings, the combination of abemaciclib and the anti-mitotic agent docetaxel was not antagonistic in vivo, and the addition of docetaxel to the triple combination of abemaciclib, trastuzumab, and tamoxifen induced the most efficacy of any of the treatment arms tested. Combinations were well tolerated in animals.

These data highlight the potential of abemaciclib to have single agent activity in addition to combined activity with anti-mitotic or targeted therapies for breast cancer.

#2829

Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures.

Karsten Boehnke,1 Bruna Calsina,1 Joaquín Amat,1 Ana Hermoso,1 Raquel Torres,1 Christoph Reinhard,2 Juan A. Velasco,2 Philip W. Iversen,2 Alfonso De Dios,2 Sean Buchanan,2 Richard P. Beckmann,2 Dirk Schumacher,3 Christian RA Regenbrecht,4 Marie-Laure Yaspo,5 Hans Lehrach,5 María José Lallena1. 1 _Eli Lilly and Company, Madrid, Spain;_ 2 _Eli Lilly and Company, Indianapolis, IN;_ 3 _Charité Universitätsmedizin Berlin, Berlin, Germany;_ 4 _cpo - cellular phenomics & oncology GmbH, Berlin, Germany; _5 _Max Planck Institute for Molecular Genetics, Berlin, Germany_.

Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing.

In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays.

Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting.

#2830

The major human metabolites of abemaciclib are inhibitors of CDK4 and CDK6.

Teresa Burke, Raquel Torres, Ann McNulty, Jack Dempsey, Stanley Kolis, Palaniappan Kulanthaivel, Richard Beckmann. _Eli Lilly and Company, Indianapolis, IN_.

Abemaciclib (LY2835219) is an ATP-competitive inhibitor of cyclin dependent kinases 4 and 6 (CDK4 and CDK6) which is currently undergoing clinical evaluation for the treatment of breast and lung cancers. A radiolabeled disposition study following a single 150-mg oral dose of [14C]LY2835219 in healthy subjects indicated that in plasma, in addition to parent drug, the presence of 5 metabolites denoted as M1, M2, M18, M20 and M22. Abemaciclib (34%), M20 (26%), M2 (13%), and M18 (5%) constituted the majority of the plasma exposure. This study investigated the in vitro biological activity of these human circulating metabolites, with the exception of the trace metabolite, M1, and compared their potencies with the parent drug abemaciclib. Specifically non-small cell lung cancer (NSCLC) cells, colorectal cancer (CRC) cells and breast cancer cell lines were evaluated for growth inhibition, cell cycle inhibition and biomarker expression following treatment with abemaciclib and the metabolites. The metabolites were also profiled and compared to abemaciclib for inhibition of CDK4, CDK6, CDK1, and CDK9 in cell-free biochemical kinase assays. The IC50 values for the inhibition of CDK4 and CDK6, for metabolites M2, M18, and M20, but not M22, were between 1 and 3 nM and were nearly equivalent in potency to abemaciclib. Likewise, metabolites M2, M20, and M18 inhibited cell growth and cell cycle progression in a concentration-dependent manner that was consistent with the inhibition of CDK4 and CDK6 since these outcomes correlated with the concentration-dependent inhibition of various biomarkers such a phospho-serine 780-Rb (pRb), topoisomerase II-alpha (Topo IIα), and phospho-serine 10-histone H3 (pHH3). In this regard, metabolites M2 and M20 showed potencies nearly identical with abemaciclib in the cancer cell lines evaluated, whereas depending on the endpoint measured, the potency of M18 was approximately 3-20-fold lower than abemaciclib. M22 showed the least potency for growth inhibition and little or no inhibition of biomarker expression or cell cycle progression at concentrations below 2 µM. Although the cell-free kinase assays showed that like abemaciclib, M2, M18, and M20 had potential to inhibit CDK9, no measurable inhibition of CDK9 by any of these compounds was observed in cancer cells, indicating that the primary targets driving cell cycle inhibition for these metabolites in cancer cells were CDK4 and CDK6 and not CDK9. Studies with abemaciclib, M2 or M20 in breast cancer cells showed that all 3 compounds induced senescence in addition to growth inhibition following 6-8 days of treatment at 200 and 500 nM. In total the results indicated that the major human metabolites of abemaciclib, M2 and M20, are effective inhibitors of CDK4 and CDK6 that are remarkably similar to abemaciclib in regards to their effects in cancer cells on growth, senescence, and other phenotypic responses.

#2831

Composite cyclin dependent kinase inhibition shows potent activity against hepatocellular carcinoma.

Yi-Hsin Liang, Yu-Yun Shao, Yong-Shi Li, Hang Lin, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. _National Taiwan University Hospital, Taipei City, Taiwan_.

Background: Almost a decade after its approval, sorafenib remains the only approved therapy for advanced hepatocellular carcinoma (HCC). Novel treatment is warranted for advanced HCC. Previous preclinical studies reported that inhibition of various cyclin dependent kinases (CDKs), whether cell cycle dependent or independent, showed activity against HCC. Dinaciclib is a potent inhibitor of CDK1, 2, 5, and 9 and has an acceptable safety profile in humans. We thus examined the efficacy of dinaciclib in HCC using preclinical models.

Methods: In a panel of HCC cell lines with high Rb expression (HuH7, PLC5, HepG2, SK-Hep1, SNU387, SNU449, SNU426 and SNU475) and low Rb expression (Hep3B and HLE), dinaciclib was examined for its effect on cell viability. The impacts of dinaciclib on cell cycle distribution, apoptosis induction, and the expressions of phospho- (p-) Rb (the target of CDK1 and 2), p-ATM (the target of CDK5), c-myc, and p-RNA polymerase II (the target of CDK9) were explored in selected cell lines including HuH7 and PLC5. The in vivo efficacy of dinaciclib on HCC was tested in mouse xenografts of HuH7 cells implanted subcutaneously. Knockdown of various CDKs using RNA interference was conducted to explore the mechanisms of dinaciclib.

Results: Dinaciclib showed potent anti-proliferative activities in all the tested HCC cell lines. The IC50s to dinaciclib by the MTT assay did not significantly differ between cell lines with high Rb expression (8.5 to 20.1 nM) and cell lines with low Rb expression (9.4 to 15.6 nM). After 48 hours of dinaciclib treatment, HuH7 and PLC5 cells showed G2/M arrest in a dose-dependent manner. Apoptosis assays including sub-G1 fraction analysis, DNA fragmentation detection, and cleaved PARP-1 confirmed the occurrence of apoptosis in HuH7 and PLC5 cells treated with dinaciclib. Dinaciclib at the concentration near the IC50 inhibited the phosphorylation of Rb, RNA polymerase II, and ATM in HuH7 and PLC5 cells; it also inhibited the expressions of multiple anti-apoptotic proteins including Mcl-1, XIAP, and survivin. In xenograft studies with HuH7 cells, mice receiving dinaciclib had a significantly slower tumor growth rate compared with mice receiving vehicles. The TUNEL assay showed that tumors treated with dinaciclib exhibited higher apoptosis activity. Mice tolerated the dinaciclib treatment well without significant body weight change. Knockdown of CDK 1, 2, 5, and 9 using RNA interference showed that CDK1 and CDK9 may contribute the most to the efficacy of dinaciclib.

Conclusions: Composite knockdown of CDK 1, 2, 5, and 9 with dinaciclib showed potent activity against HCC. (This study was supported by Ministry of Science and Technology, Taiwan [MOST-103-2314-B-002-181-MY2, MOST-103-2314-B-002-090, MOST-103-2314-B-002-092]).

#2832

Synergistic effect of combined cdk4/6 inhibitor with docetaxel in lung cancer cell lines harboring KRAS mutations.

Kyoung Hwa Son, Jung-Young Shin, Jeong-Oh Kim, JinHyoung Kang. _The Catholic University of Korea, Seoul, Republic of Korea_.

Background & Purpose LY2835219(LY), a novel CDK4/6 inhibitor, inhibits phosphorylation of RB and E2F activation, thereby arresting the cell cycle in the G1 phase and suppressing the cell proliferation and cell division. Docetaxel(DTX) is a cytotoxic anti-cancer drug, which inhibits mitotic cell division, thus induces G2/M arrest and apoptosis of various cancer cells. In present study, we evaluated the activities of CDK4/6 inhibitor alone or combined with docetaxel on the anti-proliferation, cell cycle and apoptosis in lung cancer cell lines harboring KRAS mutations.

Methods We measured the anti-proliferative activities of LY or DTX single and their combinations (DTX+LY 72h and DTX 24h->LY 48h) on cell proliferation in A549(G12S) and H727(G12V) cells using CCK-8 asssy. We evaluated the expression of CDK2, CDK4, cPARP and caspase-3 by Western blot. The cell cycle distribution and apoptosis in subG1 phase were analyzed by flow cytometry.

Results The IC50 values of the LY and DTX alone were 0.4 ± 0.2 uM and 0.9 ± 0.2 nM in A549 cells and 2.0 ± 0.7 uM and 3.1 ± 0.3 nM in H727 cells, respectively. The CI (Combination index) of the DTX->LY sequential and the DTX+LY simultaneous treatments were 0.8 and 0.9 (CI<1; synergism) in A549 cells, respectively. In H727 cells, DTX->LY showed decreased cell viability by 60% at very low concentration of DTX (3.0e-5 nM), but DTX+LY showed significantly decreased cell viability by 40% at higher concentration of DTX (IC50 values can not be measured). In A549 cells, after DTX treatment for 72h, the cell population in G2/M phase increased by 54.5% relative to control (24.6%). With LY alone, the fraction of A549 cells in G0/G1 phase increased compared to control (78.4% vs. 49.5%).

In case of DTX+LY combination, the G2/M fraction of A549 cells significantly increased by 33% vs 24.6% in a dose-dependent manner. Meanwhile, in DTX->LY treatment, cell fractions in G2/M were increased (44.5% vs 24.6%), especially those in G0/G1 phases were remarkably reduced (14.7% vs 49.5%). In addition, the subG1 fraction was accumulated in response to individual drug treatments. In case of DTX single treatment, the subG1 fractions of both A549 and H727 cells increased in a dose-dependent manner (0.7% vs. 17.4% and 2.5% vs. 28.1%, respectively).

We were not able to observe the change of subG1 fraction in DTX+LY combination treatment in A549 cells. However, in DTX->LY sequential treatment, the subG1 fraction increased in a dose-dependent manner by relative to control (23.3% vs 10.9%). The expression of c-PARP and caspase-3 was increased by the DTX alone and two different combination schedule and CDK2 and CDK4 expressions were decreased by LY alone and DTX->LY sequential treatment.

Conclusions Taken together, our results suggest that DTX->LY sequential treatment has enhanced antitumor efficacy with synergistic interaction in lung cancer cell lines harboring KRAS mutations.

#2833

Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins.

Shuling Zhang,1 Joy M. Gary,1 John K. Simmons,1 Jinfei Xu,2 Benjamin J. Gamache,1 Ke Zhang,1 Nicholas Watson,1 Alexander L. Kovalchuk,1 Aleksandra M. Michalowski,1 Jin-Qiu Chen,1 Michelle A. Herrmann,1 Tuddow Thaiwong,3 Matti Kiupel,3 Wendy Dubois,1 Joseph R. Testa,2 Beverly A. Mock1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _Michigan State University, East Lansing, MI_.

The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice.

#2834

Antitumor effect of AGM 130 (5'-OH-5-nitro-Indirubin oxime), a cyclin-dependent kinase inhibitor in colorectal cancer cells.

Hyun-Jeong Shim,1 Jun-Eul Hwang,1 Woo-Kyun Bae,1 Myung-Sook Park,1 Hyo-Jeong Yun,1 Ji-Hee Lee,2 Sang-Hee Cho,1 Ik-Joo Chung1. 1 _Chonnam Nat Univ Hwasun Hospital, Gwang-ju, Republic of Korea;_ 2 _Chonnam Nat Univ Hospital, Gwang-ju, Republic of Korea_.

The AGM 130 (5'-OH-5-nitro-Indirubin oxime) compound is derived from Indirubin, which is an ingredient of Danggui Longhui Wan and used in traditional Chinese medicine. AGM 130 can inhibit the proliferation of a variety cells by arresting the cell in the G2/M phase of the cell cycle, it is a cyclin-dependent kinase (CDK) inhibitor that has anti-proliferative activity and apoptotic effects on cancer cells. AGM 130 has improved water solubility compared to Indirubin, however still displays a low solubility in biologic fluids and a poor bioavailability. This poor water solubility limits the clinical application of AGM 130 to treat cancer. In this study, we decided to develop and evaluate a SNEDDS (self-nanoemulsifying drug delivery systems) formulation containing AGM130 for improving its solubility, dissolution rate, and bioavailability.

To check the anti-proliferative effect of the AGM 130 compound on colon cancer cell line, we performed the MTT assay with the CT-26 cancer cell. We also performed cell cycle analysis, apoptotic assays and western blotting. The CT-26 cell bearing mice were treated intraperitoneally with AGM 130 loaded PPG-cRGD, 5-fluorouracil (5-FU) or AGM 130 loaded PPG-cRGD combined with 5-FU. We measured tumor volume and tumor progression of each groups.

In vitro, AGM 130 showed significant anti-proliferative activity in CT-26 cell (IC 50, 1.9 μM). Cells treated with AGM 130 showed G2/M cell cycle arrest and also induced apoptosis compared to untreated cells. The western blotting analysis showed that AGM 130 inhibited CKD1/cyclin B1 and also CKD2/cyclin E. The greatest tumor regression (percentage tumor growth inhibition) was observed in the group treated with AGM 130 loaded PPG-cRGD combined with 5-FU. When 5-FU was administrated alone, the anti-tumor effect was inferior to the group treated with AGM 130 loaded PPG-cRGD alone.

These results suggest that intraperitoneal administration of AGM 130 loaded PPG-cRGD inhibits tumor progression in a mouse colon cancer. Thus, AGM 130, a cyclin-dependent kinase inhibitors may provide a new therapeutic approach in the treatment of colon cancer.

#2835

Voruciclib, a clinical stage oral CDK inhibitor, sensitizes triple negative breast cancer xenografts to proteasome inhibition.

Joyoti Dey, Joseph Casalini, Sally Ditzler, Matt Biery, Angela Merrell, Derek Thirstrup, Marc Grenley, Richard Klinghoffer. _Presage Biosciences, Seattle, WA_.

Triple negative breast cancer (TNBC) is a highly heterogeneous disease, notoriously challenging to treat with standard chemotherapy options, and therefore is an area of intense focus for discovery of novel effective combination therapies. Here we used a previously described technology platform called CIVO, which enables assessment of multiple drugs and drug combinations simultaneously in living tumors, to identify drug combinations that result in synergistic anti-tumor activity in the HCC1187 model of TNBC. Our study focused on agents that combine with Voruciclib, a novel clinical stage oral CDK inhibitor with potent activity (<100 nM) against CDKs 1, 4, and 9. Among the drug combinations investigated, robust localized anti-tumor activity as measured by cleaved caspase 3 (CC3) positive apoptotic cells, was observed upon combined tumor exposure to Voruciclib and the proteasome inhibitor Bortezomib. In contrast, exposure to either Voruciclib or Bortezomib as single agents showed little anti-tumor activity. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies where tumor regression was induced by the Voruciclib/Bortezomib combination, but no significant impact on tumor progression was observed in xenografted subjects treated with either single agent. The ability of TNBC cells to withstand stressors such as chemotherapy may be due in part to activation of adaptive survival pathways including the unfolded protein (UPR) and endoplasmic reticulum (ER) stress responses. As observed in previous reports, exposure of HCC1187 cells to Bortezomib alone led to an increase in two markers of the cytoprotective arm of the UPR/ER stress pathway: XBP-1s and GRP-78/BIP. Consistent with the possibility that Voruciclib impedes the cytoprotective UPR/ER stress response induced by Bortezomib, exposure to the drug combination substantially reduced protein expression of both XBP-1s and GRP-78 with concomitant induction of cPARP. Voruciclib also neutralized upregulation of these same proteins by the classic ER stress inducing agent Tunicamycin. These studies provide a foundation for further investigation of breast cancer relevant anti-cancer agents that induce UPR/ER stress responses in combination with Voruciclib for treating TNBC patients.

#2836

Characterization of the mechanism of action for abemaciclib with antiestrogen combined therapy in human breast cancer cell lines.

Raquel Torres,1 Bruna Calsina,1 Ana Hermoso,1 Carmen Baquero,1 Cecilia Mur,1 Karsten Boehnke,1 Joaquín Amat,1 Alfonso De Dios,1 Xueqian Gong,2 Sean Buchanan,2 Richard Paul Beckmann,2 Maria Jose Lallena1. 1 _Eli Lilly & Company, Madrid, Spain; _2 _Eli Lilly & Company, Indianapolis, IN_.

Breast cancer is the second most common cancer worldwide after lung cancer. About 70% of breast cancers express estrogen receptor α (ER+) and/or progesterone receptor (PR+), and these biomarkers are indicative of hormone dependence. However up to 50% acquire resistance to hormone therapy [1, 2]. Estrogen independent ER+ breast cancer depends on CDK4 for tumor growth and CDK4 inhibitors have emerged as a promising approach to treat this type of tumors [3]. Abemaciclib is a cell cycle inhibitor with selective activity against CDK4 and CDK6 and it is being evaluated in advanced clinical trials for its potential to reduce metastatic ER+ breast cancer growth. We have evaluated combination of abemaciclib with an anti-estrogen therapy in an in vitro breast cancer panel. Phenotypic characterization of sensitive cell lines was carried out by monitoring cell proliferation, senescence, and apoptosis markers using flow cytometry and high content imaging approaches. Using an in vitro panel with a diversity of breast cancer cell lines, a synergistic effect of abemaciclib in combination with the ER down-regulating drug fulvestrant was observed based on Bliss score. This combination treatment demonstrated effective growth inhibition in ER+ cells and exhibited synergism in MCF-7, T47D and ZR-75-1. The mechanistic analyses revealed that the combination of abemaciclib with fulvestrant promoted a decrease in cancer cell proliferation due to G1 phase arrest at doses tested. This growth inhibition was accompanied by increased hallmarks for cell senescence as observed by markers such as SA-β-galactosidase staining or morphological changes. Subsequently, an increase in biomarkers for apoptosis was also observed. These changes occurred in a time dependent manner and were significantly greater with the combination than fulvestrant single agent treatment. We conclude the combination of abemaciclib with fulvestrant better prevented proliferation of breast cancer cell lines by blocking cell proliferation and lead to induction of senescence and apoptosis as compared to fulvestrant treatment alone in ER+ cells.

Bibliography

[1] American Cancer Society, Cancer Facts & Figures 2014.

[2] Dixon J.M. (2014) New Journal of Science. Volume 2014, Article ID 390618.

[3] Miller TW et al. (2011) Cancer Discov. Volume 1 (4): 338-51.

#2837

The CDK4/6 inhibitor has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma.

Bo Mi Ku,1 Jiae Koh,1 Yeon-Hee Bae,1 Jong-Mu Sun,2 Se-hoon Lee,2 Jin Seok Ahn,2 Keunchil Park,2 Myung-Ju Ahn2. 1 _Samsung Biomedical Research Institute, Seoul, Republic of Korea;_ 2 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea_.

Head and neck squamous cell carcinoma (HNSCC) is a rare lethal human malignancy with no effective therapy. It is critical to identify new therapeutic strategies for NHSCC. Deregulation of CDKN2A (p16) or CCND1 (cyclin D1) occurs commonly in HNSCC and induces sustained cyclin-dependent kinase (CDK) 4/6 activation. The selective CDK4/6 inhibitor, LY2835219 inhibited CDK4/6-dependent Ser780 phosphorylation in retinoblastoma (RB) and induced cell cycle arrest and growth inhibition. In an animal xenograft model of HNSCC, LY2835219 treatment led to reduced tumor growth. However, because single-agent treatment with LY2835219 showed a limited effect in HNSCC, a combinational strategy is necessary for effective therapy. At the molecular level, we found that LY2835219 inhibited activation of AKT and ERK, but not mTOR. The combination of LY2835219 with mTOR inhibitor was found to be more effective than either drug alone in vitro and in vivo. Taken together, our findings suggest that a combination treatment with LY2835219 and mTOR inhibitor is a promising therapeutic approach for HNSCC.

#2838

The role of cyclin D1-associated kinase activity in the regulation of autophagy and senescence in gastric cancer cells.

Leandro Vargas, Sindy Bravo, Claudio Valenzuela, Nelson E. Brown. _University of Talca, Talca, Chile_.

Introduction: Cyclin D1 regulates cell cycle progression through its ability to bind and activate cyclin-dependent kinases 4 and 6 (CDK4/6). Recent work carried out in mammary epithelial cells (MECs) propagated in vitro has revealed that the functional status of cyclin D1 modulates a balance between senescence and autophagy, two processes commonly disrupted in human cancers. Thus, in contrast to the commonly held assumption that autophagy is an effector process of senescence, inhibition of autophagy was accompanied by an induction or exacerbation of senescence in MECs deficient in cyclin D1-associated kinase activity, a reflection of the pro-survival function of autophagy in these cells. Materials and methods: In the present study, we set out to confirm these findings in gastric cancer cell lines (AGS, MKN-45 and KATO III) that may differ in their ability to induce senescence. To this end, we first treated AGS (cyclin D1+; pRB+; p53+) cells with Palbociclib (a specific inhibitor of cyclin D-CDK4/6 complexes), Spautin-1 (a specific inhibitor of autophagy), or a combination of these drugs, and assessed the effect of the treatment on the ability of these cells to proliferate or undergo senescence. Results and discussion: Unlike what was previously reported, our results suggest that autophagy is not required for the implementation of the senescent phenotype in AGS cells treated with Palbociclib. Nonetheless, senescent cells in which autophagy was disrupted were larger and often displayed multiple nuclei, suggesting the existence of different senescence programs depending on autophagy function.

#2840

**CDK4/6 inhibitor PD-0332991 (palbociclib) promotes cell death and synergizes with CPT-11 in colorectal cancer under hypoxia** in vitro **.**

Jun Zhang, Lanlan Zhou, Shuai Zhao, David Dicker, Wafik S. El-Deiry. _Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA_.

We recently reported that a family of nucleoside analogues (sangivamycin-like molecules) can sensitize tumor cells to TRAIL through dual blockade of CDK1 and GSK3-beta (Mayes et al., Cancer Research, 2011). We further reported that CDK inhibitors can destabilize HIF1-alpha regardless of VHL or p53 status or the presence of hypoxia (Warfel et al., Cell Cycle, 2013). In order to translate this knowledge into a cancer therapeutic strategy, we investigated the effects of CDK inhibition in colorectal cancer (CRC) cell lines with or without chemotherapy. PD-0332991 (Palbociclib) is a specific inhibitor of CDK4/6 that has been tested in numerous clinical trials for breast cancer, NSCLC, GBM, lymphoma, leukemia, in combination with 5-FU and oxaliplatin in solid malignancies (NCT01522989) or with cetuximab in head and neck cancer (NCT02101034). Palbociclib was approved by the FDA in 2015 in combination with letrozole as initial endocrine therapy for post-menopausal women with ER(+)/Her2(-) breast cancer. Little is known about the effects of CDK4/6 inhibition in CRC. We investigated the therapeutic effect and anti-proliferative mechanism of CDK4/6 inhibition in CRC. We used CellTiter-Glo assays to detect CRC cell viability and determined IC50 values (50% inhibitory concentration) of single drug through nonlinear regression analysis by GraphPad Prism software. The combination index (CI) of multiple drug combinations was identified with Compusyn analysis. We found that Palbociclib promotes cell death of CRC cells under hypoxia but not under normoxia where Palbociclib inhibited cell proliferation via the pRb pathway. These results suggest that the CDK4/6 inhibitor could regulate cell fate of CRC via different molecular mechanisms under hypoxia versus normoxia. We further found that Palbociclib can upregulate ERK/MAPK signaling under hypoxia, as compared with normoxia. The IC50 values of Palbociclib were generally higher under hypoxia (Mean ± SD: 10.54 ± 3.35 μM, N=5) versus normoxia (Mean ± SD: 6.61 ± 0.85 μM, N=5) in CRC cell lines. Thus, hypoxia promotes resistance of CRC cells toward the cytotoxic activity of the CDK4/6 inhibitor. We found that Palbociclib synergizes with CPT-11 much better than with either 5-FU or oxaliplatin against CRC cell lines with different molecular subtypes. Based on our findings that Palbociclib can promote cell death of CRC cells under hypoxia and synergizes with CPT11, further investigation is needed to assess the novel combination therapy against CRC. 

### Senescence, Cell Death, and Unfolded Protein Response

#2841

Continuous androgen receptor stimulation in thyroid cancer cells induces irreversible senescence.

Anvita Gupta,1 Melanie Jones,2 Timmy O'Connell,1 Dorota Halicka,1 Jiangwei Li,1 Hong Zhao,1 Augustine Moscatello,1 Zbigniew Darzynkiewicz,1 Raj Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _United States Military Academy Preparatory School, West Point, NY_.

Cancer of the thyroid gland accounts for 3.8% of all cancer cases in the United States as indicated by the SEER report by the NCI. The incidence of thyroid cancer has increased three-fold over the last thirty years and the American Cancer Society estimates that there will be approximately 62,450 new cases and 1,950 deaths due to the disease in the country in 2015. Papillary Thyroid Cancer (PTC) is the most common endocrine malignancy with a three-fold higher incidence in women than in men. However, PTC exhibits increased aggressiveness with poor prognosis in men diagnosed with the disease. These incongruent observations led us to explore the role of androgen and androgen receptor in this disease. We found an approximately 70% decrease in median AR expression (p<0.0001) in 24 PTC patient tissue samples, compared to matched, normal thyroid tissue. Preliminary data from our lab indicate that androgen receptor (AR) acts as a negative regulator of growth as evidenced by a statistically significant 48% decrease in proliferation over 72 hours upon 5α-dihydrotestosterone (DHT) addition to 8505c anaplastic/PTC cells stably transfected with AR (clone 84E7). Transcriptional profiling using RNAseq and gene ontology analysis, on 48 hour DHT treated 84E7 cells revealed significant changes in gene expression associated with proliferation (474 genes, p=2.4E-24) and cell cycle progression (129 genes, p= 6.54E-6). Continuous AR activation (3 to 6 days) resulted in G1 growth arrest, not accompanied by cell death, but rather a flattened, vacuolized cell morphology, indicative of senescence. This was substantiated by an increase in SA-βGal positivity from background 2.47% to approximately 65.5%, which was attenuated by the AR antagonist, flutamide. Three to six day DHT exposure resulted in increased total RNA and protein content measured using Acridine Orange and Sulforhodamine B, respectively. Senescent 84E7 cells failed to resume growth when transferred into DHT-free medium for 3 days, suggesting a permanent growth arrest. Reactive oxygen species (ROS), implicated in Rb-mediated senescence, were found to be doubled by day 6 of AR activation, compared to control cells. Additionally, using flow cytometry, we found increased p21, p27, cyclin D1, FOXO1, and phosphorylation of FOXO1 by days 3-6, suggesting that AR-dependent senescence is mediated via p21 and p27 by FOXO1/3 proteins. Furthermore, cytokine profiling of the senescence-associated secretory phenotype (SASP) would aid in defining the pro-tumorigenic or tumor-suppressive potential of androgen-induced senescent thyroid cancer cells. Our study elucidates the induction of senescence as a novel function of AR activation in thyrocytes and may indicate a protective role of AR activation in the decreased incidence of thyroid cancer in men.

#2842

5-aza-2'-Deoxycytidine induces cellular senescence in non-small cell lung cancer.

Masashi Furukawa, Yuka Mimura, Hiroyuki Tao, Yusuke Mimura, Kazunori Okabe. _Yamaguchi Ube Medical Center, Ube, Japan_.

Background: Lung cancer is one of the refractory malignancies and the leading cause of cancer-related death worldwide. Although prognosis of lung cancer was improved by the anticancer drugs and molecular targeted therapies, the results are not sufficient. Cellular senescence is irreversible cell cycle arrest. The drug-dependent induction of cellular senescence in neoplastic cells is considered an important tumor suppressive mechanism. 5-aza-2'-deoxycytidine (5-aza-dC) causes cellular senescence in solid tumors. Hypermethylation of CpG islands is well known as a major inactivation mechanism of tumor suppressor genes such as E-cadherin. Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to epidermal growth factor receptor inhibitors. This study was designed to assess the relationship of E-cadherin expression cellular senescence induced by 5-aza-dC in non-small cell lung cancer (NSCLC) cell lines.

Materials and Methods: We investigated two NSCLC cell lines; H1299 and A549 reported they have hypermethylation in promoter lesion of E-cadherin. There are two clinically approved DNA methyltransferase inhibitors (DNMTi), 5-aza-dC and 5-azacytidine (5-aza-CR). We also used Sodium Butyrate (NaB). We treated them with each drug for 120 hours. Flow cytometry analysis, immunofluorescence staining for E-cadherin and senescence associated beta-galactosidase assays and immunoblotting for senescence related proteins assessed NSCLC cell outgrowth and morphology.

Results: Both cell lines with 5-aza-dC or NaB treatment changed morphology and increased size but decreased cell numbers. 5-aza-CR causes apoptosis in both cell lines. In A549, E-cadherin expression increased after 5-aza-dC treatment. There is no E-cadherin expression in H1299 with or without treatment. Both cell lines with 5-aza-dC were positive for Senescence-associated beta-galactosidase staining, H3K9me3 and gamma-H2AX.

Conclusion: Taking together these findings strongly suggest the ability of 5-aza-dC to induce cellular senescence in NSCLC cells. The cellular senescence induced by a 5-aza-dC approach has to be considered a therapeutic option for NSCLC and an important phenomenon to further investigate in the next future.

#2843

TASC1, a selective anti-senescence therapeutic which potently and selectively counteract resistance to chemo- and radiotherapy.

Marjolein P. Baar, Diana A. Putavet, Joris Pothof, Jan H.J. Hoeijmakers, Peter L.J. de Keizer. _Erasmus MC, Rotterdam, Netherlands_.

Resistance to therapy is a major limitation towards the cure of irresectable cancer.

Cellular senescence, induced by chemotherapy and radiotherapy, can drive therapy resistance in vivo. Senescence is a tumor suppressive mechanism, but senescent cells can secrete a range of proteins that ironically promote tumor growth, migration and metastazation and therapy resistance. Recent evidence has shown that genetic removal of senescent cells indeed causes a strong reduction in tumor growth and metastasis formation. Unfortunately however, therapeutic options to remove senescent cells are currently lacking.

Here, we show the development and optimization of a biochemical compound, TASC1, that potently and selectively kills senescent cells in vitro and in vivo and lowers organ toxicity in a mouse model employed to address the off-target effects of cancer therapy. This encouraged us to test the effect of TASC1 against cancer therapy resistance. We focused on Glioblastoma multiforme (GBM), the most aggressive and lethal stage of glioma (III-IV) with a median survival of less than 15 months. Treatment of GBM involves a combination of surgery, radiotherapy and adjuvant chemotherapy (Temozolomide). Response to radiotherapy is generally strong, but resistance occurs rapidly, after which currently no successful follow-up treatment exists. We observed radiation therapy to induce senescence in astrocytes and senescence-like effects in GBM cells, without affecting their progression.

Strikingly, while both TASC1 and radiation at various doses independently failed to reduce growth of these GBM cells, the combination of TASC1 with radiation showed a potent synthetic lethal response. Similar effects were seen for other types of cancer and their respective therapies. Thus, TASC1 is a potent adjuvant to (re)sensitize therapy-resistance metastatic cancer cells to treatment.

#2844

Cell senescence and antitumor potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids.

Bianca Rocha-Sales,1 Paula Rezende-Teixeira,1 Evandro Luís Oliveira Niero,1 Camila Lauand,1 Marisa Ionta,2 Simone SL Hanemann,2 Glaucia M. Machado-Santelli1. 1 _University of Sao Paulo, Sao Paulo, Brazil;_ 2 _Federal University of Alfenas, Alfenas, Brazil_.

Brazilian flora is considered one of the most diverse in the world and represents a source of new molecules with bioactivity against several diseases. 7-epiclusianone, a prenylated benzophenone was isolated from Garcinia brasiliensis, a plant named bacupari in folk medicine. Previous studies showed that this compound has dose-dependent cytotoxic effect in several cell lines derived from human cancers and antiproliferative effect by inducing cell cycle arrest in G1/S and apoptosis in A549 lung cancer cell line. The present study aimed to analyze the cytotoxic and/or antiproliferative potential of 7-epiclusianone in human breast cancer cell lines cultured in monolayer and as spheroids. Monolayer cell cultures are commonly used for testing drug effects largely because of their easy maintenance, but they do not represent the spatial interactions of cells within a tumor. Spheroids in 3D cell cultures can overcome some of those limitations thus mimicking the architecture of solid tumors. Initially the 3D conditions were established for both cell lines MCF7 and Hs578T. Spheroids were morphologically characterized by light and transmission electron microscopy. MCF-7 spheroids showed typical epithelial organization with cohesive cells, in accordance with higher expression levels of E-cadherin compared to monolayer. In Hs578T spheroids, cells assumed fibroblast-like morphology concentrically organized, low E-cadherin expression and synthesis of extracellular matrix components. The IC50 values of 7-epiclusianone were 20 μM for Hs 578T cells and 6 μM for MCF-7 monolayers cell cultures. At this concentration, the compound treatment arrested monolayer cell cultures in G0/G1. 7-epiclusianone reduced the mRNA levels for cyclins D1 and E in line MCF-7, while only cyclin E mRNA in Hs 578T. The compound did not change the microfilaments organization or the nuclear integrity, as observed by laser scanning confocal microscopy. Interestingly, 7-epiclusianone treated cultures exhibit higher senescence indexes while apoptotic cells were not detected. Altogether, these data suggest that 7-epiclusianone is a promising molecule against breast cancer cells. The three-dimensional culture was more resistant to treatment with the compound than the monolayer, therefore more comprehensive studies are needed to understand better the effects of 7-epiclusianone on this type of culture. (Supported by FAPESP and CNPq)

#2845

PRMT5-PTEN molecular pathway regulates senescence and self-renewal of Glioblastoma stem-like cells.

Yeshavanth Kumar Banasavadi-Siddegowda, Luke Russell, Jianying Zhang, Vrajesh A. Karkhanis, Jaime Imitola, Robert Baiocchi, Balveen Kaur. _The Ohio State University, Columbus, OH_.

PURPOSE: Glioblastoma (GBM) represents the most common and aggressive histologic subtype among malignant astrocytoma and is associated with poor outcomes because of heterogeneous tumor cell population including mature non-stem like cell and immature stem-like cells within the tumor. Thus, it is critical to find new target-specific therapeutic modalities. Protein arginine methyltransferase enzyme 5 (PRMT5) regulates many cellular processes through its methylation activity and its overexpression in GBM is associated with more aggressive disease. Previously, we have shown that silencing of PRMT5 expression in differentiated GBM cell lines results in apoptosis and reduced tumor growth in mice. Here we report the critical role of PRMT5 in differentiated GBM non-stem-like cells (non-GSC) grown in serum and undifferentiated GBM stem-like cells (GSC) grown as neurospheres in vitro.

METHODS: GSC grown in stem-cell media and non-GSC grown in serum were transfected with specified siRNAs. At appropriate time, these cells were tested for in vitro proliferation, self-renewal capacity, apoptosis, cell cycle progression, mRNA expression and protein expression. Also, using intracranial mouse xenograft, tumor forming ability of PRMT5-intacta and depleted GSC and non-GSC was determined.

RESULTS: Our results uncover a very significant role for PRMT5 in GSC self-renewal capacity and proliferation. PRMT5 knockdown in non-GSC led to apoptosis, knockdown in GSC led to G1 cell cycle arrest through upregulation of p27 and hypophoshorylation of Rb protein, leading to senescence. Comparison of impact of PRMT5 on cellular signaling by Human Phospho-Kinase Array and ChIP-PCR revealed that unlike non-GSC, PRMT5 controls both AKT and ERK activity by direct repression of PTEN in GSC. In vivo transient depletion of PRMT5 decreased intracranial tumor size and growth rate in mice implanted with both primary tumor derived GSC and non-GSC.

CONCLUSION: This is the first study to identify PTEN as a potential downstream target of PRMT5 and vital to support both mature and immature GBM tumor cell populations.

#2846

Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence.

Tareq Saleh, Theresa Thekkudan, Moureq R. Alotaibi, David A. Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Most lung cancer cases are diagnosed in advanced, inoperable stages and are treated with chemoradiation; while chemoradiation is effective in suppressing tumor progression, recurrence following treatment is not infrequent. As there is no well-accepted preclinical model for tumor dormancy and disease recurrence, we hypothesize that the promotion of transient autophagy and senescence could be developed as a model of tumor growth and recovery following treatment. Studies were performed utilizing H460 NSCLC cells as well as a primary NSCLC cell line isolated and grown in our laboratory from a stage IV adenocarcinoma. After treatment with etoposide (1 uM), growth arrest was accompanied by the robust induction of autophagy and senescence. This growth arrest was transient and was followed by proliferative recovery in the course of several days. Genetic and pharmacological inhibition of autophagy failed to interfere with the ability of the cells to regrow, indicating a non-cytoprotective function of autophagy in response to etoposide (in contrast to the cytoprotective function of autophagy exhibited in response to radiation). Quantification of senescence over time based on C12FDG staining and flow cytometry demonstrated that the reversal of growth arrest coincided with a decline in the extent of senescence. To more precisely define the source of the recovered cells, senescent and non-senescent but growth arrested cells were separated by flow cytometry based on their relative β-galactosidase expression and replated. Both cell populations demonstrated the ability to re-emerge from the growth-arrested state and recover proliferative capacity. These observations suggest that senescence is ultimately a transient process in that at least a subpopulation of tumor cells can and will recover proliferative capacity. Furthermore, the reversibility of therapy-induced senescence (TIS) might prove to be a useful model both in cell culture and in vivo to study tumor dormancy and disease recurrence. Studies are currently in progress to determine the impact of senescence inhibition on tumor recovery as well as sensitivity to subsequent therapy given that recurrent disease tends to be relatively refractory to further treatment.

#2847

Phenyl 2-pyridyl ketoxime induces cellular senescence-like alterations via NO production in human diploid fibroblasts.

Hyun-Jin Jang,1 Eunbi Jo,1 Young-Ho Chung,1 JunSoo Park,2 Sung-Jun Park,1 Ik-Soon Jang1. 1 _Korea Basic Science Institute, Daejon, Republic of Korea;_ 2 _Yonsei University, WonJu, Republic of Korea_.

Phenyl-2-pyridyl ketoxime (PPKO) was found to be one of small molecules enriched in the extracellular matrix of near-senescent human diploid fibroblasts (HDFs). Treatment of young HDFs with PPKO reduced the viability of young HDFs in a dose and time-dependent manner, and resulted in senescence-associated β-galactosidase (SA-β-gal) staining and G2/M cell cycle arrest. In addition, the levels of some senescence-associated proteins, such as phosphorylated ERK1/2, caveolin-1, p53, p16ink4a, and p21waf1, were elevated in PPKO-treated cells. To monitor the effect of PPKO on cell stress responses, reactive oxygen species (ROS) production was examined by flow cytometry. After PPKO treatment, ROS levels transiently increased at 30 min but then returned to baseline at 60 min. The levels of some antioxidant enzymes, such as catalase, peroxiredoxin II, and glutathione peroxidase I, were transiently induced by PPKO treatment. SOD II levels increased gradually, whereas the SOD I and III levels were biphasic during the experimental periods after PPKO treatment. Cellular senescence induced by PPKO was suppressed by chemical antioxidants, such as N-acetylcysteine, 2,2,6,6-tetramethylpiperidinyloxy and L-buthionine-(S,R)-sulfoximine. Furthermore, PPKO increased nitric oxide (NO) production via inducible NO synthase (iNOS) in HDFs. In the presence of NOS inhibitors, such as L-NG-nitroarginine methyl ester and L-NG-monomethylarginine, PPKO-induced transient NO production and SA-β-gal staining were abrogated. Taken together, these results suggest that PPKO induces cellular senescence in association with transient ROS and NO production and the subsequent induction of senescence-associated proteins.

#2848

Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition.

Moureq R. Alotaibi, Khushboo Sharma, Tareq Saleh, Lawrence Povirk, Eric Hendrickson, David Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

#2849

RNA-binding protein FXR1 negatively regulates senescence by destabilizing mRNA CDKN1A and stabilizing noncoding RNA telomerase RNA component.

Mrinmoyee Majumder,1 Nallasivam Palanisamy,2 Shuo Qie,1 Terry Day,1 Alan J. Diehl,1 Viswanathan Palanisamy1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Department of Urology, Detroit, MI_.

RNA-binding proteins (RBPs) regulate numerous aspects of co- and post-transcriptional gene expression. RBP fragile X-related protein 1 (FXR1) belongs to a family of RNA-binding proteins that includes functionally similar Fragile X mental retardation 1 (FMR1) and Fragile X-related 2 (FXR2). FMR1 is significantly studied in Fragile X Syndrome (FXS) where the gene is non-functional due to mutation or aberrant methylation. FXR1 protein is highly expressed in multiple cancers including lung and oral cancers. Here, we demonstrate that RBP FXR1 plays an essential role in the growth of head and neck squamous cell carcinomas (HNSCC) by blocking cellular senescence. FXR1 MEF cells were stained positive for senescence associated beta-galactosidase straining. We report a major function of FXR1 as it promotes the stability of Telomerase RNA Component (TERC), a non-coding RNA and simultaneously destabilizes CDKN1A mRNA, and blocks cellular senescence. FXR1-deficient HNSCC cells show an increase in different cyclin dependent kinase inhibitors (p21, p27), a decrease in p-AKT, and these cells also undergo a G0/G1 cell cycle arrest which are the early onsets of cellular senescence. FXR1 binds and stabilizes TERC RNA for telomere maintenance. On the contrary, FXR1 binds and destabilizes CDKN1A mRNA. By an independent assay we also show that the senescence phenomenon was only observed by a combined up and downregulation of CDKN1A and TERC, respectively which was only obtained by FXR1 knockdown. Thus, FXR1 forms a molecular link between CDKN1A and TERC for cell cycle control and telomere length, respectively, to repress cellular senescence in HNSCC.

#2850

Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia.

Ganchimeg Ishdorj,1 Sara EF Kost,1 Yunli Zang,1 Spencer B. Gibson,2 James B. Johnston2. 1 _CancerCare Manitoba, Winnipeg, Manitoba, Canada;_ 2 _University of Manitoba, Winnipeg, Manitoba, Canada_.

Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. In the present study, we demonstrate that the leukemia cell telomere length shortens over time were CLL are followed over year, indicating that telomere shortening in these cells is an ongoing process. When CLL cell metaphases were examined by Q-FISH, telomere length shortening was found to affect all chromosomes equally. To determine whether these telomere changes were related to alterations in the shelterin complex, protein levels of the six shelterins were determined in CLL cells and normal pooled B cells. In general, apart from TIN2, the shelterin protein levels were increased in CLL cells and TRF2 levels were higher in IgVH unmutated cells than in mutated cells. TIN2 protein levels were dramatically reduced in CLL cells as compared to normal B cells and an alternative isoform of TIN2 was observed, both in IgVH mutated and unmuted cases. TIN2 targeted RT-PCR followed by sequencing revealed a deletion of the entire exon 2, totaling 105 bp. This abnormality appeared in monoclonal B-cell lymphocytosis (MBL) indicating that this was an early event. In addition, a subgroup of patients showed an increase in the spliced form of TIN2, when followed over time, suggesting that this might provide the cell with a survival advantage. A protein translated from the spliced variant RNA is expected to be deleted for 35 amino acids at the N-terminus, which is the binding site for TRF2. As TIN2 maintains telomere structure by forming a complex of TRF1 and TRF2 with the telomere, this should not occur with the splice variant. Indicating a pathological role of spliced TIN2 in CLL, TRF2 was localized in both the nuclei and cytoplasm in CLL cells compared to localization only to the nucleus in normal B cells. To confirm the function of the spliced TIN2, we have overexpressed the both full length and spliced clones of TIN2 in HEK293 cells and are presently determining the localization of TRF2 and the ability to form a TRF2/TIN2 complex. Thus, these studies demonstrate a unique splice variant of TIN2 in CLL, which potentially may influence the formation of the shelterin complex and contribute to telomere shortening in this disease.

#2851

EGFR-TERT cooperation in the development of EGFR-TKI treatment induced pulmonary fibrosis.

Rongrong Wei, Wanqing Liu. _Purdue University, West Lafayette, IN_.

Background: Previous studies have shown that epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer can lead to the development of pulmonary fibrosis (PF). In other studies, it has been shown that reduced telomerase reverse transcriptase (TERT) function increases susceptibility to PF. In this work, we hypothesize that TERT is associated with the pathogenesis of EGFR-TKI induced PF via important signaling pathways.

Methods: HCC827 cells (EGFR L858R) was treated with gefitinib and TERT expression level was detected following the treatment. To investigate the molecular changes following gefitinib treatment, we also performed a 84-TF qPCR- microarray and a qPCR microarray for 84-key secreted proteins of cytokines and chemokines. The level of interleukin-6(IL-6) was further measured with ELISA in cell medium following the gefitinib treatment. Fibrogenic markers were detected after co-culturing HCC827 with lung fibroblast IMR-90 cells.

Results: TERT gene expression was significantly decreased over 90% after gefitinib treatment. Early growth response protein 1(EGR-1) and E2F transcription factor 1(E2F1) were significantly down-regulated as major TFs that are responsive to gefitinib treatment. Both EGR-1 and E2F1 have been shown to regulate TERT expression in previous studies. In addition, TFs including JUN and most REL members involved in the NF-κB pathway, were significantly up-regulated after gefitinib treatment, which are known to lead to the activation of the senescence-associated secretory phenotype (SASP) pathway. IL-6 mRNA as well as protein secretion were both significantly up-regulated after gefitinib treatment. After co-culturing HCC827 and IMR-90 for 7 days and treating with gefitinib, the growth of IMR-90 cells were increased compare to the control group. Fibrogenic markers, e.g. COL1A1 (Collagen, Type I, Alpha 1), α-SMA (alpha smooth muscle actin), FSP-1(Fibroblast-specific protein 1), etc. were all up-regulated.

Conclusion: Our data suggest that EGFR inhibition leads to lung epithelial cell senescence via inhibiting TERT function and activating of the SASP pathway, which further activates fibroblasts and induces fribrogenesis. Continued studies are warranted to further understand the molecular signaling in this mechanism.

#2852

CITED2, an emerging regulator in hypoxia- and anoikis- mediated cancer cell growth.

Ming-Han Kuo, Chia Ee Chan, Yu-Ting Chou. _National Tsing Hua University, HsinChu, Taiwan_.

In solid tumors, hypoxia and anoikis are two common phenomena occurred in the central of region, inducing a series of genes to switch metabolic pathways, prevent apoptosis and promote continuous growth of cancer cells. Herein, we reported that CITED2 (CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain 2) responded to hypoxia and anoikis induction, preventing lung cancer cells from cell cycle arrest. We found that hypoxia induced CITED2 through a HIF2α dependent pathway; moreover, three-dimensional (3D) growth of lung cancer cells induced the expression of CITED2. 3D growth condition elicited anoikis-mediated cell cycle arrest, which was further potentiated by hypoxia stimulation. Knockdown of CITED2 or HIF2α in lung cancer cells enhanced anoikis, causing G1/S cell cycle arrest. Soft agar analysis displayed that both HIF2α and CITED2 are necessary for anchorage-independent cell growth under the hypoxic condition. Spheroid assay indicated that HIF2α-CITED2 signaling axis plays an important role in spheroid formation. Pearson correlation analysis showed that CITED2 was associated with HIF2α expression in lung adenocarcinoma. Kaplan Meier analysis revealed that CITED2 expression was correlated with poor survival outcomes in patients with lung adenocarcinoma. Thus, our findings support the notion that CITED2 plays a critical role in hypoxia- and anokis- mediated cancer cell growth with the potential as a prognostic biomarker for predicting lung cancer progression.

#2853

Apoptosis and proliferation in micropapillary structures of colorectal polyps and carcinomas.

Madhura Patankar, Sara Vayrynen, Anne Tuomisto, Markus Makinen, Tuomo Karttunen. _Institute of Diagnostics, Oulu, Finland_.

Background: Micropapillary structures (MIP) can be defined as focal piles of epithelial cells in columnar epithelium. MIP's are found in many cancers, but they are characteristic for serrated colorectal carcinomas. Suprabasal cells of MIP show no contact with extracellular matrix (unpublished).

Objective: To evaluate whether MIP cells in colorectal polyps and cancers would show evidence for anoikis inhibition we assessed proliferation and apoptosis indexes (PI and AI, respectively) in MIP and non-MIP epithelium.

Materials and methods: We stained human colorectal lesions: non-serrated adenomas (n=15), serrated polyps (n=29), conventional adenocarcinomas (n=32), and serrated adenocarcinomas (n=30) with antibodies to Ki67 and M30. To obtain PI and AI two independent observers counted proportions of positive cells separately in the cells of MIP and in non-MIP epithelium.

Results: In carcinomas, AI was lower in the cells of MIP (M30; median 0, range 0-40) than in the non-MIP cells (median 2, range 0-40; p<0.001, Wilcoxon). Similarly, PI was lower in the MIP cells (Ki67; MIP: median 17, range 0-93; non-MIP: median 30, range 1-93; p<0.001). Similar patterns were evident in subsets of serrated lesions, in both carcinomas (p<0.001) and polyps (p<0.001), and in non-serrated carcinomas (p<0.004). Apoptosis/proliferation (AI/PI) ratio was lower in the MIP cells than in the non-MIP epithelium in both cancers and their precursor lesions (P<0.01).

Conclusion: Apoptosis rate was lower in MIP's than in other tumor epithelium indicating ability of these cells to survive without matrix contact, i.e. Anoikis is inhibited in them. Lower PI could suggest that cells in the MIP are in some kind of quiescent stage. However, significantly lower AI/PI ratio in the MIP infers that these structures may form a fast growing subpopulation of tumor cells. Further studies are in progress to dissect mechanism of formation of MIP and matrix independent survival of tumor cells in them.

#2854

CRISPR-knockout of HuR renders pancreatic cancer cells incapable of growth in vitro and in vivo.

Edwin Cheung, Shruti Lal, Nicole C. Mambelli-Lisboa, Mahsa Zarei, Saswati Chand, Carmella Romeo, Kevin O'Hayer, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. _Thomas Jefferson University, Philadelphia, PA_.

Pancreatic ductal adenocarcinoma (PDA) is the most prevalent type of pancreatic cancer and will soon become the second leading cause of cancer related deaths in the U.S. Studies show that the nuclear localized mRNA-binding protein HuR (ELAVL1) is activated in PDA cells, with cytoplasmic translocation associated with increased tumor size and poor prognosis. Previous in vitro and in vivo studies have established HuR's role as a PDA cell survival mechanism. Thus, we explored the phenotypic effect of completely eliminating HuR expression from PDA cells through the use of clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology to target and disrupt the HuR genomic sequence. Since INDELs are induced randomly, we designed 3 gRNAs to target HuR at different loci. Gene disruption was determined via sequencing and validated through protein and mRNA expression, where homozygous knockouts (HuR-/-) had undetectable HuR expression as compared to wild-type (HuR+/+), heterozygotes (HuR+/-), and CRISPR/Cas9 negative control. Sanger sequencing confirmed homozygous knockouts with a frame shift mutation on both alleles. When HuR knockout cells were exposed to chemotherapeutic stress including mitomycin C, oxaliplatin, and gemcitabine, no HuR expression (nuclear or cytoplasmic) was detected via immunofluorescence. Phenotypically, HuR-/- cells resulted in increased apoptosis and necrosis as measured via trypan blue assay, and accordingly, had increased caspase 3 activity, a marker of a cell death. HuR-/- cells, when treated with mitomycin C, oxaliplatin, gemcitabine, and glucose deprivation exhibited decreased long and short-term cell survival as compared to control cells. HuR-/- cells, pulse-labeled with bromodeoxyurdine (BrdU), had a higher proportion of cells in S phase and fewer cells in G2/M phase. HuR deletion enhanced premature mitotic entry thereby preventing efficient repair of DNA damage, leading to cell death. Importantly, CRISPR knockout of HuR showed marked impairment in tumor growth in mouse xenografts. The differences in median tumor volume with HuR-/- xenografts was significant as compared to xenografts in mice with HuR(+/+) cells (0.0 mm3 vs 378.0 mm3, P < 0.005). Taken together with our past work in patient samples, this pre-clinical model demonstrates that HuR is essential for PDA growth in vivo. Future work will develop strategies to target HuR either as a monotherapy or in combination with other chemotherapies.

#2856

Inhibition of glycolysis sensitizes cancer cells to metformin-induced anoikis.

Yong Yi, Zhi-Xiong Jim Xiao. _Center of Growth, Metabolism, Aging, College of Life Science, Sichuan University, Chengdu, China_.

Metformin is a widely used medicine to treat type 2 diabetes. Clinical studies have shown that metformin can reduce cancer incidence, yet the molecular mechanism of which is not totally understood. The p53-related p63 plays a critical role in regulation of cell proliferation, apoptosis, differentiation and development. We and others have shown that the major isform of p63 proteins, ΔNp63α, is important in regulation of squamous carcinoma cell proliferation, survival and cancer metastasis (Bergholz et al.,Oncogene 33:212-224, 2014;Li et al.,CDDis,2013, e943; doi:10.10384). In this study, we show that metformin can induce instability of ΔNp63α protein, leading to subsequent anoikis. In addition, glucose restriction or inhibition of glycolysis significantly sensitizes cells to metformin. Furthermore, we show that integrin β1 is involved in this process. Taken together, this study reveals a novel molecular mechanism by which metformin inhibits squamous carcinoma formation in vitro and in vivo.

#2857

The yeast homolog of the mammalian oncogene, Bax Inhibitor-1, regulates the unfolded protein response by altering the ER microenvironment.

Savannah Benko, Melissa Brown, Morgan McCarthy, Matthew Sanborn, William Cavedon, Lukas Ritzer, B. Michael Berry, Nicanor Austriaco. _Providence College, Providence, RI_.

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. The yeast homolog of BI-1, yeast Bax inhibitor-1 (BXI1), encodes a protein that belongs to the Bax Inhibitor (TMBIM) family of proteins. The crystal structure of a prokaryotic member of the family, BsYetJ, has revealed that the Bax inhibitor proteins are pH sensitive calcium leaks. Our studies have shown that yeast Bxi1p is localized to the ER and is involved in the unfolded protein response (UPR) that is triggered by ER stress. We have recently found that Bxi1p regulates the UPR downstream of endonuclease Ire1p clustering but upstream of HAC1 mRNA splicing. Studies with an ER localized calcium reporter and an ER redox reporter suggest that Bxi1p accomplishes this by altering the ER microenvironment. [Our laboratory is supported by grant NIGMS R15 GM110578, awarded to N. Austriaco.]

#2858

Endoplasmic reticulum stress as possible target for therapy in glioblastoma.

Natalia M. Peñaranda Fajardo, Coby Meijer, Frank A. E. Kruyt. _University of Groningen, Groningen, Netherlands_.

Introduction: Glioblastoma (GBM) is the most aggressive form of brain cancer with an overall survival of patients of only 1-2 years after diagnosis. The current standard treatment is largely ineffective due to high resistance and infiltrative nature of GBM. Cellular heterogeneity in the tumor and the presence of cancer stem cells in GBM (GSCs) are considered the main contributors to aggressiveness. Recently, different molecular subtypes have been found in GBM. Proneural (PN) and Mesenchymal (MES) GBM are most distinct and MES tumors are more aggressive and infiltrative. Drugs that induce Endoplasmic Reticulum Stress (ER stress)/Unfolded Protein Response (UPR) activation provide a potential novel therapy for cancer treatment. In this study we aim to characterize and explore the potential of ER stress-inducing drugs to target GBM.

Methods: GBM neurospheres PN and MES were tested for sensitivity to the ER stress inducer Thapsigargin (Tg) by MTS. TGFβ was used to induce mesenchymal transition in PN GBMs. Limiting dilution assay was performed to analyze the spheroid formation capacity upon ER stress induction. Activation of UPR was determined by analyzing protein expression of key signals by Western Blotting or XBP1 splicing assay (RT-PCR). Caspase Glo 3/7 assay was performed as read-out for apoptosis.

Results: GBM MES neurospheres cells were 2-fold more sensitive to the ER stress inducing drug Tg compared to PN cells. The reduction of cell viability upon ER stress induction coincided with induction of apoptosis (caspase 3/7 activation). Consistently MES GBM cells showed stronger increase in apoptosis activation. Tg-induced ER stress resulted in 2- 4-fold reduction in spheroid formation capacity of MES GBM cells and higher Tg concentration also reduced spheroid formation capacity in one of the PN GBM cell lines. Interestingly, TGFβ-induced mesenchymal transition sensitized particularly PN GBM cells to Tg. The activation of UPR was confirmed by induction of GRP78 and CHOP expression. In MES GBM cells particularly activation of the ATF6-UPR branch by Tg ER stress induction was detected and XBP1 splicing activation appeared to be stronger in PN cells. No significant differences were observed in PERK activation among the different GBM subtypes.

Conclusion: ER stress induction appears to be effective for eradicating GBM neurospheres, particularly the aggressive MES subtype. ER stress also targets the GSC compartment in GBM. ATF6 is notably activated in MES GBM cells, while XBP1 seems more activated in PN GBM cells upon Tg-induced ER stress. The relationship between an UPR branch activation and the differential sensitivity for the ER stress induction will be further explored. Overall, targeting the ER stress response appears a promising approach to target GBM.

#2859

Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models.

Virginia A. Espina,1 Solomon Yeon,1 Joshua N. VanHouten,2 John Wysolmerski,2 Lance A. Liotta1. 1 _George Mason University, Manassas, VA;_ 2 _Yale University, New Haven, CT_.

The defining feature of premalignant mammary lesions is proliferation of cells within the intraductal niche. Survival in this stressful duct microenvironment requires sophisticated mechanisms to avoid apoptosis caused by hypoxia, nutrient deprivation, metabolic acidosis, and intracellular calcium overload. Two major survival mechanisms of breast ductal carcinoma cells (DCIS) are 1) the upregulation of autophagy and 2) the enhanced export of calcium via plasma membrane calcium-ATPase isoform 2 (PMCA2) efflux pumps. We hypothesize that calcium efflux, through PMCA2, and activation of autophagic catabolism are critical interdependent mechanisms for the survival of pre-invasive human breast neoplastic cells.

To assess the proteomic response to autophagy inhibition and/or elevated intracellular calcium, 41 signal transduction proteins/post-translationally modified proteins related to proliferation, autophagy, and calcium pathways were quantified by reverse phase protein microarrays (RPPA) in time course studies of T-47D metastatic ductal breast cancer cell line, and MCF-10A non-tumorigenic breast cell line, both of which express low basal levels of PMCA2, and in human primary DCIS organoid cultures. Cell lines were treated with chloroquine (50µM), or 1,25 dihydroxyvitamin D3 (Calcitriol) (50nM or 100nM), or chloroquine + Calcitriol, and harvested at periodic intervals over 197 hours. Stoichiometric time course studies (24 hours) were evaluated for Calcitriol as a single agent in both T-47D and MCF-10A cell lines. Cell morphology, adhesion, proliferation and autophagic flux in the DCIS organoids were assessed by Immunohistochemistry and RPPA.

Calcitriol alone did not increase proliferation based on PCNA expression within DCIS ducts. Chloroquine markedly increased Cleaved PARP (Chi Sq p=0.004) and CAMKinase II (p=0.0068). Chloroquine (50µM) plus Calcitriol (100nM) increased E-Cadherin (p=0.0379) and phosphoPAK1/2 (p=0.0163), while PMCA2 levels were not significantly different between treatment groups.

Autophagy inhibition and calcium efflux may be exploited therapeutically by co-administration of chloroquine and Calcitrol, to inhibit lysosomal catabolism and increase intracellular calcium levels, respectively, switching them from autophagic survival into apoptotic death.

#2860

Loss of expression of the metastasis suppressor CREB3L1 is associated with high-grade metastatic breast cancer and poorer prognosis.

Paul Mellor,1 Alison Ward,1 Deborah Anderson2. 1 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada;_ 2 _Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada_.

The unfolded protein response (UPR) is required for proper protein folding in the endoplasmic reticulum under conditions of stress such as those encountered in the tumor microenvironment. Recently, a number of studies have suggested a role for the unfolded protein response in the development of cancer, more specifically in regulating the balance between cell death, dormancy and the growth of cancer cells in the tumor microenvironment. This study examined the role of CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis.

Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low- or non-metastatic cell lines. When metastatic cells were transfected with CREB3L1 they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under non-adherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, CREB3L1 expressing cells not only failed to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and ChIP on Chip analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis.

To determine if these findings translated to human breast cancer, real-time PCR analysis of CREB3L1 expression in human breast cancer tissues of differing grade (n=213) was performed. Expression of CREB3L1 was elevated in low and medium grade tissue but substantially reduced in high grade tissue, compared to normal. Analysis of the on-line TCGA database of breast cancer samples identified a strong inverse relationship between CREB3L1 gene methylation and the loss of CREB3L1 mRNA expression. Low expression of CREB3L1 was associated with a poorer prognosis with a shorter relapse-free survival for luminal A and triple negative breast cancer patients.

These data suggest that CREB3L1 plays an important role in suppressing tumorgenesis and loss of expression is required for the development of a metastatic phenotype. In addition CREB3L1 expression may be a useful marker for determining patient prognosis.

#2861

Validation of PERK as an oncology target: A role for the unfolded protein response in cancer.

Ken Dellamaggiore, Petia Mitchell, Ji-Rong Sun, Jeffrey Jones, Tony Muchamuel, David Hollenback, Seifu Tadesse, Shon Booker, Fang-Tsao Hong, Adrian Smith, Mark Rose, Pedro Beltran, James R. Lipford. _Amgen, Inc, Thousand Oaks, CA_.

The Unfolded Protein Response (UPR) is a cellular stress response to stressors that induce accumulation of unfolded proteins in the endoplasmic reticulum (aka ER stress). The UPR protects cells from ER stress by increasing the capacity of the ER and attenuating bulk translation. Intense or unresolved ER stress induces apoptosis through pro-apoptotic factors like CHoP. The UPR is activated in tumors, especially those of hematological origin. PERK, a UPR sensor-kinase, is highly active in these settings and might be an attractive target in oncology.

We have generated multiple potent, selective PERK inhibitor scaffolds. Low doses of PERK inhibitor (< pPERK IC50) activate the downstream pathway, whereas higher doses return the pathway to baseline, resulting in a bell-shaped activity curve for all pathway readouts. The activation phase results in robust, selective killing of tumor cells in vitro and in vivo, likely through sustained translation inhibition and CHoP induction. However, application in the clinic will be challenging due to irreversible toxicity to pancreatic islets at constant, high doses and difficulty managing human dosing through a bell curve.

Emerging data might provide a solution to these challenges. PERK IP-kinase assays demonstrate that compound binding at any dose activates PERK and this activity is retained after compound removal. Exposure modeling in vitro demonstrates that transient dosing followed by compound removal results in a conventional sigmoidal dose-response curve for viability. Intermittent dosing in vivo results in CHoP induction and tumor growth inhibition even at very high doses of PERK, consistent with PERK activation following compound clearance. These findings suggest that optimized scheduling might drive robust tumor growth inhibition with reduced risk of toxicity and facilitate a standard clinical dose escalation.

#2862

The balance of divergent IRE1α signaling regulates pancreatic neuroendocrine tumor growth.

Jenny Y. Qi,1 Paul C. Moore,1 Maike Thamsen,1 Rajarshi Ghosh,1 Micah J. Gliedt,1 Dustin J. Maly,2 Bradley J. Backes,1 Feroz R. Papa,1 Scott A. Oakes1. 1 _UCSF, San Francisco, CA;_ 2 _University of Washington, Seattle, WA_.

The Unfolded Protein Response (UPR) is an intracellular signaling pathway that communicates the protein folding status of the endoplasmic reticulum (ER) to the nucleus to maintain ER homeostasis. Hypoxia, nutrient deprivation, proteasome dysfunction, or sustained demands on the secretory pathway--conditions often encountered by solid tumor cells--lead to the accumulation of misfolded proteins in the ER and cause "ER stress." However, the precise role of UPR signaling in the development and maintenance of cancer remains controversial. We reasoned that pancreatic neuroendocrine tumors (PanNETs) might be one class of solid tumor that may be particularly sensitive to protein folding stress due to their high secretory activity.

The Master UPR regulator IRE1α is an ER transmembrane kinase/endoribonuclease (RNase) that acts as a critical life-death switch depending on the level of ER stress. We analyzed primary human PanNET samples for activation of UPR components and found strong evidence of ER stress and IRE1α hyperactivation in this tumor type. We used a variety of chemical-genetic tools to selectively manipulate IRE1α's homeostatic and apoptotic outputs in a PanNET xenograft model and found that the precise balance of IRE1α signaling is critical for tumor growth in vivo. Disrupting this balance by pushing the pathway in either direction is detrimental to PanNET survival. Moreover, targeting IRE1α with highly selective small molecules in a preclinical mouse model phenocopied the results of genetic manipulation. Our results indicate that IRE1α is an important regulator of PanNET cell survival and growth, and provide a strong rationale for therapeutically targeting this kinase in PanNETs and other cancers that experience high levels of ER stress.

#2863

Mechanisms underlying estrogen-induced inflammation in estrogen-deprived breast cancer cells.

Ping Fan,1 Fadeke A. Agboke,2 Amit Kumar Tyagi,1 V. Craig Jordan1. 1 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _University of Georgetown, Washington DC, DC_.

Inflammation is critical for estrogen (E2) to undertake apoptosis in E2-deprived breast cancer cells. However, it remains unclear how E2 induces inflammation. Here, we demonstrate that E2 increases lipid metabolism and accumulates unfolded proteins in the endoplasmic reticulum in E2-deprived breast cancer cells. These two major alterations result in different inflammatory responses. E2 rapidly increases lipid metabolism related genes such as fatty acid desaturase 1 (FADS1) and CCAAT/enhancer binding protein beta (CEBPB) to modulate adipose inflammation related factors, e.g. IL6/IL6R after two hours treatment, which regulate the cellular proliferation or differentiation. Simultaneously, accumulation of the unfolded protein activates unfolded protein response (UPR) sensors, protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol-requiring protein 1 alpha (IRE1α) within few hours after exposure to E2. Importantly, inhibition of PERK, rather than IRE1α, is able to completely prevent E2 from induction of late event inflammatory genes associated with apoptosis, such as tumor necrosis factor (TNF) superfamily members, BCL2L11 (Bim), and HMOX1,which are activated by E2 after 48 hours. Consistently, nuclear factor-kappa B (NF-κB) is activated when TNFα is maximal after E2 treatment. Further examination revealed that an anti-inflammatory agent, dexamethasone (Dex), activates glucocorticoid receptor (GR) transcriptional activity and blocks E2-induced apoptosis. These effects can be reversed by the knockdown of GR. A notable mechanistic change is that Dex effectively blocks the phosphorylation of PERK and produces equivalent inhibitory effects on the apoptosis-associated inflammatory genes as the PERK inhibitor. However, Dex has an additional potential to activate lipid metabolism and are additive with E2 to elevate FADS1 and adipokine IL6/IL6R. All of these data, for the first time, describe the mechanisms underlying E2-induced inflammation in E2-deprived breast cancer. It provides important information to restrict the clinical use of glucocorticoids, which will undermine the beneficial effects of E2-induced apoptosis in estrogen deprived breast cancer patients.

#2864

Role of ribosomal protein, Rpl22 in regulation of acute lymphoblastic leukemia.

Nehal R. Solanki-Patel, Noa Greenberg, Shu Rao, Suraj Peri, Michael Slifker, Charles Mullighan, David Wiest. _Fox Chase Cancer Center, Philadelphia, PA_.

Inactivation of ribosomal proteins (RP) is known to cause diseases called ribosomopathies, which are often associated with abnormal hematopoiesis and an increased risk for development of leukemia. Our laboratory has recently reported an important link between inactivation of one such ribosomal protein, Rpl22, and poor survival in T cell acute lymphoblastic leukemia (T-ALL) patients. Preliminary bioinformatic analysis shows that RPL22 is lost in ~10% of pediatric T-ALL patients, who exhibit a more aggressive disease course. Deletion of the RPL22 locus is also enriched in the early T-cell precursor (ETP-ALL) subset of T-ALL patients, which exhibit a more aggressive disease course. Thus, RPL22 loss appears to be a marker for poor outcome in pediatric T-ALL patients. While RP inactivation has previously been linked to increased cancer risk, the mechanistic basis for this linkage remained unclear. We have recently demonstrated that inactivation of Rpl22 promotes leukemic transformation by activating NF-κB and inducing the stem cell gene, LIN28B. Nevertheless, the molecular link between Rpl22 inactivation and the induction of NF-κB activity remained unclear. We have now determined that inactivation of Rpl22 activates NF-κB signaling by exacerbating ER stress responses. Indeed, Rpl22 loss results in hyperactivation of the PERK-EIF2α-ATF4 stress pathway, which is known to activate NF-κB. We have demonstrated that knockdown of PERK using shRNA abrogates the activation of NF-κB in Rpl22 mutant cells and returns Lin28B expression levels to baseline. This reduction in Lin28B eliminates the predisposition to transformation exhibited by Rpl22 mutant cells, as evidenced by decreased formation of colonies in soft agar. Taken together, these data suggest that RPL22 inactivation may serve as a negative prognostic indicator in T-ALL and that pharmacologic targeting of ER stress pathways may represent a novel therapeutic alternative in this molecularly-defined subclass of T-ALL.

#2865

Dual action glycotherapy for triple negative breast cancer.

Dipak K. Banerjee,1 Aditi Banerjee,1 Zhenbo Zhang,1 Jesus E. Serrano,1 Eva C. Romero,1 Neysharie Sanchez,1 Krishna Baksi2. 1 _University of Puerto Rico, San Juan, PR;_ 2 _Universidad Central del caribe, Bayamon, PR_.

Triple negative breast cancer (TNBC; ER-/PR-/HER2-) accounts for 15% of all breast cancer with a disproportionate share of mortality. The patients are younger and pre-menopausal. The cancers are poorly differentiated, and most fall into the basal subgroup of breast cancers. The long-term survival is extremely low because of lack of specific treatment guidelines. Thus, the patients are managed with standard treatment. This causes a high rate of local and systemic relapse, and make patients resistant to existing targeted therapies (endocrine/biologics/adjuvant/neo-adjuvant). Anthracycline/taxane combination therapy and PARP inhibitor are currently being explored but not known what would be the outcome or if the patients would achieve a pathological complete response (pCR). Protein glycosylation has been claimed as an important feature helping cancer cells escaping immune surveillance, facilitate tumor invasion, and increased malignancy with increased tumor burden and poor prognosis as well as enhanced angiogenesis. Based on the glycomics profile of human breast cancer cells and tumor specimen, we have hypothesized that targeting asparagine-linked (N-linked) protein glycosylation would evolve a new generation therapeutic preventing breast tumor progression and eliminating the disease. Consequently, when treated with a homolog of protein N-glycosylation inhibitor tunicamycin the progression of a double negative breast cancer was inhibited significantly (J. Biol. Chem. 286, 29127-29138, 2011). We have now tested a triple negative breast tumor xenograft in athymic nude mice Balb/c (nu/nu) and the result is reduction of tumor progression (~65% in one week) at tunicamycin concentration of 0.25 mg/Kg given orally twice a week. Human triple negative breast cancer cells (MDA-MB-231/MDA-MB-468) are equally susceptible to tunicamycin action. The cells are arrested in G1 and exhibit ER stress followed by induction of apoptosis mediated by unfolded protein response (upr) signaling. Western blotting and qRT-PCR support increased expression of the ER stress master regulator GRP78. But, the immunofluorescence microscopy could not detect GRP78 on the surface of control or tunicamycin treated cells. The result is identical in cells cultured in the absence of serum. GRP78 fluorescence however, is detectable in cells either fixed with ice-cold methanol or permeabilized with digitonin. We, therefore, conclude that GRP78 is not expressed on the outer-leaflet of the cell surface of the triple negative human breast cancer cells MDA-MB-231. But, its intracellular expression is anti-tumorigenic. Supported in part by grants from NSF-EPSCoR RII Track 1 Grant # EPS-1002410 (DKB) and NIH/NIMHD 2G12MD007583 (KB)

#2866

Sodium channel γENaC mediates salt induced inflammatory stress in cancer cells.

Suneetha Amara,1 Venkataswarup Tiriveedhi2. 1 _Mercy Hospital, St Louis, MO;_ 2 _Tennessee State University, Nashville, TN_.

Chronic inflammation is known to play a critical role in the development of cancer. Recent evidence suggests that high salt in the tissue microenvironment induces chronic inflammatory milieu. Further, high salt intake has been correlated with higher incidence of cardiovascular diseases and cancers. In this report, using breast cancer cell lines, MDA-MB-231 (highly invasive), MCF-7 (poorly invasive), and a normalized breast epithelial cell line, MCF10A, we determined the molecular basis of the potential inflammatory effect of high sodium chloride (NaCl) in the extracellular environment. Dose response studies demonstrated a significantly reduced cell viability (>50%) with external NaCl concentration above 0.25 M. Combined treatment of high NaCl (0.15 M) with sub-effective interleukin (IL)-17 (0.1 nM) induced reactive nitrogen (RNS) and oxygen (ROS) species release along with upregulation of the enzymes iNOS, SOCS and SOD1. Similar effect was not observed with equi-molar mannitol. Western-blot and qRT-PCR analysis demonstrated an elevated expression of an inflammation-specific epithelial sodium channel, γENaC, in MDA-MB-231 (7.1±1.4 fold, p<0.05), MCF-7 (3.9±0.8 fold, p<0.05) and MCF10A (2.2±0.4 fold, p<0.05) cell lines following combined treatment of high NaCl with sub-effective IL-17. This was significantly abrogated by lipid raft depletion and ERK-1/2 knock-down. These culture conditions have also induced cellular expression of cytokines, IL-6 (3 fold), TNFα (6.2 fold); chemokines, CCL5 (2.3 fold), CXCL-12 (2 fold), MIP-1δ (2.7 fold); and angiogenic growth factor, VEGF (2.5 fold). Taken together, these data suggest that high NaCl in the cellular microenvironment induces a γENaC mediated chronic inflammatory response with a potential pro-carcinogenic effect.

### Transcriptional Regulation and Gene Expression in Human Malignancies

#2867

Functional role of FHL2 in the splicing machinery of liver cells.

Ye Cao, Minghua Liu, Yi Yang, Yinfeng Zhang, Kwok Wing Tsui. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is a highly lethal cancer with increasing worldwide incidence. The four-and-a-half LIM domain protein 2 (FHL2) has been identified as an oncogene / tumor suppressor gene in a tissue/cell specific manner by acting as an adaptor protein to form different complexes and involving in various signaling pathways. To clarify the intriguing functions of FHL2, we have identified a batch of interacting partners of FHL2 by immunoprecipitation and liquid chromatography coupling with mass spectrometry. Many of these proteins, including SNRNP70, DAZAP1, SFPQ, TIAL1 and so on, are involved in spliceosome components and mRNA surveillance. The interaction among several proteins and FHL2 has been confirmed by co-immunoprecipitation. To further elucidate the functional role of FHL2 in the splicing process in liver cancer cells, we have studied the transcriptomes of FHL2 overexpression and FHL2 knockdown liver cells, whereas empty vector and scrambled siRNA were used as controls respectively. Followed by bioinformatics analysis, we found significant concordance between the immunoprecipitation and transcriptome datasets. Selected genes were further validated by real-time PCR such as FGFR4. In summary, FHL2 is probably regulating the mRNA splicing by interacting with components of the splicing machinery and modulating splicing related genes at the transcriptional level.

#2868

Mechanisms of transcriptional regulation of FOXO3 by cofactors upon PI3K/AKT/mTOR inhibition.

Vasilis Hristidis,1 James Cownie,1 Ana Bosch,2 Joanne Soong,1 Pau Castel,1 Maurizio Scaltriti,1 Baselga José1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Lund University, Sweden_.

The PI3K/AKT/mTOR pathway is frequently deregulated in breast cancer. Specific inhibition of its key signaling nodes results in cell death and tumor growth arrest, but also in activation of feedback loops that ultimately limit therapeutic activity.

The subcellular localization and activity of Forkhead box O3 (FOXO3) transcription factor is heavily regulated by the PI3K pathway. Phosphorylation of FOXO3 by AKT inhibits its transcriptional functions leading to cell survival and growth.

Although our understanding of the transcription function of FOXO3 downstream of the PI3K pathway has increased significantly in recent years, it is still unclear how FOXO3 acts in concert with other cofactors of the transcription machinery.

We conjectured that inhibition of the PI3K/AKT/mTOR signaling by specific agents targeting the pathway at different nodes leads to unique FOXO3-mediated transcriptional programs unveiling a complex network of regulation. We therefore sought to identify proteins that differentially co-immunoprecipitate with FOXO3 upon PI3K, AKT or mTOR blockade.

To target the pathway at different levels, we used BYL719, a PI3Kα inhibitor; MK2206, an allosteric AKT inhibitor; and RAD001, an allosteric mTORC1 inhibitor. Upon treatment, we immunopreciptated endogenous FOXO3 and searched for FOXO3 interactors by mass spectrometry analysis. In parallel RNA samples from each treatment were analyzed using gene expression microarrays.

Gene expression analyses revealed that each specific compound originated distinct transcriptional signatures. Accordingly, mass spectrometry analysis revealed novel FOXO3 co-interactors engaging in different transcriptional complexes at each treatment conditions. We identified some of these proteins as CUL1, the core component of the SCF (SKP1-CUL1-F-BOX protein) E3 ubiquitin-protein ligase complex, the cyclin-dependent kinase 5 (CDK5), the upstream binding transcription factor (UBTF), and the core component of RNA polymerase I POLR1C (polymerase RNA I polypeptide C). We performed coimmunoprecipitation analyses to confirm that these cofactors are bona fide FOXO3-interacting proteins. We then analyzed the effects of overexpressing these proteins on FOXO3 transcriptional activity. Luciferase assays demonstrated an increase in FOXO3 transcriptional activity when CUL1, UBTF, CDK5 or POLR1C were overexpressed. The overexpression of cofactors also induced the expression of FOXO3 target genes such as TRAIL or IRS2. Furthermore, overexpression of these co-interactors enhanced the binding of FOXO3 to its target gene promoters. We are now engaged in both elucidating how these cofactors can modulate the transcriptional activity of FOXO3 upon PI3K/AKT/mTOR inhibition and understanding the therapeutic implications of these FOXO3-containing complexes.

#2869

FLASH-dependent post-translational regulation of the EMT activator ZEB1.

Camille Abshire, Ana Maria Dragoi. _LSUHSC-Shreveport, Shreveport, LA_.

Understanding the mechanisms of metastasis initiation and tumor persistence despite aggressive treatments is paramount to developing new viable therapeutic targets. Critical to tumor progression is initiation of epithelial-to-mesenchymal transition (EMT), a cellular process characterized by loss of E-cadherin, a critical cell-cell adhesion molecule, gain of migratory properties and chemoresistance. Transcriptional repression of E-cadherin and other genes responsible for maintaining the epithelial phenotype by ZEB1/ZEB2 family is one of the most significant events occurring at the interface between non-metastatic/metastatic cancer and chemo-sensitive/chemo-resistant cancer. Using a novel interdisciplinary high-throughput screen methodology we uncovered novel regulators of E-cadherin. Caspase 8 - associated protein 2 (CASP8AP2 or FLASH) a protein involved in apoptosis and transcriptional control was identified through this screen and we discovered that FLASH depletion resulted in increased E-cadherin levels at the plasma membrane through loss of ZEB1 expression. Subsequent examination of ZEB1 protein expression revealed that ZEB1 is rapidly degraded in FLASH-depleted cells. This process is blocked by proteasome inhibitors suggesting that post-translational modifications (PTMs) and in particular ubiquitination of ZEB1 is responsible for its fast turnover and decreased protein expression in the absence of FLASH. Indeed, ubiquitinated ZEB1 was detected in FLASH-depleted cells in the presence of proteasome inhibitors. Although ZEB1 is degraded through the ubiquitin pathway, its rapid turnover in the absence of FLASH suggests that protective mechanisms are in place to delay and/or prevent this process. Even more, we determined that the atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 is involved in ZEB1 degradation in the absence of FLASH and is upregulated in cells depleted for FLASH. Altogether our data suggests that FLASH can stabilizes ZEB1 indirectly by transcriptional repression of the ubiquitin ligase responsible for its degradation and directly by binding to ZEB1 and masking the recognition site of the ubiquitin ligase. Even more, absence of FLASH reprograms the cells into a more epithelial-like phenotype which is less invasive and is resistant to TGFβ-induced loss of E-cadherin. Not only is FLASH-dependent post-translational regulation of ZEB1 novel, but is also conserved in multiple cells lines from various tissues, making this mechanism an original therapeutic target for cancer metastasis.

#2870

Serine/arginine rich proteins show different distribution patterns in stratum of oral epidermoid carcinoma.

Cynthia J. Ceja-Lopez,1 Reyna Lara-Martínez,1 Lourdes Segura-Valdés,1 Abelardo Meneses-García,2 Jorge Alberto Guadarrama-Orozco,2 Luz Ruíz Godoy Rivera,2 Erika Ruíz-García,2 Luis Felipe Jiménez García1. 1 _Universidad Nacional Autonoma de México, México D.F., Mexico;_ 2 _Instituto Nacional de Cancerología, México D.F., Mexico_.

Serine/arginine proteins increased their activity during the alternative splicing in cell nucleus. The nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. SR proteins are related with splicing during different types of cancers and metástasis. We search for phosphorylated SR proteins and the nuclear pattern and speckle formation in human oral carcinomas.

Methods: To address nuclear pattern and speckle formation, we performed a series of immunostaining experiments, with SC35 antibody for phosphorylated srsf2 on sections derived from oral epidermoid tumor from 33 patients. The images were obtained with a microscope (Leica TCS SPE/CTR 4000). For imaging cytometry analyses, a Leica LAS AF Lite software (Leica Microsystems) was used to capture images automatically; images and recognition of nuclear speckles were quantitatively analyzed using analysis software (ImageJ). ANOVA analysis was performed for the cellular and staining correlation.

Results: There are significant differences in morphology and distribution of intranuclear mottled pattern of SR proteins in each tissue layer between oral epithelial without neoplasia and oral squamous cell carcinoma (P < 0.0001 ). We also found that number, size and pattern (p <0.05) is different between the stratum layers of the oral squamous cells, showing more transcriptional activity in the granulosum stratum related with a decreased in nucleus numbers. In oral squamous cells the pattern changes become homogeneous and similar to the basal layer of normal tissue.

We found that is in the granulosum stratum of oral squamous carcinomas where the SR proteins shows more transcriptional activity. This finding could be related with the increased metabolism of this layer.

#2871

Dual role of EpCAM cleavage in adhesion attenuation and transcription enhancement for cell migration.

Ya-Ting Hsu,1 Pawel A. Osmulski,1 Yao Wang,1 Yi-Wen Huang,2 Lu Liu,3 Jianhua Ruan,3 Victor X. Jin,1 Nameer B. Kirma,1 Maria E. Gaczynska,1 Tim H.M. Huang1. 1 _UTHSCSA, San Antonio, TX;_ 2 _Medical College of Wisconsin, Milwaukee, WI;_ 3 _UTSA, San Antonio, TX_.

Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a regulatory molecule internalized into the nucleus for transcriptional control of gene expression. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the cleavage of the extracellular fragment EpEX culminating in removal of cell-surface EpCAM as monitored with recognition atomic force microscopy (AFM). As a result, internalization of the cytoplasmic domain EpICD leads to formation of transcription factor complexes with LEF1 that regulate gene transcription for enhancing mobility functions of cancer cells. Comprehensive probing with AFM further reveals increased elasticity and decreased adhesiveness of these cells, implicating acquisition of an epithelial-mesenchymal transition phenotype. While EpCAM cleavage contributes to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells.

#2872

Cytoplasmic localization of PU.1 with mutated NPM1 causes myeloid differentiation arrest.

Xiaorong Gu,1 Reda Mahfouz,1 Ji Zhang,2 Francis Enane,1 Zhenbo Hu,3 Tomas Radivoyevitch,1 David Wald,4 Maria Paola Martelli,5 Brunangelo Falini,5 Yosef Landesman,6 Jaroslaw Maciejewski,1 Yogen Saunthararajah1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Department of Ophthalmology, The Second Affiliated Hospital of Soochow University, SuZhou, JiangSu, China;_ 3 _Affiliated Hospital of Weifang Medical University, Weifang, China;_ 4 _Case Western Reserve University, Cleveland, OH;_ 5 _Institute of Hematology, University of Perugia, Perugia, Italy;_ 6 _Karyopharm Therapeutics, Newton, MA_.

The most frequently mutated gene in de novo acute myeloid leukemia (AML) is nucleophosmin (NPM1). How mutated NPM1 (mNPM1) confers clonal advantage is unknown. We hypothesized that mNPM1 interferes with the function of one or more master transcription factors (TF) essential for myeloid differentiation. We used mass-spectrometry to comprehensively analyze the protein interactome of endogenous NPM1 and mNPM1 in AML cell nuclear and cytoplasmic fractions, and examined the cell fate implications of observed interactions. NPM1 was found in abundance in both nuclear and cytoplasmic fractions of wtNPM1 AML cells, but only in the cytoplasm of mNPM1 AML cells. In the nucleus of wtNPM1- and cytoplasm of mNPM1-AML cells, the NPM1/mNPM1 interactome was enriched for the master TF driver of monocytic differentiation PU.1 and the RARA co-factors STAT1 and CNOT1. Accordingly, by both Western blot (WB) of cell fractions and immunofluorescence (IF) microscopy, PU.1 was localized in the cytoplasm of mNPM1 AML cells together with mNPM1. Another master differentiation-driving TF highly expressed in AML cells, CEBPA, was not in the NPM1 interactome nor localized to cytoplasm. Nuclear export of NPM1 is dependent on the nuclear export protein XPO1 (CRM1). Selinexor (KPT-330) is an anti-cancer drug that inhibits protein trafficking to cytoplasm by covalently binding to the XPO1 cargo binding pocket. Selinexor 20-50nM relocated both mNPM1 and PU.1 into the nucleus in mNPM1 AML cells, demonstrated by IF and cell fraction WB. Importantly, this nuclear relocation prompted terminal monocytic differentiation of mNPM1 AML cells. The RARA ligand ATRA at 10nM transferred RARA and STAT1 but not mNPM1/PU.1 into the nucleus and triggered terminal granulocytic rather than monocytic differentiation, presumably enabled by high baseline intra-nuclear CEBPA. mNPM1 AML is distinguished by high HOX gene expression. To evaluate a potential cause-effect relationship, Hox expression was examined in Pu.1 knock-out hematopoietic precursors retrovirally transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER). Addition of estrogen to translocate Pu.1-ER into the nucleus substantially decreased Hox expression accompanied by terminal monocytic differentiation. Thus, nuclear export of PU.1 and RARA cofactors with mNPM1 causes the differentiation arrest that defines AML. Low nanomolar concentrations of selinexor transfer PU.1 to the nucleus and trigger terminal monocytic differentiation, while low nanomolar concentrations of ATRA transfer RARA/STAT1 to the nucleus and trigger terminal granulocytic differentiation. These are p53-independent pathways of cell cycle exit distinct from p53-dependent apoptosis that mediates chemotherapy effect. Thus selinexor and/or ATRA differentiation therapy may offer the ~50% of mNPM1 AML patients with chemo-refractory disease a needed novel, mechanistically rational treatment option.

#2873

Identification of FXR1-associated protein complexes in lung cancer.

Jun Qian,1 Yong Zou,1 Megan Hoeksema,1 Bradford Harris,1 Heidi Chen,2 Pierre Massion1. 1 _Vanderbilt-Ingram Cancer Center, Nashville, TN;_ 2 _Department of Biostatistics, Nashville, TN_.

RNA-binding proteins (RBPs) are the master regulators of mRNA processing and translation and are often aberrantly expressed in cancer. We have recently identified Fragile X Mental Retardation-Related 1 (FXR1) as a novel cancer gene in non-small cell lung cancer (NSCLC) and its expression is correlated with poor prognosis in multiple human cancers. FXR1 encodes an RNA binding protein (RBP) that belongs to the family of fragile X-related proteins including the fragile X mental retardation protein (FMR1) and FXR2. Inactivation of FMR1 expression is the cause of the Fragile X syndrome in humans. Little is known for the function of FXR1 in human cancers. In this study, endogenous FXR1 or a Flag-tagged FXR1 was immunoprecipitated from H520, a lung squamous carcinoma cell line, and analyzed by shotgun proteomics. The Flag-tagged FXR1 was also transfected into human HEK293 cells and followed by co-immunoprecipitation and shotgun proteomic analysis. In total we found 206 proteins enriched in H520 with more than two-fold change spectral counts over the IgG control. Of the 206 proteins, 49 were detected in HEK293 cells as well and 157 were only detected in H520. KEGG pathway analysis indicated enrichment of proteins involved in ribosomal function, RNA transport and proteasome. To identify lung cancer related proteins, we interrogated the mRNA expression of 206 proteins in The Cancer Genome Atlas (TCGA) lung cancer dataset (1013 tumors,109 normal), in a combined NSCLC dataset (1392 tumors, 240 normal) from the Gene Expression Omnibus (GEO) and in a previously reported dataset of putative FXR1 target mRNAs and found 75 altered genes in common. Among these, we found that 24 genes were not only unregulated in all NSCLC samples (n=2405, FDR<0.007) but also positively correlated with FXR1 (p<0.05). Gene ontology analysis revealed 22 genes were enriched in cellular macromolecule metabolic process including six genes in cell cycle regulation and seven genes in mRNA processing (p=0.0017). Disease association analysis showed six genes were enriched in immunologic deficiency syndromes (p=0.002), consistent with post-transcriptional suppression of tumor necrosis factor-alpha (TNFα) by FXR1 and further implying a role for FXR1-associated complex in the suppressed immune response pathway we previously identified in squamous cell carcinoma of the lung. Lastly, survival analysis indicated that this 25-gene signature (BUB3,CSNK2A1,DARS,DHX9,FXR1,HDAC2,HNRNPAB,HSP90AA1,KPNB1,LMNB1,MCM4,MSH6,NUP205,PRKDC,PSMC5,PSMD3,SFPQ,SMARCC1,SMC4,SNRPD1,STK38L,TNPO1,UCK2,XPO1 and XRCC5) was significantly associated with worse overall survival in NSCLC (HR=1.84, p=0.01, adjusted for age, gender, stage and smoking history). Together, these data suggest the 24 proteins/mRNAs are closely associated with FXR1 in lung cancer and may form critical core complexes that are associated with NSCLC tumorigenesis. This work was supported by RO1 CA102353 to PPM.

#2874

Genetic suppression reveals DNA repair-independent antagonism between BRCA1 and COBRA1 in mammary gland development.

Xiaowen Zhang,1 Sreejith J. Nair,1 Huai-Chin Chiang,1 Yao Wang,1 Md Jamiul Jahid,2 Jianhua Ruan,2 Victor X. Jin,1 Yanfen Hu,1 Rong Li1. 1 _UTHSCSA, San Antonio, TX;_ 2 _UTSA, San Antonio, TX_.

Breast cancer susceptibility gene BRCA1 is well known for its function in double strand break (DSB) DNA repair. While BRCA1 is also implicated in transcriptional regulation, the physiological significance remains unclear. Cofactor of BRCA1 (COBRA1) is a BRCA1-binding protein and an important regulator of transcription elongation. Here we used mouse genetics to interrogate functional interaction between BRCA1 and COBRA1 during mammary gland development. Tissue-specific deletion of Cobra1 reduced mammary epithelial compartments and blocked ductal morphogenesis, alveologenesis, and lactogenesis, demonstrating a pivotal role of COBRA1 in adult tissue development. Remarkably, these developmental deficiencies due to Cobra1 knockout were largely rescued by additional loss of full-length Brca1. Furthermore, Brca1/Cobra1 double knockout restored developmental transcription at puberty, altered luminal epithelial homeostasis, yet remained deficient in homologous recombination-based DSB repair. Thus our genetic suppression analysis uncovers a previously unappreciated, DNA repair-independent function of BRCA1 in antagonizing COBRA1-dependent transcription program during mammary gland development.

#2875

Genes associated with neuronal development, including splicing regulatory factors, increase in expression across epithelial-mesenchymal transition.

Yevgenia L. Khodor, Daisy Riquelme, Frank Gertler, Christopher B. Burge. _Massachusetts Institute of Technology, Cambridge, MA_.

The program by which epithelial cells lose their polarity and close contacts with neighboring cells and acquire migratory and invasive properties is known as epithelial-mesenchymal transition (EMT). Initially identified as a critical developmental process, EMT is now understood to occur during cancer, where it has been shown to confer resistance to senescence and apoptosis. Previous work indicates that cancer cells that escape the site of the primary tumor hijack at least a portion of the EMT program to increase their invasive properties and metastasize. Earlier work in the field has focused on cell signaling and transcriptional regulation changes that lead to EMT, but changes in the gene expression and post-transcriptional regulation of the cell have only recently become the focus of study. We characterized gene expression and splicing across EMT in the luminal epithelial MCF7ras SLUG+SOX9 breast cancer cell line by collecting samples at 2-day intervals across its 6-day transition and profiling the RNA by RNA-seq. We found over 4,000 significantly changing genes, and that the majority of gene expression and splicing changes occurred in the first two days of the transition, with few changes noted between days 4 and 6. Genes that changed could be broadly categorized into those whose expression decreased, increased, or peaked in the middle of the transition before decreasing. Intriguingly, genes whose expression increased during EMT were enriched for gene ontology terms related to cell signaling and neuronal development. We specifically observed increased expression of mRNAs encoding splicing factors and other RNA-binding proteins (RBPs) associated with neuronal development, in contrast to the majority of RBPs whose expression decreases in EMT. We are currently exploring various aspects of this program, including effects of increased expression of the neuronal-associated splicing regulatory factor quaking (QKI). These may include downregulation of the Notch pathway via effects on splicing of NUMB, a Notch inhibitor. We are currently validating the role of QKI on this and other RNA processing changes and will report our findings.

#2876

The androgen receptor utilizes protein kinase docking sites to constitutively activate Elk1.

Rayna Rosati,1 Mugdha Patki,1 Selvakumar Dakshnamurthy,2 Venkatesh Chari,1 Thomas McFall,1 Manohar Ratnam1. 1 _Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 2 _Wayne State University, Detroit, MI_.

The ETS domain transcription factor Elk1 tethers the androgen receptor (AR) to chromatin, enabling sustained activation of a set of genes critical for growth in established prostate cancer cell lines. This process does not entail the transient hyper-activation of immediate early genes typical of activation of Elk1 through phosphorylation by ERK1/2. We compared the structural requirements for the association of AR and Elk1 with that for activation of Elk1 by phosphorylation using mammalian one- and two-hybrid assays. The critical polypeptide segments were mapped by systematic deletion mutagenesis. The amino-terminal A/B domain of AR, which lacks the ligand-binding domain (LBD), was adequate for association with Elk1 and to activate Elk1-AR target genes in LNCaP prostate cancer cells. The AR A/B domain also supported hormone-independent growth of LNCaP cells reflecting the ability of overexpressed natural splice variants of AR, which also lack LBD, to support prostate tumor growth. The association of AR with Elk1 was optimal in the absence of ERK1/2 and serum response factor (SRF), which are the known binding partners of Elk1. We identified two sites on Elk1 that are critical for its association with the AR A/B domain as well as the whole AR molecule. One of those sites spans amino acid residues 307 to 330, overlapping the D box, which is one of two ERK docking sites. The other site on Elk1 mapped to amino acid residues 372 to 397, overlapping the FXFP motif, which comprises the downstream ERK docking site. The results suggest that AR utilizes ERK docking sites to associate with Elk1 and is exclusive of the interaction of Elk1 with ERK1/2 or SRF. A nuclear receptor could thus function as a transcription factor co-activator by binding at protein kinase docking sites.

#2877

Effects of metabolic stressors on cellular differentiation.

Juan Manuel Schvartzman, Craig B. Thompson. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Poor differentiation (the divergence of tumor cell morphology from the cellular morphology of the normal tissue of origin) is associated with unfavorable responses to chemotherapy and poor clinical outcomes in solid tumors. Recurrent somatic mutations in the genes encoding for the metabolic enzymes isocitrate dehydrogenase (IDH) 1 and 2 have recently been identified in patients with acute myeloid leukemias, chrondrosarcomas, cholangiocarcinomas and glioblastomas. Point mutations in IDH lead to production of 2-hydroxyglutarate (2HG). 2HG acts as a potent inhibitor of enzymes involved in the removal of chromatin marks associated with transcriptional repression (DNA and Histone H3K9 methylation). Excess 2HG leads to impaired differentiation but very little is known about the underlying mechanisms. Here we use a well established model of cellular differentiation, the differentiation of 10T1/2 fibroblasts into myofibers by the lineage-specifying transcription factor MyoD to study the mechanisms by which IDH mutations impair differentiation. We show that 5-azacytidine induced differentiation of 10T1/2 cells is impaired when mutant IDH2 is expressed and that IDH2 mutations impair the ability of MyoD expression to reprogram 10T1/2 cells into myofibers. We propose that the metabolic landscape of undifferentiated cells determines the ability of the lineage determining factor MyoD to trigger differentiation and that the undifferentiated state of cancer cells is dependent on their unique metabolic profile. Further work aims to a) define the effects of mutant IDH1/2 on the ability of MyoD to program muscle differentiation, b) determine the nature of the tumorigenic effects of IDH1/2 mutations in 10T1/2 cells and c) identify molecular pathways that can overcome differentiation blocks upon activation. Insight from these experiments will outline the mechanism(s) by which metabolic alterations, in the form of 2HG, impair differentiation and drive tumorigenesis. They will also serve as a platform for the identification of metabolic alterations that trigger differentiation. These mechanisms could prove to be novel and readily targetable processes in an array of poorly differentiated and aggressive cancer subtypes where other pharmacological approaches have failed.

#2878

The tumor-permissive role of KHSRP in colorectal cancer: Mechanistic insights from a gene expression study.

Francesco Caiazza,1 Robert Power,1 Kate Killick,1 Des Higgins,1 Walter Kolch,1 Laura Breen,2 Sinead Aherne,2 Sudipto Das,3 Darran O'Connor,3 Miriam Tosetto,1 Blathnaid Nolan,1 David Fennelly,4 Kieran Sheahan,4 Glen Doherty,1 Elizabeth J. Ryan1. 1 _University College Dublin, Dublin, Ireland;_ 2 _Dublin City University, Dublin, Ireland;_ 3 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 4 _Saint Vincent's University Hospital, Dublin, Ireland_.

Inflammation plays a key role in the development of Colorectal Cancer (CRC), but the underlying regulatory mechanisms are yet to be fully elucidated. The K-homology splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein which regulates translation of transcripts containing ARE sequences in their 3'-UTR, affecting RNA fate at different levels. KHSRP has been implicated in different functions associated with cancer cell biology, such as inflammation and cell-fate determination. Importantly, KHSRP regulates expression of pro-tumorigenic and anti-inflammatory cytokines, potentially impacting on the tumor microenvironment. We have previously shown that knockdown of KHSRP expression in CRC cell lines regulates cell proliferation and clonogenic survival. KHSRP is also required for the secretion of the pro-angiogenic cytokines, IL-8 and VEGF, impacting on endothelial tube formation. To understand the global impact of KHSRP in regulation of CRC cell function we used Affimetrix Human Transcriptome array to investigate gene expression upon KHSRP silencing in SW480 CRC cells. A total of 210 genes were differentially expressed, 155 of which were up-regulated (100 coding and 55 non-coding genes) and 55 were down-regulated (34 coding and 21 non-coding genes). Among these were genes involved in RNA biology, inflammation, immunity, cell signaling and tumor-stroma interaction, shedding light on the potential mechanisms of action of KHSRP in epithelial cells. Representative targets have been selected for validation and further mechanistic studies. These data builds upon our previous findings suggesting a role for KHSRP in creating a tumor-permissive microenvironment in CRC.

#2879

PIGU modulates radioactive iodine uptake in differentiated thyroid cancers.

MORAN AMIT, shorook Na'ara, Tomer Charas, Ziv gil. _Rambam Medical Center, Rappaport School of Medicine, the Technion Israel Insitute of Technology, Ha, HAIFA, Israel_.

Radioactive iodine (RAI) treatment has a significant role in the management of differentiated thyroid carcinoma after surgical treatment. Although there is significant variability between patients, in response to RAI treatment, the mechanism responsible for this phenomenon is unknown. PIGU is an endoplasmic reticulum protein and a component of the GPIT complex, which plays a crucial role in post-translational modification of proteins such as sodium iodide symporter (NIS). Immunohistochemical analysis revealed significantly lower expression of PIGU in papillary carcinomas compared to matched normal thyroid tissue (P<0.001). To study the role of PIGU in RAI uptake of differentiated thyroid cancer, we stably expressed PIGU and mock plasmid in the papillary cancer cell line (K1-PIGU and K1, respectively). Immunofluorescence analysis showed a significant increase in perinuclear expression of PIGU in the K1-PIGU cancer cells compared to controls. Similarly, flow cytometry analysis showed a significant increase in membranous expression of NIS in PIGU transfected cancer cells compared to controls. There were no significant differences in proliferation, invasion or motility between K1-PIGU and K1 cell lines. PIGU expression significantly increased I125 uptake compared to controls both in vitro (1.13 and 1030 ppm, respectively, p< .001) and in vivo (153 and 1.2x105 ppm, respectively, p< .001). Mitogen-activated protein kinase inhibition showed clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine. To address the mechanism underlying PIGU down regulation in RAI resistant tumors, we treated K1 and KI-PIGU cells with U0126 MEK inhibitor. This inhibition resulted in a significant increase in PIGU expression and I125 uptake in vitro. Taken together, our results revealed a pivotal role for PIGU in RAI sensitivity of thyroid cancer cells, by modulation of NIS expression.

#2880

The discovery of novel GSN alternative splicing in head and neck squamous cell carcinoma.

Daria A. Gaykalova,1 Dylan Kelley,1 Theresa Guo,1 Craig Bohrson,1 Ilse Tiscareno,1 Veronika Zizkova,1 Michael Considine,1 Ludmila Danilova,1 Emily Flam,1 Justin Bishop,1 Julie Ahn,1 Samantha Merritt,1 Marla Goldsmith,1 Chi Zhang,1 Wayne Koch,1 William Westra,1 Zubair Khan,1 Michael Ochs,2 Sarah Wheelan,1 Elana Fertig,1 Joseph Califano3. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Johns Hopkins University, Ewing, NJ;_ 3 _University of California, San Diego, San Diego, CA_.

Alternative splice events (ASES) are significant components of potential oncogenic pathways alterations and play a critical role in malignant cell transformation in a variety of solid and liquid tumors, including head and neck squamous cell carcinoma (HNSCC). However, high throughput analyses performed to date have not considered ASEs. Therefore, they have detected a limited number of genetic alterations for HNSCC, which incompletely describe the HNSCC specific pathway alterations. The heterogeneous nature of these alterations has made the discovery of reliable HNSCC biomarkers and therapeutic targets for this disease challenging.

We performed alternative splice events (ASEs) analysis to enhance our understanding of HNSCC biology. To define ASEs specific to HNSCC we designed a novel bioinformatics pipeline from RNA-sequencing data of HNSCC tumors and independent normal samples. Evaluating the top scoring candidates, we have found several highly promising ASE candidates, including GSN, Gelsolin, an actin-binding protein, a key regulator of actin filament assembly and disassembly.

Previously published literature proposes that GSN demonstrates tumor-suppressor properties by reducing cell proliferation in vivo and in vitro via suppression of protein kinase C (PKC, part of the PI3K pathway which is altered in HNSCC). The alternative splicing event involves an insertion of 110 bp from the 14th intron. This insertion contains a stop codon in frame, and the splice variant gives rise to a truncated (562 amino acids(aa)) protein with only 4 Gelsolin domains (instead of the full-length 731 aa protein with 6 Gelsolin domains). Using RNA-Seq data we demonstrated that 40% of tumor samples harbor the GSN-ASE. QRT-PCR confirmed that while total expression of GSN is decreased in HNSCC samples, GSN is expressed in the alternative truncated form only in HNSCC tumors and not normal tissues. Accordingly, total GSN expression is also seen to be downregulated in breast, lung and colon cancers.

Evaluation of TCGA data confirmed the pre-dominant expression of the truncated GSN isoform over the wt GSN in HNSCC, bladder urothelial carcinoma, colon adenocarcinoma, lung SCC, breast invasive carcinoma, cervical SCC and endocervical adenocarcinoma. Moreover, we confirmed that that the expression of the truncated GSN is important for the migration and invasion of the cancer cells in vitro. These data suggest that alternative splicing plays an important role in the GSN gene for multiple tumor types.

#2881

Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma.

Harish Potu,1 Luke F. Peterson Peterson,1 Malathi Kandarpa Kandarpa,1 Anupama Pal Pal,1 Hanshi Sun Sun,1 Paul W. Harms Harms,1 Peter C. Hollenhorst Hollenhorst,2 Ugur Eskiocak Eskiocak,3 Moshe Talpaz Talpaz,1 Nicholas J. Donato Donato1. 1 _University Of Michigan, Ann Arbor, MI;_ 2 _Indiana University Bloomington, Bloomington, IN;_ 3 _University of Texas Southwestern, Dallas, TX_.

Transcription factors are frequently deregulated in cancer cells and may be good therapeutic targets, but few successful targeting strategies have been reported. Deubiquitinases (DUBs) are specialized enzymes that regulate the ubiquitin (Ub) content on many proteins and DUB expression and activity are elevated in a number of cancers where they can act to alter tumor suppressor and/or oncoprotein levels. We previously described Usp9x activity and expression in melanoma; here we sought to investigate its role in primary melanoma and metastatic disease. Usp9x was upregulated in tumor cells compared to normal melanocytes and Usp9x expression and activity were found to be essential for 3D growth and melanoma tumor expansion in vivo. We defined the Usp9x ubiquitinated protein landscape and demonstrate that Usp9x regulates Ets-1, a cancer-promoting transcription factor. Usp9x binds, deubiquitinates and thereby stabilizes Ets-1 protein, and primary tissue and tumor analysis demonstrated elevated and coincident Usp9x/Ets-1 protein expression in melanoma compared to normal skin or benign nevi. Usp9x knockdown or Usp9x inhibition with small molecule G9 reduced Ets-1 protein levels and blocked tumor growth in vitro and in vivo. Conversely, Usp9x overexpression in melanoma cells increased Ets-1 protein levels and enhanced 3D tumor growth in vitro and in vivo, which were all reversible by treatment with G9. We conclude that Usp9x is essential for Ets-1 protein stability and may be therapeutically exploited with small molecule Usp9x inhibitors to reduce Ets-1-dependent gene expression and tumorigenicity.

#2882

HnRNP Q1, a novel translational regulator for Aurora-A protein synthesis in colorectal cancer.

Chien-Hsien Lai, Liang-Yi Hung. _Institute of Bioinformatics and Biosignal Transduction, National Cheng-Kung University, Tainan, Taiwan_.

HnRNP Q1 is a multifunctional RNA-binding protein and has an abundant expression level compared with hnRNP Q member. In colorectal cancer, hnRNP Q has been reported to associate with cell proliferation, but the underlying mechanism is still unclear. By RNA-immunoprecipitation assay following next-generation sequencing (NGS), a group of cell cycle-related genes was identified to be targeted by hnRNP Q1, including Aurora-A kinase. Aurora-A is a cell cycle dependent kinase involved in regulating centrosome duplication, maturation and spindle assembly during mitosis. Abnormally elevated Aurora-A will cause mitotic defects and aneuploidy, which might lead to genomic instability and tumorigenesis. Our previous work has demonstrated that Aurora-A can be translational up-regulated in colorectal cancer. In order to investigate the role of hnRNP Q1 in Aurora-A expression, GFP-hnNRP Q1 was overexpressed in colorectal cancer cells. We found that Aurora-A protein, but not the mRNA level, was up-regulated in those GFP-hnRNP Q1-overexpressing cells. By biotin pull down and Biacore analysis, we revealed that hnRNP Q1 binds to the 5'-UTRs of Aurora-A mRNA directly, and their interacting domain was mapped. Importantly, we found that hnRNP Q1 can enhance Aurora-A protein synthesis through IRES-mediated translational regulation in a cell cycle-regulated manner by ribosomal protein S6 immunoprecipitation, polysome fractionation and bicistronic reporter assay. CCK-8 assay, colony formation and in vivo xenograft assay all suggested that overexpression of hnRNP Q1 can promote cell proliferation and tumorigenesis of colorectal cancer cells. In addition, the expression level of hnRNP Q1 is positively correlated with Aurora-A in clinical colorectal cancer tissues. Taken together, our data indicate that hnRNP Q1 is a novel trans-acting factor that binds to Aurora-A 5'-UTRs and regulates its translation, which may increase cell proliferation and contribute to the tumorigenesis of colorectal cancer.

#2883

Impact of novel CDKN2A/p16INK4a 5'UTR variants predisposing to melanoma on p16 translational regulation.

Alessandra Bisio,1 Elisa Latorre,1 Virginia Andreotti,2 Brigitte Bressac-de Paillerets,3 Mark Harland,4 Odile Cabaret,3 Julia Newton-Bishop,4 Lorenza Pastorino,2 William Bruno,2 Roberto Bertorelli,5 Veronica De Sanctis,5 Chiara Menin,6 Gilberto Fronza,7 Paola Queirolo,8 Giovanna Bianchi Scarrà,2 Robert C. Spitale,9 Alessandro Provenzani,1 Alberto Inga,1 Paola Ghiorzo2. 1 _CiBIO (University of Trento), Povo, Italy;_ 2 _Laboratory of Genetics of Rare Hereditary Cancers, DiMI, University of Genoa, Genoa, Italy;_ 3 _Service de Génétique, Institut Gustave Roussy, Villejuif, France;_ 4 _Section of Epidemiology and Biostatistics, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom;_ 5 _NGS Core Facility, CiBIO (University of Trento), Povo, Italy;_ 6 _Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, Padua, Italy;_ 7 _Mutagenesis Unit, IRCCS AOU San Martino -IST, Genoa, Italy;_ 8 _Medical Oncology Unit, IRCCS AOU San Martino-IST, Genoa, Italy;_ 9 _Department of Pharmaceutical Sciences, University of California, Irvine, CA_.

CDKN2A/p16INK4a is an important tumor-suppressor gene whose dysregulation is associated with melanoma. We have recently demonstrated that the p16INK4a expression can be modulated by IRES-dependent mRNA translation and this regulation might have an important role during tumorigenesis. We also identified YBX1 as RNA binding protein targeting and stimulating p16 mRNA translation efficiency. The identified IRES function was particularly active in hypoxia and inhibition of mTOR. This post-transcriptional regulatory mechanism would potentially be a target for mutational inactivation during melanomagenesis. Indeed, we previously showed that p16INK4a 5'UTR variants found in melanoma patients with a family predisposition can have a negative effect on p16 translation. Single Nucleotide Variants (SNVs) have been investigated during routine testing of CDKN2A/p16INK4a in Italian, English and French melanoma patients followed by GenoMEL, the International Melanoma Genetics Consortium and a total of 17 germline variants were identified in a cohort of nearly 6000 patients. These p16INK4a 5'UTR 17 variants were studied with multiple approaches, that included mono- and bi-cistronic reporter assays, western blot of endogenous protein, and quantification of allelic representation after polysomal profiling to investigate their impact on p16INK4a mRNA translation regulation. We devised a classification score based on the concordance between functional assays and the extent of dysfunction displayed by each variant compared to the wild type. Variants are classified as neutral (score 0) when no difference was observed in at least 3 assays. This applied to: c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T. Variants were considered as potential mutations when a defect was measured in either one or two assays (score 1-2; c.-42T>A and c.-67G>C variants). Finally, we classified variants as causal mutations when three or more assays showed impairment (score >3). This applied to: c.-27del23, c.-56G>T, c.-93-91delAGG, as well as to c.-21C>T and c.-34G>T, which were already considered as a causal mutations. We have also determined the structure of the wild type p16INK4a 5'UTR by Selective Hydroxyl Acylation or SHAPE assay. The variant c.-42T>A encompassing the predicted YBX1 binding site was also studied and shown to induce a local change in conformation. The structure of all p16INK4a 5'UTR variants examined so far is being investigated as well as their impact on the interaction of RNA binding proteins such as YBX1, SRSF1 and RBM4. Our data indicate that the sequencing of the entire p16INK4a 5'UTR should be included in routine screening of melanoma families as nearly half of SNVs tested so far displayed a negative impact on the p16 mRNA translation efficiency.

#2884

Role of Kaiso in intestinal tumorigenesis.

Manish K. Tripathi, Albert B. Reynolds. _Vanderbilt University Medical Center, Nashville, TN_.

p120-catenin is a master regulator of classical cadherin stability and essential for epithelial homeostasis. p120 physically interacts with transcription factor Kaiso (ZBTB33) suggesting a direct or indirect role in transcription, but its precise function with respect to Kaiso is largely unknown. Kaiso belongs to the ZBTB family of transcription factors, most of which have important roles in development and/or cancer. Interestingly, Kaiso is strongly expressed in the intestinal crypt and abruptly downregulated as the cells move up onto the villus and terminally differentiate. Kaiso is constitutively upregulated in nascent adenomas initiated by loss of the tumor suppressor APC, suggesting a link between canonical Wnt signaling, Kaiso upregulation and intestinal tumorigenesis. Here, using ChipSeq and proteomics we describe several candidate Kaiso interactions with a group of proteins that include the tumor suppressor BRCA1, CHD2 (a chromatin remodifier) and the Ets1 oncoprotein, along with preliminary evidence for functional significance relevant to intestinal tumorigenesis.

#2885

Somatic mutation of STAT3 leads to the preferential Th17 differentiation in human naïve CD4-positive cells and favor TCR-mediated proliferation.

Ramona Crescenzo, Valentina Fragliasso, Marcello Gaudiano, Marco Pizzi, Giorgio Inghirami. _Weill Cornell Medicine, New York, NY_.

Intro: Mature Th17 lymphocytes play a key role in the host defenses against bacteria and fungi although unchecked Th17 activation can lead to autoimmune disorders, inflammation and cancer. The engagement of CD4+ naïve T-cells via IL6 and TGFβ favors a Th17 differentiation program, which requires the presence of IL21 and IL23 to reach a complete maturation and the maintenance of Th17 phenotype. The Th17 differentiation requires multiple lineage defining transcription factors (i.e. RORα, RORγt, and STAT3) and the activation of STAT3 signaling, which regulates a plethora of factors (i.e. RORγt, IL23R), is required for the full execution of the Th17 program. We have recently shown that JAK1/STAT3 activating mutations in Anaplastic Large cell Lymphomas (ALCL) are oncogenic, but it is unknown their protumorigenic contribution in modulating/derailing the host microenviroment and the host responses.

Methods: 50 FFPE ALCLs were investigated with pSTAT3, GATA3, RORγ and T-Bet antibodies using a dual color approach. CD4+ CD62L+ CD25- CD45RAhi CD4+ naïve cells of healthy donor were sorted and cultured (7 days) in KBM (IL23 (25ng/ml), IL6 (25ng/ml), IL21 (25ng/ml), IL1β (12,5ng/ml), TGFβ (5ng/ml) + 1% FBS and anti-CD3/CD28 beads (1:50). Forced expressions of WT or mutated Y640F (YF) STAT3 were achieved via lentiviral transduction. STAT3 qRTPCR, luciferase assay, ATPlite, and ELISA were used as readouts on samples collected at day 7 post-transduction.

Results: IHC stains showed that pSTAT3+ ALCL preferentially co-expressed nuclear RORγ (p<000.1). Conversely, GATA3+ ALK- ALCL samples were preferentially STAT3-/RORγ- (12/14). Forced expression of STAT3 led to a higher expression of IL23R, RORγt and IL17, compared to controls cells. No statistically differences were seen in cell proliferation or metabolic levels in these conditions. Naïve YF STAT3 CD4 cells, when cultured w/o IL2 (100U/ml) + IL6 + anti-CD3/CD28 beads, were able to efficiently proliferate up to 20 days overcome controls and WT STAT3 cells. Lastly, transfection of HEK293T cells with YF STAT3 in the presence of an IL17 promoter reporter cassette, led to a significant higher luciferase expression levels than control or WT STAT3 transfected cells.

Conclusions: Our data demonstrated that the ectopic expression of STAT3 leads to a robust Th17 differentiation of CD4 naïve T-cells and YF STAT3 sustains their growth overtime in the presence of TCR signaling. The preferential expression of RORγ in pSTAT3+ ALCL (ALK+ and mutated STAT3 ALK-ALCL) suggest that the deregulated activation of STAT3 can regulate the plasticity of the neoplastic cells favoring the recruitment of inflammatory host cells within the neoplastic lesions. Additional studies are underway to define the lymphoma-host interactions and the role of JAK TKI in controlling the lymphoma growth and in modulating the pro-tumorigenic inflammatory phenotype of Patient Derived Tumor Xenograft

#2886

KLF8 phosphorylation at Serine 48 by ERK2 is critical for its stability and function.

Satadru Lahiri, Heng Lu, Debarati Mukherjee, Lin Yu, Jihe Zhao. _University of Central Florida, Orlando, FL_.

Krüppel-like factor 8 (KLF8) is a member of Krüppel like family transcription factor which was found to be upregulated in different types of cancers, primarily breast and ovarian cancer. It transcriptionally activates a host of downstream effectors like CyclinD1, EGFR, MMP9, and EPSTI1 to promote cell proliferation, cancer cell invasion and migration. Additionally, KLF8 transcriptionally represses E-cadherin to induce epithelial to mesenchymal transition and oncogenic transformation of cell. As such, KLF8 plays a central role in cancer progression. Several types of post-translational modifications including sumoylation, acetylation, ubiquitylation and PARylation have been discovered that regulate KLF8's function KLF8 has been shown to consistently migrate as doublet on SDS-PAGE. However, the reason for this motility shift of KLF8 has remained mysterious. We performed a series of experiments to determine the mechanism(s) behind this motility shift of KLF8. Truncation mutation showed that deletion of the first 50 amino acid residues, but not other regions, caused disappearance of the top band of the doublet, suggesting that the N50 region is responsible for the motility shift. Phosphatase treatment resulted in the same change in wild-type KLF8 protein, indicating that phosphorylation in the N50 region is causal to the motility shift. Further deletion and point mutation in this region identified Serine 48 as a novel phosphorylation site for KLF8. Interestingly, these experiments also identified KLF8 N terminal 31-40 region to play an important role in regulating the S48 phosphorylation and the motility shift. Our study identified that phosphorylation at Serine 48 is critical in maintaining KLF8 stability. Finally, we demonstrated that abolishing KLF8 S48 phosphorylation inhibits KLF8 mediated cancer cell migration. Kinase inhibitors treatment showed that ERK2 is the kinase responsible for KLF8 Serine 48 phosphorylation. Furthermore we identified that phosphorylated KLF8 acts as a mask to protect unphosphorylated KLF8. Overall this study identifies a novel phosphorylation site of KLF8 and a hitherto unknown link between ERK2 and KLF8 that is critical for maintaining its overall stability and function.

#2887

Autonomous nucleolar mechanism of colon cancer cell survival through induction of perinuclear ribosomal protein translation.

Tattym E. Shaiken, Antone R. Opekun. _Baylor College Medicine, Houston, TX_.

Hypertrophic and irregularly shaped nucleoli are characteristic of gastroitinal cancers with poor prognosis. The role of nucleolar hypertrophy in cancer cell survival has not been studied well. By suppressing several cellular pathways we observed a common cellular response mechanism that involves acceleration of perinuclear ribosomal protein synthesis and multifocal nucleoli hypertrophy. Paradoxically, the induction of perinuclear ribosomal protein biosynthesis was observed by inhibition of protein translation, suppression of protein degradation and mitochondrial activity. Acceleration of perinuclear protein translation in combination of nuclear hypertrophy was detected not only in actively dividing cells but also in quiescent, cell cycle arrested cells, and when cytotoxic effects were manifested. Intensification of perinuclear ribosomal protein translation and nucleolar hypertrophy by Rotenone-mediated damage of mitochondria supported a century long Otto Warburg observation that most cancer cells predominantly produce energy by high rate of glycolysis. Cell fractionation permitted detection of a perinuclear wall that provides a structural basis for the perinuclear protein translation. We found that that rapid- and slow-growing colon cancer cells significantly differ by abundance of ribosomes in the perinuclear region. Therefore, the perinuclear set of protein translation is an important mechanism of cancer cell survival that rescues ribosome biogenesis and leads to the drug resistance through nuclear transformation.

#2888

Identification of an NKX3.1-G9a-UTY regulatory network that controls proper prostate differentiation.

Clementine Le Magnen, Aditya Dutta, Antonina Mitrofanova, Andrea Califano, Cory Abate-Shen. _Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY_.

Maintenance of cell differentiation is tightly coordinated by specific gene expression programs, which are often deregulated during tumorigenesis. While the homeodomain-containing transcription factor, NKX3.1, plays essential roles in both prostate differentiation and prostate cancer, the mechanisms governing these events are still unclear. Here, we aim to elucidate detailed mechanisms governing Nkx3.1-driven prostate epithelial differentiation and explain how deregulation of such can lead to prostate tumorigenesis.

Using an Nkx3.1 mutant mouse model, we find that loss-of-function of Nkx3.1 in the prostate results in the down-regulation of genes associated with prostate differentiation as well as the up-regulation of genes that are not normally expressed in prostate. Interestingly, these mice also develop pre-malignant lesions resembling prostatic intraepithelial neoplasia (PIN). Conversely, gain of function of Nkx3.1 in a fully differentiated non-prostate epithelium is sufficient to induce prostate differentiation in renal grafts in vivo. Using a DNA binding mutant, we show that these activities are mediated via NKX3.1 homeodomain in vitro and in vivo. Exploiting mass spectrometry to identify NKX3.1-interacting proteins, we find that NKX3.1 interacts with the histone methyltransferase G9a and that this interaction is required for induction of prostate differentiation in vitro and in vivo. Finally, we show that these NKX3.1-driven activities are mediated, at least in part, by regulation of target genes such as the male-specific factor UTY (KDM6c).

We propose that the NKX3.1-G9a-UTY network is essential for proper prostate differentiation whereas disruption of these activities predisposes to cancer.

#2889

Loss of 4E-BP1 function promotes EMT and metastasis via translational activation of snail.

Qing Ye, Weijia Cai, Qing-Bai She. _University of Kentucky, Lexington, KY_.

The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular mechanisms underlying the translational activation for cancer metastatic progression remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer snail followed by repression of E-cadherin expression and promotion of cell migration and invasion as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates snail expression and inhibits cell migratory/invasive capacities and metastasis. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the eIF4F translation initiation also profoundly attenuates snail translation/expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings reveal a novel role of 4E-BP1 in the regulation of EMT and metastasis through translational control of snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking snail-mediated metastatic potential of cancer.

#2890

ERCC5 variant rs2296147 T-allele creates a predicted TP53 binding site and up-regulates transcript abundance in normal bronchial epithelial cells, while rs17655 C-allele is linked to miRNA binding site variant and down regulates.

Xiaolu Zhang,1 Erin L. Crawford,1 Thomas M. Blomquist,1 Sadik A. Khuder,1 Jiyoun Yeo,1 Albert M. Levin,2 James C. Willey1. 1 _The University of Toledo, Toledo, OH;_ 2 _Henry Ford Health System, Detroit, MI_.

Background: Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair (NER) and dysregulation of ERCC5 is associated with increased lung cancer risk. This study was conducted to characterize cis-acting genetic variants responsible for inter-individual variation in ERCC5 transcript regulation in normal bronchial epithelial cells (NBEC).

Methods: We determined genotypes at putative ERCC5 cis-regulatory single nucleotide polymorphic sites (SNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. Using a recently developed targeted sequencing method, ERCC5 allele-specific transcript abundance was assessed in NBEC RNA from 55 individuals heterozygous for rs1047768 and 21 subjects heterozygous for rs17655. Syntenic relationships among alleles at rs751402, rs2296147 and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We assessed association of NBEC ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at putative ERCC5 promoter cis-regulatory SNPs rs751402 and rs2296147.

Results: Genotype analysis revealed higher inter-individual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655 (p<0.0001 and p=0.0005 respectively). By haplotype analysis, mean expression was higher at the rs1047768 alleles syntenic with rs2296147 T allele compared to rs2296147 C allele (p=0.0030). Sequence analysis predicts that T allele at SNP rs2296147 creates a TP53 binding site. Mean expression was higher at rs17655 G allele (p<0.0001) which is syntenic with G allele at a linked SNP rs873601 (r2=0.74). C allele at SNP rs873601 is predicted to create a miRNA binding site.

Conclusions: These data support the conclusion that T allele at SNP rs2296147 creates a TP53 binding site and up-regulates ERCC5 while C allele at SNP rs873601 creates a miRNA binding site and down-regulates ERCC5. Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs is likely associated with variation in NER function in NBEC and lung cancer risk.

#2891

High throughput single cell gene expression profiling by multiplex qPCR.

Steven T. Okino, Michelle Kong, Jason Ma, Joshua Fenrich, Luca Boveri, Yann Jouvenot, Yan Wang. _Bio-Rad Laboratories, Hercules, CA_.

Single cell gene expression analysis is a powerful technique that provides a unique and insightful perspective on biological pathways and processes. Here we present a robust workflow that enables fast and accurate analysis of up to 100 genes in isolated single cells. Our workflow is highly sensitive, by assessing RNA reference standards we find that a single RNA transcript is detected with about 80% efficiency. We used this workflow to study differentiation in cultured NTera2 cells (NT2), a human embryonic stem cell model system. We analyzed untreated NT2 cells, and NT2 cells treated with low and high doses of retinoic acid (RA) for 10 days to initiate differentiation to a neuronal lineage. The expression levels of 16 genes were quantified in 164 single cells by multiplex real-time qPCR with two technical replicates. The entire experiment, from cultured cells to results, can be completed in 2-3 days and requires four 384-well qPCR plates for gene expression quantification. We find that control cells and cells treated with a high dose of RA (10 uM) are relatively homogeneous in the expression levels of the targeted genes. However cells treated with a low dose of RA (0.25 uM) exhibit significant heterogeneity with respect to gene expression; about half of the cells are similar to the high-dose RA cells, the other cells exhibit a wide range of partial differentiation. Interestingly, we find that LEFTY2 expression is almost exclusive to the low dose RA cells and strongly correlates with partial differentiation. A time-course study analyzing cell populations reveals that LEFTY2 is only transiently expressed in differentiating NT2 cells with peak expression at 3 days of high dose RA treatment. These findings imply that, in NT2 cells, LEFTY2 is a potential biomarker of early differentiation. In summary, we present an accurate, sensitive and robust single cell analysis procedure that uses standard reagents and platforms. We envision that this workflow will enable researchers to investigate cell heterogeneity in biological pathways in a cost-effective way.

#2892

FOXD3 is a novel repressor of the expression of short isoform of DCLK1 (DCLK1-S) from IntronV(β)-promoter of hDCLK1-gene, and is epigenetically silenced in hCRCs: prognostic/diagnostic implications of FOXD3/DCLK1-S expression in hCRCs.

Malaney R. O'Connell,1 Shubhashish Sarkar,1 Gurinder Luthra,1 Yoshinaga Okugawa,2 Yuji Toiyama,3 Denise Ward,1 Ajay Goel,2 Pomila Singh1. 1 _University of Texas Medical Branch, Galveston, TX;_ 2 _Baylor University Medical Center, Dallas, TX;_ 3 _Mie University, Mie, Japan_.

DCLK1 is a specific marker of colon and pancreatic cancers in mice, and is elevated in human colon adenocarcinomas (hCRCs). Downregulation of DCLK1, results in loss of cancer stem-cell-markers and tumorogenic-potential of human colon cancer cells (hCCCs). We recently reported a novel finding that hCRCs express short-transcripts of DCLK1 (DCLK1-S) from an alternate promoter located within IntronV of DCLK1-gene, while normal human colons express the canonical long transcript (DCLK1-L) from 5'(α)-promoter. We and others have demonstrated that 5'(α)-promoter is hypermethylated in hCRCs, resulting in epigenetic silencing and loss of expression of DCLK1-L in hCRCs. Although 5'(α)-promoter is differentially methylated in normal human colons vs hCRCs, methylation status of IntronV(β)-promoter does not change. We therefore hypothesized that differential expression of DCLK1-S in normal colons vs hCRCs is perhaps due to differences in transcriptional activity of the promoter in normal vs cancer cells. To test our hypothesis, we used several in silico and molecular biology approaches, and report for the first time that FOXD3 is a potent transcriptional inhibitor of the IntronV(β)-promoter, resulting in the absence of DCLK1-S expression in normal human colons. Our results suggest that FOXD3 gene becomes methylated during colon carcinogenesis, causing loss of FOXD3 expression, and results in the expression of DCLK1-S in hCRCs. In order to examine pathophysiological relevance of the loss of FOXD3 and gain of DCLK1-S expression in hCRCs, we measured relative levels of FOXD3/DCLK1-S by qRT-PCR in a cohort of 92 CRC patients, in relation to overall survival and clinicopathological parameters. Patients expressing high-DCLK1-S/low-FOXD3 had significantly worse overall-survival compared to patients expressing low-DCLK1-S/high-FOXD3. High expression of DCLK1-S, in conjunction with low expression of FOXD3, was a stronger independent prognostic factor than expression of high levels of DCLK1-S alone. Conclusions. Based on our studies, we report for the first time that FOXD3 is a potent repressor of IntronV(β)-promoter of hDCLK1-gene in normal cells, and that loss of FOXD3 expression due to hypermethylation and silencing of FOXD3 gene during colon carcinogenesis, results in the expression of DCLK1-S in hCRCs, representing an important biomarker of hCRCs. Our findings also suggest a prognostic/diagnostic value of measuring relative expression levels of DCLK1-S/FOXD3 in tumors of CRC patients. It is speculated that loss of DCLK1-L and FOXD3 expression, associated with increased expression of DCLK1-S can be used as an early diagnostic marker of epigenetic changes, associated with colon carcinogenesis in humans.

#2893

PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo.

Pablo Iglesias, Marcos Seoane, Isabel Castro-Piedras, Victor M. Arce, Jose A. Costoya. _Molecular Oncology Laboratory MOL, Departamento de Fisioloxía, Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CIMUS), IDIS, Universidade de Santiago de Compostela, Santiago de Compostela, Spain_.

Although PARP-1 has been traditionally related to DNA repair, in the last years an increasing number of studies have linked this polymerase to other cellular processes such as metabolism and cell mitosis.

In this study we wanted to investigate the biological importance of the interaction between PARP-1 and the transcription factor E2F-1, and more specifically in scenarios where the activity, or hyperactivity, of E2F-1 is of critical importance such as embryonic development or oncogenesis. In this regard, we have found that the treatment either with enzymatic inhibitors of PARP-1 has an effect on the transcriptional activity of E2F-1 as well as the proliferative rate of treated cells. This effect is significantly increased with the treatment with gossypol, a specific inhibitor of PARP-1 protein-protein interactions occurring through the BRCT domain, as in the case of E2F-1. This effect is also observed in vivo since the severity of histological malformations in Rb-/- embryos is significantly reduced in Parp-1-/- Rb-/- embryos, which phenotype closely resembles that of E2f-1-/- Rb-/- mice. Finally, we also found that the deletion or inhibition of PARP-1 in an animal model of gliomagenesis helps the cell to block oncogenic stimuli derived from E2F-1 hyperactivity, by reactivating critical signalling pathways involved in oncogene-induced senescence.

In summary, the importance of the relationship between PARP-1 and E2F-1 in different biological contexts leads us to believe that the search for ways of altering or disrupting this interaction could be a novel strategy for the development of molecules of therapeutic potential.

#2894

Impact of honokiol on sialylation in breast cancer cells.

Padmamalini Thulasiraman, Galen Garriga. _University of South Alabama, Mobile, AL_.

Metastasis is a process in which malignant cells spread form the tumor of origin and colonize at distant organs. One of the diagnostic markers of metastatic progression is the overexpression of a transmembrane protein called Mucin 1 (MUC1). High levels of MUC1 have been found in tumor tissues, including breast cancer, and have been implicated in reduced survival rate. After translation of the protein, carbohydrate groups are attached to MUC1, a process called glycosylation. The resulting glycocalyx, which serves as a protective layer on epithelial surfaces, is involved in cell-cell interactions, signaling and metastasis. Glycosylation patterns differ on MUC1 in normal breast tissue in comparison with MUC1 in breast tumors. Not only do breast tumors have simpler and fewer carbohydrate chains than MUC1 from normal breast epithelial tissue, they also have increased levels of the carbohydrate derivative, sialic acid. Altered addition of sialic acid (sialylation) has been associated with cancer transformation and metastatic progression of the disease. The enzyme, α-2,3 sialyltransferase (ST3Gal I) which adds the α-2,3-linked sialic acids to MUC1 is increased in breast tumors and has been correlated with poor prognosis. Shortening of the carbohydrates units on MUC1 in breast tumors unmasks peptides that would otherwise be covered by the carbohydrates in normal MUC1, and current studies seek to identify antibodies that would directly target MUC1 on the cancer cells and in turn prevent cancer metastasis. Plant derived natural product honokiol is being studied for its anti-tumor activities. To assess the effect of honokiol on ST3Gal I, we treated breast cancer cell lines, MDA-MB-231 and MCF-7 cells with honokiol and analyzed its effect on ST3Gal I mRNA and protein expression using RT-PCR and Western Blotting, respectively. Honokiol suppressed ST3Gal I at the mRNA and protein level in both cancer cell lines. To investigate specifically whether honokiol reduces free sialic acid, we treated mammary carcinoma cells with honokiol and using the Sialic Acid Kit, we observed reduction in sialic acid in honokiol-treated mammary carcinoma cells. Future immunoprecipitation studies are underway to investigate specifically whether honokiol suppresses sialylation of MUC1. We propose that these studies will lead to the understanding on how sialylation of MUC1 alters the cellular signaling processes involved in the transformation of breast cancer and eventually to the spread of the disease.

#2895

Radiation-induced alternative splicing as detected in total and polysome-bound mRNA in glioblastoma stem-like cells.

Amy Wahba,1 Michael C. Ryan,2 Uma Shankavaram,1 Kevin Camphausen,1 Philip J. Tofilon1. 1 _NIH/NCI, Bethesda, MD;_ 2 _In Silico Solutions, Falls Church, VA_.

The vast majority of human genes undergo alternative splicing, which is dependent on tissue type, developmental stage, and environmental context. Given that alternative splicing regulates gene expression and is a major source of diversity within the proteome, we used RNAseq to determine the effects of ionizing radiation on the generation of alternative transcripts in the human glioblastoma stem-like cell line NSC11. For these studies, total cellular mRNA, reflecting the transcriptome, and polysome-bound mRNA, reflecting the translatome, were collected from control and irradiated (2Gy, 1h) NSC11 cells and subjected to RNAseq to identify all transcripts; SpliceSeq was then used to visualize and quantitate splice variations induced by radiation. We found that after irradiation 122 genes were differentially spliced in the transcriptome, which indicates that radiation induces alternative splicing, while 357 were differentially spliced in the translatome suggesting that radiation also increases the translation of specific alternative transcripts. However, 33 genes were in common between the transcriptome and translatome suggesting that radiation increases the level and translation of these alternative transcripts. IPA analyses of the radiation-induced alternative transcripts in the translatome identified DNA Double-Strand Break Repair by Homologous Recombination, Ceramide Degradation, and Sphingosine and Sphingosine-1-phospahate Metabolism as the top 3 canonical pathways enriched in this dataset. The top 3 molecular and cellular functions in this analysis were Cellular Assembly and Organization, DNA Replication, Recombination, and Repair, and Cell Cycle. The same analysis of alternative transcripts in the transcriptome did not show an enhancement of DNA damage and repair networks after radiation, suggesting that it is the polysome loading of these transcripts that is changed after irradiation. Further analyses of alternative transcripts in the translatome using EnrichR demonstrated that the top 3 pathways, analyzed using the Reactome 2015 curated pathway database, were Homologous Recombination Repair of Replication-Independent Double-Strand Breaks, Homologous Recombination Repair, and Double-Strand Break Repair. Finally, GeneCodis was used to analyze the biological processes enhanced in the radiation-induced translatome. The top 3 biological processes were Regulation of Transcription, DNA-Dependent, Negative Regulation of Transcription, DNA-Dependent, and Double-Strand Break Repair. These results indicate that radiation affects alternative splicing at both the level of mRNA abundance and translation and suggest that these processes may play a role in radiation-induced translational control of gene expression.

#2896

4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity.

Prashant Khade, Paraskevi Giannakakou. _Weill Medical College of Cornell University, New York, NY_.

Taxane based chemotherapy is commonly used to treat a wide variety of solid tumors including breast, prostate, lung and ovarian. Taxanes bind to and stabilize microtubules inhibiting their function in interphase and mitosis, and leading to cancer cell apoptosis. Although taxanes are widely used clinically and their target, tubulin, is ubiquitously expressed, patient response is heterogeneous and the molecular mechanism of clinical drug resistance remains elusive. In addition to their antimitotic effects, taxanes affect several signaling pathways whose perturbation by microtubule stabilization may determine cell sensitivity or resistance. Our recent work has identified one such pathway by showing that Taxol treatment inhibits the translation of HIF-1α mRNA by inducing its sequestration to P-bodies where it is silenced. However, the signaling cascade that is initiated in response to microtubule damage leading to translation suppression of HIF-1α is not understood. Using polysome analysis we showed that Taxol treatment inhibited translation at the initiation step of protein synthesis, evidenced by the increase in the 80S peak. Post-translational modifications of various translation initiation factors are implicated in the regulation of translation initiation. Hence, we analyzed phosphorylation states of several initiation factors, including eIF4E, eIF2α, 4E-BP1, 4E-BP2 and rpS6. We didn't observe any change in the phosphorylation state of these initiation factors except for 4E-BP1. We observed that Taxol treatment led to 4E-BP1 hyper-phosphorylation, at a unique previously unrecognized, site. Using isogenic cell lines with acquired mutations at the taxane binding site we conclusively showed that microtubule disruption was required for the hyper-phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 is known to activate cap-dependent translation. Instead, our data using polysome profiling revealed that Taxol-induced 4E-BP1 hyper-phosphorylation was associated with translation suppression. We confirmed these results by performing the cap-binding assay, which demonstrated that Taxol treatment had no effect on cap-dependent translation. Taken together these results suggest a novel biological function for 4E-BP1 hyper-phosporylation. Further, treatment with the mTOR inhibitor, Torin-1, had no effect on Taxol-induced 4E-BP1 hyper-phosphorylation, suggesting mTOR pathway independence. Interestingly, treatment with the CDK inhibitor Roscovitine, prevented both Taxol-induced 4E-BP1 hyper-phosphorylation and Taxol-induced cell death. Although we do not yet know which of the three CDKs exerts the inhibitory effect on taxanes, our data suggest that there is a functional link between 4E-BP1 hyper-phosphorylation and taxane activity. We are currently investigating the role of CDK1, CDK2 and CDK5 in this pathway to decipher the novel biological function of 4E-BP1 hyper-phosphorylation.

### Translational and Therapeutic Relevance of Perturbations of Gene Regulation in Malignancy

#2897

Recurrent transcriptome alterations across multiple cancer types.

Bogumil Kaczkowski,1 Yuji Tanaka,2 Hideya Kawaji,1 Albin Sandelin,3 Robin Andersson,3 Masayoshi Itoh,1 Timo Lassmann,4 Yoshihide Hayashizaki,5 Piero Carninci,1 Alistair R.R. Forrest,6 FANTOM5 Consortium. 1 _RIKEN Center for Life Science Technologies (CLST), Yokohama, Japan;_ 2 _RIKEN Advanced Center for Computing and Communication, Yokohama, Japan;_ 3 _Department of Biology, University of Copenhagen, Copenhagen, Denmark;_ 4 _Telethon Kids Institute, the University of Western Australia, Perth, Australia;_ 5 _RIKEN Preventive Medicine and Diagnosis Innovation Program, Wako, Japan;_ 6 _Harry Perkins Institute of Medical Research, the University of Western Australia, Nedlands, Australia_.

Genes that are frequently deregulated in cancer are clinically attractive as both potential pan-cancer diagnostic markers and therapeutic targets. Here we compared Cap Analysis of Gene Expression (CAGE) profiles from 225 cancer cell lines and 339 corresponding primary cell samples to identify transcripts that are recurrently deregulated in a broad range of cancer types. CAGE is a 5' sequence tag technology that globally determines transcription start sites (TSS) in the genome and their expression levels. This allowed us to assess novel aspects of the cancer transcriptome.

First, we identified (at promoter resolution) hundreds of protein coding and long non-coding transcripts that are commonly de-regulated in cancer cell lines. Next, we showed that promoters that overlap repetitive elements (especially SINE/Alu and LTR/ERV1 elements) are often upregulated in cancer. In particular, a specific repeat family, REP522 (largely palindromic, unclassified interspersed repeat of ~1.8Kb in size), was strongly enriched for the most up-regulated promoters. To our knowledge this is the first report implicating REP522 activation in cancer. Then, taking advantage of the fact that CAGE data can be used to estimate the activity of enhancers from balanced bidirectional transcription we identified 90 enhancer RNA producing regions that are recurrently activated in cancer cell lines. With ENCODE ChIA-PET data, we linked 16 of the cancer-activated enhancers to promoters of known cancer related genes.

Finally, to confirm that our results are relevant to clinical tumors we performed complementary analysis in RNA-seq data from 4,055 tumors and 563 normal tissues profiled by The Cancer Genome Atlas (TCGA) and we identified a core set of pan-cancer biomarkers (of both coding and non-coding transcripts) that are recurrently perturbed in both the FANTOM5 and TCGA datasets.

In summary, our extensive transcriptome analysis identified a comprehensive set of candidate biomarkers with pan-cancer potential, and extended the perspective of enhancers and repetitive elements that are recurrently activated during carcinogenesis.

#2898

Elucidating the role of BHLHE40/DEC1/SHARP2/STRA13 in prostate cancer.

Zsofia Kiss, Paramita Ghosh. _UC Davis, Davis, CA_.

Background: Basic-helix-loop-helix proteins (BHLH) are transcription factors that play important roles in critical developmental processes in many organisms by repressing the transcription of various target genes. Previous studies showed that overexpression of the BHLHE40 (also known as DEC1, STRA13 or SHARP2) transcriptional repressor results in growth suppression, cell cycle arrest, and cellular senescence indicating that BHLHE40 may have important tumor suppressor functions. Studies showed that the expression of BHLHE40 is indeed downregulated in some tumors, intriguingly however, it is also overexpressed in many such as lung cancer, breast cancer. Our overall goal is to elucidate the function of BHLHE40 in prostate cancer (CaP) and gain an understanding of how it is regulated.

Methods: Initial studies were based on qPCR analysis to compare the expression levels of BHLHE40 in different cell lines ranging from androgen-dependent LNCaP cells to the castration resistant C4, C4-2, C4-2b, R1, Rv1, PC3 and PC3 cells that stably express the androgen receptor (AR) (PC3-AR). Further analysis were done by treating LNCaP and C4-2 cells with 1 nM dihydrotestosterone (DHT) in media containing fetal bovine serum (FBS) , or charcoal stripped serum (CSS). Western blot analysis was done to check the level of BHLHE40 protein in these cell lines. Knock-down of AR, or the use of AR inhibitors Enzalutamide and/or Bicalutamide were used to further assess the regulation of BHLHE40 by the AR.

Results: qPCR comparison of BHLHE40 transcript levels in LNCaP versus C4, C4-2, R1, Rv1, PC3 cells showed that while LNCaP cells have high transcript levels of BHLHE40, the CRPC cells all had very low transcript levels of this transcription factor. When comparing LNCaP to PC3 and PC3-AR cells, it was interesting to note that PC3-AR cells showed significantly higher transcript levels of BHLHE40 than PC3 implying that BHLHE40 may be regulated by the AR pathway. Treatment with 1 nM DHT on these cells showed that BHLHE40 increases over time following DHT treatment. In contrast, inhibition of AR by Enzalutamide (2 μM) showed reduced BHLHE40 levels, although Bicalutamide (10 μM) did not significantly altered the expression level of BHLHE40 in LNCaP cells.

Conclusions: Our results indicate that BHLHE40 is regulated by the AR pathway, although whether this is direct or indirect is yet to be determined. This observation is significant, since it indicates that the AR may be utilizing this transcription factor to suppress the transcription of other genes.

Future studies will attempt to determine if BHLHE40 may have an androgen-responsive-element (ARE) in its promoter and via ChIP assay to identify possible binding sites for AR in conjunction with luciferase assay to assess androgen-receptor mediated regulation.

#2899

Selinexor inhibits NF-κB activity by sequestering IkB-a in the nucleus and blocking IkB-a degradation.

Trinayan Kashyap, Christian Argueta, Boris Klebanov, TJ Unger, Benjamin Link, Maxwell Werman, Margaret Lee, Sharon Shacham, Yosef Landesman. _Karyopharm Therapeutics, Newton, MA_.

Background: Selinexor is a small-molecule therapeutic that inhibits XPO1 mediated nuclear export, resulting in nuclear accumulation of tumor suppressor proteins (TSPs) and subsequently cancer cell death while sparing normal cells. It has been previously demonstrated that the inhibitor of NFκB, IκB-, is localized to the cytoplasm by XPO1 in several cancer cell lines and that treatment of cancer cells with selinexor reduces NF-κB transcriptional activity. The mechanism of NF-κB inhibition by selinexor, however, is not fully understood. We hypothesized that nuclear retention of IκB- and down-regulation of IκB- kinase (IKK) in response to selinexor treatment would inhibit NF-κB transcriptional activity.

Methods: U2OS (Osteosarcoma) cells were treated with selinexor in the presence or absence of TNF stimulation and whole protein lysates were analyzed by Western blotting. IκB- localization was evaluated by immunofluorescence microscopy and NF-κB transcriptional activity by ELISA (Thermo Scientific).

Results: TNFα induced the phosphorylation of NF-κB p65 subunit on serine 536 and IκB- on serine 32/36 through IKK. This resulted in the dissociation of IκB-α from NF-κB and led to IκB-α degradation via the 26S proteasome. Free NF-κB could now migrate into the nucleus and initiate transcriptional activation supporting tumorigenicity and inflammation. The IKK kinase is a complex made of two kinases (IKKα and IKKβ) and one regulatory subunit, NEMO/IKKγ. We found that selinexor treatment blocked IKK activity through the down regulation of the IKK (IKKγ) gamma subunit protein levels. This inhibition was dose dependent and prevented IκB- phosphorylation, thereby protecting IκB- from degradation. The protection of intact IκB- from degradation and its forced nuclear accumulation through XPO1 inhibition enabled inhibition of NF-κB transcriptional activity even in the presence of TNFα. Selinexor did not alter the protein levels of IKK or IKK.

Conclusions: Selinexor blocks TNFα induced degradation of IκB- by reducing the levels of IKK. In addition, selinexor increased nuclear IκB- levels through the inhibition of nuclear export. This blocked NF-κB activity and enhanced cancer cell death. We are currently investigating the beneficial effects of combining selinexor with proteasome inhibitors, which are known to prevent IκB- degradation.

#2900

Sumoylation of the retinoic acid receptor alpha protein represses transcriptional activity and contributes to retinoic acid resistance in glioma stem cells.

Virginia W. Rodriguez, Rolanda Bailey, Mark Gilbert. _NIH-NCI, Bethesda, MD_.

Introduction: Glioblastoma (GBM) is the most deadly form of brain cancer. Despite treatment with tumor resection followed by radiation and chemotherapy, the median survival rate is less than 15 months. Glioma stem-like cells (GSCs) are thought to be treatment resistant and drive tumor growth. Retinoids have been successfully used to terminally differentiate the cancer stem cell population in some leukemias, but show only modest success in GBM despite the presence of RARA in tumor cells. Resistance to retinoic acid (RA) can develop due to mutations in the retinoic acid receptor alpha (RARA) protein that prevent its phosphorylation. This study evaluated the retinoic acid receptors to determine if receptor dysfunction is the underlying cause of resistance in GBM. Methods: Several GSCs derived from both primary (GSC923) and recurrent (GSC827, GSC604) GBM tumors and normal mouse neural stem cells (MNSC) were analyzed for RARA protein expression by Western blots using an RARA specific antibody. 2D Westerns were used to measure RARA phosphorylation in response to RA treatment. MNSC were treated with RA in the presence or absence of MG132, a proteasomal inhibitor, to determine if MNSC RARA protein was sensitive to proteasomal degradation. RARA transcriptional activity was measured using a promoter luciferase construct containing a specific retinoic acid response element (RARE). Immunoprecipitations using either Sumo1, Sumo 2/3 or RARA identified the type of sumo peptide covalently attached to the endogenous GSC RARA protein. Results: MNSCs express the predicted 51 kDa RARA protein; however, GSC827 and GSC923 both express two high molecular weight forms that were approximately 62 and 73 kDa. In response to RA, the MNSC RARA became phosphorylated, but the GSC827 RARA remained hypophosphorylated. Treatment with RA induced the proteasomal degradation of MNSC RARA as expected. In contrast, both forms of the GSC RARA proteins were resistant to ligand-induced proteasomal degradation. In response to RA, the RARA protein in three glioma stem cell lines showed a 40-60% decrease (p=0.008) in transcriptional activity compared to MNSC. Immunoprecipitation showed that the Sumo1 peptide is covalently attached to the GSC RARA protein, occurring before the treatment with retinoic acid. Conclusions: We demonstrated that aberrant posttranslational modification of the RARA protein contributes to retinoic acid resistance in glioma stem cells. Covalent attachment of the Sumo1 peptide likely prevents RARA phosphorylation and ligand-induced proteasomal degradation. The Sumo1 peptide serves as a scaffold to bind nuclear corepressor proteins. Future exploration of the role of the Sumo1 modification may uncover underlying mechanisms of inherent retinoic acid resistance in GSCs potentially leading to successful differentiation therapy in GBM.

#2901

Nuclear β-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression.

Piera Tocci, Roberta Cianfrocca, Laura Rosanò, Valentina Caprara, Rosanna Sestito, Valeriana Di Castro, Anna Bagnato. _Regina Elena National Cancer Institute, Rome, Italy_.

In epithelial ovarian cancer (EOC), endothelin-1 (ET-1)/endothelin A receptor (ETΑR) axis has been reported to be involved in invasiveness, epithelial-to-mesenchymal transition, chemoresistance and metastatic dissemination and is thereby associated with poor prognosis. In these cells, ET-1 is able to induce the nuclear translocation and stability of hypoxia-inducible factor-1α (HIF-1α) the principal transcriptional factor that allows cellular adaptation to hypoxia. The importance of co-factors that trigger an epigenetic regulation of HIF-1α within tumor is not well understood. Here we elucidate that ET-1/ETAR-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signaling. In EOC cells, ET-1 induced vascular-endothelial growth factor (VEGF), the pro-agiogenic target downstream of HIF-1α through HIF-1α nuclear accumulation. Activation of ET-1/ETAR axis, by mimicking hypoxia, promotes the nuclear association between β-arr1 and HIF-1α on the hypoxia responsive elements (HRE) binding sites. By ChIP assay, nuclear β-arr1 was able to interact with HIF-1α, by promoting the recruitment of p300 acetyltransferase. The formation of this functional complex resulted in enhanced histone acetylation, and gene transcription of HIF-1α downstream targets, such as ET-1 and VEGF, required for cell invasion. The induced release of ET-1 and VEGF by EOC cells, promoted angiogenic effects in endothelial cells, as evaluated by cell migration and tube-like structure formation assays. Interestingly ETAR/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit required for invasiveness and angiogenic responses. These effects were abrogated by the silencing of β-arr1 or HIF-1α or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. In a murine orthotropic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation, suggesting that ET-1, together with VEGF, represents one of such factors that mediates tumor-endothelial cell communications, favoring a permissive environment for metastatic spread. Collectively these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ETAR pathway, mediating epigenetic modifications, to an extent comparable with hypoxia, directly involved in metastatic progression. Targeting ET-1 axis by macitentan may represent a new opportunity to target the orchestration mediated by nuclear β-arr1/HIF-1α complex in EOC, demonstrating that this approved small molecule interfering with stromal compartment, expressing ETBR, and tumor cells, expressing mainly ETAR, can be used in clinical setting for improved therapeutics in EOC.

#2902

Assays for androgen receptor activity using cell based ARE reporter systems.

Waqas Azeem,1 Margrete R. Hellem,1 Jan R. Olsen,1 Yaping Hua,1 Kristo Marvyin,1 Lisha Li,2 Yi Qu,1 Biaoyang Lin,2 Xisong Ke,1 Anne M. Oyan,1 Karl-Henning Kalland1. 1 _Department of Clinical Science, University of Bergen, Bergen, Norway;_ 2 _Zhejiang-California International NanoSystems Institute, Hangzhou, China_.

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer. Inhibition of its ligand by androgen deprivation therapy (ADT) is consequently the main medical treatment of invasive prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration resistant prostate cancer (CRPC). Prostate cancer cells escape from ADT using a variety of mechanisms. The AR and target genes have therefore become even more focused therapeutic targets in aggressive and disseminated prostate cancer. AR and its classical target genes, such as KLK3 (PSA) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, several androgen response elements (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence recording. The 241B promoter sequence reporter was selected among the constructed ARE reporters on the basis of higher activity in initial screenings. The AR positive and androgen responsive prostate cancer cell line, LNCaP, and the prostate transit amplifying epithelial cell line, EP156T-AR with exogenous AR, were transduced with the lentiviral 241B-mCherry fluorescence reporter. Flow cytometry, multi-well reader and fluorescence microscopy results corresponded when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, and with the anti-androgen, abiraterone. An SV40-promoter GFP reporter element was next cloned into the 241B-mCherry reporter. Constitutive expression of GFP facilitated normalization of ARE driven mCherry fluorescent signals. Furthermore, AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors and used in co-transfection and co-transduction experiments in AR negative cell types. The developed ARE reporter system will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR.

#2903

Nuclear localization of a secretion protein NAG-1/GDF15.

Kyung-Won Min, Gabriel Silva, Seung J. Baek. _Univ. of Tennessee, Knoxville, TN_.

Nonsteroidal anti-inflammatory drug (NSAID)-activated gene (NAG-1, also known as GDF15) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily, which exert their biological activity by binding to membrane receptors as a secretion protein. Abnormal regulation of TGF-β signaling can contribute to various diseases such as cancer. Although there is sequence similarity between NAG-1 and other TGF-β superfamily members, NAG-1 does not bind to the TGF-β receptors; therefore the biological consequence of NAG-1 remains to be elucidated. Here, we report that NAG-1 has two putative non-canonical nuclear localization signals (NLSs) that mediate NAG-1 nuclear localization. Nuclear import assay shows that NAG-1 enters the nucleus through nuclear pore complex (NPC) in the presence of ATP, indicating nuclear entry of NAG-1 is active transport. Furthermore, we found the nuclear export signal (NES) recognized by CRM1 at near N-terminal region of NAG-1, that is conserved across various mammalian species. Impeding NES via chemical or genetic approaches promotes nuclear accumulation of NAG-1. We also found that nuclear NAG-1 dampens TGF-β-mediated cell migration and invasion via the inhibition of DNA binding of Smads complex. Overall, our data suggest that nuclear NAG-1 affects TGF-β-induced metastasis activity in cancer cells.

Grant support: This work was supported by grant from the University of Tennessee Center of Excellence in Livestock Diseases and Human Health.

K Min, Current address: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA

#2904

The alternative NF-kB2/RELB pathway is activated by LTB/LTB receptor and NIK to promote cell migration and metastasis-related gene expression in HNSCC.

Rita Das, Tsu-Fan Cheng, Jamie Coupar, Anthony Saleh, Paul E. Clavijo, Xinping Yang, Zhong Chen, Carter VanWaes. _NIH/NIDCD, Bethesda, MD_.

Head and neck squamous cell carcinomas (HNSCC) are highly inflammatory and preferentially migrate and metastasize to lymph nodes. In this study, we find that LTβ/LTβR/NIK signaling mediates alternative RELB/NF-kB2 activation, which promotes activation of important cancer related genes and migration. We find that LTβ and LTβR are over expressed in subsets of HNSCC tissues and cell lines, and LTβ activates the alternative NF-κB pathway, enhancing nuclear translocation of RELB and NF-kB2/p52. Knockdown of LTβR decreased its target kinase NIK, and downstream NF-kB subunits RELB and NF-kB2/p52 protein expression. Knockdown of NIK protein decreased RELB and p52 protein expression, while LTβ treatment stabilized NIK, RELB and NF-kB2/p52 expression. Consistent with this, knockdown of LTBR and NIK functionally decreased NF-kB reporter gene activity, while treatment of LTB partially restored the NF-kB reporter activity. Notably, knockdown of NIK and RELB by siRNA inhibited cell migration in HNSCC. Since NIK is known as central regulator of the activation of the non-canonical pathway, we tested the effects of a NIK inhibitor 1,3[2H,4H]-Isoquinolinedione on NF-kB function and cell migration. NIK inhibitor decreased NIK protein in the cytoplasm, downstream nuclear expression of RELB and NF-kB2/p52 proteins by Western blot and RELB localization by immunofluorescence staining. We have found evidence for LTβ induction of migration/metastasis and cell death genes MET, BIRC3,and SERPINE1, and NIK knockdown inhibited cell migration and the inducible expression of MET, BIRC3, SERPINE 1 by LTB. Our data reveal the mechanisms how LTβ and NIK activation mediated alternative NF-kB pathway and contribute to migration and metastasis of HNSCC.

Supported by NIDCD intramural Project ZIA-DC-000016, 73 and 74.

#2905

Induction of hTERT and increased proliferative potential in conditionally reprogrammed normal bronchial epithelial cells.

Daniel J. Craig, Rose T. Zolondek, Xiaolu Zhang, Jiyoun Yeo, Erin L. Crawford, James C. Willey. _University of Toledo, Toledo, OH_.

Background. Based on increasing evidence from this laboratory and genome wide association studies (GWAS), single nucleotide polymorphisms (SNPs) responsible for inter-individual variation in normal bronchial epithelial cell (NBEC) cis-regulation of antioxidant, DNA repair, and cell cycle control genes are key determinants of lung cancer risk. Thus, there is a need for NBEC culture methods that enable extended population doublings without genetic alteration to enable experimental investigation of putative cis-regulatory SNPs in NBEC. Toward this goal, we assessed the effect of previously reported conditional reprogrammed culture (CRC) conditions on regulation of human telomerase reverse transcriptase (hTERT) transcript abundance and proliferative potential in NBEC. Methods. NBEC were obtained by bronchoscopic brush from eight individuals after obtaining informed consent to an IRB-approved protocol. NBEC were incubated in three different culture conditions: bronchial epithelial cell growth media (BEGM) only, co-cultured with irradiated mouse embryonic fibroblasts (IRR-MEF) + Rho kinase inhibitor (ROCKi) in BEGM, and conditioned BEGM + ROCKi. Media were changed every three days and cells were passaged and sub-cultured after ten days. Human telomerase reverse transcriptase (hTERT) was measured in three individuals after each passage in triplicate via qPCR. The proliferative capacity of all eight individuals was assessed using cell count and morphology at passage >3. Results. Co-culturing NBEC with IRR-MEF in BEGM supplemented with ROCKi produced a highly proliferative cell population while maintaining lineage commitment evident after removal of CRC conditions. Transcript abundance of hTERT was elevated 6.5-fold in NBEC in co-cultured conditions and 4.3-fold in NBEC in conditioned media compared to BEGM alone. Cell count in CRC conditions were up to 22-fold higher compared to BEGM alone. Cells were passaged and sub-cultured up to passage 4, followed by being frozen down in cell culture freezing media for further assessment. Conclusion. NBEC hTERT transcript abundance was up-regulated and cell population proliferative potential was extended in CRC conditions. It is likely that hTERT functions to protect the ends of linear chromosomes in dividing cells, enabling increased cell divisions while maintaining normal genome. These cell populations will be used in future studies to assess the effect of putative cis-regulatory single nucleotide polymorphisms (SNPs) on gene expression in NBEC.

#2906

Targeting FoxA1 in estrogen receptor-positive breast cancer: biological characterization of kinase regulators.

Simon J. Johnston, Kelly A. Holmes, Jason S. Carroll. _Cancer Research UK, Cambridge Institute, University of Cambridge, Cambridge, United Kingdom_.

Estrogen receptor (ER) is the driving and defining transcription factor in 75% of breast cancers and is directly inhibited by targeted therapies such as tamoxifen. DNA interactions and transcriptional potential of ER depend on the pioneer factor, FoxA1, which thereby plays an essential role in determining tumour growth and progression - even when resistance to existing drugs has developed. The aim of this study is to characterize kinase regulators of FoxA1 function in order to identify druggable targets for the indirect inhibition of ER.

To identify kinase regulators in ER+ breast cancer, primary screening of multiple cell lines was performed using an siRNA library of the human kinome. After secondary screening of ER+ specific growth-regulatory kinases using a stable luciferase reporter of FoxA1 activation, 50 putative kinase regulators of FoxA1 were identified. These were cross-referenced with kinase inhibitors of FoxA1 activation discovered via a separate cell-based compound screen and competitive-binding kinase displacement assay.

This two-pronged screening approach revealed 6 common putative FoxA1-regulatory kinase enzymes. These were characterized by chromatin immunoprecipitation (ChIP) of FoxA1 and ER with quantitative PCR of archetypal ER enhancers, 48 hours after siRNA knockdown of the kinase. This revealed 3 kinases whose inhibition prevented FoxA1 and ER from accessing the chromatin, and 1 kinase that had the opposite effect.

Mass spectrometry-based proteomic analysis of endogenous FoxA1-protein interactors using ChIP is underway to assess the change in FoxA1 co-factors following inhibition of these 4 candidate kinases. We used a proteomic purification method called RIME which permits analysis of phosphorylation events on FoxA1. We are currently assessing whether silencing of specific kinases influences phosphorylation of the putative substrate, FoxA1.

In summary, the identified kinases demonstrate promising FoxA1 specificity, and may provide druggable targets for the inhibition of ER via FoxA1. This study has potential clinical value for patients with tumours that have become resistant to existing ER-targeted therapies.

#2907

Eugenol potentiates the effect of cisplatin on cancer stem-like cells through targeting the NF-κB pathway.

Syed S. Islam, Al-Sharif Ibtehaj, Sultan Ahlam, Abdelilah Aboussekhra. _King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia_.

Triple-negative subtype of breast cancer (TNBC) contains exclusively tumorigenic and often endowed with self-renewing and resistance to cytotoxic chemotherapy, such as, paclitaxel resulting in relative increases in CSCs phenotype. Signaling through NF-kB may be essential for the CSCs self-renewal and could present potential target for novel treatment in breast cancer patients. We explored the possible involvement of NF-kB pathway that contribute to the functions and maintenance of breast CSCs and targeting this pathway by cisplatin combining with a natural product eugenol both in vitro and in vivo, using estrogen dependent (MCF-7) and independent (MDA-MB-231, MDA-MB-468 and BT-20) cell lines and xenografted both subcutaneous and orthotopic (fat pad) sites in nude mice. Chromatin immunoprecipitation (ChIP) assay was utilized to assess the NF-kB associated DNA binding capacity in promoter region. Surprisingly, neither cisplatin nor eugenol was capable of complete diminishing the CSCs populations. Cisplatin alone administration partially diminished apoptosis, mammospheres formation, aldehyde dehydrogenase 1 (ALDH1) activity, invasion and growth on nude mice. We observed cisplatin-induced activation of NF-kB associated with survival and regrowth of mamospheres. For effective elimination of CSCs phenotype by cisplatin, we cotreated cells with eugenol. Combination of eugenol and cisplatin significantly eradicated cisplatin induced NF-kB DNA binding capability assed by EMSA and ChIP assays, which was associated with abrogated apoptosis, mammospheres formation, ALDH1 activity and invasion. In vivo, combination therapy reduced the tumor size in synergistic manner. This was possibly due to induction of apoptosis, inhibition of proliferation, angiogenesis and downregulation of cisplatin-induced expression of proteins of NF-kB target genes. Although treatment of mammospheres by cisplatin partially induced the CSCs population through interleukin (IL)-6 pathway, however, only coadministration of eugenol and cisplatin reduced the numbers of CSCs to virtually undeletable levels in vitro and in vivo. Our data suggest that eugenol may be well suited to increase targeting of breast CSCs by cisplatin.

#2908

The clinical significance of Nrf2 overexpression in esophageal cancer.

Yuki Kitano, Yoshifumi Baba, Kensuke Yamamura, Hironobu Shigaki, Kousuke Mima, Junji Kurashige, Masaaki Iwatsuki, Yasuo Sakamoto, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. _Graduate School of Medical Science Kumamoto University, Kumamoto, Japan_.

Background: NF-E2-related factor 2 (Nrf2) is a key transcription regulator for antioxidant and detoxification enzyme and is overexpressed in various cancer cells. In this study, prognostic significance of Nrf2 expression in esophageal cancer was investigated. In addition, we examined the relationship between the expression of Nrf2 and cell proliferation using in vitro assay.

Methods: We immunohistochemically evaluated the protein expression of Nrf2 in the surgically resected specimens in 83 esophageal cancer patients. And ten esophageal cancer cell lines were utilized in vitro assay.

Results: We examined the protein expression of Nrf2 in 10 esophageal cancer cell lines. TE-11 cell had the highest expression of Nrf2, while KYSE30 had the lowest expression of Nrf2. Importantly, there was no correlation between the protein level and mRNA level of Nrf2 in these cell lines. The cell proliferation was inhibited in TE-11 cells, and not inhibited in KYSE30 cells by treatment with Nrf2-siRNA. Moreover, Flow Cytometry Assay revealed that knockdown of Nrf2 by siRNA arrested the cell cycle at G1 phase in TE-11 cells. In immunohistological study, Nrf2 was predominantly identified in the nucleus of esophageal cancer cells and the expression level of Nrf2 was higher in the tumor than normal esophageal epithelium (P<0.001). There was no relationship between Nrf2 protein expression and clinicopathological characteristics. The overall survival of the high-Nrf2 expression group was significantly poorer than that of low-Nrf2 expression group (P=0.004).

Conclusions: The expression of Nrf2 is up-regulated in esophageal cancer cell lines and resected tumor specimen. Our in vitro assay revealed that Nrf2 appeared to support cancer cell proliferation. Therefore, strategies to pharmacologically manipulate the level and / or activity of Nrf2 may have potential to reduce tumor growth and improve the prognosis of esophageal cancer patients.

#2909

Correlation between the expression of Sox2, Oct4 and deltaNp63 in clinical samples of resected esophageal cancers.

Yoshikuni Inokawa,1 Kenichi Inaoka,2 Fuminori Sonohara,1 Hisaharu Oya,1 Masamichi Hayashi,1 Yukiko Niwa,1 Naoki Iwata,1 Daisuke Kobayashi,1 Masahiko Koike,1 Yasuhiro Kodera,1 Shuji Nomoto2. 1 _Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 2 _Aichi Gakuin University School of Dentistry, Nagoya, Japan_.

Background. DeltaNp63 is the oncogenic isoform in several squamous cell carcinomas including esophageal cancer (EC), however, the mechanisms of regulation of the expression are still unclear. Sox2 and Oct4 are well known transcription factors which function in cancer stemness. We supposed that these two molecules work as transcription factors on deltaNp63, and verified the expression of these genes in cases of resected squamous cell EC.

Methods. In 78 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, messenger RNA (mRNA) expression of deltaNp63, Sox2 and Oct4 in EC tissues and corresponding normal tissues was analyzed by quantitative real-time reverse transcription-polymerase chain reaction assay. The mRNA expression was standardized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expressions were compared in terms of clinicopathological factors and prognosis of postoperative survivals.

Results. Peason correlation analysis showed significant correlations between expression of deltaNp63 and Sox2 in tumor tissues (r=0.67, P<0.001), and between deltaNp63 and Oct4 (r=0.71, P<0.001). The deltaNp63 expressions in tumor tissues were significantly higher than that in corresponding normal tissues (P<0.001), whereas Sox2 and Oct4 did not show significant differences between cancer and non-cancerous tissues. Univariate analysis identified poor differentiation, lymphatic involvement and higher ratio (Tumor/Normal) of expression of Oct4 as significant prognostic factors for poor overall survival, however, lymphatic involvement was the only independent prognostic factor in multivariate analysis (hazard ratio: 5.34; P<0.001).

Conclusions. Expression of deltaNp63 in EC tumor tissues showed positive correlation with expression of Sox2 and Oct4. Therefore, these two transcription factors may regulate the expression of deltaNp63. The deltaNp63 is not a strong prognostic factor, however, Oct4 might be a significant predictor for poor prognosis for overall survival in squamous cell EC.

#2910

Overcoming therapy resistance in SOX2-expressing head and neck squamous cell carcinoma cells by targeting the downstream BCL2-pathway.

Hui Wang,1 Angela Quiesser,2 Andreas Schröck,3 Sven Perner,4 Claudia Lengerke5. 1 _University Hospital Basel, Department of Biomedicine, Basel, Switzerland;_ 2 _University Hospital of Bonn, Department of Prostate Cancer Research and Institute of Pathology, Bonn, Germany;_ 3 _University Hospital of Bonn, Department of Otorhinolaryngology/Head and Neck Surgery, Bonn, Germany;_ 4 _Pathology of the University Hospital of Luebeck and the Leibniz Research Center Borstel, Luebeck and Borstel, Germany;_ 5 _University Hospital Basel, Department of Biomedicine and Clinic for Hematology, Basel, Switzerland_.

SOX2 (SRY (sex determining region Y)-box 2) is a transcriptional master regulator that confers "stemness" in early embryogenesis and acts as an oncogene in various cancers. Recently, SOX2 expression has been shown in several solid tumor types. However, the SOX2 expression pattern and the correlation with histopathologic status and clinical outcome are highly variable among tumors, suggesting distinct roles of SOX2 in individual tumors. In head and neck squamous cell carcinoma (HNSCC), we reported that expression of SOX2 is detectable and associates with high-risk disease in non-HPV human HNSCC. Functionally, SOX2 overexpression enhanced apoptosis resistance in response to treatments with cisplatin and other apoptosis-inducing agents, indicating SOX2 as a mediator of therapy resistance in human HNSCC. On the molecular level, induction of the anti-apoptotic protein BCL2 was observed following SOX2-induction. Here we explore if the anti-apoptotic effects of SOX2 are functionally mediated through up-regulation of BCL2 expression and explore BCL2 as a potential direct downstream target of SOX2.

We tested SOX2 and BCL2 expression in different human HNSCC cell-lines by qPCR and respectively immunoblot. To analyse the functional effect of SOX2, SOX2 overexpressing and control SCC25 cells generated via transduction with lentiviral particles were analysed in in vitro apoptosis assays after treatments with staurosporine, TRAIL or cisplatin. Furthermore, to analyse the influence of BCL2 in SOX2-overexpressing cells, treatment with the BCL2 inhibitor ABT-199 was used.

We could confirm the strong inductive effect of SOX2 overexpression on BCL2 gene and protein levels in the human SCC25 cell line. In line, cells treated with SOX2 overexpressing lentiviruses showed enhanced apoptotic resistance as compared to control-transduced cells. Notably, HNSCC cells were less sensitive to ABT-199 as previously reported for lymphatic leukemic cells (5-10 μM versus 0.1-1 μM for EC50). However, ABT-199 treatment in low dosage (1 μM) was indeed able to sensitize SOX2-overexpressing human HNSCC cells to apoptosis, indicating up-regulation of BCL2 as an important mechanism by which SOX2 confers treatment resistance in these cells. Moreover, analysis of the BCL2 promoter revealed a potential SOX2-binding site, and a direct association of SOX2 protein with this site could be confirmed by chromatin immunoprecipitation assays (CHIP). We are currently further exploring the effect of ABT-199 on the therapy resistance of SOX2-overexpressing cells using in vivo models.

Taken together, our findings indicate that, in human HNSCC cells, SOX2 plays an important role in drug resistance by elevating BCL2 expression. In patients with SOX2-expressing HNSCC, combinatorial treatment with ABT-199 may enhance response to conventional chemotherapies by re-sensitizing cells to apoptosis induction.

#2911

Gene knockout of NANOG and NANOGP8 mediated by CRISPR/Cas9 system decreases the malignant potential of prostate cancer cells.

Norihiko Kawamura,1 Keisuke Nimura,2 Atsunari Kawashima,1 Takeshi Ujike,1 Akira Nagahara,1 Kazutoshi Fujita,1 Motohide Uemura,1 Yasufumi Kaneda,2 Norio Nonomura1. 1 _Department of Urology, Osaka University Graduate School of Medicine, Suita, Japan;_ 2 _Division of Gene Therapy Science, Osaka University Graduate School of Medicine, Suita, Japan_.

Introduction & objectives :

NANOG is an essential transcription factor for self-renewal and pluripotency of embryonal stem cells. Recently, it has been reported that NANOG is expressed in various somatic cancers, including prostate cancer, and drive tumor development, and that increased NANOG expression in human prostate cancer tissues is correlated with an increased Gleason score. NANOG (hereinafter NANOG1) has many pseudogenes, and only the NANOGP8 pseudogene encodes the full-length NANOG1 protein with high sequence similarity. NANOGP8 is reported to be expressed in most types of cancer as a primary contributor of NANOG mRNA expression and to increase the malignant potential. However, the proportion of NANOG protein expression that comes from NANOG1 and NANOG8 in cancer cells is not known because of the high similarity between them. Therefore, a causal role of NANOG1 and NANOGP8 in prostate cancer cells is not clear.

Materials & methods :

We established NANOG1-/- and NANOGP8-/- prostate cancer cell lines from DU145 cells using CRISPR/Cas9, and also we established NANOG1-rescued cells and NANOGP8-rescued cells from each NANOG knockout cells for rescue experiments. We examined cancer properties associated with malignant potential in these cells and DU145 cells, including self-renewal, sphere-formation, migration, drug resistance and tumorigenic potential.

Results :

Colony formation assays were performed to examine the role of NANOG1 and NANOGP8 in self-renewal. The colony-forming capacity of NANOG1-/- and NANOGP8-/- cells was decreased compared to parental cells. Similary, the sphere-forming capacity of NANOG1-/- and NANOGP8-/- cells decreased to approximately 50% compared to the parental cells. Wound-healing assays were performed to examine the migration capacity, and migration was decreased in NANOG1-/- and NANOGP8-/- cells by 40-60%. MTS assays 48 hours after docetaxel administration were performed to evaluate the effect of NANOG1 and NANOGP8 on drug sensitivity. NANOG1-/- and NANOP8-/- cells showed increased sensitivity to docetaxel. NANOG1 and NANOGP8 knockout did not inhibit in vitro cell proliferation, but in vivo tumorigenic potential decreased significantly. These phenotypes were recovered in NANOG1- and NANOGP8-rescued cell lines.

Conclusion :

These results indicate that both NANOG1 and NANOGP8 proteins are expressed in prostate cancer cell lines, and both genes equally contribute to the high malignant potential of prostate cancer cells.

#2912

The AP-1 transcription factor JunB promotes multiple myeloma cell proliferation, survival and drug resistance in the bone marrow microenvironment.

Fengjuan Fan,1 Sonia Vallet,1 Muhammad Hasan Bashari,1 Mostafa Jarahian,1 Eugenio Morelli,2 Dirk Hose,1 Latifa Bakiri,3 Claudia Ball,1 Hanno Glimm,1 Martin Sattler,4 Hartmut Goldschmidt,1 Giovanni Tonon,5 Pierfrancesco Tassone,2 Erwin F. Wagner,3 Dirk Jäger,1 Klaus Podar1. 1 _National Center for Tumor Diseases (NCT) Heidelberg, Heidelberg, Germany;_ 2 _Magna Graecia University and T. Campanella Cancer Center, Catanzaro, Italy;_ 3 _Spanish National Cancer Research Centre, Madrid, Spain;_ 4 _Dana-Farber Cancer Institute, Boston, MA;_ 5 _San Raffaele Scientific Institute, Milan, Italy_.

Introduction: The activator protein-1 (AP-1) transcription factor has been implicated in a multitude of physiologic processes, but also tumorigenesis. In multiple myeloma (MM), the role of AP-1 is largely unknown.

Experimental procedures: MM cell lines and primary tumor cells were co-cultured with primary bone marrow stromal cells (BMSCs) or BMSC lines. AP-1 expression was measured by western blot analysis and real-time PCR. AP-1 activation was determined using TransAM AP-1 assay kit. To investigate the upstream regulators of JunB, cytokine array and specific inhibitors were used followed by 3H-thymidine incorporation, western blot and TransAM AP-1 assays. To delineate the specific functional role of JunB in MM pathogenesis, we used pLKO.1- JunB shRNA (shJunB) and pLKO.1- scrambled shRNA (SCR) vectors for constitutive knockdown, as well as pMSCV-JunB-ER-IRES-GFP and empty vectors for inducible overexpression, together with 3H-thymidine incorporation, alamarBlue, flow cytometry and western blot analysis, as well as gene expression profiling (GEP). To evaluate the functional role of JunB in vivo, a MM xenograft mouse model was used.

Results: Co-cultures of MM cells with BMSCs rapidly and strongly induced sustained expression and activation of JunB, but not of other AP-1 family members. Induction of JunB was predominantly mediated by soluble factors secreted by BMSCs rather than direct MM-BMSC contact. Indeed, IL-6 stimulation of MM.1S cells resulted in rapid and strong upregulation of JunB. Conversely, anti-IL-6 receptor antibody tocilizumab blocked BMSC-induced JunB expression and activation. Pharmacologic inhibition identified the requirement of the MEK/ERK and NF-κB pathways for BMSC-induced JunB expression. Functionally, significant inhibition of proliferation was observed in MM cells carrying pLKO.1- shJunB, but not pLKO.1-SCR. Importantly, knockdown of other AP-1 family members had minor effects on MM cell proliferation. Moreover, GEP performed on MM.1S- shJunB cells co-cultured with BMSCs as well as data analysis of a patient cohort using Gene Set Enrichment Analysis (GSEA) suggested a key role for JunB in the regulation of Mcl-1 and c-Myc expression. Furthermore, knockdown of JunB overcame resistance of MM cells to dexamethasone. Conversely, 4-OHT treatment of MM cell lines transduced with JunB-ER but not control vector induced significant JunB/AP-1 luciferase activity and protected MM cells against bortezomib-induced apoptosis and ER stress. Confirming our in vitro data, preliminary results show significant inhibition of tumor growth in a xenograft mouse model inoculated with inducible Tet-shJunB-GFP but not Tet-SCR-GFP MM.1S cells upon treatment with doxycycline.

Conclusion: Taken together, our data demonstrate for the first time an important and surprising role of JunB/AP-1 in MM tumorigenesis and strongly propose it as a novel therapeutic target in MM.

#2913

The SOX11 transcription factor is critical for basal-like breast cancer growth and migration and is associated with poor survival.

Jonathan Shepherd,1 Abhijit Mazumdar,1 Anna Tsimelzon,2 Susan G. Hilsenbeck,2 Powel H. Brown1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX_.

Introduction: Basal-like breast cancers (BLBCs) are aggressive breast cancers that are associated with poor survival and have distinctive mRNA expression profiles compared to non-BLBC tumors. Through an integrated screen of transcription factors (TFs) we identified SOX11 as uniquely critical for growth in BLBC, but not essential in estrogen-receptor (ER)-positive breast cancer cells. In this study we examine the role of SOX11 regulating growth and other BLBC phenotypes, including increased migration, invasion and poor survival.

Methods: SOX11 was identified through an integrated primary screen comparing mRNA expression of TFs, DNA sequences of differentially expressed BLBC genes, and differential binding of nuclear proteins to oligonucleotide sequences of transcription factor binding sites, and a secondary siRNA screen of growth in a panel of breast cancer cell lines. Further growth studies were performed using siRNA to SOX11 and measuring 2D-growth, or 3D-growth of cell lines grown in soft-agar. Immuno-fluorescent staining of phosphorylated histone H3 (pH3) was used to measure the effect of SOX11 depletion on proliferation. Migration and invasion assays were performed with membranes coated in Matrigel (invasion) or uncoated (migration). Survival analyses were performed with patients dichotomized by median SOX11 expression level, and log rank statistical comparisons as well as a multivariable Cox Proportional Hazards model to investigate the prognostic value of SOX11.

Results: SOX11 depletion resulted in reduced growth of ER-negative breast cancer cell lines, including ER-negative/ Her2-positive cells; whereas, growth of ER-positive breast cancer cells or ER-negative, non-transformed breast cells was not affected. In MDA-MB-468 cells, SOX11 depletion resulted in a 60% reduction of pH3-positive cells. Additionally, depletion of SOX11 in MDA-MB-231 or MDA-MB-468 cells reduced migration and invasion 40-60%, while over-expression of SOX11 in MCF7 cells increased migration nearly 12-fold. High SOX11 is correlated with worse prognosis, whether analyzed for all patients or subsets of patients divided by ER or HER2 status. In the multivariable analysis accounting for tumor size, grade, lymph node status, and PAM50 class, high SOX11 expression was associated with increased risk of breast cancer related death with a hazard ratio of 1.42 and p-value < 0.001.

Conclusions: SOX11 is a critical regulator of multiple BLBC phenotypes, including growth, migration, invasion, and expression of signature BLBC genes. Growth reduction of BLBC cells following SOX11 depletion is a result of decreased proliferation. High SOX11 expression was also found to be an independent prognostic indicator of poor survival in women with breast cancer. These results identify SOX11 as a potential target for the treatment of BLBC, the most aggressive form of breast cancer.

#2914

Biphenylene and bipyridine connected benzofuran compounds as novel regulators of kRAS transcription.

Harshul Batra,1 Mohammad K. Islam,2 David E. Thurston,2 Khondaker M. Rahman,2 Tracy A. Brooks1. 1 _The University of Mississippi, Oxford, MS;_ 2 _King's College London, London, United Kingdom_.

Pancreatic cancer is the fourth most deadly cancer with 5 year survival rate of ~6%. One of the major attributes of pancreatic cancer is kRAS mutations. kRAS is a proto-oncogene with intrinsic GTPase activity, and is responsible for cell proliferation, division, and apoptosis. kRAS mutations are observed in >95% of pancreatic adenocarcinoma and in 30 % all human tumors. When mutated it leads to continuous activity and uncontrolled proliferation which results in increased tumorigenicity and poor prognosis. Downregulating kRAS expression has shown to halt proliferation and leads to cellular death in pancreatic cancer models, but to date no small molecule capable of such transcriptional silencing has been described. A novel series of molecules with either biphenylene or bipyridine spacer connecting the terminal benzofuran ring was synthesized using solution phase chemistry. Tertiary amine side chains were incorporated to the C2-position of the benzofuran ring to improve the DNA binding affinity of these compounds. The synthesized compounds were purified using a "catch and release" method employing the sulfonic acid based resins. The eleven novel compounds were screened in parallel for their ability to stabilize G-quadruplex structures in the kRAS promoter, and to downregulate kRAS promoter activity. Two of the compounds increased the thermal profile of non-canonical DNA formations in the promoter region by >5 °C; however, on the whole these compounds did not seem to function as G-quadruplex-stabilizing agents. Eight compounds significantly decreased luciferase expression under the explicit control of the kRAS promoter by up to 75%, by compound BF 4.3, through an as-yet unknown mechanism of action. The compound series was examined for the inhibition of cellular viability in two mutant kRAS pancreatic cancer cell lines - Panc-1 and MIA PaCa-2; in both cell lines the compounds containing the bipyridine spacer had greater cytotoxic effects with IC50's of ~30 μM, consistently. qPCR is being used to confirm the decrease in promoter activity in native cellular conditions. These agents are intriguing in both their novel scaffold and their unexpected activity decreasing kRAS expression in a mechanism unrelated to higher order DNA structures. Ultimately, down regulation of kRAS transcription will provide a novel therapeutic approach for pancreatic cancer and improve the prognosis of this highly lethal disease.

#2915

Penfluridol suppresses glioblastoma tumor growth by inhibiting sonic hedgehog signaling.

Alok Ranjan, Sanjay K. Srivastava. _Texas Tech Uni. Health Sciences Center, Amarillo, TX_.

Glioblastoma (GBM) is the most common brain tumor with poor survival rate. The main obstacle in the treatment of glioblastoma patients is the presence of blood brain barrier, which restricts the movement of the drugs to reach the brain. Penfluridol (PF) is known to cross blood brain barrier and is a clinically approved drug for schizophrenia patients. It is demonstrated and established by us that penfluridol suppresses the growth of metastasized breast cancer cells in brain giving us the rationale to evaluate it against glioblastoma (Alok Ranjan, Parul Gupta and Sanjay Srivastava, Cancer Research 2015). Penfluridol significantly reduced the viability of U87-MG, T98G and U251 MG glioblastoma cells with an IC50 of 6µM, 5.5µM and 9µM respectively after 24 h of treatment and induced apoptosis as exhibited by FITC/Annexin assay and cleavage of caspase 3 as well as PARP. It has been shown that GLI1, a transcription factor belonging to sonic hedgehog signaling is overexpressed in GBM and responsible for tumor progression. Our results demonstrated that penfluridol treatment reduced the expression of GLI1 in U87MG, T98G and U251 MG cells in concentration-dependent manner. In addition, overexpression of MGMT, a DNA repair enzyme has been linked to temozolomide (TMZ) resistance in GBM therapy. Combination of PF and TMZ induced more apoptosis and reduced the expression of GLI1 and MGMT as compared to PF or TMZ treatment alone. Our results further demonstrated that oral administration of PF inhibited the growth of GBM tumors by 66% and 72% in subcutaneous and intracranial GBM tumor model respectively. Immunohistochemical analysis of tumor tissue and western blot analysis of tumor lysate indicated down regulation of GLI1, MGMT and increase in cleavage of caspase 3, confirming in vitro finding. Apoptosis induction effect of PF treatment in tumor tissues was further confirmed by Tunel staining. Taken together, these results indicate that overall GBM tumor growth suppression by penfluridol was associated with inhibition of Gli1 and induction of apoptosis

#2916

TAF15 as an important mediator of RNA polymerase II-dependent drug tolerance.

Kohei Kume, Kohei Ito, Takeshi Iwaya, Satoshi S. Nishizuka. _Iwate Medical Univ., Morioka, Japan_.

Background: Cancer relapse after chemotherapy is thought to originate from drug-tolerant cancer cell subpopulations. We studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Previous DTC formation-based compound screening identified an RNA polymerase (RNAP) II inhibitor, α-amanitin (α-AMA), suggesting the existence of an RNAPII-dependent drug tolerance in cancer cells. However, the mechanism underlying this regulation is unclear.

Materials and Methods: DTCs derived from the gastric cancer cell line MKN45 were used as an in vitro model for gastrointestinal tumor recurrence. For the generation of the DTCs and those of untreated colonies (i.e., control colonies), cells were sparsely disseminated (100 cells/cm2) into the presence or absence of cisplatin at 50% colony inhibitory concentration. To identify the DTC formation responsible genes, a DNA microarray analysis was performed followed by gene ontology analysis and quantitative RT-PCR (qRT-PCR) validation. For gene knockdown experiments with those that have been identified as DTC-formation responsible genes, cells were treated with small interference RNAs (siRNAs) for 48 hrs and then sparsely disseminated into the microplates in the presence of cisplatin for the DTC formation.

Results: In terms of the RNAPII-dependence of the DTC formation, we focused on RNAPII-binding protein-coding genes in the list of the top 2.5% of specifically induced genes in DTCs. The transcriptional screening identified only one gene, TATA-binding protein-associated factor 15 (TAF15). The level of TAF15 mRNA was 2.1-fold higher in DTCs than in the control colonies. A protein family consisting of fusion in sarcoma (FUS), Ewing's sarcoma (EWS), and TAF15 is called the FET (FUS/EWS/TAF15) family, and they play various roles in transcription as well as RNA processing. Among the FET family proteins, the TAF15 protein level was specifically increased in DTCs, suggesting that TAF15-specific function may contribute to DTC formation. Next, we examined whether TAF15 protein depletion by siRNA affects DTC formation. The TAF15 depletion preferentially inhibited DTC formation despite the fact that the degree of inhibition to the control colony formation was limited. With an anti-TAF15 siRNA treatment, the original small, round-shaped MKN45 cells exhibited spindle-like morphological changes within 48 hrs. Interestingly, α-AMA treatment also induced similar morphological changes, suggesting that RNAPII inhibition caused TAF15 depletion. Consistent with the morphological changes, both TAF15 mRNA and protein levels decreased in response to α-AMA treatment.

Conclusion: These results suggest that TAF15 is an important mediator of RNAPII-dependent drug tolerance and one of the potential molecular therapeutic targets of α-AMA in the context of cancer relapse after chemotherapy.

#2917

Therapeutic targeting of the mitochondria: An evaluation of the transcriptional link between the antioxidant response and autophagy.

Kaytee L. Pokrzywinski, Francesca Mascia, V. Ashutosh Rao. _US Food and Drug Administration, Silver Spring, MD_.

Mitoquinone (MitoQ), a mitochondrially targeted chemotherapy induces autophagy via excess reactive oxygen species (ROS) production. Elevations in ROS stimulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in breast cancer cell lines. Nrf2 is a nuclear transcription factor that activates the antioxidant response element when under oxidative stress. The aim of this study was to evaluate transcriptional and protein changes associated with Nrf2 activation to identify potential links between oxidative stress, autophagy and cell death. By suppressing Nrf2 and its targeted antioxidant enzymes we should induce elevated ROS levels, which can thereby alter cellular homeostasis and impact the ultimate fate of cancer cells. In the breast cancer cell line MDA-MB-231 RNA was extracted after 24 hours of Nrf2 or non-target pool (NTP) silencing and 18 hours of MitoQ treatment. RNA was sequenced using an Illumina HiSeq and differential expression was analyzed using DESeq in the statistical program R. To avoid bias, raw read counts were filtered to remove low counts possibly due to constraints of the instrumentation. To validate efficient silencing of Nrf2 we confirmed that Nrf2 transcript levels decreased and that there were observed reductions in transcripts containing the Nrf2 transcriptional promoter, the antioxidant response element (ARE) including FTH1, FTL, HMOX1, SRXN1, TXNRD1, NQO1, GCLC, GCLM, GSR and SQSTM1. From there we validated RNAseq data with qPCR using 25 random transcripts and found significant positive correlation between the two methods. Comparisons were first made between the MitoQ and DMSO treatment by silencing (NTP or Nrf2). 2775 genes were differentially expressed (DE) for MitoQ vs DMSO NTP siRNA and 1869 genes were DE for MitoQ vs DMSO Nrf2 siRNA both at an α < 0.05 and Log2 fold change cut off > |1|. We then compared the degree of overlap in DE gene lists between the NTP and Nrf2 siRNA comparisons to identify genes affected only by Nrf2 silencing in combination with MitoQ treatment. 1405 mRNAs were common between silenced and non-silenced sample sets, only 464 mRNAs were due to Nrf2 silencing alone. We are currently performing pathway analysis on these transcripts to determine the role of Nrf2 silencing with redox-active therapies. We are also evaluating autophagic protein levels at later time points, 24 to 48 hours, to further evaluate the potential signaling between oxidative stress and the autophagic response driven by Nrf2.

#2918

Interaction between MUC1 and STAT1 drives IFITM1 overexpression in aromatase inhibitor-resistant breast cancer cells.

Asona Lui, Joan Lewis-Wambi. _University of Kansas Medical Center, Kansas City, KS_.

Aromatase inhibitors (AIs) successfully treat many estrogen receptor expressing breast cancers by depleting circulating estrogen levels. Unfortunately, many patients eventually develop resistance to this treatment. In order to elucidate the mechanism(s) of AI-resistance, our lab has developed an AI-resistant breast cancer cell line MCF-7:5C which was clonally derived from parental MCF-7 cells following long-term estrogen deprivation. Unlike MCF-7 cells, the AI-resistant MCF-7:5C cell line grows robustly in the absence of estrogen and is highly aggressive. We have previously reported that interferon induced transmembrane protein 1 (IFITM1) is overexpressed in MCF-7:5C cells and in AI-resistant breast tumors. Additionally, we have shown that loss of IFITM1 leads to cell death. What remains to be addressed is how IFITM1 expression is regulated in AI-resistant breast cancer cells. IFITM1 is a type 1 interferon (IFN) stimulated gene which is not expressed in normal tissue and is only induced by the type1 IFNs (IFNα/β). In previous studies we have found that MCF-7:5C cells produce elevated levels of IFNα, which binds to the type 1 IFN receptor, IFNAR, and induces JAK/STAT signaling, resulting in the overexpression of IFITM1. In this study, we further characterized the mechanisms of IFITM1 overexpression in AI-resistant MCF-7:5C cells. We have found that mucin 1 (MUC1) associates with STAT1 and stabilizes its phosphorylated form, thereby enhancing IFITM1 expression. This interaction is disrupted by treatment with the JAK inhibitor, Ruxolitinib. MUC1 is a transmembrane O-glycosylated protein that is overexpressed in 90% of breast cancers and known to promote the survival of the epithelium through formation of mucous, interaction with transcription factors and inhibition of apoptosis. A luciferase reporter determined that MUC1 contributes directly to IFITIM1 transcription. MUC1 expression is hormonally controlled and is normally enhanced by estrogen stimulation. In this study we found that MUC1 expression is dysregulated in AI-resistant MCF-7:5C cells and is instead reduced by estrogen stimulation. In contrast with the AI-sensitive MCF-7 cells, western blot and immunofluorescent staining showed that MUC1 expression was enhanced by treatment with the pure anti-estrogen, fulvestrant. Additionally, Annexin V/PI staining and PARP cleavage indicated that loss of MUC1 expression induced cell death in MCF-7:5C cells. We found that treatment with fulvestrant protects MCF-7:5C cells from this phenomenon, indicating that loss of MUC1 induces apoptosis in an ERα dependent manner. MCF-7:5C cells are sensitive to apoptosis following exposure to estrogen, which can be enhanced by loss of IFITM1 expression. We report that loss of MUC1 expression similarly enhances estrogen-induced apoptosis, suggesting that the communication between MUC1 and STAT1 also influences estrogen signaling in AI-resistant breast cancer.

#2919

Ultrasound-mediated neuropilin-1 shRNA minicircle delivery inhibits tumour growth in an orthotopic human pancreatic adenocarcinoma model.

Pratiek N. Matkar,1 Krishna K. Singh,1 Gerald J. Prud'homme,1 David W. Hedley,2 Howard Leong-Poi1. 1 _St. Michael's Hospital, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation in several pathological disorders, including cancer via endothelial-to-mesenchymal transition (EndMT), thereby implicating EndMT as a potential therapeutic target. Previous studies have demonstrated angiogenic co-receptor, neuropilin-1 (NRP-1) as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the therapeutic benefits of targeting NRP-1 in PDAC remain unexplored. We therefore hypothesized that targeting NRP-1 in PDAC may inhibit TGFβ1-mediated EndMT, tumour perfusion and tumour growth.

Methods: Orthotopic tumours were generated in RNU athymic rats from BxPC-3 human pancreatic cancer cells using the implantation technique. Animals were delivered at day 28 post surgery with either empty minicircle-DNA (150 μg) or shNRP-1 minicircle-DNA (200 μg) using ultrasound targeted microbubble destruction (UTMD). For UTMD, ultrasound was transmitted using a phased array transducer (S3, Sonos 5500) at 1.3 MHz and a transmit power of 0.9 W (120 V, 9 mA), and at a pulsing interval of 5 s. Contrast enhanced ultrasound perfusion imaging was performed on day 28 (gene delivery) and day 56 (end-point) with standardized analyses. Tumour tissues and remote organs were collected for evaluation of tumour volume and other downstream analyses like real-time polymerase chain reaction (RT-PCR), immunoblotting, immunohistochemistry and Masson's trichrome staining (for fibrosis).

Results: RT-PCR and immunoblotting demonstrated successful silencing of NRP-1 in shNRP-1 minicircle group. Immunohistochemistry demonstrated a typical ductal morphology and substantial collagen content within the tumours. Histological assessments further revealed reduced NRP-1 expression and reduced fibrosis in shNRP-1 minicircle group. Significant changes were observed in EndMT markers at transcript level and protein level in shNRP-1 minicircle delivered animals compared to empty minicircle. At day 56, profound reductions were observed in tumour volumes in shNRP-1 minicircle delivered animals. Tumour area and perfusion were found to be greater and more complete throughout the tumour in empty minicircle group, while reduced area and perfusion was observed in shNRP-1 minicircle group. At day 56, tumour microvascular blood flow and blood volume were markedly reduced in the shNRP-1 minicircle group.

Conclusions: Overall, these in vivo results suggest that loss of NRP-1 leads to reduced fibrosis and tumour growth in PDAC possibly through limiting EndMT and NRP-1 mediated angiogenesis, making NRP-1 as a potential therapeutic target against PDAC.

#2921

The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells.

Mandeep K. Takhar,1 Mark P. Labrecque,1 Kevin J. Tam,2 Anne Haegert,2 Robert H. Bell,2 Manuel Altamirano-Dimas,2 Colin C. Collins,2 Gratien G. Prefontaine,1 Michael E. Cox,2 Kevin L. Bennewith,3 Timothy V. Beischlag1. 1 _Simon Fraser University, Burnaby, British Columbia, Canada;_ 2 _The Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 3 _The BC Cancer Agency, Vancouver, British Columbia, Canada_.

The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies.

#2922

Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells.

Mark Labrecque,1 Mandeep Takhar,1 Rebecca J. Nason,1 Stephanie Santacruz,2 Kevin Tam,3 Shabnam Massah,1 Anne Haegert,3 Robert Bell,3 Manuel Altamirano-Dimas,3 Colin Collins,3 Frank Lee,1 Gratien Prefontaine,1 Michael Cox,3 Timothy Beischlag1. 1 _Simon Fraser University, Burnaby, British Columbia, Canada;_ 2 _University if British Columbia, Vancouver, British Columbia, Canada;_ 3 _The Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it's presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.

#2923

Preclinical validation of Omomyc cell-penetrating peptides as a viable in vivo anti-Myc therapy.

LAURA SOUCEK. _VHIO, BARCELONA, Spain_.

Deregulated Myc is associated with most human cancers suggesting that its inhibition would be a useful therapeutic strategy. Indeed, we have shown that Myc inhibition displays extraordinary therapeutic benefit in various transgenic mouse models of cancer (i.e. skin, lung, pancreatic cancer and glioma), without eliciting resistance to therapy, and causes only mild, well-tolerated and reversible side effects in normal tissues. For these studies we employed a dominant negative inhibitor of Myc, called Omomyc, which is an effective inhibitor of Myc transactivation function both in vitro and in vivo. Omomyc has so far been utilized exclusively as a transgene and served as a successful proof of principle. Here we present our current results based on the direct use of Omomyc itself as a peptide displaying unexpected and efficient cell-penetrating activity (OmomycCPP). When tested in vitro, OmomycCPP causes growth arrest and/or death of several cancer cell lines. In vivo, upon intranasal administration, it rapidly biodistributes to the lung and brain, as well as to other tissues of the animals, and - like its transgenic counterpart before - has a notable therapeutic impact on KRasG12D-driven Non-small Cell Lung Cancer (NSCLSC).

In summary, our results show that OmomycCPP represents a novel and viable pharmacological strategy to inhibit Myc in vivo, a strategy that has the potential to be translated rapidly to the clinic.

#2924

Metastasis-associated protein 1 induces tamoxifen resistance in MCF7 breast cancer cells.

Min-Ho Lee, Dahae Koh, Mi-Ock Lee. _Seoul National Univ. College of Pharmacy, Seoul, Republic of Korea_.

Tamoxifen is most commonly used to treat estrogen receptor-alpha positive breast cancer, but its therapeutic benefit is limited by the development of drug resistance. Metastasis associated protein 1 (MTA1) is a cancer progression-related gene and has been reported to be overexpressed in a variety of human cancers including breast cancer. Here, we showed that transient overexpression of MTA1 in MCF7 breast cancer cells results in tamoxifen resistance, whereas si-RNA mediated knockdown of MTA1 was more susceptible to tamoxifen-induced growth inhibition. Moreover, stable overexpression of MTA1 in MCF7 cells promoted cell proliferation and reduced tamoxifen sensitivity. To explore the mechanisms of MTA1-mediate tamoxifen resistance, we used microarray analysis to profile the gene expression patterns of MTA1 overexpress MCF7 stable cells after tamoxifen treatment. From microarray data analysis, 538 genes were selected as associated with MTA1-mediated tamoxifen resistance (P < 0.05, LPE test; cutoff ≥ 1.5-fold). The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis revealed that the predominant biological processes associated with these genes are M phase, negative regulation of cell proliferation, mitosis and M phase of mitotic cell cycle. Further, we found that cell-cycle regulator Cyclin D1 and cyclin B1 protein levels were increased in MTA1 overexpression MCF7 stable cells. Analyses of publicly available patient data sets indicated that MTA1 levels are higher in patients after endocrine therapy failure (recurrence), compared to those of responders (non-recurrence). These results suggest that MTA1 may contributed tamoxifen resistance through the regulation of cell cycle components.

#2925

Anti-tumor activity of a MYC-targeting dicer substrate siRNA in combination with BRD4/CDK7 inhibitors.

Edmond Chipumuro, Zakir Siddiquee, Shanthi Ganesh, Serena Shui, Anee Shah, Boyoung Kim, Dongyu Chen, Purva Pandya, Rachel Storr, Weimin Wang, Hank Dudek, Cheng Lai, Marc Abrams, Bob Brown. _Dicerna Pharmaceuticals, Cambridge, MA_.

MYC is a well-characterized driver of numerous tumor types. Since the protein encoded by this gene is challenging to target via conventional modalities, progress in new therapeutic agents has been slow despite decades of research. RNA interference technology has enabled the inhibition of previously-undruggable genetic targets at the mRNA level, and has advanced to clinical development for several indications. DCR-MYC, a lipid nanoparticle (LNP)-formulated Dicer substrate siRNA (DsiRNA) targeting MYC mRNA, is currently in Phase Ib/II clinical trials and showing promising results. In this study we used an improved EnCore LNP and MYC DsiRNA, which demonstrated MYC mRNA silencing activity and efficacy in mouse models of human hepatocellular carcinoma (HCC). Small molecule inhibitors that target BRD4; JQ1 and CDK7; THZ1 has previously reported anti-proliferative effects in various cancer types and efficacy in several tumor mouse models including HCC. Treatment with both JQ1 and THZ1 induced cell cycle arrest and apoptosis in various cancer cells by repressing MYC expression. Here we observed striking anti-proliferative effects in vitro when MYC-DsiRNA was combined with THZ1 in cancer cells. In addition, when mice bearing HCC tumors were treated with MYC-DsiRNA combined with either THZ1 or JQ1, the antitumor efficacy was additive or synergistic relative to either single agent alone. We observed significantly more MYC mRNA knockdown in the tumors that had the combination treatment compared to the tumors that received either of the single agent treatment. These preclinical data suggests the possibility of a significant and practical benefit of combining MYC-DsiRNA and small molecule inhibitors that target an epigenetic regulator BRD4 (JQ1) and a global gene regulator CDK7 (THZ1).

#2926

The regulatory mechanism of human LY6K related to carcinogenesis and metastasis in breast cancer.

Hyun Kyung Kong, Da Som Son, Jong Hoon Park. _Sookmyung Women's University, Seoul, Republic of Korea_.

The human lymphocyte antigen 6 complex locus K (LY6K) belongs to the lympocyte antigen 6/urokinase-type plasminogen activator receptor (Ly-6/uPAR) superfamily which are associated with development of the immune system and carcinogenesis. LY6K is a cancer biomarker and a therapeutic target that induces invasion and metastasis by activating the Raf-1/MEK/ERK signaling pathway and that stimulates cytotoxic T lymphocytes. However, the molecular mechanisms including genetic and epigenetic that determine human LY6K transcription are complete unknown. To elucidate the genetic mechanisms involved in human LY6K gene regulation and expression, multiple cis elements were predicted using TRANSFAC software, and the LY6K regulatory region was identified using the lucifearse assay in the human LY6K gene promoter. We performed chromatin immunoprecipitation, electrophoretic mobility shift analysis, and supershift assays to investigate the transcription factor activity on the LY6K promoter, and the effect of a single nucleotide polymorphism (SNP) and CpG site methylation on AP-1 transcription factor binding affinity. AP-1 and the CREB transcription factor bound to LY6K promoter within -550/-1, which was essential for LY6K expression but only the AP-1 heterodimer, JunD and Fra-1, modulates LY6K gene transcriptional level. A decrease in LY6K was associated with the SNP242 C allele, a polymorphic G/C-SNP at the 242 nucleotide in the LY6K promoter region (rs2585175), or methylation of the CpG site, which was closely located with the AP-1 site by interfering with binding of the AP-1 transcription factor to the LY6K promoter. In summary, AP-1 activation is important role in promoting LY6K gene expression that regulates cell mobility of breast cancer cells, whereas the SNP242 C allele or methylation of the CpG site may reduce the risk of invasion or metastasis by interfering AP-1 activation. And DNA methylation and histone modification in the 5'-flanking region of the LY6K gene plays an important role in LY6K expression in both breast cancer cells and breast cancer. An understanding of genetic and epigenetic changes in LY6K may contribute to the diagnosis of carcinogenic risk and to an enhanced prediction of outcome in patients with breast cancer. 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Mechanisms of Drug Resistance 3

#2927

miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cell.

Mads H. Rasmussen,1 Iben Lyskjær,1 Rosa R. Jersie-Christensen,2 Line S. Tarpgaard,3 Bjarke Primdal-Bengtson,1 Morten M. Nielsen,1 Jakob S. Pedersen,1 Tine P. Hansen,2 Flemming Hansen,1 Jesper V. Olsen,2 Per Pfeiffer,3 Torben F. Ørntoft,1 Claus L. Andersen1. 1 _Aarhus University Hospital, Aarhus, Denmark;_ 2 _University of Copenhagen, Copenhagen, Denmark;_ 3 _Odense University Hospital, Odense, Denmark_.

Oxaliplatin (oxPt) resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Here, we have used in vitro models to show that miR-625-3p functionally induces oxPt resistance in CRC cells, and have identified signalling networks affected by mir-625-3p. The p38 MAPK activator MAP2K6 was shown to be a direct target of miR-625-3p, and, accordingly, was downregulated in patients not responding to oxPt therapy. miR-625-3p resistance could be reversed in CRC cells by anti-miR-625-3p treatment and by ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, by reducing p38 MAPK signalling using either siRNA technology, chemical inhibitors to p38 or by ectopic expression of dominant negative MAP2K6 protein we induced resistance to oxPt. Transcriptome, proteome and phosphoproteome profiles revealed inactivation of MAP2K6-p38 signalling as a possible driving force behind oxPt resistance.

Our study shows that miR-625-3p induces oxPt resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

#2928

Accumulation of tumor cells with retained heterozygous BRCA1 status during platinum therapy: A probable mechanism of attenuation of tumor response in BRCA1-driven ovarian cancer.

Ekaterina Sh. Kuligina,1 Tatyana V. Gorodnova,1 Anna P. Sokolenko,1 Aleksandr O. Ivantsov,1 Aglaya G. Iyevleva,2 Evgeny N. Imyanitov1. 1 _N. N. Petrov Research Inst. of Oncology, St. Petersburg, Russian Federation;_ 2 _St. Petersburg Pediatric Medical University, St. Petersburg, Russian Federation_.

Approximately 15-25% ovarian cancer (OC) patients carry germ-line mutation in BRCA1 or BRCA2 genes. BRCA deficiency underlies the pronounced sensitivity of tumor cells to platinum-containing cytotoxic therapy. However, significant variations in platinum sensitivity are observed even within BRCA1/2 mutation-driven carcinomas. It has been shown, that chemoresistant tumor tissues could be characterized by the restoration of the BRCA1 function via additional somatic genetic events. This investigation aimed to analyze the role of BRCA status within the group of patients receiving preoperative platinum-based therapy. We addressed the question, whether intratumoral LOH status of BRCA1 gene may be altered during neoadjuvant therapy.

At first, we tested for Slavic founder BRCA mutations 225 ovarian cancer patients, who started their treatment with a few cycles of platinum-based therapy, and underwent debulking surgery afterwards. 34 BRCA1 and 1 BRCA2 mutation carriers were identified. Complete clinical response was documented in 12/35 (34%) mutation carriers and 8/190 (4%) non-carriers (P = 0.000002). Histopathologic response was observed in 16/35 (46%) women with the germ-line mutation versus 42/169 (25%) patients with the wild-type genotype (P = 0.02). Surprisingly, somatic loss of heterozygosity (LOH) for the remaining wild-type BRCA1 allele was detected only in 7/24 (29%) post-neoadjuvant therapy residual tumor tissues as compared to 9/11 (82%) BRCA1-associated OC, which were not exposed to systemic treatment before the surgery (P = 0.009). We hypothesize that the low frequency of LOH is related to the selection of tumor clones with somatically preserved BRCA1 gene during the therapy. We obtained for the LOH-analysis paired biological specimens from 8 patients; the pairs included cytological slides prepared at diagnosis and corresponding surgical material removed after neoadjuvant therapy. The restoration of BRCA1 heterozygosity was revealed in 4 out of 6 post-treatment tumor samples presenting with LOH at diagnosis. Another two ovarian carcinomas did not demonstrate somatic LOH before the treatment, and their status did not change during the neoadjuvant therapy. Thus, even though chemonaive OC specimens demonstrate somatic loss of the wild-type BRCA1 allele, the intratumoral BRCA1 heterozygosity could be restored in the platinum-exposed tissue, probably via expansion of LOH-negative clones.

Conclusion: The obtained data confirm high sensitivity of BRCA-driven ovarian cancer to platinating agents and provide evidence for a rapid selection of tumor cell clones without LOH during the platinum-based therapy.

#2930

The hostile microenvironment developed in 3D culture conditions induces Trastuzumab resistance involving cancer stem cells.

Cristina E. Rodríguez, Gabriel L. Fiszman, Elisa D. Bal de Kier joffe. _Inst. of Oncology A. H. Roffo, University of Buenos Aires, Ciudad de Buenos Aires, Argentina_.

HER2 is overexpressed in 20% invasive breast tumors and correlates with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used to treat HER2+ tumors; however more than half of the patients are resistant or acquire resistance during treatment. Multicellular tumor spheroids are a 3D cell growth model that mimics the structure of in vivo avascular tumors, with heterogenic cell subpopulations developed due to differential oxygen and nutrient supply through the spheroid. We have previously demonstrated that HER2+ cells cultured as spheroids are more resistant to Tz than monolayers. We also observed that in spheroids Tz inhibited basal apoptosis and was capable of inducing autophagy, leading to Tz resistance. In addition, cancer stem cells (CSC), widely associated with chemotherapy resistance, were specifically targeted by Tz.

The aim of this study was to analyze the resistance acquired in 3D and the impact of the CSC developed in these conditions.

Since Tz-treated BT474 (HER2+) spheroids overexpressed the autophagy marker LC3, correlating with resistance to the treatment, we first evaluated these cells viability after autophagy inhibition with 3MA. We found that these resistant cells became susceptible to Tz after autophagy inhibition, decreasing viability by 33% (p<0.05). In an attempt to recreate the hostile microenvironment developed in 3D, we treated BT474 cells with CoCl2 to induce a pseudo hypoxia state, similar to that found on the inner core of spheroids. In these conditions, cells were resistant to Tz treatment and interestingly, when we inhibited autophagy, we also re-sensitize these cells to Tz, being indistinguishable from controls.

To further study this 3D-induced resistance, we analyzed the CSCs subpopulation in spheroids, studying the CD44+CD24low cell phenotype by flow cytometry. We found that 15 days Tz treatment increased CD44+CD24low subpopulation by 1.5 fold compared to controls (p<0.05).

Then we analyzed HER2 expression in the spheroids and found 2 populations, HER2high and HER2low (37% vs 63%, respectively p<0.05). Interestingly, after 15 days Tz treatment, HER2high subpopulation increased to 50%.

Thus, we decided to study the 3D architecture in MCF7 cells, with no HER2 amplification and unresponsive to Tz inhibition. We analyzed HER2 by flow cytometry, observing HER2low expression in 82% of the cells that was reduced to 61% in Tz treated spheroids (p<0.05). Despite unresponsiveness of MCF7 cells to Tz as bulk spheroid, we detected a 30% reduction in the CD44+CD24low subpopulation after treatment.

In conclusion, the hostile microenvironment in 3D has a key role in the development of resistance to Tz and could be overcome by autophagy inhibition. These conditions could favor an increase of CSCs unresponsive to Tz despite HER2 expression. Moreover, MCF7 results suggest that Tz is able to target CSCs developed in 3D.

#2931

Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation.

Nayoung Kim,1 Ahye Cho,2 Hideo Watanabe,3 Yoon-La Choi,2 Meraj Aziz,4 Michelle Kassner,4 Je-Gun Joung,5 Angela KJ Park,1 Joshua Francis,6 Joon Seol Bae,5 Soo-min Ahn,7 Kyoung-Mee Kim,7 Joon-Oh Park,8 Woong-Yang Park,5 Myung-Ju Ahn,8 Keunchil Park,8 Hongwei Holly Yin,4 Jeonghee Cho9. 1 _Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea;_ 2 _SungKyunKwan University, Seoul, Republic of Korea;_ 3 _Department of Medicine, Division of Pulmonary, Critical Care and Sleep Medicine, New York, NY;_ 4 _Cancer and Cell Biology Division, Translational Genomics Research Institute, Scottsdale, AZ;_ 5 _Samsung Genome Institute, Seoul, Republic of Korea;_ 6 _Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA;_ 7 _Department of Pathology, Samsung Medical Center, Seoul, Republic of Korea;_ 8 _Samsung Medical Center, Seoul, Republic of Korea;_ 9 _DanKook University, Cheonan, Republic of Korea_.

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic alterations responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the resistant clones and RNAi-based systematic synthetic lethal screening revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, we detected increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs refractory patients by immunohistochemistry. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs in a subset of lung cancer patients and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

#2932

Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer.

Katsuhiro Kita,1 Giuseppe Galletti,1 Kyle Cleveland,1 Chao Zhang,1 Isabel Barasoain,2 J. Fernando Díaz,2 Doron Betel,1 Manish A. Shah,1 Paraskevi Giannakakou1. 1 _Weill Medical College of Cornell University, New York, NY;_ 2 _Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superíor de Investigaciones Científicas CIB-CSIC, Madrid, Spain_.

Successful clinical application of taxanes (microtubule-stabilizers) is limited due to intrinsic or acquired drug resistance. Thus, it is critical to unveil the molecular mechanisms of taxane resistance to significantly improve clinical outcomes. Retrospective analysis of the TAX-325 gastric cancer (GC) trial revealed that the addition of docetaxel (DTX) to cisplatin/fluorouracil increased progression-free plus overall survival, in intestinal (INT) but not in diffuse (DIF) GC subtypes. Our preclinical data confirmed that DIF GC cell lines are intrinsically resistant to taxanes. The incidence of DTX resistance in DIF GC cell lines was 2.5 times higher than INT GC cell lines. Drug efflux, tubulin posttranslational modification and differential β-tubulin isotype expression were ruled out as potential mechanisms of intrinsic taxane resistance. Thus, a new molecular mechanism must underlie the intrinsic taxane resistance in DIF GC. To quantify the kinetics of taxol binding to cellular MTs, we treated both DIF and INT groups of GC lines with the fluorescein-conjugated paclitaxel analog, Flutax-2. The Flutax-2 staining intensity of cellular MTs was assessed by live-cell confocal microscopy at different time points. Following a 3h incubation, there was less than 20% decrease in Flutax-2 intensity in the sensitive cell lines, compared to 59~89% decrease in the resistant cell lines. These data suggested different binding kinetics between sensitive and resistant cells. We added 1μM Flutax-2 to sensitive/resistant cells' native cytoskeletons for 0~60 seconds to determine the association rate (kon) of Flutax-2 binding to MTs. The kon of Flutax-2 in the sensitive cell lines was significantly higher in sensitive (5.8×104M-1s-1) versus resistant cells (0.3~2.6×104M-1s-1). Next, we measured the dissociation rates (koff) by competing the pre-bound Flutax-2 with DTX (0~600 seconds). Although 20~40% faster koff was observed in resistant cells, it appeared that the association rate of Flutax-2 was the dominant factor of differential taxane binding to MTs in GC cells. The binding mode of taxanes to MTs involves 1) binding to MT pores and 2) internalization to the high-affinity binding site at the MT lumen. To differentiate between the two, we used hexaflutax, which binds exclusively to the MT pores. We observed hexaflutax decorating radial MT arrays in sensitive but not in the resistant cells. As tubulin mutations around the high-affinity taxane binding site or tubulin posttranslational modifications/differential expression of β-tubulin isotype is not the case in DIF GC cell lines, our data suggest that modifications of the MT pore conformation or occlusion of the pore site is responsible for the intrinsic taxane resistance in DIF GC. Determining the origin of the defect at the pores will help design better MT-stabilizing drugs to overcome chemo-resistance, the major obstacle hindering overall survival of patients.

#2933

Long-term BRAF(V600E) inhibition results in a spontaneous KRAS(G12D) mutation and increased epithelial to mesenchymal transition (EMT) in papillary thyroid cancer cells (PTC).

Brian P. Danysh, Maria E. Cabanillas, Marie-Claude Hofmann. _University of Texas MD Anderson Cancer Center, Missouri City, TX_.

Objective:

Activating mutations in BRAF(V600E) are commonly observed in PTC and result in a functional dependence upon the constitutively active MAPK pathway. While targeted inhibitors are initially effective, inevitably cells develop alternative mechanisms of pathway activation. Mechanisms of primary resistance have been described in thyroid cancer cell lines, however acquired resistance has not. Our study investigates adaptive mechanisms of BRAF(V600E) inhibitor resistance and accompanying metastatic phenotypes in PTC cells following long-term vemurafenib exposure.

Materials & Methods:

KTC1 sub-cell lines (PTC cells, BRAFV600E) were developed following treatment with either 0.25 or 1.0 μM of vemurafenib, a BRAF V600E inhibitor, for >20 passages. Western blot and qRT-PCR were used to assess EMT marker expression and pathway activation. 2D and 3D invasion assays, immunofluorescence microscopy, and direct cell count growth assays were used to assess inhibitor resistance and metastatic phenotypes. Sequenom was used to detect acquired mutations.

Results:

A vemurafenib resistant (VR) subline of KTC1 cells was derived following long-term treatment with the drug. Resistance coincided with spontaneous acquisition of a KRAS(G12D) activating mutation. Our data show KRAS(G12D) driven vemurafenib-resistance significantly enhanced expression of distinct EMT markers and translated into increased growth, migration, and invasion, when compared to the control sub-lines. This gain in metastatic phenotype coincided with increase activation of both the AKT and ERK pathways.

Conclusion:

Our results suggest an acquired KRAS mutation confers BRAF V600E inhibitor resistance and a more aggressive metastatic phenotype in vitro. Our hope is that further study of the mechanisms of resistance will add to potential markers for early detection of BRAF inhibitor resistance, leading to improved patient outcomes.

#2934

GSK3β modulates chemoresistance in epithelial ovarian cancer.

Naeha Pathak, Noelle L. Cutter. _Molloy College, Rockville Centre, NY_.

Introduction: Epithelial ovarian cancer is one of the most common gynecological malignancies and the fifth most frequent cause of cancer death in women, affecting over 22,000 women annually. Nearly 15,500 affected women die from this disease annually, and chemoresistance from the commonly prescribed platinum-based drug, carboplatin, is a major contributor to this mortality. Previous studies have identified genes with CpG islands that are methylated and transcriptionally silenced in resistant epithelial ovarian cancer patients. One of these genes is GSK3β, an important regulator of apoptosis and cell growth in the Wnt pathway. Thus, understanding the role of GSK3β suppression in chemoresistance of epithelial ovarian cancer can help contribute to more effective treatments for this disease. By performing different assays our study examined the functional role that GSK3β plays in carboplatin chemoresistance.

Procedure: Human ovarian surface epithelium (HOSE) 6-3 cell line was utilized, which is characterized as sensitive to carboplatin therapy. The cells were studied in six groups: 1) untreated; 2) treated with Lithium Chloride (LiCl); 3) treated with carboplatin; 4) treated with carboplatin and LiCl; 5) treated with doxorubicin as control; 6) treated with doxorubicin and LiCl as another control. LiCl is known to suppress GSK3β gene expression. We took images of cells using a fluorescence microscope. We also performed the Neutral Red Dye assay that determines cell viability, Vybrant® MTT Cell Assay which measures amount of non-viable cells, Caspase 3 Assay which measures cell apoptosis, and we did cell counting using a hemocytometer and a light microscope. Data analysis was done by T-tests using Microsoft Excel®, with p < .05 for significance.

Summary of Data: More growth was observed in the carboplatin and LiCl group compared to the carboplatin group alone on microscopy. Neutral Red Dye assay: Compared to the cells exposed to carboplatin alone, those exposed to carboplatin and LiCl were more viable (p <0.01). Vybrant® MTT Cell Assay: the cells treated with carboplatin and LiCl showed lesser amounts of nonviable cells as compared to the carboplatin alone group (p<0.01). Caspase 3 Assay: the cells treated with carboplatin and LiCl were less apoptotic, compared to cells treated with carboplatin alone (p <0.01). Cell Counting: cells treated with carboplatin and LiCl had significantly more growth compared to the cells treated with carboplatin alone (p <0.01).

Conclusion: Our results show that cells with suppressed GSK3β had increased proliferation and reduced apoptosis, strongly suggesting that silenced GSK3β expression contributes to carboplatin resistance and GSK3β expression is vital to carboplatin chemosensitivity. Future in vivo studies could further investigate the role of GSK3β methylation to facilitate the design of potential genome-guided treatments for patients with chemoresistant epithelial ovarian cancer.

#2935

Systematic drug testing and RNA-sequencing of tamoxifen resistant breast cancer cell lines.

Susanne Hultsch, Sara Kangaspeska, Matti Kankainen, Vilja Pietiäinen, Olli Kallioniemi. _Institute for Molecular Medicine Finland FIMM, Helsinki, Finland_.

Tamoxifen, as a standard treatment of estrogen receptor (ER)-positive breast cancer, reduces breast cancer mortality by 31%. In 50% of advanced ER-positive cancers, however, de novo resistance exists and in 40% of patients with initial response acquired resistance frequently evolves. In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new opportunities to treat patients, we created seven tamoxifen-resistant breast cancer cell line models that represent the luminal A subtype (MCF-7, T-47D, ZR-75-1), which expresses ER, and the luminal B subtype (BT-474), which additionally expresses the HER2 oncoprotein. We then performed drug sensitivity and resistance testing (DSRT), exome-sequencing and network analysis on all these cancer cell lines to determine the molecular and drug response profiles specific to tamoxifen resistance. As the cells became tamoxifen-resistant, we observed increasing sensitivity towards drugs like ERK1/2-, proteasome- and BCL-family inhibitors, but each of the isogenic cell line pairs had its distinct genomic and drug response profile. We then studied the molecular profiles of the 7 drug-resistant variants by RNA-sequencing in comparison to their 4 isogenic parental cells. We could not detect any common significantly differentially expressed genes (more than 2 fold change) across all the cell lines. However, the cell lines could be grouped into two different categories: The ones with low amount of differentially expressed genes, < 800 genes in the BT-474 and ZR-75-1 cell line pairs (with a 1,2% overlap), and the ones with high amount of differentially expressed genes, >1800 genes in T-47D and MCF-7 (with 7,3% overlap). Further analysis with the Ingenuity pathway analysis tool revealed an involvement of "Fatty Acid Activation" as well as "Stearate Biosynthesis I" in this group. Additionally, we discovered that genes involved in iron metabolism (TFRC, IREB2 and FTL) or iron-regulated genes like CP and NDRG1 are deregulated. These genes and pathways thereby provide avenues to identify new drug vulnerabilities for the tamoxifen resistant cancer cells, which we are now investigating at gene and protein level e.g. by image-based phenotyping. In summary, by combining drug testing data with the RNA-sequencing results, we hope to provide a number of potential drugs as well as matching biomarkers for planning clinical trials for patients with tamoxifen-resistant breast cancer.

#2936

Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models.

Claudio Pisano, Gianluca Carletta, Fulvio D'Angelo, Antonietta Esposito, Erminia Bianchino, Assunta Riccio, Michela Festa, Francesco Cardile, Emanuele Carchia, Walter D'Acunto, Giacomo Signorino, Mario B. Guglielmi, Pasquale De Luca. _Biogem, Ariano Irpino, Italy_.

Malignant pleural mesothelioma (MPM) development is mainly correlated with exposure to asbestos and appears to be a trend toward an increase in its incidence in the years to come. Although novel therapeutic strategies are under investigation, alone or in combination with conventional therapy, MPM remains a cancer with high medical need.

With the aim to identify putative novel therapeutic targets/biomarkers expressed in specific MPM histotypes we investigated 9 different primary MPM cell lines at molecular, biochemical and pharmacological levels.

A transcriptomic analysis has been conducted comparing the gene expression in epitheliod (E-MM473), sarcomatoid (S-MM432) and biphasic (B-MM487) primary cell lines.

We found more than 1000 genes differentially regulated and statistically validated (p<= 0.05). The hierarchical clustering of the top 100 expressed genes revealed an histotype specific expression while few of them have a similar expression among the histotypes. 14 out of 100 genes were chosen based on their role in cell survival (hk1; gdf15; ifnar1/2; bmpr1/2; tgfrb; cdca8; birc5; plk1; srsf6; pin1; nox1; wt1). Their expression was confirmed via RT-PCR in all the 9 MPM cell lines (E-MM473, E-MM288, E-MM317, E-MM404, E-MM481; S-MM432, S-MM472; B-MM487, B-MM491) vs normal mesothelium cells (MET5A). Among the validated genes, gdf15 and hk1 expression strongly correlated with the epithelioid and bifasic histotype, respectively. The tumor relevance of these genes has been confirmed by siRNA interference in which hk1 and gdf15 transcript reduction significantly affected S-MM432 (>90%) and E-MM473/B-MM487 cell survival (>50%).

GDF15 belongs to the TGF-β superfamily and its role in different pathologies including cancer is controversial (Breit et al., 2011). Its overexpression has been associated to tumor chemoresistance (Chung-Ying et al.,2007) including Cisplatin (Cis) (Li et al., 2015; Yang et al. 2014). To this end, we investigated if GDF15 protein levels in MPM cells exposed to Cis correlate with its citotoxicity. Interestingly GDF15 protein level, which is high in E-MM473, medium in B-M M432 and low in S-MM487, is inversely correlated with the cells sensitivity to the treatment (i.e. E-MM473 are 2-5 times more resistant) and GDF15 silencing increases Cis-induced cytotoxicity. To assess the relationship between GDF15 levels/ Cis resistance, efficacy of Cis (4mg/kg; q7dx3w;i.v.) on stably expressing luciferase E-MM473(highGDF15) and B-MM487 (lowGDF15), orthotopically xenografted in nude mice, were investigated. While, E-MM473 showed a poor response to the Cis (TGI 58%), an impressive tumor response (TGI 95%) was observed in treated B-MM487.

Our results suggest and support the need of further clinical investigation to correlate GDF15 plasma level to the chemotherapy response in MPM patients.

#2937

Expression of osteopontin splicing isoforms in prostate cancer cells resistant to docetaxel.

Mariana Concentino Menezes,1 Amanda O'Neil,2 Isabella Guimaraes,3 Letícia Rangel,3 Etel Rodrigues Pereira Gimba4. 1 _Instituto Nacional de Câncer., Rio de Janeiro, Brazil;_ 2 _Univerisity College of Dublin, Dublin, Ireland;_ 3 _Universidade Federal do Espírito Santo, Vitória, Brazil;_ 4 _Universidade Federal Fluminense, Rio Das Ostras, Brazil_.

Resistance to chemotherapeutic drugs corresponds to main causes of treatment failure. Total osteopontin (OPN) has been described as a gene product involved on chemoresistance, besides activating several aspects of tumor progression. OPN regulates the expression of P-glycoprotein (P-gp) in prostate cancer (PCa) cells, besides promoting apoptosis evasion induced by chemotherapy using paclitaxel and estramustin. These data point OPN as a potential therapeutic target to approaches that could reduce chemotherapy tumor resistance. However, most of this data are related to total OPN. Once OPN suffers alternative splicing, producing 3 isoforms, further investigation should demostrate their specific involvement on chemoresistance. Recent data from our group indicated that OPNb and OPNc splicing isoforms activate PCa tumor progression features. Besides, PCa cells that overexpress these splice variants are more resistant to apoptosis induced by docetaxel (DXT). The current work aims to investigate the expression pattern of OPN splicing isoforms (OPN-SIs) and their relationships in DXT-PCa resistant cells. Total RNA has been extracted from PCa cell lines resistant to DXT (PC3-D8 and PC3-D12) and PC3 control cells with similar cell passages (PC3-Ag). Then, cDNa has been synthesized. OPN-SI expression levels were analyzed by quantitative real time PCR (qRT-PCR) using SYBR Green and isoform specific oligonucleotides. Relative expression levels were calculated using ∆∆CT method. Among the 3 OPN-SIs, we observed that OPNb and OPNc variants are overexpressed in relation to OPNa in DXT-PCa resistant cells PC3-D8 and PC3-D12, when compared to PC3-Ag. PC3-D12 cell line, resistant to higher DXT concentrations, expresses higher levels of OPNc, when compared to the other cell lines. Overall, our data further evidence that OPNb and OPNc overexpression in DXT-PCa resistant cells could be involved on resistance to DXT. Functional assays will be performed in order to investigate the molecular mechanisms by which these OPN-SIs mediate resistance to this chemotherapeutic drug.

#2938

Role of CD73 in promoting metastasis and resistance to 5-fluorouracil of colorectal cancer.

Xuan-hui Liu,1 Xian-rui Wu,1 Yu-feng Chen,1 Feng-wei Wang,2 Xiao-bin Zheng,1 Dan Xie,2 Bo Shen,3 Jian-ping Wang,1 Xiao-jian Wu,1 Ping Lan1. 1 _The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China;_ 2 _Sun Yat-sen University Cancer Center, Guangzhou, China;_ 3 _Cleveland Clinic, Cleveland, OH_.

Background and aim: Our previous study showed that high expression of CD73 (ecto-5'-nucleotidase) was a poor prognostic biomarker in patients with colorectal cancer (CRC). However, the underlying mechanism is unclear. Metastasis and chemo-resistance are among the main causes for the poor outcomes of CRC. The aim of this study is to evaluate the role of CD73 in the metastasis and chemo-resistance in CRC.

Methods: The expression levels of CD73 were detected using quantitative RT-PCR and Western blot in 9 CRC cell lines and 3 5-FU-resistant cell lines (HCT8-FR, HCT116-FR, and RKO-FR). CD73 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-CD73) in HCT8 and RKO. We then performed migration, invasion and MTS assays and wound healing assessment to evaluate the function of CD73 in cell progression and chemo-resistance. We conducted Cignal Finder Reporter Array to assess the main downstream signaling pathways of CD73.

Results: Increased expression of CD73 was found in all 3 5-FU-resistant cell lines. Inhibition of CD73 expression in HCT8 and RKO by si-CD73 attenuated the capacity of cell migration and invasion but increased the sensitivity to 5-FU. On the other hand, overexpression of CD73 in HCT8 and RKO increased the capacity for cell migration but decreased the sensitivity to 5-FU. CD73-induced migration and chemo-resistance was found to be associated with the activation of MAPK/ERK pathway.

Conclusions: High expression of CD73 was found to be associated with the metastasis and resistance to 5-FU via the activation of MAPK/ERK signaling pathway. This finding provides a molecular basis for the role of CD73 in the progression of CRC, suggesting a novel target for the treatment of CRC.

#2939

Heregulin-induced resistance against HER2-targeted therapies in HER2 positive breast and gastric cancer in vitro and in vivo.

Yoshikane Nonagase,1 Kimio Yonesaka,1 Satomi Watanabe,1 Koji Haratani,1 Takayuki Takahama,1 Naoki Takegawa,1 Hiroto Ueda,1 Hisato Kawakami,2 Hidetoshi Hayashi,1 Masayuki Takeda,1 Haruka Sakamoto,1 Takao Tamura,1 Kazuhiko Nakagawa,1 Junji Tsurutani1. 1 _Kinki University Faculty of Medicine, Osaka-sayama, Osaka, Japan;_ 2 _Mayo clinic, Rochester, MN_.

Background

HER2-targeted therapies have been shown clinical benefits in patients with HER2 positive breast and gastric cancers. However, resistance against such targeted therapies eventually develops in most cases. Overexpression of heregulin, a ligand for HER3, has been reported as one of the resistant mechanism against HER2-targeted therapies. Upregulation of heregulin activates HER3-PI3K-AKT signaling, leading to the resistance against HER2-targeted therapies. We investigated the effects of heregulin in cell lines and evaluate its clinical impacts in HER2 positive breast and gastric cancers.

Materials and methods

We utilized transfected or recombinant heregulin to investigate the effect of heregulin on proliferation and apoptosis of SK-BR-3 and NCI-N87 cell lines treated with lapatinib, trastuzumab, trastuzumab-emtansine and paclitaxel. Clinical data and specimens were obtained from patients with HER2 positive breast and gastric cancers, evaluating their heregulin value from both serum and tissues using ELISA and RT-PCR, respectively.

Results

Heregulin transfection and recombinant heregulin conferred resistance against treatment with lapatinib, trastuzumab, and weak resistance against trastuzumab-emtansine, but no resistance against paclitaxel in HER2 positive cell lines. Clinical data of heregulin among patients treated with HER2-targeted therapies revealed that patients with relatively high heregulin value tended to have worse prognosis.

Conclusions

Together these data, overexpression of heregulin may at least in part play a role in resistance against HER2-targeted therapies in HER2 positive cancer patients. Further investigation is warranted to elucidate the relevance of treatments targeting heregulin-HER3-PI3K signaling.

#2940

The p53 mutation R273H contributed to drug resistance of EGFR tyrosine kinase inhibitors.

Yueh-Fu Fang,1 Chun-Yu Lin,1 Peichih Lee2. 1 _Chang Gung Memorial Hospital, Taiwan, Taiwan;_ 2 _M.D. Anderson Cancer Center, Houston, TX_.

Tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) were the standard first-line treatments for lung cancers with activating EGFR mutations. Emergent drug resistance is the major problem in the patients who received treatment of EGFR TKIs. Second point mutation T790M mutations could be found in 50-60% patients who had drug resistance to EGFR TKIs. Alternative pathway of Met signaling, transformation to small cell lung cancer, over-expression of other HER family receptors or epithelial mesenchymal transit were reported in these patients who had drug resistance to EGFR TKIs. NFKB pathway played a role of early resistance to EGFR TKIs in the patients.

The p53 mutation could be found in 70-80% lung adenocarcinoma patients. These mutations could be found in lung cancer patients who had wild type or mutated EGFR. Lung adenocarcinomas with mutated p53 lose tumor suppressor function of wild type p53 and have new function from mutated p53. We thought p53 mutation may have gain of function and result in drug resistance to EGFR TKIs in lung cancer patients.

We knocked down p53 in three cancer cell line, MDA-MB-468 (p53 R273H), BT-549 (p53 R249S) and H322 (p53 R248L). We found knockdown of p53 R273H in MDA-MB-468 increased sensitivity to gefitinib. Knockdown of p53 R249S and p53 R248L showed the same response to gefitinib in cancer cell lines BT-549 and H322.

Lung cancer cell line HCC827 had EGFR exon 19 deletion and was sensitive to gefitinib. We overexpressed p53 R273H, p53 R157H and p53 R249S in HCC827 lung cancer cell line. Overexpression of p53 R273H increased resistance to gefitinib in HCC827 lung cancer cell line. Overexpression of p53 R157H and p53 R249S did not increased the resistance to gefitinib.

Conclusion: Gain of function of p53 R273H increased resistance to EGFR TKIs in cancer cell lines. Further immunohistochemistry staining and gene analysis of mutated p53 will be done in lung adenocarcinoma from lung cancer patients who received EGFR TKIs.

#2941

Increased expression of ABCB1/MDR1 could be associated with alectinib resistance in ALK-rearranged lung cancer cells.

Takahiro Tsuji, Hiroaki Ozasa, Yuichi Sakamori, Takashi Nomizo, Yoshitaka Yagi, Hiroki Nagai, Young Hak Kim, Michiaki Mishima. _Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan_.

Alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, has shown promising activity in ALK-rearranged non-small cell lung cancer (NSCLC). The mechanisms of drug resistance to alectinib have been still unclear. To explore whether overexpression of ATP-binding cassette (ABC) transporters might provide a potential mechanism of alectinib resistance in lung cancer, we established alectinib-resistant cell lines from NCI-H2228 and primary ALK-rearranged cells and evaluated the expression of ABC transporters.

We established ALK-rearranged NSCLC cell lines (KTOR 1, 2, and 3) from tumor cells in pleural effusion obtained from 3 patients with alectinib naïve ALK-rearranged NSCLC. In the 2 of 3 patients, tumor cells in pleural effusion were also obtained when disease progression was observed during alectinib treatment (KTOR1-RE and KTOR2-RE). Expression profiling of ABC transporters using quantitative real-time polymerase chain reaction (qRT-PCR) revealed that the expression of ABCB1 was significantly increased in KTOR1-RE and KTOR2-RE compared with the parental cells (4.6 and 10.8 folds), but not other ABC transporters. Then, alectinib-resistant NCI-H2228 cell lines (H2228-AR) were established by exposing the parental cells to stepwise-increasing alectinib up to 5μM for 10 months. We found that the ABCB1/MDR1 expression in H2228-AR cells were significantly increased (2.09-16.6 folds) compared with the parental cells using qRT-PCR and immunoblotting. Then, we examined the alternation of alectinib cytotoxicity by inhibition of ABCB1. Knockdown of ABCB1 expression using small interfering RNA and inhibition of ABCB1 by verapamil enhanced alectinib cytotoxicity in H2228-AR cells. Next, the levels of ABCB1 expression after short-term alectinib exposure were examined in NCI-H2228 and the three alectinib naïve ALK-rearranged NSCLC primary cell lines (KTOR 1, 2, and 3). In all the four ALK-rearranged cell lines, exposure to 100 nM alectinib for 72 hours induced increased expression of ABCB1 compared with control medium.

These data suggested that increased expression of ABCB1/MDR1 might be, in part, responsible acquired resistance and sensitivity to alectinib in ALK-rearranged NSCLC cells.

#2942

p90 Ribosomal S6 kinase mediates acquired resistance to ganetespib in KRAS mutant NSCLC.

Suman Chatterjee, Eric H-B. Huang, Timothy F. Burns. _Department of Medicine, Division of Hematology Oncology, University of Pittsburgh Cancer Institute, PITTSBURGH, PA_.

Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations and no current therapies targeting this key oncogenic driver exist. There is a critical need for novel agents targeting KRAS mutant NSCLC. Heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone required for the stability of its 'client' oncoproteins, which include a number of KRAS downstream effectors. Therefore, targeting Hsp90 could be a promising therapeutic intervention against "undruggable" KRAS driven tumors. Although single agent activity has been demonstrated with the HSP90 inhibitor ganetespib in vitro, only transient responses have been observed in the clinic in KRAS mutant patients. Our goal is to characterize the mechanisms of acquired resistance to ganetespib and develop rational combinations to overcome ganetespib resistance.

We have generated ganetespib resistant (GR) KRAS mutant NSCLC cells and demonstrated that bypass of G2/M arrest and hyper-activation of RAF/MEK/ERK and PI3K/AKT/MTOR pathways contribute to resistance. We observed increased dependence on these pathways as the GR cells were more sensitive to ERK1/2 or dual PI3K/MTOR inhibition. Moreover, the combination of ganetespib with ERK1/2 or PI3K/MTOR inhibitors was more effective than either single agent alone. Furthermore, several key mediators of G2/M progression were significantly increased in the GR cells compared to sensitive parental cell lines. Most notably, the serine/threonine kinase p90 ribosomal S6 kinase (p90-RSK), an important regulator of G2/M progression and the PI3K/AKT/MTOR pathway and a key target of ERK1/2 and PDK1 phosphorylation, were significantly upregulated both in expression and activity. Downstream p90-RSK targets and critical G2/M regulators - cdc25A, cdc25B, and cdc25C were also significantly elevated in GR cells. The p90-RSK family consists of four closely related isoforms (1-4), that are widely expressed in cancer and demonstrated to increase cell survival and proliferation. We observed that isoform-specific genetic silencing and pharmacological inhibition of p90-RSK re-sensitized GR cells to ganetespib. Conversely, overexpression of distinct p90-RSK isoforms in sensitive parental cell lines induced ganetespib resistance as well as led to bypass of ganetespib-induced G2/M arrest. The combination of ganetespib with p90-RSK inhibitor(s) enhanced cytotoxicity compared with either agent alone. These data suggest that combination of ganetespib with ERK1/2 or dual PI3K/mTOR or p90-RSK inhibitors may prevent ganetespib resistance and/or overcome acquired resistance after single agent treatment. These findings provide preclinical rationale for a potential future clinical study with an HSP90 inhibitor and a dual PI3K/mTOR inhibitor or RSK inhibitor in KRAS mutant NSCLC patients.

#2943

Hypoxia induced resistance to chemotherapy is regulated by the endothelin receptor axis.

Hyun Kyung Yu, Ho Jeong Lee, Fahao Zhang, Qiuyu Wu, Isaiah J. Fidler, Sun Jin Kim. _UT MD Anderson Cancer Center, Houston, TX_.

Introduction

Tumor cells growing in the brain are resistant to chemotherapy. We have recently demonstrated that the activation of the endothelin receptors A and B axis is involved in the induction of resistance to chemotherapy by primary brain tumor cells and by brain metastases. Treatment with a dual endothelin receptor antagonist in combination with chemotherapy produced significant therapy of orthotopically growing glioblastoma cells as well as breast cancer and lung cancer brain metastases. Since the milieu of tumors in the brain is hypoxic (0.5% - 1% O2), we investigated whether the chemo-resistance of the tumor cells is linked to hypoxia mediated activation of the endothelin receptor axis.

Materials and Methods Human glioma (LN229), breast cancer (MDA231), and lung adenocarcinoma (PC14) cell lines were cultured in DMEM supplemented with 5% FBS. The endothelin receptor A (ETAR) and/or B (ETBR) of cancer cells were knocked down by shRNA. ETAR and ETBR were activated by incubating the tumor cells with exogenous endothelin-1 for 15 minutes or blocked by treating the parental or engineered cells with ETAR-specific antagonist, BQ123 (100nM), and/or ETBR-specific antagonist, BQ788 (100nM), for 2 hours prior to the addition of paclitaxel (5ng/ml), temozolomide (100μg/ml) or cisplatinum (5μg/ml). The cultures were placed into incubators with an atmosphere of 20%, 6% or 1% oxygen for 48 hours

(MDA231 cells) or 72 hours (LN229 and PC14 cells). Tumor cells were then plated into 96-well plates to determine chemosensitivity by the MTT assay or into 6-well plates for western blots, or into chamber slides for immunohistochemical analyses. Expression of survival-related proteins such as pETAR, pETBR, pAkt, pMAPK, pNFκB, GSTA5, TWIST1 or Bcl2L1, were then determined.

Results Stimulation of parental MDA-231, PC-14, or LN229 cells with exogenous ET-1 induced significant resistance against all tested chemotherapeutic agents. The resistance was abolished only when both the ETAR and ETBR were antagonized by a combination of BQ123 and BQ788. Parental cells cultured under hypoxia were also significantly resistant to chemotherapy and treatment of these cells with BQ123 and BQ788 reversed this resistance. The effects of exogenous ET-1 on the induction of chemo-resistance or BQ123 and BQ788 on restoration of chemo-sensitivity were not found in any cancer cells devoid of ETAR and ETBR. In all cases, the chemo-resistance of tumor cells was associated with increased level of expression of proteins related to cell survival.

Conclusion These data suggest that the endothelin receptor axis plays a role in hypoxia induced resistance of cancer cells to chemotherapy. Additional studies are warranted to determine the functional changes of the endothelin receptor axis in hypoxia.

#2944

Expression of AEG-1 associated with c-Myc and PI3K activation in regulation of TS and chemoresistance of pemetrexed in non-small cell lung cancer.

Chung-Yu Chen,1 Ying-Yin Chen,1 Kuan-Yu Chen,2 Jin-Yuan Shih,2 Yih-Leong Change,2 Chong-Jen Yu,2 Pan-Chyr Yang2. 1 _National Taiwan University Hospital Yunlin Branch, Douliou, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan_.

Purpose: Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, is elevated in multiple cancers. Thymidylate synthase (TS) is an important enzyme in purine synthesis and DNA replication. In this study, we investigated the AEG-1 expression characteristics in non-small cell lung cancer (NSCLC) to determine whether its expression is correlated with TS regulation of NSCLC.

Materials and Methods: A549, H157, CL1-0, CL1-5, PC9, H1975, H520 and H292 human lung cancer cell-lines were cultured in medium with RPMI1640 + 10% FBS. Immunohistochemical stain and Western blot of AEG-1 and TS were studied in all cell-lines. The primary antibodies used were anti-AEG-1 (1:1,000; chicken polyclonal), anti-actin (1:1,000; rabbit polyclonal; Santa Cruz), and anti-TS (1:1,000; mouse monoclonal; Abcam). Total RNA was extracted using a Qiagen mRNA easy mini kit (Qiagen). Real-time PCR was performed using ABI 7900 fast real-time PCR system to detect expression of AEG-1 and TS. Cell viability was determined by standard MTS assays to recognize IC50 of Pemetrexed in lung cancer cell-lines. Transfection of siRNA for AEG-1 was carried out to knock down AEG-1. AEG-1 pcDNA (Life technologies, CA, USA) were used to quantitatively examine AEG-1 activity. Expressive change of AEG-1associated with TS and signaling pathways were analyzed by Western blot and PCR.

Results: TS and AEG-1 in H157, Cl1-0, CL1-5, H520 and H292 were higher detected by IHC stain, Western blot and RT-PCR mRNA level. The expression of TS was positive correlated with AEG-1 in lung cancer cell-lines. The IC50 values of Pemetrexed for cells were lower in the lung cancer cell-lines with lower TS gene expression. After knockdown of AEG-1 by siRNA in Pemetrexed-resistance PC-9 and A549 cell-lines, the expression of TS decreased associated with decreasing change IC50 of Pemetrexed. In contrast, transfection with AEG-1 pcDNA in PC-9 and A549 cell-lines overexpressed AEG-1 gene, TS level also enhanced associated with increasing change IC50 of Pemetrexed. AEG-1 expression can also activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway that augments binding of c-Myc thereby regulating AEG-1 transcription.

Conclusions: Our data indicated a significant correlation between TS and AEG-1 gene expression in NSCLC cell-lines. TS expression might be regulated by AEG-1 through activation of PI3K/AKT and c-Myc signaling pathways associated with development of chemoresistance to pemetrexed in NSCLC treatment

#2945

Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer.

Tanja Grimmig,1 Kerstin Kloos,1 Rebecca Thumm,1 Romana Moench,1 Christoph T. Germer,2 Ana Maria Waaga-Gasser,1 Martin Gasser2. 1 _University of Wuerzburg, Dept. of Surgery I, Molecular Oncology and Immunology, Wuerzburg, Germany;_ 2 _University of Wuerzburg, Dept. of Surgery I, Wuerzburg, Germany_.

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising procedure for the treatment of peritoneal carcinomatosis (PC). Heat shock proteins (HSPs) and other proteins involved in cellular repair mechanisms seem to induce cytoprotective processes during HIPEC therapy. Therefore, the aim of this study was to analyze the effects of HIPEC-related conditions on tumor cell proliferation and the expression of HSPs in human colon cancer.

Methods: Human colon cancer cell lines HT29, SW480 and SW620 were exposed to different temperatures (37°C, 41°C, and 43°C) as well as defined cytostatic agents (Oxaliplatin, Mitomycin C, and 5-Fluorouracil). After cellular regeneration (30 min, 24 h, 48 h and 72 h) RNA isolation and whole cell extraction was performed. Gene and protein expression analysis of HSP27, 70, 72 and 90 as well as PCNA, Ki-67, BCl-2 and BCl-Xl were carried out using RT-qPCR and Western blot. Additionally, MTS cell proliferation assays were performed 24 h, 48 h, 72 h and 96 h post treatment. Moreover, AnnexinV apoptosis assays were conducted.

Results: All colon cancer cells exposed to hyperthermic conditions showed initially up-regulated HSP gene expression. Highest expression was found after exposure to 43°C. Combined cytostatic and hyperthermic treatment demonstrated additional increase in HSP27 expression and in other HSPs to a lesser degree. Tumor cells exposed to cytostatic agents showed overall higher HSP gene expression compared to cells without chemotoxic treatment. Similar effects were detected for the expression of the proliferation marker PCNA and anti-apoptotic protein BCl-Xl. Apoptosis assay demonstrated decreased numbers of apoptotic cells at 43°C compared to normothermia. Additionally, proliferation assays revealed reduced chemosensitivity in cells treated with hyperthermia.

Conclusion: Desired effects of hyperthermia used in HIPEC therapy to achieve anti-proliferative and apoptosis inducing effects seem to be negatively influenced by cell stress mediated repair mechanisms in colon cancer. Our in vitro findings suggest analyzed HSPs to be significantly involved in this hyperthermia and chemotoxicity mediated cellular repair mechanisms. While initial increase in HSP expression can counteract cytotoxic effects during HIPEC therapy their prolonged expression may promote lasting resistance to cellular stress.

#2946

Increased ALDH7A1 expression enhances the resistance to the anticancer drugs and colony formation in lung cancer cell lines.

Yuichi Sakamori,1 Hiroaki Ozasa,1 Eiji Kunii,2 Yoshitaka Yagi,1 Takahiro Tsuji,1 Takeshi Nomizo,1 Hiroki Nagai,1 Young Hak Kim,1 Ken Maeno,2 Tetsuya Oguri,2 Michiaki Mishima1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Nagoya City University, Nagoya, Japan_.

Background: Aldehyde dehydrogenase (ALDH) forms a superfamily of enzymes that catalyze conversion of aldehydes into carboxylic acids, and they are categorized into 19 families in human. Increased ALDH activity was reported to be a potential marker of cancer stem cell (CSC) in various solid tumors including lung cancer. Furthermore, High ALDH expression has been shown to be involved in drug resistance to conventional cytotoxic drugs. Recent studies revealed that ALDH7A1 is highly expressed in prostate cancer and associated with recurrence in patients with surgically resected non-small-cell lung cancer. However, there is not a fundamental link between ALDH7A1 expression and malignant phenotype, including drug resistance and colony formation.

Material and methods: Anticancer drug resistant cell lines were established from lung cancer cell lines (PC-6, DMS53, PC-14, PC-9, NCI-H23 and NCI-H2228 cells) by exposure to various anticancer drug, including 7-ethyl-10-hydroxycamptothesin (SN-38) , gemcitabine (GEM), cisplatin (CDDP), etoposide, paclitaxel (TXL), pemetrexed, amrubicin, erlotinib and crizotinib. Protein expression was determined by western blotting and gene expression was examined by RT-PCR. ALDH enzyme activity was measured by ALDEFLOUR TM assay. Proliferation was measured using MTS assay. ALDH7A1encoding plasmid was stably transfected into PC-14 cell. These transfected cells displayed sphere formation on ultralow binding plates and survived for more than 3 weeks.

Results: We found that the levels of ALDH7A1 expression were higher in various anticancer drug-resistant lung cancer cell lines compared with the parental cells. ALDH enzyme activity was about 4-9 fold higher in SN-38, TXL, CDDP and GEM resistant cell lines (PC6/SN38, PC6/TXL, PC14/CDDP and PC14/GEM) than the parents cells. Up-regulation of ALDH7A1 expression by using a forced expression vector in PC14 cell altered cytotoxicity to SN-38 and TXL. Furthermore, overexpression of ALDH7A1in PC-14 cells enhanced sphere formation relative to the cells transfected with control vector.

Conclusion: These results suggested that increased ALDH7A1 expression in lung cancer cell lines might be one of the mechanisms of acquired resistance to anticancer agents and enhance colony formation.

#2947

MicroRNA 10b, 27a and 27b are involved in the resistance to treatment with epidermal growth factor tyrosine inhibitor (EGFR-TKI) in EGFR mutant lung cancer cell line.

Hannah Montes,1 Emma B D Reyes,1 Mukut Sharma,1 Chao H. Huang2. 1 _Kansas City VA Medical Center, MO;_ 2 _University of Kansas Medical Center, Westwood, KS_.

EGFR TKIs are now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and require treatment with another EGFR TKI or chemotherapy. The gate-keeper mutation T790M is responsible for about 50% of resistance. Rocelitinib(R) is a third generation EGFR TKI active in lung cancer cells with T790M mutation. Other mechanisms may also confer resistance in these patients. High expression of microRNA (miR)-10 is associated with worse prognosis in resected lung cancer patients with EGFR mutation. Both miR-27a and 27b are associated with increased expression of c-met which is a potential mechanism of resistance to EGFR TKI. We hypothesized that miR 10b and 27a and 27b modulate resistance in E-resistant lung cancer cells.

Material and Methods

Lung cancer cell lines with EGFR mutation HCC827 and HCC4006 were treated with 5 nM of Erlotinib (E) for 72 hours. E-resistant cells were then treated with R at 21.5 nM for 72h. The cells were harvested and microRNA profiling was performed by real time PCR. HTB177 cell line without EGFR mutation was used as control.

Results

We observed increased expression of miR-10b and miR-27a in HCC4006 compared to control cells HTB 177 cells; miR-27b is upregulated in HCC827 cell line. The increased expression of miR-10b and miR 27a were higher in CRL-2871 than the control cells 9-fold and 8-fold, respectively. MicroRNA- 27b was increased 300-fold in HCC827 compared with control.

Treatment with R reduced the proliferation in HCC827 to 30% and in HCC4006 to 50% compared to control. It did not inhibit the proliferation of HTB177 cells. R increase miR-10b in HCC4006 by 3-fold, miR-27a by 1.5-fold; in HCC827 it reduced miR-10b to 0.4, miR-27a to 0.4 and miR-27B 0.5.

Conclusion

We observed upregulation of miR-10b, 27a and 27b in lung cancer cells lines with EGFR mutation that were resistant to E treatment. Treatment of E resistant cells with R reduced proliferation and changed the expression of miR-10b, 27a and 27b. Our results suggest an epigenetic mechanism of resistance in addition to the T790M mutation. MicroRNAs may be studied as targets for developing novel approach for to treating patients with EGFR TKI resistance.

#2948

Glucose-6-phosphate dehydrogenase mediates drug resistance in diffuse large B-cell lymphoma.

Steve A. Maxwell,1 Seyed Mousavi-Fard,1 Timothy Davis,1 Deeann Wallils,2 Jim Sacchettini2. 1 _Texas A &M University Health Science Center, College Station, TX; _2 _Texas A &M University, College Station, TX_.

A leading cause of mortality in diffuse large B-cell lymphoma patients is the development of resistance to the CHOP chemotherapy regimen. We have discovered that CHOP-resistant DLBCL cells express higher G6PDH activities and lower reactive oxygen (predominantly superoxide) levels than CHOP-sensitive cells. Glucose-6-phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme of the NADPH-producing pentose phosphate shunt (PPP). The PPP is the most important pathway for recycling of cellular glutathione (GSH), a key scavenger of reactive oxygen species (ROS). In the PPP oxidative phase, glucose 6-phosphate is irreversibly converted into ribulose 5-phosphate and CO2 leading to the synthesis of NADPH, a redox cofactor for many antioxidant enzymes. The NADPH produced is used by glutathione reductase to reduce GSSG (glutathione disulfide) to GSH. We hypothesized that increased activity of the G6PDH pathway leads to higher GSH production, lower oxidative state (ROS), and CHOP-resistance. In support of our hypothesis, downregulation of G6PDH lowered NADPH and GSH levels, increased ROS, and reversed CHOP-resistance in DLBCL cells. These results indicate an important role for the G6DPH pathway in mediating CHOP resistance in DLBCL.

#2949

REV3L, the catalytic subunit of pol ζ, is involved in the progression and chemoresistance of esophageal squamous cell cancer.

Jundong Zhou,1 Sitao Zou,1 Wei-Qun Ding,2 Jinchang Wu1. 1 _Suzhou Municipal Hospital, Suzhou, China;_ 2 _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

REV3L, the catalytic subunit of DNA ploymerse zeta, is well known to participate in error-prone translesion synthesis (TLS) with less stringent and lower processivity. Recent evidence demonstrated that REV3L is involved in carcinogenesis and tumor progression. However, the function of REV3L remains unclear in esophageal squamous cell cancer (ESCC). In this study, we examined REV3L expression in ESCC tissues and its association with clinicopathological parameters. REV3L was found to be significantly up-regulated in ESCC tissues, which is correlated with lymph node metastasis and clinical stages of the patients. To further investigate the potential role of REV3L in esophageal cancer, stable ESCC cell lines with the suppression of REV3L expression were established. Down regulation of REV3L expression led to a decrease in cell proliferation and invasive tendency partly through suppressing cyclinD1 and survivin expression, and an increase in cellular sensitivity to 5-Fu by inducing G1 phase arrest and apoptosis. Therefore, REV3L plays an important role in ESCC progression and chemoresistance, and is a potential diagnostic marker and therapeutic target for ESCC.

#2950

Targeting chemo-resistant colon cancer by inhibiting embryonic resistance pathways.

Haneen K. Amawi,1 Joel Chen,1 Curt Balch,1 Karthikeyan Chandrabose,2 Piyush Trivedi,2 Amit K. Tiwari1. 1 _University of Toledo, Toledo, OH;_ 2 _Rajiv Gandhi Technical University, Bhopal, India_.

Colorectal cancer (CRC), with an estimated 2.1 million worldwide new cases, and 610,000 deaths in 2014, is the second-leading cause of cancer death in developed countries. While early detection (e.g., via colonoscopy, occult fecal blood tests) or prevention (removal of premalignant polyps) generally allows complete disease elimination, metastatic and drug-resistant disease is uniformly fatal. Even so FDA-approved, life-extending CRC chemotherapies such as camptothecin-derived alkaloidal quinolones (e.g., irinotecin, topotecin) have demonstrated potent inhibition of the DNA synthesis enzyme topoisomerase-1, although the use of these drugs is limited due to specific adverse effects and resistance mediated by ATP-dependent membrane transporters. Consequently, to improve the efficacy (e.g., specificity, attenated toxicity, chemosensitivity, etc.) of these topoisomerase inhibitors, we designed and synthesized (based on structure-activity relationships, molecular docking, and in silico studies) eight distinct analogues, based on a pyrimido[1,2:1,5]-pyrazolo[3,4-b]quinoline (PPQ) ("IND-2") structure. One of these, "IND-2," demonstrated robust anticancer efficacy, selectivity to colon cancer cells, negligible toxicity to normal cells, high water-solubility, and absent cellular efflux via multidrug transporters, but remained vulnerable to metabolic inactivation and poor bioavailability. Resultantly, based on our preliminary microarray and bioinformatics analyses of over 47,000 genes, and our findings of IND-2-facilitated transcriptional downregulation of several gene members of the embryonic WNT/EMT signaling axis, we further bioinformatically optimized and synthesized 14 IND-2 derivatives that we herein set forth to characterize in in vitro and in vivo CRC models, specifically assessing the abovementioned vulnerabilities. We hypothesize that embryonic signal pathways (e.g., WNT-β catenin, EMT, etc.) play an important role in colorectal tumorigenesis and can be successfully targeted for the development of safe and efficacious drugs for eventual human CRC treatment. Presently, we are assessing these analogs in numerous normal and cancerous CRC cell lines, to select the most promising of these candidates (based on efficacy and selectivity) for scaled-up synthesis and extensive in vivo testing, using the highly aggressive mouse APC/Min/+ CRC model. We strongly believe that the proposed work will validate embryonic signal cascades as targetable pathways that could lead to the identification of synthetic, plant-based pyrimido-pyrazolo-quinoline derivatived compounds with significant efficacy (in high-risk individuals) and/or selective (minimally toxic) for further progression into preclinical and clinical anti-CRC drug development.

#2951

A comprehensive analysis of proteins involved in innate and acquired resistance to molecularly-targeted drugs in breast cancer cells.

Hamid Montazeri. _Chapman University, Irvine, CA_.

Cancer is the second leading cause of death among Americans, and despite advances in diagnosis, new technologies for early detection, and development of potent molecularly-targeted drugs, survival rates in advanced disease have not been improving at desired rates. Perseverance of cancer cells seem to rely both on their significant heterogeneity at the onset of treatment, and the inherent plasticity (genetic instability) to adopt to environmental conditions and exogenous agents. Cancer is known as a heterogeneous disease. In fact, extensive genetic diversity has been revealed not only between different types of cancer, but also within a single tumor (as diversity in the expression of protein biomarkers). This intra-tumor heterogeneity could be a major obstacle in cancer treatment due to a wide range of responsivity to any specific anticancer agent. This study aims to analyze the heterogeneity and plasticity among a small panel of breast cancer cell lines as a reaction to exposure to a variety of molecularly-targeted agents, which could lead us to identification of the proteins that play major roles in drug resistance in cancer cells.

After initial evaluation of the LC50, we exposed five different breast cancer cell lines to high doses of erlotinib (selective ErbB1/EGFR-inhibitor), vemurafenib (pan Raf inhibitor), everolimus (selective mTORC1 inhibitor), and ruxolitinib (pan JAK inhibitor), an collected the survivors. Using an in-house designed microarray and real-time PCR analysis, we analyzed the protein expression profile of the survivors compared to the untreated population, which revealed proteins involved in innate resistance and the heterogeneity among the population. In an alternative approach, we exposed the cells to gradually increasing doses of these selected agents, affording the cells the opportunity to adapt to the treatment. The resistant cells were then analyzed in a similar fashion to reveal proteins involved in acquired resistance.

#2952

Generation and molecular profiling of drug-resistant cancer cell lines.

Fang He, Pengwei Pan, Zengjin Yuan, Jingqi Huang. _Pharmaron, China, China_.

Drug resistance is the main cause of ineffective chemotherapy in patients. Many cancers respond initially well to therapy but eventually the cancer recurs as drug-resistant disease leading to the patient death. Therefore, a better understanding of the molecular basis of the drug resistance is warranted in order to elucidate the mechanisms and markers underlying this drug-resistant phenotype In this study, we aimed to generate and characterize a panel of drug resistant cancer cell lines, providing a valuable tool with which to investigate the molecular pathways and putative markers that may be associated with this resistance phenotype in cancer.

#2953

Notch signaling is a key pathway involved in drug resistance in melanoma cells.

Gaurav Kaushik, Jonathan Sheldon, Prasad Dandawate, Dharmalingam Subramaniam, David Standing, Shrikant Anant, Joshua M.V. Mammen. _University of Kansas, Kansas City, KS_.

Background: Melanoma expresses a plastic and aggressive phenotype, lacking the majority of regulatory mechanisms due to the aberrant activation of various signaling pathways including the Notch pathway. Oncogenic BRAF mutation has the target for therapeutic interventions such as the drug vemurafinib but recent studies have indicated development of resistance. Unfortunately, however, the mechanisms of vemurafenib-induced resistance in melanoma are still poorly understood. We explored the role of Notch signaling in development of vemurafenib drug resistance in melanoma cells.

Methods: We have utilized melanoma cell lines (especially B16/F10, SKMEL-28, A2058, UACC275 SKMEL103 and M14) with and without the common hot spot BRAFV600E mutation for the studies. We performed hexoseaminidase and clonogenicity assays to determine the cell growth rate and IC50 values in the cell lines. To generate drug resistant cells, UACC275 and SKMEL-28 cells were repetitively grown in the presence of vemurafenib. For stem cells, we did melanosphere formation assay. For protein expression, we performed western blots.

Results: In the initial screening with vemurafinib, we observed a pattern of increased IC50 values in drug resistant cell lines, with UACC275 and SKMEL-28 being the more sensitive cells. Following sequential exposure, we developed vemurafinib-resistant cell lines, and observed that the IC50 values for proliferation inhibition to be ~8-10 fold higher than the parental cells. Colony forming potential of the drug resistant cells was also not affected by increasing concentrations of vemurafenib, confirming acquisition of resistance. Furthermore, the drug resistant UACC275 cells presented a smaller and round morphology compared to the usual elongated and stretched appearance of the parent lines. Additionally, we also observed a significant increase in size of melanospheres for the drug resistance cells suggesting enrichment of stem cells. We further studied the expression and activation of various notch signaling cascade proteins and observed a significant increase in the levels of cleaved Notch-2, and -4 in the drug resistant cells. Interestingly, early passages of cell culture for drug resistant cells showed decrease in cleaved Notch-2 levels in cells with significant increase in basal levels of cleaved Notch-2 levels at <10 passages. Therefore, a reductionist model of vemurafenib resistance can be developed using the UACC275 and SKMEL-28 cell line.

Conclusion: As Notch-2 and -4 levels were higher in most of the resistant cells, therefore, notch signaling may play critical role in the development of vemurafenib drug resistance in melanoma cells. Targeting specific notch in patients on chemotherapy especially on BRAF inhibitors will be a key event for better progression and treatment of melanoma.

#2954

MAP-kinase pathway activation facilitates survival of persistent FGFR1-amplified lung cancer cells upon FGFR inhibition.

Florian Malchers, Meryem Seda Ercanoglu, Roman Kurt Thomas. _University of Cologne, Cologne, Germany_.

One recurrent alteration of squamous cell lung cancer is the amplified region of the 8p12-p11 locus. The tyrosine kinase fibroblast growth factor receptor 1 (FGFR1) seems to be one of the most prominent targets of this amplification. Thus, small molecules inhibiting FGFRs have been employed to treat FGFR1-amplified squamous cell lung cancer. However, only about 16% of such FGFR1-amplified tumors respond to single agent inhibitor therapy. Several tumors exhibited insufficient tumor shrinkage, compatible with the existence of persistence of inhibitor-resistant tumor cells in the tumor. To investigate the mechanism of FGFR-inhibitor persistence we studied the lung cancer cell lines DMS114 and H1581. Both cell lines demonstrate GI50 values in the nanomolar range upon three different FGFR-inhibitors. However, both cell lines exhibit sustained cell viability within 0.5 to 5 µM inhibitor treatment, indicating a subpopulation of persistent cells. We therefore isolated these persistent cells by treating with a high dose of FGFR inhibitors. After 8 to 12 weeks the cells were completely resistant against all FGFR inhibitors tested. Genetic identity with the original cell line was confirmed by fingerprinting. We found that while parental cell lines showed depleted pERK signals, persistent cells were marked by a constant pERK level upon treatment. In H1581 cells, reactivation of the mitogen-activated protein kinase (MAPK) pathway was demonstrated by an active RAS-pulldown assay. Whole exome sequencing (WES) revealed high and focal NRAS amplification and DUSP6 depletion, leading to MAPK-pathway reactivation. Furthermore, retroviral overexpression of wild type RAS-isoforms induced FGFR-inhibitor resistance in parental H1581 cells. In DMS114 we observed MET and IGF-1R activation as possible mechanisms of persistence. Furthermore, co-inhibition of FGFR and MEK was a highly effective combination therapy to treat FGFR-inhibitor persistent cells. This study associates the MAPK-pathway as a key pathway for FGFR-dependent cell lines. Furthermore, these results suggest a beneficial FGFR / MEK combination treatment to avoid the outgrowth of FGFR-inhibitor persistent cells.

#2954A

A potent and selective TRK inhibitor ONO-5390556, shows potent antitumor activity against both TRK-rearranged cancers and the resistant mutants.

Ryohei Kozaki, Toshio Yoshizawa, Kohki Tsukamoto, Hikaru Kato, Kazuhito Kawabata. _Ono Pharmaceutical, Osaka, Japan_.

Purpose: TRKA/NTRK1, TRKB/NTRK2 and TRKC/NTRK3 belong to the neurotrophic tyrosine kinase receptor family and signals from TRK receptors play a role in neuronal survival and differentiation through activation of MAPK and AKT downstream pathways. Recently, oncogenic rearrangements of TRKA, B and C gene were identified in a variety of cancers, including lung adenocarcinoma, colorectal cancer, glioblastoma and acute myeloid leukemia. These genomic rearrangements resulted in sustained cancer cell proliferation. Recent studies indicate that targeting TRK by Entrectinib, pan-TRK, ALK and ROS1 inhibitor, may be effective in the treatment of cancers with TRK rearrangements. However, patients treated with Entrectinib showed resistance early due to NTRK1 harboring acquired mutations, G595R and G667C. ONO-5390556 is a selective pan-TRK inhibitor and shows highly potent anti-tumor activity against TRK rearranged cancers. We evaluated the anti-tumor activity of ONO-5390556 against NTRK1 rearranged cancer cells with harboring mutated G595R and G667C.

Methods: The anti-tumor activity of ONO-5390556 was evaluated in subcutaneous xenograft tumor models of KM12, human colorectal cancer cell lines expressing TPM3-TRKA. ONO-5390556 was administered orally with doses ranging between 0.2 and 2 mg/kg once a day for 14 days. In

vitro cytotoxic activity, cell viability was evaluated by WST-8 in TPM3-TRKA positive cells harboring mutated G595R and G667C. Phosphorylated proteins were detected by Western blotting.

Results: In KM12 xenograft model, treatment with ONO-5390556 at doses of 0.2, 0.6, 2 mg/kg once a day resulted in a dose-dependent inhibition of tumor growth with Tumor Growth Inhibition (TGI) of 44.4, 86.6 and 95.4%, respectively. Both G595R and G667C mutations conferred resistance to Entrectinib, but were sensitive to ONO-5390556 with an IC50 of 2.7 and 0.2 nmol/L, respectively. Interestingly, the inhibitory activity of ONO-5390556 sustained in these mutations compared with wild type (IC50 of 0.4 nmol/L). ONO-5390556 strongly inhibited phosphorylation of the TRKA mutated. Additionally, Erk phosphorylation remained strongly inhibited in KM12 cells harboring mutated G595R.

Conclusion: The oncogenic TRK fusion proteins are attractive therapeutic targets but two mutations acquired resistance has been found in the clinic. ONO-5390556 is a highly potent and selective pan-TRK inhibitor with evidence of an excellent anti-tumor activity not only in cancer cells harboring the TRKA rearrangement but also in the two acquired mutations. These results suggest that ONO-5390556 may overcome Entrectinib-resistance mutations and become a potential role for sequential therapy with first generation TRK inhibitors.

### Monoclonal Antibodies and Antibody-Drug Conjugates

#2955

Herceptarg, a novel heterodimeric biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab, exerts potent anti-tumoral activity.

Ekkehard Moessner,1 Thomas Hofer,1 Inja Waldhauer,1 Werner Scheuer,2 Ralf Hosse,1 Lydia Duerner,1 Mi He,1 Karlheinz Zick,2 Jens Fischer,2 Claudio Sustmann,2 Tina Weinzierl,1 Marina Bacac,1 Christian Gerdes,1 Pablo Umana,1 Christian Klein1. 1 _Roche Pharma Research and Early Development, Schlieren, Switzerland;_ 2 _Roche Pharma Research and Early Development, Penzberg, Germany_.

Introduction: Trastuzumab and pertuzumab are humanized antibodies recognizing different functional HER2 epitopes. Preclinical data demonstrated strong anti-tumoral efficacy when combining them (Scheuer et al., Canc Res, 2009). The CLEOPATRA trial showed that the addition of pertuzumab to trastuzumab + docetaxel strongly improves overall survival of HER2+ metastatic breast cancer patients (Swain et al., NEJM, 2015). Herceptarg is a novel heterodimeric 1+1 biparatopic common light chain IgG1 antibody based on trastuzumab and pertuzumab.

Methods: Using consensus light chains, pertuzumab heavy chain affinity maturation via phage display and knob-into-holes technology, a heterodimeric biparatopic common light chain antibody based on trastuzumab and pertuzumab was generated (Figure 1A). This bispecific antibody was characterized in direct comparison to the respective parental antibodies and their combination in vitro by surface plasmon resonance, proliferation, ADCC and CDC assays and in vivo using the orthotopic KPL-4 breast cancer xenograft model. KPL-4 cells were provided by Prof. Kurebayashi (Kawasaki Medical School, Kurashiki, Japan).

Results: In vitro, Herceptarg has the highest binding affinity for HER2 on cells, mediates potent ADCC activity, comparable or superior growth inhibition activity of breast and gastric cancer cells and CDC superior to the combination of pertuzumab and trastuzumab. In vivo, Herceptarg (10 mg/kg, q1w) mediates anti-tumoral efficacy in the orthotopic KPL-4 breast cancer xenograft model resulting in tumor regression comparable to the combination of trastuzumab and pertuzumab (10 mg/kg, q1w, each), and superior to the respective single agent therapies.

Conclusions: Taken together, these data demonstrate that Herceptarg, as a single IgG1 bispecific antibody is superior (or at least comparable) to the combination of trastuzumab and pertuzumab in vitro and in vivo and may ultimately improve outcome of breast and gastric cancer patients.

#2956

Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties.

Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Julia H. Cochran, Nagendra Chemuturi, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. _Seattle Genetics, Inc., Bothell, WA_.

As antibody-drug conjugates (ADCs) continue to emerge as an important therapeutic modality for the treatment of cancer, there is an increased effort to elucidate critical design parameters and devise improved linker technologies. The impact of drug-to-antibody ratio (DAR) on conjugate plasma pharmacokinetics (PK) is known to be an important attribute, and accelerated clearance induced by high levels of drug loading has served as a barrier to translating increased in vitro potency to in vivo xenografts. We have recently demonstrated that the incorporation of a discrete PEG24 unit into an auristatin drug-linker can greatly diminish the impact of drug loading on ADC PK. In an effort to optimize the antibody-mediated delivery of monomethylauristatin E (MMAE) as a homogeneous DAR 8 conjugate, we prepared a series of MMAE linkers using PEG units of varying lengths to identify constructs that preserve antibody PK properties and provide enhanced in vivo activity. The extent of PEGylation and linker chemistry was found to impact conjugate PK properties, biodistribution, antitumor activity, and tolerability. From that effort, a cleavable MMAE linker incorporating the glucuronide-based trigger, a self-stabilizing maleimide, and 12 PEG units emerged as the optimal design. ADCs prepared with this linker have now undergone further preclinical characterization in activity and toxicology models in which they have demonstrated an increase in therapeutic index relative to other MMAE-based ADCs.

#2957

Generation of a novel, mono-specific, antibody against short Isoform of DCLK1 (DCLK1-S): a novel biomarker of colon carcinogenesis in patients.

Shubhashish Sarkar, Malaney O. Connell, Vsevolod L. Popov, Gurinder Luthra, Pomila Singh. _UT Medical Branch, Galveston, TX_.

Double-Cortin Like Kinase I (DCLK1) marks colon cancer stem cells in mouse colons. Adenomas (Ads)/human colorectal cancers (hCRCs) express low to high levels of DCLK1. We recently reported that the canonical long isoform (DCLK1-L), transcribed from 5'(α)-promoter, is mainly expressed in normal human colons, while a novel short isoform (DCLK1-S), transcribed from IntronV (β)-promoter, is expressed in colonic tumors, with >80% hCRCs expressing significant levels of DCLK1-S (O'Connell et al., Sci Rep, 2015). We additionally reported that CRC patients, expressing high levels of DCLK1-S, had significantly lower overall survival compared to low expressers (O'Connell et al., Sci Rep, 2015), suggesting prognostic/diagnostic value of measuring DCLK1-S levels in Ads/CRCs. Since expression of DCLK1-S by CRCs was recently discovered by us, commercial antibodies (Ab) are not available for specifically identifying DCLK1-S in Ad/Carcinoma samples of patients for diagnostic/prognostic purposes. We succeeded in generating a monospecific polyclonal Ab against the few unique amino-acid sequences of DCLK1-S. By immunohistochemistry and western blot analysis, the Ab was confirmed to be specific for DCLK1-S, with no cross-reactivity for DCLK1-L, and little or no background. The DCLK1-S-Ab did not stain normal human colonic crypts, confirming absence of DCLK1-S in normal colons. In colorectal Ads, DCLK1-S was mainly located in the cytoplasm, with <10% in the nucleus, while in adenocarcinomas (AdCAs), intense staining of DCLK1-S was observed in both cytosol and nucleus, demonstrating a stage-specific increase in staining during Ad-AdCA sequence of colon carcinogenesis, with increasing localization in the nucleus. DCLK1-S-Ab and a DCLK1-L specific Ab was also used to investigate localization of the long vs short isoforms of DCLK1 in cells by immuno-electron microscopy, demonstrating important differences in their sub-cellular localization, which maybe reflective of differential biological effects. The DCLK1-S-Ab is currently being used by us to measure relative expression of DCLK1-S in Ad samples in a retrospective study, in collaboration with colleagues in the departments of pathology and biostatistics, for purposes of examining the usefulness of the Ab for prognostic purposes. Conclusion: We have generated a specific antibody against the novel short isoform of cancer stem cell marker, DCLK1, which will allow us for the first time to detect this novel cancer specific molecular biomarker in colonic tumors for prognostic purposes. Rather than lengthy and cumbersome processing, as required for most molecular markers, the Ab will allow physicians, in any setting, to make informed decisions, such as time to colonoscopy for patients, which is currently based on mostly morphological/pathological parameters, and can result in missing or over scoping patients.

#2958

A cancer-specific monoclonal antibody against podocalyxin developed by CasMab technology inhibited the tumor growth by antibody-dependent cellular cytotoxicity.

Yukinari Kato,1 Satoshi Ogasawara,1 Akiko Kunita,2 Yuki Fujii,1 Mika K. Kaneko1. 1 _Tohoku University, Sendai, Japan;_ 2 _The University of Tokyo, Japan_.

Background: Podocalyxin (PODXL) is a CD34-related sialomucin and a well-known marker of embryonic stem cells. PODXL is expressed in many cancers including brain tumors, breast cancers, and colorectal cancers. Overexpression of PODXL is an independent predictor of progression, metastasis, and poor outcome. In contrast, PODXL is highly expressed in normal cells such as vascular endothelial cells (VECs) or renal podocytes. Although many monoclonal antibodies (mAbs) against PODXL have been established, they bind to both cancer and normal cells. We recently established CasMab technology for developing cancer-specific mAbs, which could target only cancer cells, although those membranous proteins are highly expressed in both cancer and normal cells.

Methods: We immunized mice with human PODXL, which was overexpressed in a glioblastoma cell line. Cancer-specific mAbs were screened using flow cytometry against PODXL-expressing cancer and normal cells. The cancer specificity was confirmed using immunohistochemistry against breast cancer tissues. A mouse-human chimeric anti-PODXL mAb was produced and antibody-dependent cellular cytotoxicity (ADCC) was investigated in vitro. The in vivo efficacy of anti-PODXL mAbs was evaluated using xenograft models of PODXL-expressing cell lines.

Results: One clone of anti-PODXL mAbs, which was established by CasMab technology, reacted with PODXL-expressing many cancer cell lines including breast cancers, brain tumors, and malignant mesotheliomas, whereas it did not bind to VECs in flow cytometry. The anti-PODXL mAb reacted only with PODXL-expressing cancer cells, not with VECs in breast cancer tissues in immunohistochemistry. Furthermore, the anti-PODXL mAb possesses ADCC activity in vitro and anti-tumor effect in vivo.

Conclusion: The anti-PODXL mAb established by CasMab technology could be useful for targeting PODXL in cancer, although PODXL is highly expressed in many normal cells.

#2959

Peptide-linked indolino-benzodiazepine DNA-alkylating agents for use in antibody-drug conjugates (ADCs).

Manami Shizuka, Alan Wilhelm, Katie Archer, Emily Reid, Nicholas C. Yoder, Chen Bai, Nathan Fishkin, Luke Harris, Erin Maloney, Erica Hong, Rui Wu, Olga Ab, Kate Lai, Surina Sikka, Shan Jin, Jan Pinkas, Ravi Chari, Michael L. Miller. _ImmunoGen, Inc., Waltham, MA_.

As part of our efforts to expand the available toolbox of cytotoxic payloads for use in antibody-drug conjugates (ADCs), we have recently described the development of the mono-imine-containing indolino-benzodiazepine dimer, IGN-P1 (Miller, et al., AACR 2014 #652). IGN-P1 was conjugated via a protease-cleavable peptidic L-alanine-L-alanine linker to a folate receptor α (FRα)-binding antibody, and an EGFR-binding antibody. The resulting ADCs demonstrated high in vitro potency (IC50 ~4-100 pM) and specificity towards several cancer cell lines. In vivo, anti-FRα-IGN-P1 induced complete regressions in NCI-H2110 non-small cell lung cancer xenografts following a single dose of 3 µg/kg (linked payload dose, equivalent to 0.18 mg/kg Ab dose).

Here we describe the structure-activity relationship of a number of peptide-linked IGN ADCs, leading to the selection of Ab-IGN-P1 for further advancement. The cytotoxic activities of these peptide-IGN conjugates were evaluated in vitro in cancer cell lines with both high and low target antigen expression. We also assessed bystander activity and identified in vitro and in vivo catabolites. We found that the stereochemistry of the peptide linker was crucial for bystander activity and in vivo efficacy. The IGN-P1 ADC exhibited strong bystander activity, which we believe is dictating the strong antitumor activity in vivo. Treatment of low to moderate antigen-expressing models with Ab-IGN-P1 further resulted in tumor regressions at doses as low as 3-10 µg/kg linked payload.

These data underscore the potential therapeutic benefit of highly active and specific IGN-P1 conjugates, even for the challenging subset of patients with solid tumors where the target antigen is expressed at lower levels.

#2960

**Potent** in vivo **activity of site-specific indolino-benzodiazepine antibody-drug conjugates (ADCs) generated via engineered cysteine conjugation.**

Nicholas C. Yoder, Chen Bai, Alan Wilhelm, Erin K. Maloney, Olga Ab, Emily E. Reid, Manami Shizuka, Daniel Tavares, Rassol Laleau, Xiuxia Sun, Megan E. Bogalhas, Lintao Wang, Jan Pinkas, Michael L. Miller, Ravi Chari, Thomas A. Keating. _ImmunoGen, Inc., Waltham, MA_.

ADCs are widely studied for cancer therapy, with numerous agents in preclinical and clinical development embodying a wide array of targets, linker chemistries, and cytotoxic effector classes. A fourth element of ADC design that has received much attention recently is the site of conjugation of the cytotoxic molecule to the antibody. Historically, lysine- or interchain cysteine-directed conjugation has been used, but site-specific chemistries have become increasingly popular. Our previous evaluation of site-specific and lysine-linked ADCs utilizing a tubulin-acting maytansinoid effector molecule found the lysine-linked version was more active in vivo (Yoder et al., AACR 2015 #645). Here we present a comparison of engineered cysteine site-specific and lysine-linked ADCs utilizing the previously described indolino-benzodiazepine (henceforth referred to as IGN) effector IGN-P1 (Miller et al., AACR 2015 #652) which is designed to undergo proteolytic cleavage upon cell uptake to release a potently cytotoxic DNA alkylator.

We show that HC-S442C mutants of human IgG1 can be conjugated via maleimide chemistry to IGN-P1 to give stable, potent, and homogeneous ADCs with drug to antibody ratio (DAR) of 2. The in vitro potency of engineered-cysteine IGN-P1 ADCs is largely dependent on the DAR of the ADC, although some difference is observed between HC-S442C and other cysteine mutants used for conjugation.

Pharmacokinetic study of C442 maleimide conjugates suggests that the chemical linkage between effector and antibody is stable upon administration in mice. Further, and in contrast to our previous observations utilizing maytansinoid ADCs, the site-specific and Lys-linked IGN-P1 ADCs showed comparable efficacy in vivo on a molar drug basis. This effect was observed across two different antibodies targeting two different cell surface antigens. These results suggest that, in certain cases, site-specific conjugation chemistry can offer comparable activity to heterogeneous conjugation at well-tolerated doses.

#2961

deBouganin conjugated to trastuzumab overcomes multiple mechanisms of T-DM1 drug resistance.

Rachelle L. Dillon,1 Shilpa Chooniedass,1 Arjune Premsukh,1 Gregory P. Adams,2 Joycelyn Entwistle,1 Glen C. MacDonald,1 Jeannick Cizeau1. 1 _Viventia Bio Inc, Winnipeg, Manitoba, Canada;_ 2 _Viventia Bio Inc, Philadelphia, PA_.

DeBouganin is a T-cell epitope-depleted variant of the type I Ribosome Inactivating Protein (RIP) plant toxin Bouganin. DeBouganin binds and deadenylates the sarcin/ricin loop of the 28S subunit of the ribosomal RNA leading to protein synthesis inhibition and apoptosis. To demonstrate the potential advantages of deBouganin over current small molecule payloads, deBouganin was randomly chemically conjugated to trastuzumab with a DAR of approximately 2 and compared to T-DM1 both in vitro and in vivo. The trastuzumab-deBouganin conjugate (T-deB) demonstrated a tighter IC50 range of killing and an overall greater potency in vitro against most cells lines with high levels of Her2 expression as compared to T-DM1. In addition, unlike T-DM1, T-deB was unaffected by inhibitors of multidrug resistance (MDR) and overexpression of Bcl-2 family members. Moreover, T-deB potency was unchanged by Her2-Her3 dimerization in the presence of heregulin. Contrary to T-DM1 which showed only minimal cytotoxicity, T-deB was highly potent in vitro against tumor cells with cancer stem cell (CSC) properties by preventing the formation of tumorspheres. Furthermore, in a BT-474 xenograft study, T-deB was more efficacious than T-DM1 resulting in increased survival of the T-deB treated mice. Overall, the results demonstrate the potency and efficacy of deBouganin and emphasize the importance of using payloads with different MOAs. The data suggest that deBouganin used alone or in combination with other payloads could be a highly effective cancer therapeutic that would provide prolonged clinical benefit.

#2962

Development of the LCMS bioanalysis for clinical pharmacokinetics of antibody drugs using Fab-selective limited proteolysis (nSMOL).

Takashi Shimada,1 Noriko Iwamoto,1 Sho Saeki,2 Ji-ichiro Sasaki,3 Taka-Aki Sato,1 Akinobu Hamada4. 1 _SHIMADZU Corporation, Tokyo, Japan;_ 2 _Kumamoto University, Kumamoto, Japan;_ 3 _Kitasato University, Kanagawa, Japan;_ 4 _National Cancer Center, Tokyo, Japan_.

Monoclonal antibody drugs (mAbs) have become a major therapeutic strategy in cancer care. The approved mAbs have been about 40 items, and a lot of clinical trials have been now performing in the world. For precise cancer therapy, overall pharmacokinetic (PK) information in blood or tumor tissue will become the mainstream indicators for many clinicians. Therefore, versatile and robust analytical technologies for the direct detection of antibody will be important outcome in clinical applications. The bioanalysis of mAbs using LCMS is a significant platform for the selective quantitation or the feasibility of the multiplex assays for combination therapy. For regulated LCMS bioanalysis independent of a variety of mAbs or biological taxonomy sources, we have focused on the two features: antibody structure-indicated analysis, and complementarity-determining region (CDR)-targeting quantitation.

Using the previous LCMS bioanalysis by general proteomic method, the signature proteolytic peptides of the mAb should be quantified from the massive analyte and residual protease because immunoglobulin molecules are second major protein in plasma proteome. The excess peptides will be cause to decrease the selectivity and sensitivity. To address this issues, we have recently reported the novel method for the selective proteolysis on the Fab by limiting protease access to antibodies, which have named nano-surface and molecular-orientation limited (nSMOL) proteolysis. nSMOL chemistry is designed based on the unique features: (1) protease reaction on nanoparticle surface; (2) Fc immobilization onto the resin, such that the Fab is oriented outward to the solution; and (3) Fab-selective limited proteolysis by making use the difference of the protease nanoparticle diameter (200 nm) and the Ab resin pore size (100 nm). nSMOL has made it possible to minimize the sample complexity while the maintaining the sequence specificity in CDR.

We are now performing the bioanalytical validation for several mAbs in accordance with the Guideline on Bioanalytical Method Validation in Pharmaceutical Development. The results of validation study show that nSMOL is possible to cover the clinical dose, and more accurate and sensitive than ELISA and conventional proteomic approaches. For clinical feasibility test, we measured the plasma concentration of Bevacizumab in patients with recurrent non-small cell lung cancer according to nSMOL protocols.

nSMOL proteolysis coupled with LCMS bioanalysis is a novel method for the clinical PK independent of a variety of monoclonal antibodies, and have confirmed the clinical usability by the fully validated bioanalysis. This analytical approach will be applicable for many new mAbs and biosimilar drugs. Furthermore, the clinical trials based on LCMS may be expected to aid acceleration of the development of many biopharmaceuticals.

#2963

VB7-756: a Her2-specific diabody armed with deBouganin, a plant toxin with a distinct MOA.

Shilpa Chooniedass,1 Rachelle L. Dillon,1 Arjune Premsukh,1 Joycelyn Entwistle,1 Gregory P. Adams,2 Peter J. Hudson,3 Glen C. MacDonald,1 Jeannick Cizeau1. 1 _Viventia Bio Inc, Winnipeg, Manitoba, Canada;_ 2 _Viventia Bio Inc, Philadelphia, PA;_ 3 _Avipep Pty Ltd, Parkville, Australia_.

VB7-756 is Targeted Protein Therapeutic (TPT) comprised of a de-immunized form of bouganin (deBouganin), a potent, plant-derived, type I ribosome-inactivating protein (RIP), genetically linked to the C6.5 anti-Her2 diabody via a furin protease sensitive linker. To engineer the optimal diabody TPT format, several constructs were generated to assess the best diabody-deBouganin orientation. All constructs were expressed as a soluble protein in E. coli supernatant and compared with respect to expression level, stability and potency. The optimal configuration consisted of deBouganin genetically linked to the N-terminus of the VH-VL diabody via a furin protease-sensitive linker and was termed VB7-756. VB7-756 potency was analyzed against a panel of breast cancer cell lines with disparate levels of Her-2 expression and compared to that of Trastuzumab chemically linked to either DM1 (T-DM1) or MMAE (T-MMAE). Overall, VB7-756 was more potent than T-DM1 and T-MMAE with high Her-2 expressing tumor cell lines. In contrast to T-DM1, VB7-756 potency was unaffected by the Her2-Her3 dimerization mediated by heregulin. As opposed to T-DM1 and T-MMAE, which showed only minimal cytoxicity, VB7-756 was highly potent in vitro against tumor cells with cancer stem cell properties. To further differentiate the RIP mechanism of action of deBouganin from tubulin inhibitor reagents, tumor cells that escaped T-DM1 and T-MMAE treatment were incubated in the presence of VB7-756. Results revealed that, VB7-756 was cytotoxic against cancer cells surviving T-DM1 or T-MMAE treatment suggesting that deBouganin can overcome mechanisms of resistance developed by small molecule agents. Moreover, VB7-756 was also cytotoxic against tumor initiating cancer cells evading T-DM1 or T-MMAE toxicity by preventing tumorosphere formation. Overall these results demonstrate that deBouganin's distinct MOA could overcome mechanisms of resistance affecting the efficacy of small molecule drugs.

#2964

An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies.

James R. Prudent, Chad A. Hall, David J. Marshall, John Murphy, Scott Harried. _Centrose, Madison, WI_.

Purpose:

CD20 is known to be a therapeutic antibody drug target as it is expressed on the surface of most B-cell neoplasms. Here, we assessed the anti-tumor activity of EDC9, a novel extracellular drug conjugate composed of Rituximab (a well know and FDA approved anti-CD20 monoclonal antibody) conjugated to a steroidal glycoside via a long, flexible and stable linker.

Experimental Design:

The anti-cancer activity and safety of EDC9 were examined and compared to Rituximab using in vitro and in vivo techniques.

Results:

We found that EDC9 showed picomolar cytotoxic activity that was independent of effector functions and exhibited cytotoxic activity through a mechanism that was dependent on CD20 expression, as cells not expressing CD20 were resistant and Rituximab alone could compete with EDC9, rendering it inactive. After 72 hours of EDC9 treatment at levels between 100 and 200 picomolar, no cells were determined to be viable by CellTiter-Glo or high definition phase contrast imaging. Importantly, when compared on the basis of toxicity to PBMC, EDC9 was found not to be more toxic than Rituximab and the activity of EDC9 was dependent on specific steroid chemistry. In a tumor xenograft model, EDC9 provided complete long-term remission of diffuse large B-cell human lymphoma line SU-DHL-8 tumors, while Rituximab alone did not.

Conclusion:

These results support efforts to further evaluate EDC9 (a novel CD20 specific antibody drug conjugate) and may lead to phase I clinical trials in patients with certain B-cell related malignancies.

#2965

In vitro **and** in vivo **activity of a site-specific SeriMab antibody-drug conjugate (ADC) using an indolino-benzodiazepine DNA-alkylating agent.**

Dilrukshi Vitharana, Alan Wilhelm, Luke Harris, Katie Archer, Manami Shizuka, Erin Maloney, Olga Ab, Rassol Laleau, Xiuxia Sun, Jan Pinkas, Michael Miller, Ravi Chari, Thomas Keating, Nathan Fishkin. _ImmunoGen, Inc, Waltham, MA_.

Previously we have described the characterization of a proprietary class of indolino-benzodiazepine dimers, IGNs, with high potency against many cancer lines. Antibody-drug conjugates (ADCs) made with mono-imine containing IGNs were designed to only alkylate DNA and not cause DNA crosslinking. ADCs with the IGN linked via lysine residues of the antibody were shown to be highly potent and antigen specific. Here we apply our SeriMab site-specific technology platform, which employs N-terminal conjugatable aldehydes derived from oxidation of a serine residue, to link an IGN molecule to the antibody using a peptide linker. This ADC (SeriMab-IGN-P1) has exactly 2 IGN molecules per antibody conjugated to the N-termini of the heavy chains, as determined by MS analysis.

Serimab-IGN-P1 was found to maintain binding to the target antigen with comparable affinity to the unconjugated antibody by FACS analysis (Kd = 300 pM). The conjugate was highly potent (IC50 = 4 pM) against a cancer cell line with high target expression (3 x 106 antibodies bound per cell) as well as a cell line with much lower target expression (2 x 104 antibodies bound per cell, IC50 = 22 pM). SeriMab-IGN-P1 also demonstrated strong bystander killing activity against proximal target-negative cancer cells, likely due to a cell permeable metabolite identified in cells expressing the target antigen.

The unique oxime linkage in SeriMab-IGN-P1 was found to be stable in circulation in mice for 3 days, as determined by affinity capture LC-MS; the conjugate species with 2 IGNs per antibody remained predominant over this period with an estimated t1/2 of >10 days for payload release.

In vivo, SeriMab-IGN-P1 was found to be effective against human lung cancer xenografts (NCI-H2110), with anti-tumor activity observed at single doses as low as 2 μg/kg (payload dose), and complete responses observed at 5 μg/kg. This conjugate was also well tolerated in CD-1 mice at doses significantly higher than the minimum effective dose that produced tumor regression.

These data are highly encouraging for new ADC therapies using the SeriMab site-specific conjugation platform with potent DNA-acting payloads.

#2966

Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity.

Fu Li, Martha Anderson, Joshua Hunter, Jaime Miyamoto, Michelle Ulrich, Ana Kostic, Che-Leung Law, Django Sussman. _Seattle Genetics, Bothell, WA_.

SGN-LIV1A is an antibody-drug conjugate (ADC) currently being evaluated in a phase 1 clinical trial for metastatic breast cancer. SGN-LIV1A consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), conjugated to the anti-LIV-1 humanized monoclonal antibody hLIV22. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. We have previously shown that as a single agent, SGN-LIV1A displays target specific internalization and cytotoxic activity against a breast cancer cell line in vitro and also demonstrates antitumor activity in in vivo preclinical xenograft models with significant delay of tumor growth. We report here additive and synergistic effects when combining SGN-LIV1A with current chemotherapeutic modalities used in the treatment of metastatic breast cancer. Specifically, we show synergy between SGN-LIV1A in combination with either doxorubicin or Abraxane in MCF-7 breast cancer tumor model. In addition, we show additive effects when carboplatin or protein kinase inhibitors are dosed in combination with SGN-LIV1A in this tumor model. These findings support further evaluation and development of SGN-LIV1A in combination with standard of care chemotherapeutic agents for the treatment of breast cancer.

#2967

In vitro **and** in vivo **activity of site-specific antibody-drug conjugates (ADCs) with 2 and 4 maytansinoid molecules per antibody prepared through conjugation to SeriMabs (N-terminal serine engineered Abs).**

Luke Harris, Leanne Lanieri, Jose Ponte, Erin Maloney, Laura Bartle, Olga Ab, Juliet Costoplus, Lingyun Rui, Jan Pinkas, Ravi Chari, Thomas Keating, Daniel Tavares, Nathan Fishkin. _ImmunoGen, Waltham, MA_.

Site-specific attachment of cell-killing agents to antibodies directed against tumor-associated antigens has continued to be an active area of innovation in the field of ADCs. Most reports focus on homogeneous ADCs that have a DAR (cytotoxic molecules per antibody ratio) of 2. Here we describe the preparation, biochemical characterization, and biological evaluation of ADCs made through conjugation of maytansinoids (DM1, DM4) to aldehydes derived from chemically oxidized N-terminal serines (SeriMab) engineered onto the antibody heavy chain (2 DAR) or both light and heavy chain simultaneously (4 DAR). ADCs prepared with a non-cleavable linker or a cleavable disulfide linker were homogeneous 2 or 4 DAR by MS analysis, and were produced in high yield with a monomer content of >98%. Despite conjugation at the N-termini of both the light and heavy chain variable regions, FACS analysis showed the 4 DAR SeriMab conjugates maintained binding to the target antigen.

The ADCs showed antigen-specific potency in vitro on a panel of target-expressing cancer cell lines. In the disulfide cleavable linker series, the 2 DAR SeriMab conjugate was 2-5 fold less active than lysine-conjugated Ab-SPDB-DM4 (3.4 DAR), while the 4 DAR SeriMab conjugate was comparably active on an antibody basis. The SeriMab conjugates also displayed strong bystander killing. Surprisingly, in the non-cleavable linker series, the 2 DAR SeriMab conjugate was up to 17-fold more active (depending on cell line) than lysine-conjugated Ab-SMCC-DM1 (3.5 DAR), and the 4 DAR SeriMab conjugate was up to 100-fold more potent than the SMCC-DM1 conjugate on an antibody concentration basis. In a P-gp-positive multi-drug resistant cell line, the non-cleavable 4 DAR SeriMab-maytansinoid conjugate was highly active while the 2 DAR SeriMab ADCs and lysine-conjugated maytansinoid ADCs were >100-fold less potent.

The unique oxime bond formed with the non-cleavable SeriMab-maytansinoid conjugate was found to be stable in circulation in mice for >3 days as assayed by affinity capture LC-MS. Polar carboxylic acid containing metabolites were identified which may lead to high cellular retention of maytansinoid species in cancer cells, yielding higher in vitro potency than lysine-linked ADCs in some cell lines.

The in vivo anti-tumor activity of disulfide cleavable 2 and 4 DAR SeriMab-DM4 ADCs was evaluated in a clinically relevant cancer xenograft model. The 4 DAR conjugate was active at 60 μg/kg (maytansinoid payload dose) and was more active than the 2 DAR conjugate at this same payload dose.

Using the SeriMab conjugation platform we show that in vitro and in vivo activity of site-specific ADCs can be dependent on amount of cytotoxic agent attached per antibody.

#2968

**A novel bispecific CD3/CDH19 antibody construct (CDH19 BiTE) directs potent killing of melanoma cells** in vitro **and** in vivo **and is enhanced by blockade of PD-L1.**

Gordon E. Moody, Jodi Moriguchi, Shyun Li, Fei Lee, Brendon Frank, Amy Gilbert, Ryan Case, Khue Dang, Beth Hinkle, Suzanne Coberly, James Rottman, Kim Merriam, Julie Bailis, Pedro J. Beltran. _Amgen, Inc., Thousand Oaks, CA_.

CDH19 is an unconventional type 2 cadherin widely expressed in malignant melanoma with limited expression in normal tissues. RNAseq and immunohistochemical analysis of

CDH19 in human samples confirmed its over-expression in >60% of melanoma, whereas normal expression primarily occurred in tissues derived from the neural crest such as nerve fibers and autonomic ganglia. CD3 based bi-specific T-cell engagers (BiTEs) were used to determine if redirected lysis of T-cells against CDH19 would drive anti-tumor efficacy. In vitro, the CDH19 BiTEs were high affinity binders to both human and cyno CDH19, and were able to induce specific T-cell activation and cytotoxicity against a panel of melanoma cell lines. In addition, soluble CDH19 levels were detected in human serum and the effects of BiTE cytotoxicity toward melanoma cells in the presence of soluble CDH19 was investigated. In vivo studies were conducted to confirm the specificity and activity of CDH19 BiTEs in xenograft models of melanoma. The CDH19 BiTE AMG-CDH19X was able to cause tumor growth inhibition in models expressing as few as 250 receptors per cell, and inhibition of tumor growth was enhanced by the addition of a blocking antibody against PD-L1. Immunohistochemical analysis of post-treatment xenograft samples suggested that anti-PD-L1 the persistence of tumor reactive T cells, and provided rationale for combining a BiTE against CDH19 with a PD-1 or PD-L1 blocking antibody in melanoma. In summary, targeting CDH19 presents a promising novel opportunity for BiTEs in the treatment of melanoma, both alone and in combination with current standard of care.

#2970

**MEDI4276, a HER2-targeting antibody tubulysin conjugate, displays potent in** vitro **and** in vivo **activity in preclinical studies.**

John Li,1 Dorin Toader,1 Samuel R. Perry,1 Vanessa Muniz-Medina,1 Leslie Wetzel,1 Marlon C. Rebelatto,1 Mary Jane Masson Hinrichs,1 Ryan Fleming,1 Binyam Bezabeh,1 Pamela Thompson,1 Nazzareno Dimasi,1 Brandon Lam,1 Xian-Qing Yu,1 Changshou Gao,1 Rakesh Dixit,1 Steven Coats,1 Jane Osbourn,2 Herren Wu1. 1 _MedImmune LLC, Gaithersburg, MD;_ 2 _MedImmune LLC, Cambridge, United Kingdom_.

Antibody drug conjugates (ADCs) combine the specificity of antibodies with the potent cytotoxicity of small molecule drugs and have shown to provide therapeutic options for various cancers. We report herein the discovery of a HER2-targeting ADC MEDI4276 that showed potent cell killing activity in vitro in cancer cell lines that express the HER2 receptor. The observed in vitro activity translated into in vivo tumor growth inhibition in various xenograft mouse models. MEDI4276 is a homogeneous molecule with precise control of drug loading following site specific conjugation of a cytotoxic drug. The drug in MEDI4276 is MMETA, a fully synthetic analog of the tubulysin family that showed pM potency in a panel of cancer cell lines. MMETA was conjugated to the antibody via engineered cysteines with a maleimide-bearing mc-Lys protease cleavable linker. The antibody in MEDI4276 is a bivalent biparatopic antibody targeting two distinct non-overlapping epitopes on HER2 that leads to antibody-receptor clustering following binding and thus promoting internalization, lysosomal trafficking and degradation. The combination of enhanced internalization and potent cytotoxic drug allows for this ADC to kill tumor cell populations with a broader range of HER2 expression. Preclinical studies showed that MEDI4276 induced tumor regression in HER2-positive tumor models that had developed acquired resistance to T-DM1 and in a number of models with lower HER2 expression that are refractory to T-DM1 treatment. Overall, our findings underscore the potential application of MEDI4276 to treat a large patient population that is ineligible for or relapsed/refractory to current HER2-targeted therapies. MEDI4276 is currently being investigated in a Phase I clinical trial.

#2971

Optides (optimized knottin peptides) computationally designed to target the oncogenic HIPPO pathway.

Zachary R. Crook,1 Philip Bradley,1 Chris King,2 Andrew J. Mhyre,1 David Baker,2 James M. Olson1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

The HIPPO pathway plays a critical role in contact inhibition, a pathway that is commonly dysregulated in many human cancers (including liver, colon, ovarian, and lung) and which relies on the intranuclear interaction of the transcriptional coactivator YAP and the transcription factor TEAD(1-4). This pathway also plays a crucial role in recovery from injury; for example, its regulated repression allows hepatocytes to divide and replace tissue lost to a partial hepatectomy, after which its activation suppresses cell growth and prevents tissue overgrowth. While small molecule enzyme inhibitors have proven to be a revelation in cancer therapy, cell growth signaling via protein-protein interactions has proven much more difficult to drug. While Antibodies can be effective in targeting extracellular or cell surface epitopes, intracellular targets, such as the interaction between YAP and TEAD, are not amenable to antibody-based therapeutics. Optides are small disulfide-knotted peptides (knottins) and serve to bridge these capabilities; they are large enough to interfere with protein-protein interactions, but small enough to penetrate into the cytosol. Examples include imperatoxin, an activator of mitochondrial ryanodine receptors, and Tumor Paint, which contains an optimized variant of chlorotoxin conjugated to a fluorescent probe and is capable of accumulating in a wide range of tumor types.

To test whether Optides can abrogate oncogenic signaling mediated by protein-protein interactions, we created libraries of computationally designed candidates to target the TEAD/YAP interface. The library is expressed on the surface of mammalian cells, chosen for the improved fidelity of disulfide bridge connectivity observed in the mammalian secretory pathway as compared to that found in yeast. By repetitive screening against soluble TEAD protein, we are optimizing the pool of candidates for targeting TEAD. The lead Optides will be characterized for their ability to reduce YAP-TEAD interaction, and to impair YAP-mediated cell growth. Owing to the wide variety of knottin scaffolds, both natural and in silico designed, this flexible technology could be applied to other targets in order to impair oncogenic protein-protein interactions.

#2972

Development and characterization of synthetic antibodies with a human framework that directly link metabolism to immune system in cancer cells.

Jelena Tomic,1 Megan McLaughlin,1 Ron Geyer,2 Sachdev Sidhu,1 Jason Moffat1. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _University of Saskatchewan, Saskatoon, Saskatchewan, Canada_.

Deregulated cellular energetics and evasion of immune system are two hallmark features of cancer cells. In fact, increased uptake of glucose and glutamine by cancer cells elevates flux of a minor glucose pathway known as the hexosamine biosynthetic pathway (HBP), resulting in alteration of glycosylation. Addition of sugar moieties to proteins plays an important role in sensing cellular metabolism, growth factors, signaling molecules, nutrient flux and stress. Since many of these processes are de-regulated in tumorigenesis, global alterations in glycosylation via HBP flux are likely to play an important role in cancer. We hypothesize that cancer cells can "sense" stress through the HBP pathway, and, consequently, reprogram the cell surface and adapt to tougher conditions. Neo-epitopes on the cell surface that emerge from tuning the HBP pathway activity may be prime candidates as readouts that reliably report the status of metabolic reprogramming.

In order to advance the understanding of metabolic reprogramming from the perspective of the cell surface, we combined genetics and advanced protein engineering using phage-displayed synthetic human antibody libraries to generate recombinant antibodies (rAbs) that respond to changes in the activity of the HBP. Our method for discovery of surface and nutrient-associated protein sensors (SNAPS) has allowed us to identify multiple human synthetic rAbs whose activity is potentiated under different metabolic states. More specifically, these rAbs recognize cell surface features that discriminate cancer cells with increased HBP flux and effectively recruit components of the immune system to initiate tumor cell lysis. Our findings suggest that the nutrient-dependent cell surface epitopes may have functional roles in defining cancer cell identity and may link multiple cancer hallmarks.

#2973

CD269 - A promising target for amanitin based ADCs.

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

Antitumor activity of monoclonal antibodies can be dramatically enhanced by conjugation to toxic small molecules. Beside the recent approval of Kadcyla (T-DM1) and Adcetris (SGN-35) more than 30 antibody-drug conjugates (ADC) have entered clinical trials, promising to strengthen the therapeutic capabilities for cancer treatment in the next decade. Surprisingly most ADCs are based on one of few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansines. Toxins that operate through such a mechanism could suffer from limited activity in different cancer indications and in cells expressing resistance mechanisms. Accordingly the use of new drugs that function via alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. Heidelberg Pharma focuses on Amanitin, the most well-known toxin of the amatoxin family. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription at very low concentrations.

In the current study, in vitro and in vivo Data of Amanitin-ADCs targeting CD269 (B cell maturation antigen) are presented. CD269 is expressed on cells of the B cell lineage, predominantly on plasma blasts and plasma cells. It is not expressed on naïve B cells, germinal center B cells and memory B-cells (Darce et al. (2007) J Immunol 179:7276-7286). CD269 is highly expressed on malignant plasma cells like multiple myeloma, a B cell non Hodgkin lymphoma of the bone marrow (Novak et al. (2004) Blood 103:689-94). Since multiple myeloma is a usually incurable malignancy of plasma cells, new therapies are urgently needed. Using ADCs in the cure of multiple myeloma could be a promising approach, especially by using a toxin whose mode of action was not applied before, like amanitin based ADCs.

In vitro data of anti-CD269-amanitin ADC showed cytotoxicity on CD269 positive cell lines in picomolar range, while up to micromolar concentrations, no cytotoxic activity on CD269 negative cells was observed. In mouse xenograft models, anti-CD269-amanitin showed clear anti-tumorigenic potential. A comprehensive data package will be presented.

#2974

Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer.

Scott D. Collins,1 Parmita Saxena,1 Xiao Y. Li,1 Yeonju Shim,1 Lance Ostrom,1 Nicholas C. Yoder,2 Kalli C. Catcott,2 Molly A. McShea,2 Xiuxia Sun,2 Sanela Bilic,1 William R. Tschantz,1 Meghan Flaherty,1 Keith Mansfield,1 Tiancen Hu,1 Vladimir Capka,1 Markus Kurz,3 Ivana Liric Rajlic,1 Anne Serdakowski London,1 Duc Nguyen,1 Rebecca Mosher,1 Matthew J. Meyer,1 Aaron Bourret,1 Jamal Saeh,1 Scott Cameron,1 Emma Lees,1 Carl U. Bialucha1. 1 _Novartis Institutes for Biomedical Research, Cambridge, MA;_ 2 _ImmunoGen Inc, Waltham, MA;_ 3 _Novartis Pharmaceuticals, Basel, Switzerland_.

In an attempt to mine tumor versus normal mRNA expression datasets for novel tumor antigens, we identified the Cadherin-6 (CDH6) gene as frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. CDH6, also known as K-(kidney)-cadherin, is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach.

CDH6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the maytansinoid payload DM4. The antibody component of CDH6-ADC was selected from a panel of anti-CDH6 antibodies based on a multi-factorial lead selection campaign incorporating readouts of internalization propensity, in vitro cytotoxicity, as well as in vivo PK and efficacy across multiple linker-payload formats. CDH6-ADC features potent, target-dependent in vivo activity consistent with the mechanism of the anti-mitotic, tubulin-targeting sulfo-SPDB-DM4 linker-payload combination used. Specifically, treatment of CDH6-expressing ovarian cancer xenograft models with CDH6-ADC results in the time-dependent generation of intra-tumoral ADC catabolites and concomitant induction of phospho-histone H3 and cleaved caspase-3 - markers of G2/M arrest and apoptosis, respectively. CDH6-ADC induces durable tumor regressions at clinically relevant exposures in multiple human patient-derived tumor xenografts (PDX) across both ovarian and renal cancer lineages.

To gain a more thorough understanding of CDH6-ADC activity in pre-clinical models of human ovarian cancer and identify potential molecular correlates for patient stratification, we profiled CDH6-ADC in a PDX clinical trial or PCT comprising 31 individual PDX models. In this unselected population, treatment with CDH6-ADC resulted in robust anti-tumor activity. Integration of PDX response data with CDH6 target expression in both the PDX models and human clinical samples indicate CDH6 expression patterns consistent with in vivo activity are found in a substantial fraction of ovarian, renal and cholangiocarcinoma patients. Together, the encouraging pre-clinical efficacy and tolerability data support the clinical evaluation of CDH6-ADC.

#2975

Development of a probody drug conjugate (PDC) targeting CD71 for the treatment of solid tumors and lymphomas.

Shweta Singh, Amy DuPage, Annie Yang Weaver, Jason Sagert, Clayton White, Michael Krimm, Yuanhui Huang, Linnea Diep, Kim Tipton, Shouchun Liu, Jennifer Richardson, W. Michael Kavanaugh, Jonathan A. Terrett, Luc R. Desnoyers. _CytomX Therapeutics, South San Francisco, CA_.

The targets of Antibody Drug Conjugates (ADCs) have typically been selected by identifying transmembrane antigens that are highly expressed in tumors but are low or absent in normal tissues. The number of potential ADC targets meeting these requirements is limited, either because expression in tumors is not high enough for optimal efficacy, or because expression in normal tissues is too high, leading to toxicity. Probody (TM) therapeutics are antibody prodrugs designed to remain inactive until proteolytically activated in the tumor microenvironment. Probody technology therefore has the potential to enable targeting of more desirable tumor antigens with higher, more persistent and more homogeneous expression in tumors, while limiting toxicity due to interaction with these antigens in normal tissues.

CD71 (transferrin receptor) is an example of a highly desirable ADC target, because of its well-characterized ability to efficiently internalize and deliver an ADC payload intracellularly. Further, CD71 is expressed at homogeneously high levels (3+ expression by IHC) in almost all tumor types, including in metastatic disease. However, because CD71 is also expressed on multiple normal cell types, including many progenitor hematological cells, we reasoned that a CD71-targeted ADC would be challenging to develop. To enable targeting of CD71, we have developed an anti-CD71 Probody Drug Conjugate (PDC) CX-2005, which can be activated by multiple proteases in the tumor microenvironment, but which remains in a relatively inactive form while in circulation and in normal tissues. CX-2005 produces complete tumor regressions at therapeutic doses in mouse models of lymphoma, breast cancer and lung cancer. Consistent with our hypothesis that it would be difficult to develop an anti-CD71 ADC, treatment of cynomolgus monkeys with an anti-CD71 ADC at doses that were efficacious in mouse tumor models caused life-threatening depletion of CD71-expressing hematopoietic cells, including neutrophils, lymphocytes and RBCs. In contrast, these toxicities were not observed in monkeys treated with the same dose of the anti-CD71 PDC, consistent with the Probody therapeutic avoiding interaction with these normal cells.

Our data demonstrate that, in preclinical studies, Probody drug conjugates can safely and effectively target attractive tumor antigens like CD71 which have been difficult to effectively approach with traditional ADCs due to their expression on critical normal tissues. Further, our data support the development of Probody therapeutics directed against CD71 in multiple different cancers.

PROBODY is a trademark of CytomX Therapeutics, Inc.

#2976

Engineering next-generation anti-CD25 immunotoxins with improved cytotoxic activity and low immunogenicity.

Gilad Kaplan, Fred Lee, Ira Pastan. _National Cancer Institute, NIH, Bethesda, MD_.

Objective: CD25 is expressed on many T cell malignancies, including adult T cell leukemia, and is a target for cancer therapy. Immunotoxins against CD25+ cells containing a portion of Pseudomonas exotoxin A (PE38) show extremely high cytotoxic activity. Our lab has previously created a de-immunized PE immunotoxin in which toxin domain II was removed. Removal of domain II confers two major advantages: i) the immunogenic sequences found in domain II are removed, generating a less immunogenic toxin; ii) the non-specific toxicity of domain II-truncated toxins is much lower, allowing much higher doses of immunotoxin to be given safely (7-10-fold higher safe dosage). However, removal of domain II, which does not affect or enhances the cytotoxic activity of immunotoxins targeting the B cell marker CD22 or the mesothelioma marker mesothelin, lowers the activity of anti-CD25 immunotoxins 30-fold. Because lowering toxin immunogenicity and animal toxicity are critical for the success of immunotoxins in a clinical setting, we have engineered domain II truncated immunotoxins with improved activity against CD25+ cells using a novel adaptation of an established screening system.

Methods: Eighteen domain II truncation mutants were designed based upon the crystal structure of the toxin. To bypass the resource intensive protein refolding of standard immunotoxins, these domain II mutants were expressed as fusions with the ZZ protein, which is a small (13.6kD) derivative of protein A. Fusion to ZZ allowed easy production of these ZZ-domain II mutant fusion proteins in the E. coli periplasm and fast purification using IgG coated beads. The purified ZZ-domain II mutant toxins were then bound to anti-CD25 IgG by mixing (the ZZ-domain binds human Fc) and tested for cytotoxic activity on the CD25+ T-cell leukemia HUT-102 cell line. The most active domain II mutants were made into standard recombinant immunotoxins and retested.

Results: Of the 18 tested ZZ-domain II mutants, 6 were completely inactive, 8 were active at near parental levels, and 4 were as active as the parental toxin. The 4 high-activity and 2 structurally-interesting, medium-activity mutants were then produced as standard recombinant immunotoxins and retested for cytotoxic activity. Two of the 4 high-activity mutants showed a 2-4-fold increase in activity compared to parental domain II truncated immunotoxin. While still being 8-fold less active than immunotoxins containing domain II (PE38), these high-activity mutant immunotoxins will most likely have lower immunogenicity than toxins with PE38 and much less non-specific toxicity, allowing higher doses and more treatment cycles to be given. Our engineered high-activity domain II mutant immunotoxins are improved candidates for treating CD25+ cancers and for eliminating regulatory immunosuppressive T cells.

#2978

Characterization of novel antibodies for understanding MUC16 cleavage and its pathological implications in cancer.

Abhijit Aithal,1 Wade Junker,2 Sukhwinder Kaur,1 Srustidhar Das,1 Prakash Kshirsagar,1 Satyanarayana Rachagani,1 Kavita Mallya,1 Maneesh Jain,1 Surinder K. Batra1. 1 _University of Nebraska Medical CL, Omaha, NE;_ 2 _Sanguine Diagnostics and Therapeutics Inc., Omaha, NE_.

Introduction: MUC16 (CA125) is a type I transmembrane mucin that exhibits differential expression in multiple malignancies including ovarian, pancreatic and breast cancers. It was initially predicted to undergo cleavage in the penultimate and/or last sea urchin, enterokinase, and agrin (SEA) domain (among a total of 56) to generate circulating CA125. However, our recent studies demonstrated the presence of a unique cleavage site that involves 12 amino acid residues proximal to transmembrane segment. Upon ectopic overexpression, this cleaved intracellular C-terminal (C-ter) fragment promotes tumor metastasis, invasion and upregulates cancer stem cell inducing genes in cancer cells. However, physiological presence of C-ter cleavage and its role in cancer remains obscure since most available antibodies recognize the N-terminal tandem repeat domain of MUC16. The present study aims to develop novel antibodies against MUC16 C-ter fragment to dissect its role in tumor development and progression.

Methods: Monoclonal antibodies (mAbs) were generated by immunizing mice with purified C-ter (114 amino acid) domain of MUC16. MUC16 reactive hybridomas were screened by indirect ELISA. Positive hybridomas were isolated, cloned and the antibodies were purified from culture supernatant. The specificity of the newly generated anti-MUC16 antibodies was determined using immunoprecipitation, immunoblot, confocal, and FACS analysis. Finally, the reactivity of novel antibodies was evaluated on mouse and human tumor tissues by immunohistochemistry.

Results: Two mAbs (3H1 and 5E6) showed specific reactivity to recombinant MUC16 C-ter fragment in ELISA. Stringent checkpoints were included such as expression analyses in MUC16 expressing and non-expressing cell lines, histological reactivity in normal and MUC16 expressing cancer tissues and MUC16 C-ter over expression constructs to delineate the highly specific nature of these antibodies. Epitope mapping studies using MUC16 C-ter constructs transfected into HEK293T cells identified the epitope to most probably lie in a 61 amino acid region just proximal to transmembrane domain. Interestingly, in immunoblot and immunoprecipitation analysis, these mAbs identified a unique 20kDa cleavage product in some but not all MUC16 expressing cancer cell lines. This suggests the existence of a cell-context dependent cleavage mechanism for MUC16. Both mAbs preferentially recognize intracellular antigen as compared to cell surface exposed epitope. Further, these exhibited strong reactivity in pancreatic and ovarian cancer tissues in the same regions that stained positive for mAb CA125.

Conclusions: The novel mAbs exhibit unique specificity towards cleaved MUC16 C-ter in cell lysates and tissues and hence can be valuable reagents for studying the mechanisms and role of MUC16 cleavage in cancer cells.

#2979

Dual targeting of HER3 and PIK3CA has potent anti-tumor effects in pre-clinical models of HNSCC.

Nayel Khan,1 Kara S. Davis,1 Neal Godse,1 Carolyn Kemp,1 Sucheta Kulkarni,1 Diego Alvarado,2 Theresa LaVallee,2 Jennifer R. Grandis,1 Umamaheswar Duvvuri1. 1 _University of Pittsburgh Medical Center, Pittsburgh, PA;_ 2 _Kolltan Pharmaceuticals, New Haven, CT_.

Background: The phosphoinositide 3-kinase (PI3K) pathway is a frequently mutated oncogenic pathway in head and neck squamous cell carcinoma (HNSCC). This pathway is often further activated by dysregulation of receptor tyrosine kinases, such as the epidermal growth factor receptor family. HER3 has a direct phosphorylation site with the regulatory subunit of PIK3CA, and is being investigated as a potential co-target to increase anti-tumor effects of PI3K inhibition. In this study, we use the human monoclonal antibody KTN3379, which blocks HER3 in the autoinhibited configuration and thus inhibits ligand-independent and ligand, neuregulin β-1,-dependent HER3 activation, and an inhibitor of PIK3CA (BYL719) to determine if HER3 and PIK3CA can be inhibited in a synergistic fashion. KTN3379 and BYL719 are clinical stage compounds and are being evaluated in HNSCC clinical trials.

Methods and Results: Treatment with neuregulin β-1 significantly increased phosphorylation of HER3, AKT, and S6 across HNSCC cell lines, regardless of baseline level of HER3 expression. HER3 inhibition (KTN3379) consistently abrogated these effects and decreased baseline phosphorylation of HER3. Combined blockade of HER3 (KTN3379) and PI3K (BYL719) inhibited the growth of HNSCC cell lines in a synergistic fashion (CI range 0.1-0.5), in both cell proliferation and colony formation assays. This synergy appeared to be less pronounced in HPV-associated cancer cell lines. Combined treatment decreased tumor growth in xenograft models of HNSCC. Furthermore, treatment of some HNSCC cell lines with BYL719 led to upregulation of the HER3 protein in HNSCC cell lines by Western blotting analysis, suggesting a possible role of HER3 in PI3K pathway inhibitor resistance.

Conclusion: HER3 interacts with PI3KCA, and the PI3K pathway in multiple HNSCC cell lines, and combined blockade has synergistic effects in pre-clinical HNSCC models. These findings suggest that HER3 and PI3K dual inhibition may be more effective in tumors that are resistant to mono-therapy with either HER3 or PI3K inhibition alone.

### New Mechanisms of Anticancer Drug Action

#2981

DNA damaging agent-induced apoptosis is controled by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex.

Wataru Nakajima,1 June Y. Lee,1 Nicolas Maxim,1 Kanika Sharma,1 Mark A. Hicks,1 Thien-Trang Vu,1 Angela Luu,1 William Andrew Yeudall,2 Nobuyuki Tanaka,3 Hisashi Harada1. 1 _VCU Massey Cancer Center, Richmond, VA;_ 2 _Georgia Regents University, Augusta, GA;_ 3 _Nippon Medical School, Kawasaki, Japan_.

DNA damaging agents, such as cisplatin and etoposide, are employed for the treatment of a wide array of solid tumors, but the prolonged use of chemotherapeutic drugs is limited by their toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies that maximize the antitumor effect of drugs while limiting their toxicities. These agents exert anticancer effects via multiple mechanisms, yet their most prominent mode of action involves the generation of DNA lesions followed by activation of the DNA damage response and induction of the BCL-2 family-dependent mitochondrial apoptosis. However, the signaling pathways that connect to BCL-2-family-regulated cell death are not entirely clear.

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic BCL-2 family protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and as a consequence, apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex. Of note, the identified phosphorylation sites are only observed in human MCL-1; thus, the regulatory mechanism shown here may be human-specific. It is of interest in the future to analyze whether CDK2 expression/activity correlates to cisplatin-resistance in patient samples.

#2982

Antineoplastic activity of the novel CDK2/9 inhibitor CCT68127 occurs via induced anaphase catastrophe and inhibition of PEA15 phosphorylation in lung cancer.

Masanori Kawakami,1 Xi Liu,1 Lisa M. Mustachio,1 David J. Sekula,1 Shanhu Hu,2 Lin Zheng,1 Jaime Rodriguez-Canales,1 Barbara Mino,1 Pamela A. Villalobos,1 Carmen Behrens,1 Ignacio I. Wistuba,1 Sarah J. Freemantle,2 Ethan Dmitrovsky1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Geisel School of Medicine at Dartmouth, Hanover, NH_.

We reported the first generation CDK2/9/7 inhibitor seliciclib (R-roscovitine, CYC202) exerts antineoplastic effects against lung cancer by inducing anaphase catastrophe. In anaphase catastrophe, cells with aneuploidy (a hallmark of cancer) cannot cluster supernumerary centrosomes, triggering abnormal anaphase and apoptosis. This study now finds the new CDK2/9 inhibitor CCT68127 (Cyclacel) is a more potent and selective CDK2 inhibitor than seliciclib. CCT68127 exerted substantial antineoplastic effects against diverse lung cancer cells. It inhibited growth, caused cell cycle arrest, and induced apoptosis more than did seliciclib. Using a high throughput screen platform of 75 (57 KRAS wild-type and 18 KRAS mutant) lung cancer cell lines, those with KRAS mutation were significantly (p<0.05) more sensitive to CCT68127 than were KRAS wild-type cells. Anaphase catastrophe was triggered by CCT68127 treatment. Expression of 180 critical growth-regulatory proteins was studied before and after 6, 24, and 48 hours of CCT68127 or vehicle treatments in murine (LKR13; KRAS mutant and ED1; KRAS wild-type) and human (Hop62; KRAS mutant and H522; KRAS wild-type) lung cancer cells using a reverse phase protein array (RPPA). Regulated species included DNA repair, Hippo, and Rab GTPase proteins. The multifunctional growth regulator PEA15 (phosphoprotein enriched in astrocytes 15), exhibited marked Ser116 phosphorylation inhibition after CCT68127 treatment. This was independently confirmed by immunoblot analysis in murine and human lung cancer cells. When PEA15 was overexpressed in these cells, CCT68127 growth inhibition was antagonized, indicating its direct involvement in these effects. PEA15 knockdown using siRNAs significantly (p<0.05) repressed growth of lung cancer cells and enhanced growth inhibition after CCT68127 treatment. PEA15 immunohistochemical detection was explored in 235 human non-small lung cancers. PEA15 expression was reduced in lung cancers versus adjacent normal lung. Decreased PEA15 expression was significantly (p<0.05) associated with advanced stage and overall survival, establishing the translational importance of PEA15 expression in lung cancer. In summary, the novel CDK2/9 inhibitor, CCT68127, elicits marked antineoplastic effects in lung cancer. This occurs through mechanisms that engage anaphase catastrophe and reduce phosphorylation of the growth regulator PEA15.

#2983

Stat3 regulates supernumerary centrosome clustering in cancer cells via Stathmin/PLK1.

Edward J. Morris,1 Eiko Kawamura,1 Jordan Gillespie,1 Paul McDonald,1 William Muller,2 Shoukat Dedhar1. 1 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 2 _Goodman Cancer Centre, Montreal, Quebec, Canada_.

Centrosome amplification and supernumerary centrosomal content are common features of cancer cells. Such cells must cluster their centrosomes to form a bipolar spindle and achieve productive mitosis. Targeting the mechanisms that allow cells to cluster extra centrosomes is considered a promising cancer therapeutic strategy. We have now identified Stat3, a protein that is frequently activated in many types of cancers and also regulates stem cell function, as a regulator of centrosome clustering in cancer cells. A high content chemical screen for the identification of inhibitors of centrosome clustering identified Stattic, a Stat3 inhibitor, as a centrosome clustering inhibitor. Stat3 depletion in cell lines as well as in tumors in vivo resulted in significant inhibition of centrosome clustering and in decreased tumor viability and growth. Interestingly, we identify a novel, transcription-independent mechanism for Stat3-mediated centrosome clustering that requires activities of Stathmin, a Stat3 interactor involved in microtubule depolymerisation, and polo-like kinase1 ( PLK1). Furthermore, stem cell function in PLK4-driven centrosome amplified breast tumor cells is highly sensitive to Stat3 inhibitors, reflected in higher inhibitor sensitivity of tumors derived from these cells in vivo.

We have therefore identified a novel role of Stat3 in the regulation of centrosome clustering, and this role of Stat3 may be critical in identifying tumor types that are sensitive to Stat3 inhibitors.

#2984

Plumbagin induces p53-dependent apoptosis via generation of reactive oxygen species in human cancer cells.

Umasankar De, Amit Kundu, Eunbin Kim, Jonghwan Kwack, Hyungsik Kim. _Sungkyunkwan University (SKKU), Suwon, Republic of Korea_.

Plumbagin [5-hydroxy-2-methyl-1,4-naphthaquinone], a major constituent derived from Drosera and Plumbago, displays antitumor activity both in vitro and animal models, but the molecular mechanisms of p53-mediated antitumor effects have not been clearly explored. The aim of this study is to determine the anticancer effects of plumbagin on MCF-7 (wild type p53) or Ishikawa (p53-mutant type) cells. We compared the cytotoxicity, cell cycle regulation, apoptotic cell death, and generation of intracellular reactive oxygen species (ROS) in these cancer cells. Plumbagin inhibits the growth of Ishikawa (endometrial cancer cells) and SKOV-3 cells, particularly MCF-7 cells. Plumbagin significantly increased the expression of p53 and reduced Murine Double Minute 2 (MDM2) in the MCF-7 cells. Plumbagin up-regulated the expression of p21(CIP1/WAF1) causing cell cycle arrest in the G2/M-phase by down-regulating cyclin B1 and Cdc2 in MCF-7 as well as Ishikawa cells. Plumbagin altered the ratio of Bax/Bcl-2 and cytochrome c released resulting apoptotic cell death in MCF-7 cells. Furthermore, plumbagin dramatically increased the intracellular ROS level, and pretreatment with the ROS scavenger, N-acetyl cysteine (NAC), protected against growth inhibition by plumbagin, suggesting that ROS play a pivotal role in the antitumor activity in MCF-7 cells. In mice bearing the MCF-7 cell xenografts, plumbagin significantly reduced the tumor growth and weight, without apparent side effects. In conclusion, plumbagin exerted anticancer activity in MCF-7 by generation of intracellular ROS that causes induction of apoptosis via Bcl-2/Bax pathways as well as cell cycle arrest.

KEYWORDS: Plumbagin; breast cancer; apoptosis; p53, reactive oxygen species

#2985

Molecular mechanisms of dianhydrogalactitol (VAL-083) in cancer treatment.

Beibei Zhai,1 Anne Steino,2 Jeffrey Bacha,2 Dennis Brown,2 Mads Daugaard1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _DelMar Pharmaceuticals, Inc., Vancouver, British Columbia, Canada_.

Dianhydrogalactitol (VAL-083) is a unique bi-functional alkylating agent causing methylation of N7-guanine and inter-strand DNA crosslinks. VAL-083 is a small water-soluble molecule that readily crosses the blood-brain-barrier. In China, VAL-083 is approved as a chemotherapeutic drug for the treatment of chronic myelogenous leukemia (CML) and lung cancer. In the United States, VAL-083 has been evaluated in more than 40 National Cancer Institute (NCI)-sponsored phase I and phase II clinical trials. Preclinical studies and clinical trial data suggested antineoplastic effects of VAL-083 in a variety of malignancies, including lung cancer, brain tumors, leukemia, cervical cancer, and ovarian cancer. Here we report new insight into VAL-083 mechanism of action by showing that VAL-083 leads to irreversible cell cycle arrest and cell death caused by replication-dependent DNA damage.

In lung (H2122, H1792, H23 and A549) and prostate (PC3 and LNCaP) cancer cell lines, VAL-083 treatment caused irreversible cell cycle arrest in late S and G2 phase as measured by propidium iodide (PI) and immunofluorescent (IF) staining in synchronized cultures. Importantly, VAL-083 was cytotoxic to all cell lines tested (IC50 range 3.06 - 25.7 µM). Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. VAL-083 treatment led to phosphorylation of the proximal DNA double-strand break (DSB) sensor Ataxia Telangiectasia Mutated kinase (ATM), the single-strand DNA-binding Replication Protein A (RPA32), and the histone variant H2A.X (γH2A.X). Importantly, the DNA damage was specific to cells in S phase indicating that VAL-083-induced DNA cross-links translates into more severe DNA lesions during replication. Furthermore, S/G2 phase cell cycle arrest and increased phosphorylation of γH2A.X in cancer cells persisted after pulse-treatment with VAL-083, indicating irreversible DNA lesions.

Taken together, VAL-083 displayed broad anti-neoplastic activity in lung and prostate cancer cells through the induction of replication-dependent DNA damage. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity in cancer cells will offer help in identifying and predicting efficacy of combination treatments.

#2986

Targeting multiple nodes in the RTK- PI3K/AKT/mTOR signaling pathway in a p53-/- mouse model of DIPG induces G2/M phase cell cycle arrest.

Yue Linda Wu,1 Uday Bhanu Maachani,1 Melanie Schweitzer,1 Oren J. Becher,2 Melinda Wang,1 Ranjodh Singh,1 Zhiping Zhou,1 Mark M. Souweidane1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Duke University School of Medicine, Durham, NC_.

Introduction: Diffuse intrinsic pontine glioma (DIPG) is the most common pediatric brainstem tumor, but its prognosis is dismal, with a median survival time of less than one year. Prior studies have implicated amplifications in the receptor tyrosine kinase (RTK)-PI3K/AKT/mTOR signaling pathway in DIPG gliomagenesis, and platelet-derived growth factor receptor (PDGFR) is the most commonly over-expressed RTK. One treatment strategy is thus to inhibit kinases in this pathway with drugs such as dasatinib (PDGFR inhibitor), perifosine (AKT inhibitor), and everolimus (mTOR inhibitor). In this study, we aim to show that combinatorial therapy with dasatinib, perifosine, and everolimus is more effective in impeding tumor cell growth than each drug individually.

Methods: Mouse brainstem glioma cells (mBSG) were derived from a transgenic mouse model of DIPG driven by PDGFB overexpression and p53 loss. Cells were treated for 72 hours with dasatinib, perifosine, and everolimus individually and in combination, and cell viability was assayed with MTS. Western blot with cleaved-caspase 3 antibody was used to assess apoptosis. Cell cycle analysis was performed by propidium iodide flow cytometry.

Results: GI50 concentrations of each individual drug were as follows: 50 nM dasatinib, 50 µM perifosine, and 10 µM everolimus. With combined dasatinib and perifosine treatment, 31.6% of cells survived (p = 0.016 vs. dasatinib alone). The addition of everolimus to dasatinib and perifosine further reduced cell survival to 27.0% (p = 0.011 vs. combinatorial treatment with dasatinib and perifosine). Cell cycle analysis revealed that dasatinib treatment alone prominently arrested cells in G0/G1 phase while both 2-drug combinatorial treatment with dasatinib and perifosine and 3-drug combinatorial treatment with dasatinib, perifosine, and everolimus caused substantial cell cycle arrest in the G0/G1 and G2/M phases. However, Western blot analysis found that no drug treatment group effectively induced apoptosis.

Conclusion: Three-drug combinatorial therapy with dasatinib, perifosine, and everolimus was more effective in reducing DIPG cell viability than dasatinib alone, primarily through the induction of cell cycle arrest at G0/G1 and G2/M. This effect and the lack of apoptosis is consistent with previous observations that inhibition of the PI3K/AKT/mTOR signaling pathway is cytostatic rather than cytotoxic. Moreover, mutations in p53 are seen in up to 50% of patients with DIPG, which allow tumor cells to evade apoptosis despite treatment with chemotherapeutic agents. Taken together, targeting the PI3K/AKT/mTOR pathway alone appears to be insufficient. Ongoing studies aim to understand resistance mechanisms to inhibitors of this pathway and to improve therapeutic efficacy in DIPG by identifying potential synergistic drug combinations targeting alternate survival pathways.

#2987

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers.

Agamemnon A. Epenetos,1 Christina Kousparou,1 Aleksandra Filipovic2. 1 _Trojantec Ltd, London, United Kingdom;_ 2 _Imperial College London, London, UK, London, United Kingdom_.

The most common genetic alteration in human cancer involves the p53 tumor suppressor gene resulting in defective control of cell cycle arrest and death.

The p53 protein induces a cyclin-dependent kinase (cdk) inhibitor, p21 which occupies a central position in cell cycle regulation. Cdk inhibition by p21 results in a lack of progression from the G1 to the S-phase due to the prevention of retinoblastoma (Rb) phosphorylation and subsequent inhibition of transcription factors that regulate the genes involved in DNA replication and cell-cycle progression .

It is hypothesized that the re-introduction of p21 into tumor cells will regenerate the pathway to apoptosis (programmed cell death) and inhibit proliferation since it has been suggested that overexpression of p21 results in suppression of tumor growth in vitro and in vivo.Unfortunately, p21 protein alone is unable to be administered directly in vivo and progress to the cell nuclei of cancer cells since there is no active mechanism to transport the protein. However, a number of proteins are known which can translocate across the cytoplasmic membrane of mammalian cells and into the nuclei, carrying additional cargoes with them . One of these translocating proteins is a 60 amino acid peptide corresponding to the homeodomain of the Drosophilia protein Antennapedia [ANTP] .

A fusion protein (TR1) has been developed where the full-length p21 protein is attached to the antennapedia protein with a view to delivering the p21 protein into cancer cells to restore cell cycle control.

TR1 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2.Non-specific toxicity was excluded by showing that TR1 penetrated but did not kill p53- or p21- wild-type cells. TR1 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of TR1. TR1was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with chemotherapy.

TR1 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of cancer therapeutics.

#2988

Integrated functional RNAi screening and structural genomics identify cell cycle and kinetochore components co-modulating NF-κB activity as potential therapeutic targets in head and neck squamous cell carcinoma.

Anthony D. Saleh,1 Sophie Carlson,1 Shaleeka Cornelius,1 Hui Cheng,1 Scott Martin,2 Pinar Ormanoglu2. 1 _NIH-NIDCD, Bethesda, MD;_ 2 _NIH-NCATS, Bethesda, MD_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with over 600,000 new cases diagnosed each year, and a five year survival of ~50%. Recent findings in The Cancer Genome Atlas (TCGA) analysis of 279 HNSCC patient samples revealed that components of the NF-κB/REL signaling pathways are aberrantly expressed in about ~70% of cases, and implicated in expression of pro-proliferative, inflammatory, angiogenic, and therapeutic resistance genes. Alterations are prevalent in this pathway across patient samples; however, the detailed mechanisms that increase NF-κB signaling in HNSCC are not well understood. Recently, we established an NF-κB β-lactamase reporter UM-SCC-1 cell line to identify key regulators of this NF-κB oncogenic signaling. We performed genome-wide RNAi screening using the NF-κB reporter line, and upon validation, most structural components of the TNF receptor complex and downstream NF-κB pathway genes, such as IKKs, were scored highly in the screening. We integrated the functional genomics data with structural mutation and expression data from TCGA to prioritize genes that are frequently deregulated in HNSCC specimens. WNT9/FZD6, NOTCH2, and TGFβR2 and several receptors were identified to cross-activate NF-κB signaling. In addition, we observed novel genes that co-regulate NF-κB activity, which are involved in the cell cycle, such as CDC2, CDC7, WEE1 and CALM2, as well as components of the kinetochore such as NDC80, PLK1, TTK, and AURKA. Among these targets WEE1 kinase is currently under investigation as a therapeutic target in HNSCC using the WEE1 inhibitor AZD1775. To further investigate potential cross-talk between WEE1 cell cycle regulation and NF-κB activation, we have shown that treatment of HNSCC cells with AZD1775 decreases NF-κB transcriptional activation which corresponds to decrease in TNF-inducible phosphorylation of RELA. These changes also correlate with alteration in cell cycle regulation detected by flow cytometry. Mechanistic studies are currently underway to understand how WEE1 signaling connects to NF-κB activation, and what role inhibition of NF-κB signaling plays in AZD1775 activity as a sensitizer to chemo-or radiation therapy for HNSCC.

Supported by NIDCD intramural projects ZIA-DC-000073,74

#2989

An Intact G1/S checkpoint determines response to CDK4/6 inhibitor in breast cancer.

Smruthi Vijayaraghavan, Khandan Keyomarsi. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Deregulation of the cell cycle machinery is a hallmark of most cancers, making the positive regulators of cell cycle such as Cyclin dependent kinases (CDKs) as attractive and druggable targets. Palbociclib or PD-0332991, a potent CDK4/6 inhibitor is an anti-proliferative agent that induces G1 arrest, senescence and inhibits tumor growth in breast cancer. Based on the drug's success in phase II clinical trials, Palbociclib was recently awarded FDA approval for the treatment of metastatic ER+ breast cancer. However, one of the main limitations of this treatment is the associated adverse events of grade 3&4 neutropenia (62%) and grade 3&4 leukopenia (25.2%). Moreover, despite promising results in clinical trials, little is known about palbociclib's mechanism of action and mode(s) of resistance in ER+ breast cancer. A clear understanding of these mechanisms is critical to identify biomarkers and understand the biology of treatment response, drug resistance and combination strategies, and this study is aimed at addressing these gaps in knowledge.

In order to examine the mechanism of action and identify nodes of sensitivity to Palbociclib, we used a model system of two ER+ breast cancer cell lines (MCF7, T47D) and a mammary epithelial cell line (MCF10A). Results from drug response studies, cell cycle analysis and senescence assay revealed that treatment with palbociclib induces a dose-dependent sustained growth inhibition and G1 arrest specifically in the ER+ cell lines. Additionally, palbociclib induced senescence in a dose-dependent manner in the ER+ cancer cells and not in MCF10A.

Further, to interrogate if deregulation of the G1/S checkpoint can render cells resistant to palbociclib, we examined the effect of knocking down Rb and overexpressing the oncogenic low molecular weight isoforms of cyclin-E (LMW-E), which are known to induce constitutive phosphorylation of Rb, in our model system. Drug response, cell cycle analysis and senescence assay revealed that both downregulation of Rb and overexpression of LMW-E expression significantly diminished the sensitivity of breast cancer cells to palbociclib (~6 to 7 fold) and its ability to induce senescence.

Moreover, while Palbociclib is known to be a specific CDK4/6 inhibitor, drug response studies showed that downregulation of either kinase in our model system did not completely recapitulate the drug's anti-tumor effects. This suggests that Palbociclib could inhibit another target, albeit, at a higher concentration, and this would be the subject of our future studies.

Collectively, our study indicates that the CDK4/6 inhibitor, Palbociclib, acts in a dose-dependent manner to induce senescence, specifically in breast cancer cells. Further, our results suggest that the presence of an intact G1/S checkpoint is necessary for response to palbociclib, providing rationale to utilize Rb positivity and absence of LMW-E as biomarkers of response to the drug in ER+ breast cancer patients.

#2990

Sulforaphane enhances the anti-cancer efficacy of anti-androgens in prostate cancer cells (LNCaP and C4-2B) by increasing androgen receptor (AR) degradation.

Namrata Khurana, Sudha Talwar, Partha Chandra, Asim B. Abdel-Mageed, Debasis Mondal, Suresh Sikka. _Tulane University, New Orleans, LA_.

Introduction: Prostate Cancer (PC) cells utilize androgen for their growth. Therefore, androgen deprivation therapy (ADT) that blocks both systemic androgen production and androgen receptor (AR) signaling remains the mainstay of PC treatment. However, despite the efficacy of AR antagonists such as bicalutamide (BIC) and enzalutamide (ENZ), their sub-therapeutic efficacy in tumor microenvironments enable the selection and outgrowth of castration resistant PC (CRPC). Interestingly, CRPC cells continue to express AR and exploit the intratumoral androgen. Hence, alternate strategies to suppress AR expression in PC cells will be needed to enhance the efficacy of BIC and ENZ. The phytochemical, sulforaphane (SFN) is known to decrease AR protein levels. We hypothesize that co-exposure to SFN will increase the anti-cancer efficacy of BIC/ENZ.

Methods: The AR expressing PC lines, both androgen-dependent (AD) LNCaP cells and androgen-independent CRPC cells (C4-2B), were used to investigate the effects of BIC or ENZ, alone and in combination with SFN. The following cellular parameters were evaluated: (a) cell proliferation by MTT-assay; (b) clonogenic ability by colony forming unit (CFU); (c) AR expression by immunoblot analysis; (d) subcellular AR localization by immunofluorescence microscropy (IFM); (e) AR and prostate specific antigen (PSA) gene expression by qRT-PCR; and (e) PSA protein secretion by ELISA. Studies were carried out under hormone deprived (charcoal-stripped FBS) conditions and/or in presence of AR agonist, R1881.

Results: Co-exposure to SFN (10µM) significantly (p<0.05) enhanced the anti-proliferative effects of BIC (50µM) or ENZ (20µM) at 48 h post treatment, and suppressed R1881 stimulated PC growth, as well. At much lower concentrations, SFN (0.2µM) co-exposure enhanced the suppressive effects of BIC (0.5µM) or ENZ (0.2µM) on both the number and size of CFUs at 14 days. Western blot analysis revealed that SFN decreases AR protein levels in a time- and dose-dependent manner. IFM analysis indicated that SFN suppresses R1881-stimulated AR nuclear localization. Cycloheximide treatment suggested that SFN's effect on AR is primarily regulated at the translational level. Experiments with MG-132, a proteasomal inhibitor indicated that the SFN-induced AR degradation is partially regulated by the proteasomal pathway. Studies with qRT-PCR showed that SFN enhances the efficacy of BIC in suppressing PSA mRNA levels and decreased PSA protein secretion was also corroborated by ELISA.

Conclusion: Sulforaphane decreases AR levels in both LNCaP and C4-2B cells to increase the anti-cancer efficacy of AR antagonists in a synergistic manner. This implies that adjuvant therapy with this safe phytochemical may be very promising.

#2991

Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10.

Rodney Balhorn,1 Arjan J. van Adrichem,2 Saphon Hok,3 Monique C. Balhorn1. 1 _SHAL Technologies, Inc., Livermore, CA;_ 2 _University of Helsinki, Helsinki, Finland;_ 3 _Lawrence Livermore National Laboratory, Livermore, CA_.

SH7139, the first in a series of selective high affinity ligand (SHAL) therapeutics designed to treat non-Hodgkin's lymphoma, has been shown to be selectively cytotoxic to lymphoma cells over-expressing HLA-DR10. Recent efforts to elucidate the mechanisms of action of SH7139 show that the small molecule drug functions similar to both an antibody drug conjugate and a pro-drug. SH7139 is comprised of three small molecule recognition elements that, when linked together, collectively target the drug to HLA-DR10. Following its binding to HLA-DR10, SH7139 is shuttled into the interior of the lymphoma cell where the subsequent metabolism of these recognition elements releases a series of metabolites that inhibit multiple activities required for tumor cell growth and replication. Studies performed using the Burkitt's lymphoma cell line Raji have shown that SH7139 is metabolized by Raji cells, and the metabolic cleavage of two of the recognition elements (Ct and Dv) produce cytotoxic compounds that contribute to tumor cell killing. While the third recognition element, Cb, is not cleaved off the SHAL scaffold or hydrolyzed to release a cytotoxic metabolite, its structural similarity to known inhibitors of the GTPase activating protein (GAP) MgcRacGAP suggested that it might be active in this pathway. MgcRacGAP functions as a switch that stimulates by many orders of magnitude the activity of the Rac1 GTPase, which is required for cleavage furrow formation, ingression, and the completion of cytokinesis. Experiments conducted with the MgcRacGAP-Rac1 complex have shown that the intact SH7139 molecule (IC50=10.6±1.6µM) as well as SH7139 fragments containing the Cb recognition element is effective in inhibiting the GTPase activity of the MgcRacGAP:Rac1 complex. These results confirm that one mechanism action of SH7139 is the inhibition of the Rac1-dependent effector pathways that control the rounding of cells undergoing mitosis, confine Rho activation to the equator of the cell for proper cleavage furrow formation and other processes involved in the completion of cytokinesis. This research was supported by the National Cancer Institute Phase II SBIR award R44CA159843 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

#2992

Propranolol induces G0/G1/S phase arrest and apoptosis in melanoma cells via AKT/MAPK pathway.

Chengfang Zhou,1 Xiang Chen,1 Howard L McLeod,2 Todd C Knepper,2 Yijing He1. 1 _CSU, China, Changsha, China;_ 2 _Moffitt Cancer Center,USA, FL_.

Both preclinical and clinical studies implied that β-adrenoceptor-blockers (β-blockers) could inhibit the growth of melanoma. However, the underline mechanism is still unclear, especially in acral melanoma. MAPK and AKT pathway is involved in tumor survival and apoptosis. Therefore, we investigated the effect of propranolol on melanoma in the A375, two primary acral melanoma cell lines and mice model. In this study, propranolol significantly reduced cell viability and induced apoptosis by activating intrinsic mitochondrial pathway and inhibiting MAPK and AKT pathway in vitro. Meanwhile, we further highlighted that low dose propranolol could slow down the growth of tumor in immunodeficient mice engrafted with human melanoma cells. Thus, our data firstly showed that propranolol may inhibit melanoma by activating intrinsic mitochondrial pathway and regulating MAPK pathway and AKT pathway.

#2993

Dichloroacetate inhibits cell proliferation and induces apoptosis in human endometrial cancer cell lines.

Leslie H. Clark, Chunxiao Zhou, Victoria Bae-Jump. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Objective: Dichloroacetate (DCA) is a well-tolerated, oral drug used in the treatment of hereditary lactic acidosis. DCA has been found to have anti-tumorigenic activity in various non-gynecologic cancers through its inhibition of pyruvate dehydrogenase, leading to profound mitochondrial defects in cancer cells. Thus, we sought to assess the effects of DCA on human endometrial cancer cell lines.

Methods: Three endometrial cancer cell lines (KLE, Ishikawa, and ECC-1) were treated with varying concentrations of DCA from 0.1uM to 100 uM. Cell proliferation was measured using the MTT assay. Cell cycle progression was assessed by Cellometer. Apoptosis was evaluated by the Annexin V-FITC assay using Cellometer. Western immunoblotting was performed to assess effects of DCA on cell cycle and cellular stress proteins. In addition, cellular stress was measured using a reactive oxygen species assay. All experiments were performed in triplicate.

Results: DCA inhibited cell proliferation in a dose-dependent manner in all three endometrial cancer cell lines following 72 hours of exposure. The IC50 was 30 uM for KLE, 78 uM for Ishikawa, and 72 uM for ECC-1. Treatment with DCA resulted in G1 phase cell cycle arrest in all three cell lines. DCA induced apoptosis in all three cell lines. Western blotting analysis demonstrated that DCA increased cyclin D1 expression, decreased CDK4 expression, and increased PERK expression after 24 hours of DCA exposure. A dose dependent increase in reactive oxygen species was seen in all three cell lines. At a maximum dose of 100 uM DCA, ROS production compared to controls for KLE increased 3.2-fold (p=0.002), Ishikawa increased 1.8-fold (p=0.003), and ECC-1 increased 2.1-fold (p=0.002).

Conclusions: DCA effectively inhibited cell proliferation via G1 cell cycle arrest and induced apoptosis in human endometrial cancer cell lines. DCA induced significant cellular stress as evidenced by a dose dependent increase in reactive oxygen species. Further evaluation of DCA as a targeted metabolic therapy in endometrial cancer is warranted.

#2994

Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy.

Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. _University of North Carolina, Chapel Hill, NC_.

Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines.

Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways.

Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p=0.008 for ECC-1 cells and p=0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein.

Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention.

#2995

Mechanisms of high dose ascorbate inhibiting pancreatic cancer growth and metastasis.

Qi Chen, Kishore Polireddy, Ruochen Dong, Ping Chen. _University of Kansas Medical Center, Kansas City, KS_.

Purpose:

High-dose intravenous ascorbate (IVC) bypasses bioavailability barriers of oral ingestion and provides pharmacologic concentrations in tissues, and exhibits pro-oxidant anti-cancer activities. In pancreatic cancer pre-clinical models, ascorbate has been shown to sensitize the tumor to the 1st line chemotherapeutic drug gemcitabine. Here, we investigated the possible mechanisms of actions of ascorbate on pancreatic cancer growth and metastasis.

Methods

Orthotopic pancreatic cancer mouse model was utilized for evaluation of the effects of ascorbate in inhibiting tumor growth and metastasis. Molecular biological approaches were used in investigation the mechanisms of these effects.

Results

In the orthotopic mouse tumor model, ascorbate treatment alone decreased pancreatic cancer growth and metastasis. Ascorbate treatment enhanced expressions of epithelial markers and suppressed expression of mesenchymal markers in pancreatic cancer cells. MMP-2 expression and activity were decreased dose-dependently. Tumor tissues from ascorbate-treated mice had markedly increased collagen and desmoplasia in the extracellular matrix. Such fibrosis was not seen in normal tissues from the same mouse. In addition, ascorbate decreased the protein level of deacetylase HDAC6, and inhibited activity of the deacetylase Sirt-2 through depletion of NAD+. As a result, ascorbate robustly increased α-tubulin acetylation in pancreatic cancer cells. Acetylation promoted tubulin polymerization and stabilization, mimicking the cellular outcome of paclitaxel, resulted in inhibition of cell motility and mitosis.

Conclusion

Pharmacological concentrations of ascorbate affected pancreatic tumor cells and the tumor microenvironment, led to inhibition of tumor growth and metastasis.

#2996

IT-139 targets GRP78 in stressed cancer cells.

Jyothi Sethuraman,1 Tara Lee Costich,1 Tomas Vojkovsky,1 Rick Crouse,2 Valentin Cognet,3 Suzanne Bakewell1. 1 _Intezyne Technologies, Tampa, FL;_ 2 _Yale University, New Haven, CT;_ 3 _Lille University, Lille, France_.

IT-139, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is a first in class small molecule that has successfully completed a phase I clinical trial against solid refractory cancers. 11 of 41 patients achieved stable disease with manageable side effects and 8 patients showed tumor size reduction including a durable partial response. Preclinical studies suggest IT-139 down-regulates the chaperone GRP78, but the mechanism of action is not fully understood. GRP78 (also known as BiP and HSPA5) is the primary sensor and regulator of the endoplasmic reticulum (ER). In cancer cells it is preferably expressed on the cell surface, but also translocates to the mitochondria and cytosol where it regulates critical oncogenic signaling pathways, thus making GRP78 an excellent target for therapy. Here we show in vitro results of IT-139 treatment in a panel of cancer cell lines. IC50 levels measured at 72 hour varied ranging from 15 μM to 180 μM. Annexin V/PI staining by flow cytometry in HCT-116, HT-29, A375 and A549 cell lines at 24 hour show early and late apoptosis, which corresponds to their relative IC50s. UPR induction and overexpression of GRP78 has been reported to cause cell cycle arrest in G1. But in IT-139 treated cell lines HCT116, HT-29, A549 and LnCAP, cell cycle arrest occurred in G2, whereas in A375 cells IT-139 induced cell cycle arrest at G1. These data suggest an inhibition of GRP78 levels and that cell cycle arrest status is also dependent upon the cell line. Electron microscopic (EM) images of HCT-116 and HT-29 cells treated with IT-139 at 24 hours showed significant vacuolization, ER expansion and disorganization of intracellular organelles, strongly suggesting ER stress. However, there is an absence of double membrane vacuoles or autophagosome formation in response to the ER stress. Also, immunofluorescent analysis displayed no LC3-II punctate distribution, although protein immunoblots showed LC3 and p62 protein levels increasing in IT-139 treated cells. These data suggest an inhibition of autophagy. Additionally, EM images showed vacuolated mitochondria and distortion of cristae. In all cell lines treated with IT-139 even below their corresponding IC50 concentration, JC-1 staining showed an increased loss of mitochondrial membrane potential. These observations hypothetically indicate an inhibition of stress-induced GRP78. Quantitative real-time PCR showed IT-139 treatment in combination with thapsigargin exhibits a significant fold change decrease in the GRP78 mRNA expression. These results suggest that IT-139 downregulates GRP78 leading to an increase in ER stress, mitochondrial damage, decreased autophagy, increased apoptosis and cell death.

#2997

Dynarrestin, a novel dynein inhibitor that does not block ciliogenesis.

Matthias Baumann,1 Susanne Höing,1 Ting-Yu Yeh,2 Nancy Martinez,3 Peter Habenberger,1 Lea Kremer,3 Hannes CA. Drexler,4 Philipp Küchler,3 Peter Reinhardt,4 Axel Choidas,1 Mia-Lisa Zischinsky,1 Gunther Zischinsky,1 Stephanie A. Ketcham,2 Lydia Wagner,5 Peter Nussbaumer,1 Slava Ziegler,3 Bert Klebl,1 Trina Schroer,2 Hans R. Schöler,4 Herbert Waldmann,3 Jared L. Sterneckert5. 1 _Lead Discovery Center, Dortmund, Germany;_ 2 _Johns Hopkins University, Baltimore, MD;_ 3 _Max Planck Institute for Molecular Physiology, Dortmund, Germany;_ 4 _Max Planck Institute for Molecular Biomedicine, Münster, Germany;_ 5 _University of Technology, Dresden, Germany_.

Introduction: Aberrant hedgehog (Hh) pathway activation contributes to the pathogenesis of multiple cancers. Currently available Hh pathway inhibitors target Smoothened (Smo) which can acquire mutations causing drug resistance. Therefore, a major challenge is to identify compounds that inhibit Hh signaling downstream of Smoothened.

Methods: We conducted a high-throughput screening (HTS) campaign of 337,000 small molecules using a Hh-dependent differentiation assay of C3H10T1/2 cells into osteoclasts. Primary active screening hits were validated by a cell-based assay cascade including motor neuron differentiation from mouse embryonic stem cells, Gli reporter activation in ShhLight2 cells and proliferation of heterozygous Patched (PTCH+/-) medulloblastoma (MB) cells. Smo binding was monitored in membrane-based Cyclopamine competition assays. To identify the underlying target identification of promising hits, affinity chromatography was performed followed by label-free quantitative (LFQ) tandem mass spectrometry. Effects on cytoplasmic dynein were examined in Smo trafficking and in vitro microtubules motility assays as well as by live cell imaging of labeled endosome movement.

Results: We identified dynarrestin, a novel aminothiazole that potently blocks Hh signaling and the proliferation of Hh-dependent PTCH+/- MB tumor cells (IC50~70nM). Dynarrestin does not bind to Smo and is able to suppress the Hh pathway in the presence of the Smo agonist SAG and following the knockdown of the Hh pathway suppressor SUFU indicating that dynarrestin inhibits Hh signaling downstream of both Smo and SUFU. Chemical LFQ proteomics identified cytoplasmic dynein as the target of dynarrestin. Dyneins convert energy from ATP hydrolysis into motor activity and are essential for many cellular processes, including Hh signaling. Unlike other dynein inhibitory molecules, e.g. Ciliobrevins, dynarrestin inhibits Smoothened trafficking but not ciliogenesis. We further demonstrate that dynarrestin reversibly interferes with endosome movement, proper mitotic spindle orientation, and dynein-based microtubule translocation in vitro without blocking ATP hydrolysis.

Conclusions: Dynarrestin provides a novel dynein inhibitor with distinct specificity that uniquely facilitates probing dynein function. Given its multiple contributions to Hh signaling, mitosis and other cellular events, dynein remains a highly attractive target for medicinal chemistry programs aimed at exploiting and modulating its complex, poorly understood chemical cycle to interfere with different aspects of activity and function. We believe that unraveling dynein-dependent cellular processes by dynarrestin has great potential for development into novel anti-cancer drugs controlling Hh signaling downstream of Smoothened.

#2998

Molecular and cellular effects of individual and combinatorial silencing of HSP90 paralogs.

Marissa V. Powers, Robert H. Te Poele, Ravindhi L. Murphy, Emmanuel de Billy, Paul A. Clarke, Paul Workman. _The Institute of Cancer Research, London, United Kingdom_.

HSP90 is an ATP-dependent molecular chaperone critical for the folding, stability and function of over 350 client proteins, including many that are oncogenic. Pharmacologic inhibition of the essential ATPase domain inhibits HSP90 function and produces a well-documented molecular and cellular response comprising depletion of client proteins, induction of heat shock proteins and reduction of tumor cell proliferation. There are four paralogs within the HSP90 family, HSP90α, HSP90β, GRP94 and TRAP1, and it is not currently clear what precise role each one plays in the molecular and therapeutic response to HSP90 inhibitors. Here we use an siRNA approach to selectively reduce the expression of each HSP90 paralog, alone or in combination, in an attempt to dissect the individual roles of each chaperone in the molecular and cellular response associated with pharmacologic HSP90 inhibition. In the human cancer models used we found that by simultaneously silencing HSP90α and HSP90β we could phenocopy aspects of pharmacologic HSP90 inhibition including reduction of cell proliferation, induction of HSP72 and other HSF-1 target genes and depletion of oncogenic client proteins. Through analysing representative HSP90 client proteins we observed interesting patterns of paralog-dependence that were related to their overall sensitivity to pharmacologic HSP90 inhibition. Our results reveal the importance of HSP90 paralogs in particular aspects of the molecular and cellular response to pharmacologic HSP90 inhibition.

#2999

Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells.

Sheryl A. Flanagan, Jeffrey J. Ackroyd, Aaron Kramer, Jennifer Feigin, Donna S. Shewach. _University of Michigan Medical School, Ann Arbor, MI_.

Used alone or in combination with other chemotherapeutics, the antimetabolites 2', 2'-difluoro-2'-deoxycytidine (gemcitabine, dFdCyd), and 5-fluorouracil (5-FU)/fluorodeoxyuridine (FdUrd) comprise the mainstay of treatment for gastrointestinal malignancies, such as pancreatic and colorectal cancer. Gemcitabine with cisplatin is also first-line therapy for non-small cell lung cancer (NSCLC). While these treatments can prolong survival, many patients still relapse and there is need for improvement to current therapies. Cytotoxicity elicited by dFdCyd and 5-FU/FdUrd is influenced by drug metabolism/activation, DNA repair proteins, cell cycle progression, and apoptosis. Histone deacetylase inhibitors (HDACi) target histone deacetylases, which regulate gene expression through deacetylation of lysine residues on histones and some non-histone proteins. HDACis have been shown to increase expression of p21 and increase apoptosis which could enhance cytotoxicity with antimetabolites. The success of HDACis, including vorinostat and panobinostat, in the treatment of hematologic malignancies has encouraged their use in solid tumors. However, results from clinical trials in patients treated with HDACis alone or with other drugs, including antimetabolites have been mixed and the mechanism(s) underlying successful therapy with HDACis is largely unknown. In the present studies, we used solid tumor cell lines that represent tumors commonly treated with antimetabolites; A549 (NSCLC) and PANC1 (pancreatic cancer) cells, and HT29 (colon cancer) cells to investigate the effect of an HDACi with either dFdCyd or FdUrd, respectively, and we measured clonogenic survival. The data demonstrate that cytotoxicity with an HDACi and an antimetabolite is highly sequence dependent; vorinostat added concurrently with dFdCyd or FdUrd or prior to either antimetabolite resulted in antagonistic cytotoxicity, whereas the addition of the antimetabolite followed by HDACi was additive/synergistic with dFdCyd. Cytotoxicity with panobinostat and antimetabolites was also sequence dependent. Cell cycle distribution studies revealed the accumulation of cells in G1 and G2/M following HDACi addition (≥90% of cells), thus rendering S-phase specific dFdCyd less cytotoxic upon its subsequent addition. Similarly, concurrent addition of HDACi and dFdCyd produced less S-phase accumulation than observed with dFdCyd alone. These data demonstrate that administration of the HDACis and antimetabolites must be sequenced appropriately so that cell cycle effects complement rather than antagonize drug mechanisms.

#3000

Intra-tumoral accumulation of NK1.1/CD3+ cells and anti-metastasis effects of dose-intensified ONC201 in tumor-bearing mice.

Jessica Wagner, Christina L.B. Kline, Marie Baumeister, Wafik S. El-Deiry. _Fox Chase Cancer Center: Temple College of Medicine, Philadelphia, PA_.

ONC201, a novel first-in-class, orally active anti-tumor agent that upregulates the cytotoxic ligand TRAIL (Allen et al., Sci. Trans. Med., 2013; Wagner et al., Oncotarget, 2014), activates the integrated stress response leading to tumor cell upregulation of TRAIL death receptor 5 (Kline et al., Sci. Sig., in press). ONC201 is active against bulk tumor and cancer stem cells (Prabhu et al., Can. Res., 2015). ONC201 is under clinical development by Oncoceutics, and is being evaluated in multiple phase I/II clinical trials. Results of the first-in-human ONC201 study presented at the 2015 AACR-NCI-EORTC meeting (Stein et al., Abstract C138) demonstrated ONC201 to be safe in humans, to exhibit predicted PK and sustained PD characteristics, and revealed a preliminary efficacy signal. As patients were dosed on an every 3-week schedule, based on supportive preclinical data, we investigated dose-intensification of ONC201 to determine whether a higher dose/frequency schedule might impact efficacy with limited toxicity. We hypothesized that ONC201 may be effective in dose-intensified schedule and may inhibit metastases. We tested a range of ONC201 doses including 25, 50, and 100 mg/kg and frequencies including every 4, 3, 2, 1 week as well as twice a week dosing. In colon and triple-negative breast cancer we observed that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo. Frequency effects were more pronounced at lower doses and dose-dependency was more impactful with less frequent schedules. We noted a potent anti-metastasis effect of ONC201 in vivo, not previously reported, as a function of both increased ONC201 dose and frequency of administration. ONC201 inhibits cancer cell migration and invasion in vitro in a TRAIL-dependent manner. We found ONC201 more potently suppresses Akt and ERK in tumors in vivo in a dose- and frequency-dependent manner, whereas its effect on TRAIL serum levels appeared to be impacted by frequency. We observed accumulation of CD3+/NK1.1+ cells within ONC201-treated tumors in athymic nude mice that lack T-cells. Accumulation of CD3+/NK1.1 cells within ONC201-treated tumors was more pronounced with dose intensification that correlated with superior efficacy. In summary, we have uncovered a potent anti-metastasis effect of ONC201 coupled with the appearance of CD3+/NK1.1+ cells within ONC201-treated tumors. We are further evaluating the biomarker characteristics and immune function of the CD3+/NK1.1+ cells and the relationship of their intra-tumoral accumulation to observed anti-tumor effects of ONC201, including in fully immunocompetent mice. Our results suggest that clinical investigation of both dose and frequency of ONC201 administration is warranted and is being evaluated in an ongoing clinical trial (NCT02609230).

#3001

Accurins are endocytosed by KRAS-mutant cells via macropinocytosis.

Jane E. Cullis,1 Laura Taylor,1 Susan Low,2 Dafna Bar-Sagi1. 1 _NYU Langone Medical Centre, New York, NY;_ 2 _BIND Therapeutics, Cambridge, MA_.

Background:

BIND-014 is a novel prostate-specific membrane antigen (PSMA)-targeted Accurin (polymeric nanoparticle) encapsulating docetaxel. In a Phase 2 non-small cell lung cancer (NSCLC) trial, BIND-014 demonstrated promising anti-tumor activity in patients with tumors expressing KRAS mutations (mutated Kirsten ras oncogene homolog). KRAS mutations in NSCLC are generally associated with poor response to currently available drug therapy regimens, including docetaxel. Mutant KRAS stimulates an endocytic process known as macropinocytosis, in which extracellular fluid and its constituents are internalized non-selectively into cells. We hypothesized that Accurins preferentially accumulate in KRAS-mutant cells via macropinocytosis, potentially contributing to anti-tumor activity of BIND-014 in KRAS-mutant cancer.

Methods:

To determine whether BIND-014 is internalized in cells with high levels of macropinocytosis, we treated KRAS-mutant, macropinocytosis-high human NSCLC A549 cells and human bladder cancer T24 cells with PSMA targeted Accurins in which docetaxel was replaced by a fluorescent label, and visualized co-localization with fluorescently labeled markers of macropinocytosis, dextran and BSA, by confocal microscopy. In addition, we quantified the levels of labeled Accurin uptake in A549 cells by fluorescence-activated cell sorting (FACS) and compared them to labeled Accurin internalization in macropinocytosis-low, KRAS-wild-type BxPC3 cells. We further tested the macropinocytosis-dependence of labeled Accurin uptake by treating A549 and BxPC3 cells with the macropinocytosis inhibitor EIPA and measuring changes in Accurin internalization by FACS.

Results:

Confocal microscopy analysis of A549 and T24 cells co-treated with labeled Accurin and dextran or BSA showed substantial accumulation of Accurin in dextran- and BSA-positive macropinosomes. Moreover, FACS analysis of A549 cells treated with 0.1 ug and 1.0 ug of labeled Accurin revealed 60.4% and 81.8% uptake, respectively, that could be reduced to 3.6% and 3.7% with 50 uM EIPA. By contrast, BxPC3 cells showed 1.8% and 1.5% uptake of 0.1 ug and 1.0 ug labeled Accurin that was reduced to 1.3% and 0.9%, respectively, with 50 uM EIPA by FACS analysis.

Conclusions:

These data demonstrate that Accurins preferentially enter human cancer cells that display high levels of macropinocytosis and co-localize with several markers of macropinosomes. Moreover, Accurin internalization can be inhibited in macropinocytosis-positive cells with an established chemical inhibitor of macropinocytosis. BIND-014 may therefore preferentially accumulate in KRAS-mutant NSCLC tumors, thereby contributing to the clinical activity seen to date in this tumor type.

#3002

The PARP inhibitor, Rucaparib enhance the sensitivity to Trabectedin in soft tissue sarcomas cell lines and in patient xenograft model.

Laroche-Clary Audrey, Chaire Vanessa, Karanian Marie, Italiano Antoine. _Institut Bergonié, Bordeaux, France_.

Background: Soft tissue sarcomas (STS) constitute a group of rare tumors and represent less than 1% of all adult cancer. Trabectedin (trabectedin®) is a synthetic molecule derived from Caribean tunicate and is a potent antitumor agent approved for patients with advanced STS. Trabectedin binds to minor groove and interferes with the DNA repair pathway, cell cycle and interacts with transcription factors. PARP-1 and 2 are two proteins implicated in DNA repair; in response to DNA damage, PARP binds to sites of damage and catalyzes the poly (ADP-ribosyl)ation of target proteins and recruit proteins at the DNA break. The aim of this study was to evaluate if the antitumor activity of Trabectedin is potentiated by PARP inhibitor, Rucaparib, in preclinical model of liposarcoma dedifferentiated and leimyosarcoma cell lines and in a patient-derived xenograft (PDX) model.

Methods: The LPS cell lines (IB115, IB111), the LMS cells (IB136) and the PDX model have been established from human surgical specimens tumor in our laboratory, after obtaining written informed patient consent. Cells were exposed for 72h to an increasing concentration range of Trabectedin, Rucaparib or drugs in combination and viability was assessed by the MTT assay. Apoptosis was assessed by Annexin V/PI staining and cell cycle analysis was studied by DNA content by fluorescence-activated cells sorting, apoptosis and cycle cell results were analyzed using the Cell Quest Pro software (BD Biosciences, San Jose, USA). DNA strand breaks were evaluated by testing for the presence of intranuclear foci γH2ax after 48h of treatment with drugs alone or in combination by immunofluorescence and microscopy confocal analysis. Combination drugs analysis was also assessed in patient-derived xenograft mice model.

Results: As expected, sarcoma cells are very sensitive to Trabectedin (IC50 were comprised between 0.0007µM and 0.02µM), Rucaparib was less effective on proliferation with a median of IC50 at 12µM ± 5µM. The combination of Rucaparib and Trabectedin is highly synergistic, inhibiting cell proliferation, inducing apoptosis, and the accumulation of G2/M. By using the Chou-Talalay method for drug combination, we observed index combination ranging from 0.3 to 0.8. The drug combination also enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. The effect of the drug combination was corroborated in vivo by using a STS patient-derived xenograft (PDX) models in which the tumors showed sustained regression.

Conclusion: The combination of Trabectedin and Rucaparib is highly synergistic in pre-clinical models of STS and is worth to investigate in the clinical setting.

#3003

p73 DNP genotype influences effects of etoposide and 5-aza-2'-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines.

Zachary Connelly, L. Michael Carastro. _University of Tampa, Tampa, FL_.

Background: The p73 gene is a member of the p53 tumor suppressor family. A dinucleotide polymorphism (DNP) in p73 is a G4C14-to-A4T14 linked pair of transitions located in exon 2, between the P1 and P2 promoters, from which TAp73 and DeltaNp73 (DNp73) mRNA isoforms are transcribed, respectively. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. Etoposide is known to induce p73 gene expression. Others recently reported treatment of T-47D cells with 5-aza-2'-deoxycytidine (5AZA) resulted in increased TAp73/DNp73 mRNA ratios.

Hypothesis: We hypothesized that treatment with 5AZA increases the TAp73/DNp73 mRNA ratios in human cell lines and that the presence of the p73 DNP allele potentiates 5AZA-induced increases TAp73/DNp73 cellular ratios.

Methods. We treated human cancer cell lines with 50 microM etoposide for 24 hrs and/or 20 microM 5AZA for 48 hrs. RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or αNp73 mRNAs.

Results: Cell lines wild-type for p73 DNP (DU145, T47-D, MDA-MB-468) had TAp73/DNp73 mRNA ratios that were lower than those heterozygous for p73 DNP (PC-3 and MDA-MB-231) following co-treatment with 5AZA and etoposide or with etoposide alone (p<0.01 for DU145 & T47-D). PC-3 and MDA-MB-231 cell lines had TAp73/DNp73 mRNA ratios following 5AZA treatment (0.8546 and 0.7138, respectively) that were equal to or greater than the three cell lines wild type for p73 DNP: DU145, T47-D and MDA-MB-468 (0.8626, 0.6480, 0.3866, respectively).

Conclusion: Of the five cell lines used in this study, cells that harbor a p73 DNP allele had the highest TAp73/DNp73 mRNA ratios following co-treatment with 5AZA and etoposide or with etoposide alone as compared to wild-type cell p73 DNP, which is consistent with our previously reported data. Lastly, our data in this work assessing the effect of 5AZA treatment of T47-D cells on TAp73/DNp73 mRNA ratios is not consistent with a previously reported study.

#3004

Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, with nintedanib on human colorectal cancer xenografts.

Norihiko Suzuki,1 Fumio Nakagawa,2 Mamoru Nukatsuka,1 Kazuaki Matsuoka,1 Hiroshi Tsukihara,1 Teiji Takechi1. 1 _Translational Research Laboratory, TAIHO Pharmaceutical CO.,Ltd., Tokushima, Japan;_ 2 _Applied pharmacology Laboratory, TAIHO Pharmaceutical CO.,Ltd., Tokushima, Japan_.

Background:

TAS-102 (also named TFTD) is a novel antitumor nucleoside. This is a combination of an antineoplastic thymidine-based nucleoside analog, trifluridine (FTD), and tipiracil hydrochloride (TPI) at a molar ratio 1:0.5. FTD is the active antitumor component of TAS-102 and its triphosphate form is incorporated into DNA in tumor cells. TPI is an inhibitor of thymidine phosphorylase, which strongly inhibits the biodegradation of FTD. In a recent global, multicenter, randomized, double-blind Phase III study (RECOURSE), TAS-102 showed to significantly improve overall survival and progression-free survival and had a favorable safety profile in comparison to those achieved with placebo in patients with metastatic colorectal cancer refractory to standard chemotherapies. Nintedanib is an oral triple angiokinase inhibitor that simultaneously inhibits vascular endothelial growth factor receptors, platelet-derived growth factor receptors, and fibroblast growth factor receptors. It is currently being investigated in Phase III studies of advanced non-small cell lung cancer, colorectal cancer, and ovarian cancer. In this study, we evaluated the antitumor effects of TAS-102 in combination with nintedanib on human colorectal tumor xenografts in a nude mouse model.

Method:

Drug cytotoxicity was determined by the crystal violet staining method using DLD-1, HT-29, and HCT116 colorectal cancer cell lines. Drug interaction was evaluated by performing isobologram analysis. Furthermore, the human colorectal cancer cell lines were implanted into nude mice subcutaneously, and TAS-102 (150 mg/kg/day) and/or nintedanib (20 or 40 mg/kg/day) were orally administered twice daily from Days 1 to 14. Growth inhibitory activity was evaluated based on tumor volume.

Results:

The combination of TAS-102 and nintedanib exerted an additive effect on growth inhibition of DLD-1 and HT-29, and a sub-additive effect on HCT116 in vitro. TAS-102 and nintedanib monotherapies were both active on all of the evaluated colorectal cancers in vivo. Tumor growth inhibition by combination therapy (150 mg/kg/day TAS-102 plus 40 mg/kg/day nintedanib) was 61.5%, 67.6%, and 67.5% for DLD-1, HT-29, and HCT116 xenografts, and that was significantly superior to that of either monotherapy for all evaluated cancer xenografts (P < 0.05). These results demonstrate that combination therapy with TAS-102 and nintedanib is significantly more effective than either monotherapy in colorectal cancer xenografts. This combination therapy appeared to be well tolerated because neither a reduction in body weight of more than 20% nor drug-related death was observed.

Conclusion:

Combination therapy of TAS-102 and nintedanib is significantly more effective than either monotherapy in preclinical models and should be considered for investigation in metastatic colorectal cancer patients.

#3005

The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity.

Kaytee L. Pokrzywinski, Thomas G. Biel, Dmitry Kryndushkin, V. Ashutosh Rao. _US Food and Drug Administration, Silver Spring, MD_.

Reactive oxygen species (ROS) are natural byproducts of mitochondrial oxidative phosphorylation. Dysfunction in electron shuttling leads to enhanced ROS production that is notorious for inflicting damage to macromolecules. Mitochondrial DNA (mtDNA) is particularly sensitive to ROS as it is located in close proximity to the respiratory chain. Additionally, mtDNA lack histones which are known to provide protection from ROS. Mitochondria also have limited DNA repair mechanisms making damage to mtDNA more detrimental to mitochondrial physiology. Recent evidence suggests that oxidative insult, leads to mtDNA degradation to prevent the accumulation of mutagenic base lesions, which is enabled by the high redundancy of mtDNA. The primary objectives of this study were to determine the impact of redox-active mitochondrial-targeted chemotherapeutic agents on mitochondrial physiology and mtDNA integrity. All experiments were performed in two cancer cell lines [MDA-MB-231 (breast) and H23 (lung)] that were treated with 2 uM of the mitochondrial-targeted redox-active compounds: MitoTempol [C4 (MT4)], MitoQuinone (MQ) and MitoChromanol-Acetate (MCA) for 1 or 24 hrs. Mitochondrial status was determined by measuring mitochondrial membrane potential, superoxide production, oxygen consumption rate (OCR) and protein levels of the respiratory chain complexes using the JC-1 and MitoSOX assays, Seahorse XF analysis, and immunoblotting, respectively. Next, mtDNA integrity was analyzed by quantifying the degree of mtDNA fragmentation using PCR amplification of a 10kb fragment compared to a short 100bp fragment. Moreover, changes in the mtDNA replication machinery were assessed using immunoblotting. MQ and MCA induced a loss in mitochondrial function and mtDNA replication machinery as indicated by a reduction in membrane potential, OCR and protein levels. Furthermore, increases in superoxide levels and mtDNA fragmentation were observed with MQ and MCA treatment. Finally, we assessed mitochondrial content using two methods. The first method being the ratio of mitochondrial to nuclear DNA, determined by qPCR of short (~100bp) nuclear and mitochondrial sequences. The second method was the ratio of mitochondrial outer membrane proteins to cytoplasmic housekeeping proteins, determined by immunoblotting. Both mitochondria DNA and outer membrane proteins decreased with MQ and MCA treatment suggesting that redox-active mitochondrial-targeted agents may reduce the mitochondrial content in cancer cells.

#3006

Arsenic trioxide inhibits overexpression of NAMPT in oral cancer.

Jin Zhi Wang,1 Yan Liu,2 Xin Yue Wang,1 Li Chi Han,2 Fu Yin Zhang,1 Ke Liu,3 Bin Xiang1. 1 _Dalian Medical University, Dalian, China;_ 2 _Dalian University, Dalian, China;_ 3 _University of New Mexico, Albuquerque, NM_.

Purpose: Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme for a rate-limiting step in the nicotinamide adenine dinucleotide (NAD) salvage pathway. Tumor cells are reliant on the NAMPT salvage pathway for NAD regeneration, thus making this enzyme an attractive therapeutic target for cancer treatment. Overexpression of NAMPT has been observed across a broad range of solid tumors including ovarian, breast, and prostate cancers. However, it is unknown regarding the expression and function of NAMPT in oral cancer. Arsenic trioxide (ATO) has been approved for the treatment of acute promyelocytic leukemia, and accumulating evidence indicate that it is a promising therapeutic drug for certain solid malignant tumors. Up to now, the effect of ATO on NAMPT in oral cancer remains unclear. In the present study, we investigated the expression of NAMPT in different differentiated degree oral cancer and examined the effects of ATO on NAMPT in oral cancer cell line.

Materials and methods: Tissue microarray (TMA) was performed by immunohistochemistry to examine the NAMPT expression in well differentiated (n=39), moderately differentiated (n=6), and poorly differentiated (n=5) tissue samples collected from surgical specimens of the oral cancer patients. Human oral squamous cell carcinoma Tca-8113 cells were treated with ATO for 48 hrs. PrestoBlue activity assay was used to detect cell activity and NAMPT mRNA expression was measured by RT-PCR.

Results: In paracancer tissue, NAMPT was mainly distributed in cytoplasma and lightly located in nuclear. Intensely increased staining of NAMPT was found in all differentiated grade oral cancer by immunohistochemistry. Moreover, while increased NAMPT are mainly located in cytoplasma in well-differentiated cancer, the marked increase of NAMPT occurred both in cytoplasma and nuclear in moderate- and poor-differentiated cancer. TMA results showed that comparing with normal oral tissue, the protein expression of NAMPT increased by 2.20, 2.66, and 3.03 fold in well-, moderate-, and poor- differentiated oral cancer, respectively. ATO (1, 2.5, 5, and 10 µmol/L) pretreatment of Tca8113 cells significantly reduced the cell activity in an ATO concentration-dependent manner. More importantly, NAMPT mRNA expression was markedly decreased 8.60%, 26.92%, 71.01%, and 78.26%, respectively, compared with control cells.

Conclusion: Our novel findings suggest that increased NAMPT expression were highly associated with cellular differentiation in oral cancer, indicating that NAMPT may participate in the development of oral cancer. ATO can inhibit cell activity by depressing NAMPT transcription in oral cancer. These findings may have implications for exploring the NAMPT pathway for oral cancer treatment.

#3007

Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer.

Balqis A. Ikhmais, Buthaina Hussein, Ian Stratford, Sally Freeman, Joaana Neill. _The University of Manchester, Manchester, United Kingdom_.

Introduction:

NRH quinone oxidoreductase 2 (NQO2) is one of the mammalian enzymes responsible for the two-electron reduction of quinones to hydroquinones. NQO2 can use a variety of dihydronicotinamide analogues as co-factors. In particular, the co-factor, EP0152R has been used in Phase I clinical trials to support evaluation of the NQO2-mediated reductive activation of CB1954. As well as being a prodrug-activating enzyme, there is evidence to show that NQO2 may be involved in cancer progression as a consequence of its ability to act as a "nanny" protein, alter cyclin D expression and modulate the activity of NF-ĸB. Thus, we have synthesized a series of novel 4-aminoquinolines to study their ability to inhibit NQO2 in ovarian cancer cells (OVCs). We then propose to compare the results of pharmacological inhibition with those obtained by genetic down-regulation of NQO2 level.

Methods:

NQO2 level in a panel of OVC cell lines was determined by using western blot analysis and Cytochrome C based spectrophotometric enzyme activity assay.

Novel compounds have been synthesized and assessed for their inhibitory potency against recombinant human NQO2 (rhNQO2), using DCPIP spectrophotometric assay.

MTT cytotoxicity assay was utilized for several purposes: Firstly, to determine the toxicity of known and novel NQO2 inhibitors in the SKOV3 cell line; and secondly, to evaluate the sensitivity of OVC cell lines to CB1954 in the presence of EP0152R, and subsequently measure the intracellular NQO2 inhibitory potency of novel compounds, by combining them with CB1954 and EP0152R.

Results and conclusion:

In the OVCs, NQO2 protein level and enzymatic activity showed an excellent correlation; however, expression and activity differed widely among the OVC cell lines. The NQO2 levels were highest in SKOV3 and lowest in the TOV112D cell lines.

The sensitivity of OVCs to CB1954 was significantly increased when combined with EP0152R; supporting the notion that NQO2 mediates the toxicity of CB1954. This was confirmed when a strong correlation was observed between the cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Further, overexpressing NQO2 in TOV112D cells resulted in a significant enhancing of its responsiveness to treatment with CB1954.

The novel 4-aminoquinolines inhibited the activity of recombinant NQO2 when used in the nano-molar range. The inherent cytotoxicity of the inhibitors (as measured by the IC50 following 24 and 96 hours exposure to SKOV3 cells) ranged between 1.7 to >100µM. There was no relationship between toxicity and the ability of the compounds to inhibit the activity of rhNQO2. However, some of these compounds showed functional activity as NQO2 inhibitors in cells; this was revealed by their ability to inhibit the toxicity of CB1954. Several compounds were able to do this at sub-µM, non-toxic concentrations and therefore they may be able to act as molecular probes for the (multi-) functional activity of NQO2 in cells.

#3008

Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer.

Ondrej Kalous,1 Dylan Conklin,1 Kanthinh Manivong,1 William Wayne,2 Kelly Hanestad,2 Jude Canon,2 Robert Loberg,2 Gregory Friberg,2 Erick Gamelin,2 Florian D. Vogl,2 Gloria Juan,2 Angela Coxon,2 Dennis Slamon,1 Richard Finn,1 Marc Payton2. 1 _UCLA, Division of Hematology and Oncology, Department of Medicine, Jonsson Comprehensive Cancer Center, Santa Monica, CA;_ 2 _Amgen, Inc., Thousand Oaks, CA_.

BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was > 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53mut IGROV-1 cells showed minimal regrowth compared to TP53wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P < .0001 vs. vehicle alone). Notably, co-administration of AMG 900 with docetaxel enhanced efficacy and induced a delay in tumor regrowth compared to docetaxel alone. Single-agent-treated mice showed minimal body weight loss (BWL), whereas combination-treated mice showed moderate BWL (average < 10%) that was largely reversible (2 of 12 animals removed due to toxicity). CONCLUSIONS: AMG 900 alone or in combination with chemotherapy such as paclitaxel may be a promising clinical strategy to treat patients with ovarian cancer.

#3009

Targeting G-protein coupled receptor-related pathway in mucinous tumors.

Ashok K. Dilly, Yong J. Lee, Herbert J. Zeh, David L. Bartlett, Mohammad Haroon A. Choudry. _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Introduction: Pseudomyxoma peritonei (PMP) is an indolent malignancy characterized by excessive production of Mucin 2 (MUC2; a gel-forming secreted mucin). We have previously demonstrated mucinous tumor growth inhibition in a patient-derived murine xenograft model of PMP using the cyclooxygenase-2 (COX-2) inhibitor celecoxib. We evaluated the downstream mechanism for celecoxib-mediated MUC2-inhibition.

Methods: In vitro and in vivo studies were conducted using MUC2-secreting LS174T cells, PMP explant tissue and a unique intraperitoneal patient-derived murine xenograft model of PMP (PDX model of PMP).

Results: LS174T cells treated with COX-2 inhibitor celecoxib (5-20 µM) for 24 hours demonstrated a significant reduction in MUC2 protein expression measured by flow cytometric analysis. Similarly, in the PDX model of PMP chronic oral therapy with celecoxib (20 mg/kg/d) for 28 days significantly inhibited MUC2 protein expression in harvested tumor tissue. In the LS174T cells, PGE2 (a COX-2 product) induced MUC2 mRNA (real-time PCR) and protein expression (flow cytometry) and this was inhibited by the G-protein coupled-EP4 receptor inhibitor AH23848 as well as siRNA for CREB (cAMP response element-binding protein). Using chromatin immunoprecipitation assay in LS174T cells, we found that treatment with celecoxib or protein kinase A inhibitor (fragment 6-22 amide) significantly decreased CREB-transcription factor binding to the MUC2 promoter.

Conclusions: These data suggest that celecoxib inhibits MUC2 expression via the G-protein coupled receptor/ cAMP/ Protein kinase A/ CREB pathway. Since activating G-protein (GNAS) mutations are commonly found in PMP and may be responsible for its unique mucinous phenotype, our data provide a rationale for targeting this pathway to control mucin production in this malignancy.

#3010

Proteomic analysis of off-target toxicities following treatment with the tyrosine kinase inhibitor sunitinib.

Alice C. O'Farrell,1 Adam Lafferty,1 Ian Miller,1 Rhys Evans,1 Maurice Cary,2 David Murray,1 Marina Alamanou,3 Girish Mallya Udupi,3 Liam Shiels,1 William M. Gallagher,4 Annette T. Byrne1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Pathology Experts GmbH, Basel, Switzerland;_ 3 _UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin, Ireland;_ 4 _OncoMark Ltd, Dublin, Ireland_.

Background: Sunitinib is a multi-targeted agent approved for treatment of a number of cancers. However, since approval, data has continually emerged relating to toxicity profiles including hypertension, hand-foot syndrome and fatigue. Underlying mechanisms are unresolved. It has been hypothesised that the multi-parameter toxicity profile is related to 'on-target' kinase inhibition in 'off-target' tissues.

Methods: To interrogate off-target effects, the Zeptosens Reverse Phase protein Array (RPA) platform was used to assess tissues obtained from mice treated with sunitinib (40 mg/kg) for 4 weeks. Additional tissue was collected for histological analyses and pathophysiologic changes assessed.

Results: RPA data analyses on all organs collected (heart, kidney, liver, brain, intestine and skin) revealed the presence of differentially expressed proteins associated with damage and/or stress. Of note, Cyclin D1, MEK and phosphorylated-p21(CIP/WAF1) expression increased by 1.8-fold (p < 0.05), 2.2-fold (p < 0.1) and 1.7-fold (p < 0.1) respectively in the kidneys of sunitinib treated mice. These proteins are associated with kidney damage after chemotherapy, and induction of genes associated with renal immune response, inflammation and apoptosis. Mild proteinuria was observed in sunitinib treated animals, however no gross change in renal glomerular ultrastructure was visible via electron microscopy. Phosphorylated-FGFR1, cleaved Caspase-7, and phosphorylated-cMYC expression decreased by 0.45-fold (p < 0.05), 0.4-fold (p < 0.1) and 0.8-fold (p < 0.1) respectively in the skin of sunitinib treated mice. Down-regulation of these proteins has been associated with a perturbation of cutaneous wound healing (p-FGFR1/cleaved Caspase-7). Mice treated with high dose sunitinib (80 mg/kg) showed overt signs of skin toxicity. Histopathologic studies of the thyroid gland revealed decreased thyroid epithelial cell height (p < 0.05). Nevertheless, serum levels of triiodothyronine and thyroxine remained unchanged. An orthogonal validation study (Western blotting) is ongoing based on gene ontology, pathway analysis and observed fold changes in critical signalling pathway proteins involved in cellular stress.

Conclusions: Implementation of a combined histopathologic/ RPA approach may provide a sensitive method to mechanistically elucidate the off-target toxicity sequelae associated with TKIs approved in the oncology setting.

Work was supported by an EU funded Industry Academia Pathways and Partnerships Marie Curie Award (AngioTox) Grant Number 251528. RPAs were performed at Bayer Technology Services, Leverkusen, Germany. 

### Novel Targets and Pathways

#3011

Development of selective small-molecule inhibitors of cellular MALT1 protease activity.

Fredrik G. Oberg, Sofia Karlstrom, Ian Henderson, Ellen Hewitt, Anders Kallin, Susanne Sedig, Jimmy Lindberg, Anna-Karin Sohlenius-Sternbeck, Richard Bethell, Mark Albertella. _Medivir AB, Huddinge, Sweden_.

Background: The aim of this study was to develop potent, selective and cell-permeable inhibitors of MALT1 protease activity. MALT1 is an Arg-specific protease that cleaves multiple substrates, acting as a key component in T- and B-cell receptor signaling in lymphocytes. MALT1 protease activity is reported to mediate normal and disease-associated lymphocyte proliferation and survival as part of the CARMA-1/BCL-10/MALT1 (CBM) signaling complex, making MALT1 an attractive therapeutic target. Constitutive MALT1 activity is a hallmark of Diffuse Large B Cell Lymphoma of the activated B cell type (ABC-DLBCL). This aberrant activation is observed as a consequence of mutations in CARMA-1/CARD11, and in multiple regulatory proteins upstream of the CBM complex.

Methods: MALT1 enzyme assay with Ac-Leu-Arg-Ser-Arg-AMC as substrate was used to measure Ki. Selectivity towards other Arg-specific proteases was measured by inhibition of Trypsin and Thrombin. For determination of cellular potency the ABC-DLBCL cell line OCI-LY3, carrying the L251P mutation of CARMA-1/CARD11, was used. A MALT1 cell assay to measure A20 substrate cleavage was adapted to capillary electrophoresis and quantification. MALT1 down-stream signaling was measured by inhibition of IL-6 expression.

Results: Published inhibitors of MALT1 were profiled for potency, selectivity, DMPK properties and activity in cell-based assays, and showed several drawbacks such as low MALT1 activity, low cell permeability and/or selectivity. We developed several novel reversible small molecule inhibitors of MALT1 with Ki <20 nM and high selectivity against Trypsin and Thrombin. Compounds show inhibition of intracellular MALT1 activity with potencies <300nM in the A20 cleavage assay, as well as inhibition of IL-6 expression. Intracellular inhibition of MALT1 activity requires both potent inhibition of MALT1 and cell permeability (estimated by Caco-2 permeability), e.g. compound A with MALT1 Ki=16nM, Trypsin Ki>100μM, Thrombin Ki>100μM, and Caco-2 Papp=1x10-6cm/s shows intracellular A20 cleavage IC50=1100nM, whereas compound B with MALT1 Ki=47nM and Caco-2 Papp=5.6x10-6cm/s, displays intracellular A20 cleavage IC50=600nM. We observe a clear dissociation between intracellular inhibition of MALT1 and inhibition of cell proliferation of DLBCL cell lines of ABC-type with constitutive MALT1 activity, e.g. compound C (MALT1 Ki=35nM, Trypsin Ki>100μM, Thrombin Ki>100μM and intracellular A20 cleavage IC50=260nM) inhibits the proliferation of OCI-LY3 cells and TMD8 cells with CC50 values of 73μM and 96μM respectively, after 5 days exposure.

Conclusion: Inhibition of MALT1 protease activity did not translate into significant anti-proliferative effects in DLBCL cell lines. These new MALT1 inhibitors provide the

opportunity to test other therapeutic hypotheses involving the targeting of MALT1 protease activity.

#3012

ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells.

Marie D. Baumeister,1 Jessica Wagner,2 Varun V. Prabhu,3 Christina LB Kline,4 Bora Lim,5 Josh E. Allen,6 David T. Dicker,4 Wafik S. El-Deiry4. 1 _Temple University School of Medicine, Philadelphia, PA;_ 2 _Temple University, Philadelphia, PA;_ 3 _Penn State Hershey Cancer Institute, Hershey, PA;_ 4 _Fox Chase Cancer Center, Philadelphia, PA;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _Oncoceutics, Inc., Philadelphia, PA_.

Breast cancer is a major cause of cancer related death. TRAIL has been of interest as a cancer therapeutic, but resistance has been observed in breast cancer and only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. Small molecule ONC201 functions through the ATF4/CHOP pathway to upregulate TRAIL receptor DR5 (Kline et. al., Sci. Sig., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Sci. Trans. Med., 2013). We recently reported that ONC201 depletes colorectal cancer stem cell (CSC) markers and prevents formation of colonospheres. ONC201 also prevents colorectal CSCs from initiating growth of xenografted tumors (Prabhu et. al Can. Res., 2015). ONC201 has completed its first-in-human clinical trial in advanced solid tumors (Stein et al., 2015 AACR-NCI-EORTC meeting) and is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We investigated ONC201 efficacy in TNBC (TRAIL-sensitive), BRCA1-deficient, and non-TNBC (TRAIL-resistant) cells. We demonstrate IC50 values for ONC201 in the low μM range for TNBC or BRCA1-deficient (n=6) and non-TNBC cells (n=5), doses that are achievable based on human PK. Importantly, ONC201 induces apoptotic death in both TNBC and non-TNBC cells. ONC201 depletes Aldefluor+ putative breast CSCs in vitro whereas paclitaxel does not. ONC201 inhibits growth of TNBC, BRCA1-deficient and non-TNBC CSC-like mammospheres while paclitaxel chemotherapy does not. We show ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We investigated effects of ONC201 on mechanisms of TRAIL resistance in breast cancer cells, including expression of inhibitor of apoptosis (IAP) family proteins. We found that ONC201 mediates a decrease in XIAP, c-IAP1, and c-IAP2 expression in the breast cancer cells used. We also examined levels of DR5 using flow cytometry, as a mechanism of TRAIL resistance in breast cancer cells involves receptor endocytosis from the cell surface (Zhang et al., Mol Cancer Res., 2008). Interestingly, we observed greater increases in cell surface DR5 in TNBC cells than non-TNBC cells after ONC201 treatment. Our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC, BRCA1-deficient, and non-TNBC subtypes. ONC201 is efficacious against breast cancer cells regardless of the TRAIL sensitivity of the cells, and this efficacy may be mediated through mechanisms involving downregulation of IAP proteins and upregulation of cell surface death receptors. Furthermore, ONC201 may target paclitaxel-resistant breast CSCs. Our findings suggest unique targeting of multiple non-TNBC, BRCA1-deficient and TNBC cells and develop a preclinical rationale for the use of ONC201 as a treatment for breast cancer.

#3013

Identification of PHF5A as a common cellular target of splicing-modulating chemical probes.

Teng Teng, Xiaoling Puyang, Shouyong Peng, Jacob Feala, Betty Chan, Jennifer Tsai, Benjamin Caleb, Craig Karr, Eunice Park, Laura Corson, Yoshiharu Mizui, Peter Smith, Nicholas Larsen, Lihua Yu, Markus Warmuth, Ping Zhu, Agustin Chicas. _H3 Biomedicine INC., Cambridge, MA_.

Recent discoveries that splicing factors such SF3B1, U2AF1, SRSF2 are frequently mutated in multiple hematological malignancies including chronic lymphocytic leukaemia and myelodysplastic syndromes have generated interest in therapeutic approaches to target the splicesome dependency in cancer cells bearing mutations in splicing factors. Previously, several structurally unrelated natural compounds including pladienolide, herboxidiene, and FR901464 have been shown to exert potent anti-proliferative effects in cancer cells grown in vitro. Further optimization has led to the discovery of natural product analogs (e.g. E7107) with anti-tumor efficacy in vivo in xenograft models. Target identification has revealed the SF3B complex of the splicesome as the common action site for these compounds. Recent work has demonstrated biological and genetic evidence that single amino acid substitution (R1074H) in SF3B1 completely abolished the anti-proliferative effect of pladienolide derivative E7107, suggesting that SF3B1 is the direct binding partner for pladienolides. However, the same SF3B1 R1074H mutation does not provide equal level of protections for cells treated with herboxidiene derivatives, indicating differential mechanism of action for these two classes of splicing modulators. To identify targets for herboxidiene-like compounds, we have generated resistant HCT116 clones upon continuous administration of herboxidiene derivative H3B-37045 in vitro. Whole exome sequencing from 6 resistant clones revealed a common Y36C mutation in SF3B subunit component PHF5A (SF3B14b). Over-expression of PHF5A Y36C but not the wild-type form in parental HCT116 cells confirmed the protective effect of this mutation to H3B-37045. Surprisingly, PHF5A Y36C expression also conferred resistance to the pladienolide derivative E7107, which indicates that, unlike the SF3B1 R1074H mutation, PHF5A resides within a common node of action site among different splicing modulators. RNA-seq, biochemical and structure homology-modeling analysis suggested that PHF5A Y36C mutation disrupted the action of splicing modulators through interfering with the compounds' interaction with the SF3B complex. Detailed analysis of the function of the Y36C mutant and wild-type PHF5A in the SF3B complex is currently ongoing. Understanding the function of PHF5A in splicing and the molecular mechanism of Y36C mutation shall provide new insights of the biological role of splicesome, and guide the development of next generation splicesome inhibitors.

#3014

ONC201 anti-cancer effects against solid tumors are mediated through eIF2α kinases HRI and PKR but are PERK-independent.

Christina Leah B. Kline, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

ONC201 is a first-in-class small molecule inducer of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. We recently reported that ONC201-mediated induction of the TRAIL pathway is largely promoted by a preceding early activation of the integrated stress response pathway (ISR) (Kline et al., Sci. Sig., in press, 2015). ONC201 engages the ISR to exert its broad-spectrum anti-cancer effects through pro-apoptotic and anti-proliferative activities. ONC201 upregulates ATF4, the key indicator of ISR activation, in a manner that is dependent on two eIF2α kinases: heme-regulated inhibitor (HRI) and double-stranded RNA activated protein kinase (PKR). Of note, the activation of ATF4 and downstream cell death signaling by ONC201 is PERK-independent. We now further elucidate how the novel dual engagement of HRI and PKR by ONC201 contributes to the anti-cancer effects of ONC201. ONC201 treatment results in early phosphorylation of eIF2α in a manner that is dependent on HRI. On the other hand, we have shown that ONC201-induced downregulation of cyclin D1 is dependent, at least in part, on PKR, but not on eIF2 α phosphorylation per se. This occurs potentially via PKR-mediated ubiquitination and subsequent proteasome degradation of cyclin D1. Treating cells with ONC201 and the proteasome inhibitor MG132 prevents ONC201-induced decrease of cyclin D1. Despite distinctions in their regulatory protein domains, HRI and PKR have both been shown to respond to oxidative stress. Treating cells with ONC201 in the presence of the antioxidant N-acetylcysteine abrogated ATF4 induction. Our findings document a unique activity of ONC201 on protein translation, via activation of HRI and PKR, as well as effects on protein stability through the proteasome leading to anti-cancer effects.

#3015

Plitidepsin targets the GTP-bound form of eEF1A2 in cancer cells.

Alejandro Losada, Maria J. Munoz-Alonso, Juan F. Martínez-Leal, Juan M. Dominguez, Carlos M. Galmarini. _PharmaMar S.A., Madrid, Spain_.

Plitidepsin (APL), an antitumor agent originally isolated from the marine tunicate Aplidium albicans, is being tested in multiple myeloma (MM) patients in a phase III pivotal trial in combination with dexamethasone and a phase I trial in combination with bortezomib and dexamethasone. eEF1A2 is one of two isoforms of the alpha subunit of the eEF1 complex. In mammals, eEF1A2 has a selective pattern of expression in those tissues that do not express the A1 isoform, namely brain and muscle. Nonetheless, eEF1A2 is aberrantly expressed in many cancers, including solid tumors (1-3) and MM (4), and has been shown to hold pro-oncogenic activities (5). Here we analyze the interaction of plitidepsin with its target, eEF1A2. DARTS assays, either with whole cell extracts or with purified eEF1A2 protein, indicate that plitidepsin binds to eEF1A2 and protects it from digestion by subtilisin (EC 3.4.21.62). Moreover, a [14C]-APL binding-guided fractionation of K562 cell extracts through differential centrifugation and three chromatographic steps demonstrated that eEF1A2 is the only cellular protein that can be retrieved through specific binding to plitidepsin. A saturation binding experiment with [14C]-APL and purified GTP-bound eEF1A2 (from rabbit muscle) allowed us to calculate a Kd of around 80 nM for the interaction, while a dissociation experiment revealed a residence time of around 9 minutes. Indeed, we have found that plitidepsin exclusively binds to the GTP-bound form of eEF1A2. HeLa-APL-R cells, ≥1000 fold more resistant to plitidepsin than parental HeLa wt cells (6), are now shown to have lower eEF1A2 mRNA and protein levels than parental cells. Furthermore, when eEF1A2 levels were restored to normal in HeLa-APL-R cells through ectopic expression of an eEF1A2-GFP construct, they were rendered partially sensitive to plitidepsin. Interestingly, transfected cells recovered most of the signaling events typically induced by the drug in HeLa wt cells. NCI-H460 (lung) and HGC-27 (stomach) cancer cell lines were rendered resistant to plitidepsin following the same procedure described in (6) for HeLa-APL-R cells. When we analyzed the levels of eEF1A2 in this two new cell lines we observed that both of them were lacking eEF1A2. Altogether, our results demonstrate that plitidepsin targets the pro-oncogenic eEF1A2 protein, a new druggable target for anticancer therapy.

(1)Sun et al, 2014, Biochem Biophys Res Commun 450:1-6

(2)Xu et al, 2013, Clin Exp Metastasis 30:933-44

(3)Anand et al, 2002, Nat Genet 31:301-5

(4)Li et al, 2010, PLOS One 5, e10755

(5)Lee and Surh, 2009, Ann N Y Acad Sci 1171:87-93

(6)Losada et al., 2004, Br J Cancer 91:1405-13

#3016

KPT-9274 inhibits cellular NAD and synergizes with NAD depleting enzymes to induce cancer cell death.

Christian Argueta, Trinayan Kashyap, Margaret Lee, Yosef Landesman, Sharon Shacham, William Senapedis, Erkan Baloglu. _Karyopharm Therapeutics, Inc., Newton, MA_.

Nicotinamide adenine dinucleotide (NAD) is an essential metabolite and important cofactor of several biological processes (i.e. metabolism and genomic stability) that undergo significant alterations during malignant transformation. In cancer cells, NAD undergoes quick turnover through the high metabolic demands of rapidly proliferating cells and increased activity of NAD consuming enzymes, such as sirtuin 1 (SIRT1) and poly ADP ribose polymerase 1 (PARP1). NAD can be generated de novo from tryptophan or regenerated by nicotinamide phosphoribosyl transferase (NAMPT) or nicotinate phosphoribosyl transferase 1 (NAPRT1) in NAD salvage pathways. However, cancer cells do not utilize the de novo or NAPRT1 pathways effectively and instead rely on the NAMPT-dependent salvage pathway to generate NAD, making NAD depletion a promising anti-cancer therapy. We have previously described the PAK4 allosteric modulator, KPT-9274, which can also inhibit the enzymatic activity of NAMPT. KPT-9274 and other NAMPT inhibitors rapidly deplete cellular NAD levels, ultimately leading to ATP depletion and cell death. Similarly, several studies have shown that hyper activation of NAD consuming enzymes can lead to apoptosis. The purpose of this study is to determine whether NAD inhibition by KPT-9274 can synergize with NAD depleting enzymes to enhance the cytotoxic effects of KPT-9274 in cancer cells.

Methods: Celltiter-Glo was used to measure ATP levels and viability of cells. NAD/NADH-Glo was used to measure total NAD levels in cells. Gene and protein expression was measured using quantitative PCR and western blot analysis, respectively. Protein knockdown was accomplished using RNAi.

Results: We have identified an orally bioavailable dual inhibitor of PAK4 and NAMPT, which demonstrated potent anti-cancer activity in a variety of cell lines both in vitro and in vivo. We have identified a mechanistic combination that increase the antitumor activity of KPT-9274, through the activation of NAD consuming enzymes such as SIRT1 and PARP1. Specifically, we found that SRT1720 (an activator of SIRT1) synergizes with KPT-9274 to increase cancer cell death while RNAi of SIRT1 diminishes the efficacy of KPT-9274. In addition, activation of PARP1 by DNA damaging agents (e.g. gemcitabine) significantly enhances the effectiveness of KPT-9274 mediated cell death. Finally, we show that PARP activation through DNA-damaging agents enhances the cytotoxicity and anti-tumor properties of KPT-9274 in xenograft models.

Conclusions: Here we report that KPT-9274 synergizes with NAD depleting enzymes to induce cancer cell death in vitro and in vivo. This noteworthy enhancement to the anticancer activity of KPT-9274, together with its previously described PAK4 inhibition, support the continued development of this orally bioavailable small molecule in combination with current therapies.

#3017

Non-classical activation of GLI1 as a therapeutic target for squamous cell lung cancer.

Sahba Kasiri,1 Chunli Shao,1 Baozhi Chen,1 Alexandra Wilson,1 Paul Yenerall,1 Brenda Timmons,1 patrick dospoy,1 Suzie Hight,1 Luc Girard,1 Hui Tian,1 Carmen Behrens,2 Ignacio Wistuba,2 Adi Gazdar,1 James Kim1. 1 _The University of Texas Southwestern Medical Center at Dallas, Dallas, TX;_ 2 _The University of Texas M.D. Anderson Cancer Center, Houston, TX_.

The Hedgehog (Hh) signaling pathway is critical for embryonic developmental processes and its deregulation is implicated in a wide variety of tumor types. However, the role of the Hh signaling pathway in the initiation and growth of non-small cell lung cancer is largely unknown. The purpose of this study is to investigate the role of GLI1, a major Hh pathway transcription factor, in lung squamous cell carcinoma (SCC) and to test the therapeutic potential of targeting GLI1.

GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential.

We have demonstrated that GLI1 has a critical role in SCC progression. GLI1 expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not change GLI1 expression level in SCC cell lines. However, inhibition of PI3K/mTOR and MAPK signaling pathways down regulated GLI1 expression. These results suggested that GLI1 expression is dependent on PI3K/mTOR and MAPK pathway activity rather than Hh ligand. PI3K/mTOR inhibitor, or arsenic trioxide (ATO), a direct inhibitor of GLI proteins, significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. We have also observed a significant growth reduction in SCC xenografted tumors treated with combination of PI3K inhibitor and ATO.

Our findings suggest that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC.

#3018

Cotargeting MEK and CDK4/6 to treat pancreatic adenocarcinoma.

Joel D. Maust, Diane Simeone, Judith Sebolt-Leopold. _University of Michigan, Ann Arbor, MI_.

Hyperactivation of KRAS and inactivation of CDKN2A [p16] play a prominent role in tumor initiation and progression in a broad spectrum of human cancers. In pancreatic adenocarcinoma, KRAS mutation occurs in 90-95% of cases and is often coupled with inactivation of CDKN2A (>90% incidence), typically by homozygous deletion. Based on the high incidence of these genetic events, the present study addresses the hypothesis that dual targeting of MEK and CDK4/6 represents a viable therapeutic strategy for the treatment of pancreatic adenocarcinoma. A panel of primary and high passage xenograft models was screened for in vitro synergy to the antiproliferative effects of the MEK inhibitor trametinib and the CDK4/6 inhibitor palbociclib. Two models that emerged as highly sensitive to this combination treatment strategy, L3.6pl and UM59, both exhibited G1 cell cycle arrest that was significantly more pronounced in response to the combination compared to either single agent. In vivo studies were subsequently carried out to evaluate the efficacy of this combination in tumor-bearing mice. Mice implanted subcutaneously with L3.6pl or UM59 cells were treated daily by oral gavage for ten days with trametinib (3 mg/kg), palbociclib (100 mg/kg), or the combination of these two agents at these doses. Consistent with in vitro synergy observations, both models proved to be exceptional responders in vivo to this combination treatment strategy, showing a robust response to the combination not seen with either single agent. These results suggest that the combined inhibition of MEK and CDK4/6 may offer a valuable therapeutic strategy for the treatment of pancreatic adenocarcinoma. Studies are ongoing to identify molecular determinants of response in pancreatic tumors that are highly sensitive to this combination approach to guide future patient enrichment strategies.

#3019

High throughput drug screening identified spleen tyrosine kinase as a novel therapeutic target in head and neck cancer with potent in vitro and in vivo activity.

Morgan Black,1 Laurie Ailles,2 Ren Sun,2 Alessandro Datti,3 Frederick Vizeacoumar,3 Nicole Pinto,4 Kara Ruicci,4 John Yoo,1 Kevin Fung,1 Danielle MacNeil,1 David A. Palma,1 Eric Winquist,1 Joe S. Mymryk,4 Paul C. Boutros,2 John W. Barrett,1 Anthony C. Nichols1. 1 _London Health Sciences Centre, London, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Ontario, Canada;_ 3 _Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada;_ 4 _University of Western Ontario, London, Ontario, Canada_.

Background and Significance: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and survival remains poor highlighting the need for novel treatments for the treatment of HNSCC. High throughput drug screening has shown the potential to discover novel therapeutics in other cancers.

Methods: Twenty-eight HNSCC cell lines, including 5 HPV-positive lines, were characterized with whole exome sequencing and copy number arrays and screened with 1505 potential anti-cancer agents on a robotic liquid handling platform at a single dose (4uM). The most potent hits were confirmed with 10-point dose response curves. Novel therapeutic targets were further investigated with mechanistic and xenograft studies.

Results: Drug screening identified 10 agents with broad activity across our cell line panel. One of the most potent agents was ER27319 maleate, reported to be a spleen tyrosine kinase (Syk) inhibitor. We confirmed that this molecule inhibited Syk phosphorylation specifically at tyrosine residues 525/526. Additionally, ER27319 maleate as well as a more clinically relevant Syk inhibitor, Fostamatinib, were observed to significantly impaired cellular migration and invasion. Additionally, siRNA knockdown of Syk in HNSCC cells was found to decrease HNSCC cell line growth. Finally, inhibition of Syk was observed to control HNSCC tumour growth in vivo in cell line-derived xenografts.

Conclusions: High throughput drug screening of HNSCC cell lines identified Syk as a novel target and confirmed potent in vitro and in vivo activity. Further preclinical evaluation is planned with a panel of patient-derived xenografts. Should these results yield significant activity, we will aim to repurpose approved Syk inhibitors to improve outcomes for patients suffering from HNSCC.

#3020

Targeting both canonical and non-canonical NF-kB pathways by the IAP antagonist birinapant potentiates bortezomib anti-myeloma activity.

Liang Zhou,1 Shuang Chen,1 Yu Zhang,1 Maciej Kmieciak,1 Yun Leng,2 Lihong Li,2 Hui Lin,1 Joel Turner,3 Daniel Sullivan,3 Yun Dai,1 Steven Grant1. 1 _VCU Massey Cancer Center, Richmond, VA;_ 2 _Beijing Chaoyang Hospital of Capital Medical University, Beijing, China;_ 3 _H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL_.

Inhibitor of apoptosis proteins (IAPs e.g., XIAP, cIAP1, cIAP2) inhibit apoptosis through diverse mechanisms. Recent attention has focused on novel functions of cIAP1/2, including activation of the canonical and non-canonical NF-κB pathways and inhibition of the extrinsic apoptotic pathway through the ripoptosome. These considerations have stimulated the development of IAP antagonists such as the bivalent IAP inhibitor birinapant (TL-32711). Proteasome inhibitors such as bortezomib are highly active in multiple myeloma (MM), and act, at least in part, by inhibiting NF-κB, raising the possibility of cooperative interactions with IAP antagonists. Synergistic induction of apoptosis was observed in human MM cell lines, including drug-naïve U266 cells, their bortezomib-resistant counterparts (PS-R), and multiple other human MM lines. Birinapant (500 nM) sharply down-regulated cIAP1/2 in MM cells, accompanied by reduced expression of TRAF2, RIP1, and p-IKKβ, as well as robust TRAF3 up-regulation, in association with down-regulation of NIK, p-IKKα, and p52, consistent with canonical and non-canonical NF-κB pathway inactivation, respectively. Notably, U266 cells overexpressing TRAF2 or in which TRAF3 was knocked down by shRNA displayed significant resistance to the birinapant/bortezomib regimen, indicating that disruption of both canonical and non-canonical NF-κB signaling contributes functionally to anti-myeloma activity. Moreover, Bcl-2 or Bcl-xL overexpression and dominant-negative FADD markedly blocked apoptosis, suggesting the involvement of both intrinsic and or extrinsic apoptotic pathways. 3D co-culture studies with HS-5 stromal cells revealed no protection of MM cells from the birinapant/bortezomib regimen, indicating circumvention of micro-environmental forms of drug-resistance. Markedly enhanced cell killing by the combination was also observed in primary CD138+ MM cells and primitive CD138-/19+/20+/27+ progenitors, but not normal CD34+ cells. HS-5 cells also failed to protect primary CD138+ MM cells from this regimen. Finally, co-administration of birinapant and bortezomib significantly reduced tumor burden and enhanced animal survival compared to single-agent treatment with minimal toxicity in NSG mice inoculated with U266 cells. Together, these findings indicate that targeting cIAP1/2 by birinapant significantly increases bortezomib anti-MM activity in MM cells, including bortezomib-resistant and primary MM cells, circumvents microenvironment-related resistance, and mechanstically involves concommitant inhibition of the canonical and non-canonical NF-κB pathways as well as activation of the intrinsic and extrinsic apoptotic pathways. Finally, the regimen is well tolerated and active in in vivo animal models. Collectively, these findings support exploring an IAP/proteasome inhibitor strategy in MM.

#3021

Development of small molecule direct AMPK activators for the treatment of cancer.

Yasumichi Hitoshi, Yonchu Jenkins, Yingwu Li, Elmer Sampang, Xiang Xu, Guodong Dong, Jianing Huang, Nan Lin, Dane Goff, Simon Shaw, Luke Boralsky, Rajinder Singh, Sarkiz D. Issakani, Donald G. Payan. _Rigel Pharmaceuticals, Inc., South San Francisco, CA_.

5'-AMP-activated protein kinase (AMPK) is a key sensor of cellular energy status and is critical for maintaining energy homeostasis under conditions of nutrient stress. During cellular transformation, metabolic reprogramming enables the aberrant growth and proliferation of tumor cells. Both positive and negative roles for AMPK in tumor cell proliferation and survival have been reported. However, only a limited number of studies addressed this question with potent direct AMPK activators. AMPK exists as heterotrimers composed of the catalytic subunit α and reguratory subunits β and γ. We expressed the full length of all three human AMPK subunits in insect cells, purified the heterotrimer complexes, and used them for biochemical screening and characterization of AMPK activators. The purified complexes displayed basal activity, which was further enhanced by AMP. The compounds we identified potently activated the complexes in vitro at AC(2X)s (the concentration that gives a twofold activation) of 0.001-0.3 µM. Importantly, the compounds up-regulated substrate phosphorylation (pS79 Acetyl-CoA Carboxylase) and/or auto-phosphorylation (pT172 AMPKα) in multiple cancer cell lines including HepG2 hepatoma cells, A549 liver kinase B1 (LKB1) null lung cancer cells, and MOLM14 myeloid leukemia cells, indicating activation was irrespective of functional status of LKB1, which is a key AMPK-activation kinase. Activation of AMPK by the compounds was also confirmed using native AMPK isolated from normal tissues and tumor cells. We further investigated anti-proliferative effects of the compounds and found that up-regulation of AMPK kinase activity was correlated with anti-proliferative effects in A549 and MOLM14, but not in HepG2, suggesting that positive effects of direct AMPK activators could be cell-type dependent. Interestingly, we identified compounds that display comparable AMPK activation in HepG2 and A549 yet possessed divergent activities on proliferation across a panel of tumor lines. Analysis of cellular signaling across several of these tumor lines with this set of the compounds revealed dose-dependent effects on mTORC1 substrates, feedback signaling to PI3K and mTORC2, and inhibition of kinases downstream of RAF. Direct activation of AMPK could be a good therapeutic strategy for the treatment of subsets of cancers.

#3022

BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML).

Arne Scholz,1 Thomas Oellerich,2 Akhtar Hussain,2 Sarah Lindner,2 Ulrich Luecking,1 Annette O. Walter,1 Peter Ellinghaus,1 Ray Valencia,1 Franz von Nussbaum,1 Dominik Mumberg,1 Michael Brands,1 Stuart Ince,1 Hubert Serve,2 Karl Ziegelbauer1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Dept of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany_.

PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc, plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy.

In contrast to pan-CDK inhibitors, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report the preclinical activity of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor (AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02) currently being investigated in Phase I clinical trials in advanced cancer (NCT01938638) and acute leukemia (NCT02345382) in various in vitro, ex vivo and in vivo models of AML.

BAY 1143572 inhibited the proliferation of 7 MLL-rearrangements positive and negative AML cell lines with a median IC50 of 385 nM (range 230-1100 nM) and induced apoptosis. Furthermore, BAY 1143572 showed potent in vitro activity in 8 out of 10 non-MLL-rearranged patient derived AML samples incl. NPM1 mutant and Flt3-ITD positive samples derived from intermediate and high risk patients. Moreover, we elucidated the dynamic changes of the cellular proteome/phosphoproteome upon pharmacological and genetic PTEFb inhibition and identified PTEFb interaction partners in various AML in vitro models. These analyses uncover the oncogenic PTEFb-dependent signaling networks and substantiate the molecular rationale for the use of PTEFb inhibitors in this indication.

When applied in vivo, BAY 1143572 exhibited single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats upon once daily oral administration. Of note, partial or even complete remissions could be achieved in several models. Furthermore, intermittent dosing schedules with up to 4 days treatment pauses were feasible in terms of efficacy and tolerability.

Using MOLM-13 xenografts in mice and rats to address the in vivo MoA of BAY 1143572, a transient inhibition of RNA polymerase II phosphorylation, MYC mRNA and protein levels, MCL-1 mRNA and protein levels, and an induction of apoptosis was documented.

In conclusion, our data provide the rationale for the initiation of clinical development of BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential for the treatment of AML patients. A phase I clinical trial to determine the safety, tolerability and recommended Phase 2 dose in this indication is ongoing (NCT02345382).

#3023

Cytoplasmic sequestration and autophagic degradation of ErbB receptors in HER2-driven cancer cells by small molecule Sigma1 modulators.

Christina M. Maher, Jane Y. Tong, Charles G. Longen, Mercedes I. Lioni, Jeffrey D. Thomas, Xing Tan, Logan Tyler, Fernando U. Garcia, Felix J. Kim. _Drexel University College of Medicine, Philadelphia, PA_.

Epidermal growth factor receptors (EGFR/ErbB) drive cell growth, survival, metastasis, and resistance in a range of cancers. Heterodimerization of ErbB1/EGFR, ErbB2/HER2, and ErbB3/HER3 drives aggressive tumor growth through hyperactivation of cancer cell survival and growth signaling pathways. The increased protein production required to sustain these activities renders cancer cells acutely dependent on support factors, such as chaperones and scaffolds, that maintain protein homeostasis. ErbB receptors are integral membrane proteins and as such are synthesized in and transported through the secretory pathway, which comprises the endoplasmic reticulum (ER), Golgi, and associated compartments and vesicles. Nascent ErbB receptors are stabilized and chaperoned through this pathway, in part, by heat shock protein 90 family members, HSP90 and GRP94. Sigma1 (also known as sigma1 receptor) is a unique integral membrane protein found primarily in the ER. Emerging lines of evidence suggest that Sigma1 may function as a chaperone or possibly a scaffolding protein. We find that the levels of Sigma1 protein are elevated and aberrantly distributed in HER2-amplified breast tumor biopsies compared to benign breast tissue. These data indicate that the status and potentially the physiological role of Sigma1 are altered in malignancy and that Sigma1 may be a valid drug target in the treatment of HER2-driven breast cancers. Previously, we discovered that certain selective small molecule modulators of Sigma1 could be used to induce the unfolded protein response (UPR) and autophagy in a panel of cancer cell lines. Here, we demonstrate that these responses to Sigma1 modulators can be exploited to alter the trafficking, stability, and thus signaling of ErbB receptors in cancer cells. In vivo, Sigma1 modulators suppress the growth of xenografted HER2-amplified breast tumors. In the tumors, as well as in vitro cell culture, ErbB1-3 all are eliminated in response to treatment with prototypic small molecule Sigma1 modulators. This corresponds with suppression of downstream PI3K/Akt signaling and with induction of UPR and autophagy. Using high resolution microscopy and organelle fractionation techniques, we confirmed that the Sigma1 modulators induce cytoplasmic sequestration and subsequent degradation of ErbB receptors in ubiquitin-enriched autophagosomes. This process is blocked by cotreatment with autolysosome inhibitor, bafilomycin A1, suggesting that autophagy is the primary mechanism of Sigma1 modulator induced ErbB receptor degradation. Altogether, these data suggest that Sigma1 is a unique, ligand-operated scaffolding protein that contributes to the trafficking and stability of ErbB receptors in HER2-driven cancer cells. Furthermore, these data suggest that Sigma1 is a druggable component of the protein homeostasis regulatory apparatus of cancer cells.

#3024

Targeting TMEPAI/PMEPA1 inhibits triple negative breast cancer cell growth and metastasis by enhancing growth suppressive TGF-β signaling.

Prajjal Kanti Singha, Srilakshmi Pandeswara, Manjeri A. Venkatachalam, Pothana Saikumar. _UT Health Science Center at San Antonio, San Antonio, TX_.

Triple negative breast cancers (TNBC) that lack estrogen receptor (ER), progesterone receptor (PR) and hormone epidermal growth factor receptor 2 (HER-2/neu) are aggressive and cause high mortality among breast cancer patients. Transforming growth factor beta (TGF-β) dependency for their aggressive behavior (growth and metastasis) has been well established in many of these tumors. Although targeting TGF-β signaling has major potential to treat TNBCs, however it carries the risk of disturbing the tumor suppressive effects of TGF-β in early tumors and its homeostatic control of normal tissues. Hence there is a need to identify novel targets to impede tumor progression without compromising the beneficial effects of TGF-β signaling. To satisfy this unmet need, earlier we identified that transmembrane prostate androgen induced (TMEPAI/PMEPA1), a direct target gene of TGF-β could act as a "molecular switch" that converts TGF-β from a tumor suppressor to promoter in TNBC. Thus, we undertook the present study that identified a novel compound that blocked TMEPAI expression.

We identified terpenoid derivatives (TD) which inhibited the expression of TMEPAI, enhanced TGF-β signaling and blocked proliferation, migration and invasion of several triple negative breast cancer cells in vitro. Our results showed that TD increased phosphorylation of Smad2/3 and increased PTEN, p21 and p27 proteins that cause growth suppression. Concomitantly, TD decreased Akt phosphorylation and reduced Snail and Slug that are required for cell growth and metastasis. Interestingly, TD did not affect the growth of normal human mammary epithelial cells and failed to cause associated molecular changes that can result in growth suppression. Notably, using mice models (nude mice and syngeneic BALB/c mice), we showed that TD reduced tumor burden significantly with little or no toxicity. Consistent with its ability to inhibit Akt phosphorylation and induction of Snail and Slug proteins, TD suppressed lung metastasis of TNBC in mice model. Importantly, reduced tumors derived from TD treated mice exhibited increased expression of pSmad2/3, PTEN, p21, p27 and reduced expression of pAkt compared to tumors obtained from vehicle treated mice. These results suggest that drugs that target TMEPAI will restore homeostatic functions of TGF-β to inhibit growth and metastasis of TNBC and thus prevent new tumor development.

#3025

Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential.

Paul Clarke,1 Christina Esdar,2 Aurelie Mallinger,1 Kai Schiemann,2 Dennis Waalboer,1 Simon Crumpler,1 Christian Rink,1 Frank Stieber,2 Michel Calderini,2 Olajumoke Adeniji-Popoola,1 Maria-Jesus Ortiz-Ruiz,1 Rahul S. Samant,1 Paul Czodrowski,2 Djordje Musil,2 Daniel Schwarz,2 Klaus Schneider,2 Michael Busch,2 Mark Stubbs,1 Rosemary Burke,1 Robert TePoele,1 Sharon Gowan,1 Felix Rohdich,2 Florence Raynaud,1 Richard Schneider,2 Oliver Poeschke,2 Andree Blaukat,2 Klaus Urbahns,2 Paul Workman,1 Wolfgang Kaufmann,2 Stephanie Simon,2 Suzanne A. Eccles,1 Trevor Dale,3 Dirk Wienke,2 Julian Blagg1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Merck KGaA, Darmstadt, Germany;_ 3 _Cardiff University, Cardiff, United Kingdom_.

Background

The Mediator complex-associated kinases CDK8 and CDK19 are cyclin C-dependent enzymes that, with MED12 and MED13, form the kinase module of the Mediator complex. CDK8 expression correlates with activation of β-catenin in colon and gastric cancers and has also been associated with increased mortality in colorectal, breast and ovarian cancers. CDK8 is located in a region of chromosome 13 known to undergo copy number gain in ~60% of colorectal cancers and inducible shRNA-mediated knockdown of CDK8 protein reduces the growth of colorectal cancer human tumor xenograft animal models harboring CDK8 gene amplification.

Results

Here we report the discovery and evaluation of CCT251545, a potent, selective and orally bioavailable small molecule chemical probe for CDK8 and CDK19 that we identified from a cell-based WNT pathway screen [1]. We also report a structure-based design approach to the discovery of CCT251921, a potent, selective and orally bioavailable inhibitor of CDK8, with equipotent affinity for CDK19, that has optimised pharmacokinetic and pharmaceutical properties suitable for preclinical development. Furthermore, we describe the discovery of MSC2530818, a structurally differentiated back-up candidate with equivalent pharmacological profile to CCT251921, from a high throughput screen versus CDK8 and subsequent structure-based design. Taking advantage of these two structurally distinct and highly selective dual CDK8/19 modulators we were able to reliably define on-target effects of targeting both CDK8 and CDK19 in the cellular context and in in vivo animal models. We describe gene expression profiles resulting from dual inhibition of CDK8 and CDK19 to demonstrate robust modulation of WNT signalling and additional pathways, including stress and immune response, consistent with the multiple contexts in which Mediator complex is known to regulate gene transcription. We show that both CCT251921 and MSC2530818 exhibit potent cell-based and in vivo inhibition of STAT1SER727 phosphorylation, a target engagement biomarker of CDK8 inhibition, and further demonstrate in vivo antiproliferative activity in human tumour xenograft animal models of colorectal cancer and acute myeloid leukaemia at exposures where pharmacodynamics biomarker modulation is evident. Recent observations suggest CDK8 as a novel anticancer therapeutic target; here we will disclose, for the first time, comprehensive preclinical efficacy, toleration and safety findings for both CCT251921 and MSC2530818 which will inform on the potential for dual CDK8/19 inhibition in the clinical setting.

References

1. Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980.

#3026

**Galunisertib inhibits ovarian cancer cell fibrosis and metastasis** in vitro **.**

Qing Zhang, William A. Cliby. _Mayo Clinic, Rochester, MN_.

Background:

In ovarian cancer (OC), the intraperitoneal fibrosis that accompanies metastasis is a common scenario and associated with significant morbidity and gut dysfunction. Accumulating evidences indicate that Transforming Growth Factor β (TGF-β) signaling pathway plays a key role in cancer associated fibrosis and metastasis. Several TGF-β inhibitors, including Galunisertib (LY2157299) which is a TGF-β receptor I inhibitor, are currently in clinical trials for fibrosis-related diseases and cancer. Our hypothesis is that targeted anti-cancer therapies can block the growth and spread of cancer or specifically address the fibrosis accompanying metastatic peritoneal cancer. In this study, we investigated the efficacy of Galunisertib in inhibiting OC fibrosis and metastasis in vitro.

Methods:

Five OC cell lines, including SKOV3ip, HeyA8, OVCAR8, OV2008, and PEO4, and one cancer-associated fibroblast (CAF) cell line, TRS3, were tested in this study. We utilized a co-culture system that has successfully been used to study the interactions between cancer cells and local environment, including CAFs. Twenty-three candidate TGF-β signaling- and fibrosis-related genes were selected based on previous studies. RNA and protein from both fibroblast cells and OC cells were collected for RT-qPCR and western blot analysis. MTT assay, Wound healing assay and Matrigel invasion assay were used to study OC cell proliferation, migration and invasion. Chou-Talalay assays were performed to study the interactions between Galunisertib and Carboplatin and Paclitaxel.

Results:

RT-qPCR results showed that Galunisertib treatment suppresses the expression of both TGF-β receptor I and TGF-β1 which is the most abundant isoform of TGF-β in OC. Western blot results showed that Galunisertib treatment reverses the TGF-β1-induced SMAD2/3 phosphorylation, confirming the efficacy of Galunisertib in TGF-β signaling pathway blockage. Intriguingly, Galunisertib treatment inhibits the expression of candidate fibrosis-related genes, like ACTA2, TIMP3, COL5A1, COL11A1, and VCAN in both OC and CAF cells. Would healing and Transwell invasion assay also demonstrated that Galunisertib treatment effectively reduces both basal and TGF-β1-induced cell migration and invasion (p<0.05). MTT assay showed that Galunisertib is able to inhibit the proliferation of both OC and CAFs cells with IC50 values range from 100-400 μm. Additionally, Galunisertib shows strong synergistic effects with both Carboplatin and Paclitaxel in Chou-Talalay assays.

Conclusion:

Galunisertib treatment suppresses the expression of TGF-β-dependent fibrosis-related genes from OC cell lines and CAFs. Coincident with this inhibition of fibrosis related genes, galunisrtib reduces cell proliferation, migration, and invasion. These data suggest that targeting the TGF-β pathway is a viable therapeutic approach to reduce both symptoms related to OC-related fibrosis and cancer metastasis in OC.

#3027

Dual inhibition of PKC and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma.

Guillaume Carita,1 Estelle Frisch Dit Leitz,1 Ahmed Dahmani,1 Chloe Raymondie,1 Nathalie Cassoux,1 Sophie Piperno-Neumann,1 Fariba Némati,1 Ensar Halilovic,2 Sebastien Jeay,2 Andrew Wylie,2 Caroline Emery,2 Sergio Roman-Roman,1 Marie Schoumacher,1 Didier Decaudin1. 1 _Inst. Curie, Paris, France;_ 2 _Novartis, Cambridge, MA_.

Uveal melanoma (UM), although rare in incidence, is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified and therefore novel therapeutic approaches are urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway. This has led to the use of PKC inhibitors as a rational targeting strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models, several PKC inhibitor-based combination studies were performed using the PKC inhibitor AEB071 (Sotrastaurin). When combined with AEB071, the targeted agents CGM097 (p53-MDM2 inhibitor), RAD001 (Everolimus, mTORC1 inhibitor) and MEK162 (Binimetinib, a MEK inhibitor) demonstrated greater activity in the UM patient-derived xenograft models than their activity as single agents. Importantly, tumor regressions were observed in several UM models with AEB071 + RAD001 and AEB071 + CGM097 co-treatments. Follow-up in vitro studies in UM cell lines using AEB071 combined with either CGM097 or RAD001 provided a more detailed mechanistic understanding of their combination activity and confirmed their ability to induce cell death. Together, these preclinical studies reveal that combining PKC and p53-MDM2 inhibitors or PKC and mTORC1 inhibitors may provide significant clinical benefit for patients with UM.

#3028

ERK signal suppression and sensitivity to CH5183284/Debio 1347, a selective FGFR inhibitor.

Yoshito Nakanishi, Hideaki Mizuno, Hitoshi Sase, Toshihiko Fujii, Kiyoaki Sakata, Nukinori Akiyama, Yuko Aoki, Masahiro Aoki, Nobuya Ishii. _Chugai Pharmaceutical Co., Ltd., KAMAKURA, Japan_.

Drugs that target specific gene alterations have proven beneficial in the treatment of cancer. Because cancer cells have multiple resistance mechanisms, it is important to understand the downstream pathways of the target genes and monitor the pharmacodynamic markers associated with therapeutic efficacy. In a previous study, we analyzed the signaling pathway of an FGFR (fibroblast growth factor receptor) fusion kinases, FGFR3-BAIAP2L1. A Rat-2 cell line stably expressing FGFR3-BAIAP2L1 exhibited potent tumorigenic activity. Gene expression analysis revealed strong up-regulation of genes downstream of the MAPK pathway, and up-regulation of the MAPK pathway was validated by western blotting; in contrast, the PI3K/AKT pathway was not activated by FGFR3-BAIAP2L1. Therefore, we suggested that MAPK pathway activation is essential to the tumorigenic activity of FGFR3-BAIAP2L1. In this study, to generalize the MAPK pathway dependency of FGFR, we characterized the pathway modulation by CH5183284/Debio 1347, a selective FGFR inhibitor, in several FGFR genetically altered cancer cell lines, such as FGFR1 gene amplified, FGFR2 gene amplified or mutated, and FGFR3 gene mutated or rearranged cell lines. We performed a transcriptome analysis to characterize the response of various cancer cell lines to CH5183284/Debio 1347, a MEK inhibitor, or a PI3K inhibitor. FGFR and MEK inhibition produced similar expression patterns, and the ERK gene signature was altered in several FGFR inhibitor-sensitive cell lines. Consistent with these findings, CH5183284/Debio 1347 suppressed phospho-ERK in every tested FGFR inhibitor-sensitive cell line. Because the MAPK pathway functions downstream of FGFR, we searched for a pharmacodynamic marker of FGFR inhibitor efficacy in a collection of cell lines with the ERK signature and identified dual-specificity phosphatase 6 (DUSP6) as a candidate marker. Suppression of DUSP6 protein level was also confirmed in mice xenograft models. Although a MEK inhibitor suppressed the MAPK pathway, most FGFR inhibitor-sensitive cell lines are insensitive to MEK inhibitors and we found potent feedback activation of several pathways via FGFR. We therefore suggest that FGFR inhibitors exert their effect by suppressing ERK signaling without feedback activation. In addition, DUSP6 may be a pharmacodynamic marker of FGFR inhibitor efficacy in FGFR-addicted cancers.

*CH5183284/Debio 1347 was discovered by Chugai Pharmaceutical Co., Ltd. and is being developed by Debiopharm International S.A. under an exclusive worldwide license.

#3029

Dual targeting of ARK5 and CDK4 pathways with ON 123300 as a therapeutic strategy for colorectal carcinoma.

Saikrishna A. Divakar,1 M.V. Ramana Reddy,2 Stephen C. Cosenza,1 Stacey J. Baker,1 Vinee Purohit,3 Venugopal Gunda,3 Pankaj K. Singh,3 E. Premkumar Reddy1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Icahn School of Medicine at Mount Sinai, White Plains, NY;_ 3 _University of Nebraska Medical Center, Omaha, NE_.

Introduction: This study describes the development of a novel dual specificity kinase inhibitor, ON 123300, which exhibits potent activity against colorectal cancers both in vitro and in vivo. While overexpression of Cyclin D1 is closely correlated with the proliferative rate of these tumor cells, metastatic colorectal cancers over-express ARK5, a member of the AMPK family and mediator of AKT activation. In this study, we show that ON 123300, which inhibits both CDK4/6 and ARK5, is a potent inducer of apoptosis of colorectal cancer cells when compared to palbociclib, a highly selective inhibitor of CDK4/6 kinases that does not target ARK5.

Results & Conclusions: We examined the effects of palbociclib and ON 123300 on cell cycle progression, modulation of Rb and PI3K/AKT pathways, and induction of apoptosis in multiple colorectal cancer cell lines. Comparative kinase inhibition assays showed that while palbociclib and ON 123300 exhibited equivalent inhibition against CDK4/CDK6, ARK5 activity was inhibited only by ON 123300. When DLD1 and SW480 cells were incubated with increasing concentrations of palbociclib or ON 123300, both compounds were equally efficient in their ability to inhibit phosphorylation of all three members of the Rb family of proteins. However, when the phosphorylation status of proteins associated with the PI3K/AKT pathway was measured by western blot, ON 123300 showed concentration-dependent inhibition of mTOR, AKT, 4EBP1 and S6RB phosphorylation while palbociclib had little or no effect on the phosphorylation of these proteins. Cells treated with palbociclib rapidly accumulated in the G0/G1 stage of the cell cycle with increasing drug concentrations. Although cells treated with ON 123300 also arrested in the G0/G1 phase at lower concentrations (01-0.5 uM), with increasing concentrations of drug there was an accumulation of cells with sub-G1 DNA content, suggesting induction of apoptosis. ON 123300-treated cells showed cleavage of PARP and Caspases (3, 7 and 9) as well as inhibition of FOXO1 phosphorylation, which was not observed in cells treated with palbociclib. Since ARK5 belongs to the AMPK family of kinases, we next examined the effects of ON 123300-mediated ARK5 inhibition on metabolic changes of tumor cells that over-express this gene. Treatment of SW-480 colorectal cancer cells with ON 123300 resulted in an increase in glucose uptake, profound inhibition of glutamine uptake and reduced ATP production. A detailed metabolomic study revealed significant alterations in the levels of metabolites associated with glutamine metabolism. Nude mouse xenograft assays using Colo-205 cells revealed strong inhibition of tumor growth following 100 mg/kg of ON 123300 given QD or QOD, with little evidence of toxicity as measured by change in body weight. Thus, dual inhibition of ARK5 and CDK4 pathways could be an effective therapeutic strategy for the treatment of colorectal cancers.

#3030

CT179: A novel small molecule Olig2 inhibitor for the treatment of glioblastoma.

Gordon Alton,1 Graham Beaton,1 Susan Knowles,1 Greg Stein,1 Rajesh Mukthavaam,2 Santosh Kesari3. 1 _Curtana Pharmaceuticals, Austin, TX;_ 2 _UCSD, San Diego, CA;_ 3 _John Wayne Cancer Institute, Santa Monica, CA_.

Glioblastoma (GBM) is an orphan disease with a high unmet medical need. Despite optimal treatment, the median survival is only 12 to 15 months in GBM patients of which only 4% achieve 5-year survival. OLIG2 is a transcription factor that is almost exclusively expressed in the CNS that has been linked to the tumorigenesis of GBM cells and their resistance to radiotherapy. Importantly OLIG2 is a key transcription factor that maintains the stemness of the glial stem cells, drives proliferation and invasion and recurrent tumor growth. Inhibitors of this pathway are therefore of interest as potential treatments for this unmet need.

CT-179 is a small molecule inhibitor that was initially designed to prevent homodimerization of Olig2. CT-179 is highly cytotoxic to OLIG2 expressing GBM cells with a high therapeutic index for non-OLIG2 expressing cell types. This inhibitor demonstrates functional inhibition of the OLIG2 pathway through specific reduction in nuclear Olig2 protein levels in treated cells. A cell pathway analysis indicates that CT-179 impacts GBM proliferation through cell cycle arrest and induction of apoptosis. Secondarily an immunosuppression mechanism-of-action may also be relevant. CT-179 penetrates the CNS to attain high brain concentrations at levels far in excess of its cytotoxicity to GBM cells. Efficacy studies reveal dose dependent reductions of tumor growth which is enhanced with radiotherapy. CT-179 demonstrates impressive extension of survival in orthotopic human PDX GBM. Safety studies indicate that there is a large therapeutic window. The compound has a long half-life, low metabolism and has excellent formulation properties and PK supporting once per day oral dosing. CT-179 show promise as both a cytotoxic agent and as potential radiosensitizer for use in the treatment of GBM.

#3031

Bufalin regulates Cbl-b levels in HeLa cells through protein synthesis inhibition.

Mariya S. Liyasova, Stanley Lipkowitz. _National Cancer Institute, Bethesda, MD_.

Cbl-b is a member of ubiquitin ligases (E3s) family. The major function of CBL family E3s is to negatively regulate signaling through ubiquitination and degradation of receptor tyrosine kinases and kinase associated receptors. Cbl-b plays a crucial role in the regulation of the immune response, as it negatively regulates the costimulatory pathway of CD8 T cells and also suppresses natural killer cell function. Thus Cbl-b down-regulation or inhibition may lead to increased adaptive and innate antitumor immunity and can be used as a treatment strategy for a wide variety of tumors.

Bufalin is a major component of Chan Su, traditional Chinese medicine derived from the toad venom. Bufalin is a C-24 steroid with the wide variety of biological activities, including cardiotonic, anesthetic and antitumor activities. Bufalin has been shown to inhibit growth and induce cell cycle arrest and apoptosis of various cancer cells. Low doses of bufalin can be safely used for prolonged periods of time without severe side effects, while high doses can cause cardiac arrhythmia, seizure and coma.

The purpose of the current study was to investigate if bufalin regulates the levels of Cbl-b and establish the mechanism of Cbl-b regulation. By Western blotting of HeLa cell lysates we found that bufalin decreases protein levels of Cbl-b in a time- and dose-dependent manner without affecting the levels of Cbl. The greatest decrease was observed with 50 nM bufalin after 24 hours treatment. Doses of bufalin as low as 4 nM were effective in Cbl-b down-regulation. Real-time PCR analysis showed 2.9-fold increase in the amount of Cbl-b mRNA upon 50 nM bufalin treatment for 24 hours, suggesting that Cbl-b regulation by bufalin occurs post-transcriptionally. Proteasomal, but not lysosomal inhibitors treatment attenuated the negative effect of bufalin on Cbl-b levels. Co-treatment of HeLa cells with 50 nM bufalin and 50 μg/ml cycloheximide, protein synthesis inhibitor, did not result in any further decrease in Cbl-b levels compared to cycloheximide treatment alone. By measuring the nascent protein synthesis with Click-It metabolic labeling reagents, bufalin was shown to block protein synthesis of the majority of short-lived proteins. These data taken together indicate that bufalin treatment decreases Cbl-b levels in HeLa cells by blocking protein synthesis. We found that 50 nM bufalin treatment for up to 8 hours increased the phosphorylation of eIF2α, translation initiation factor, at Ser51. Phosphorylation of eIF2α known to block translation might explain the effect of bufalin on Cbl-b levels and other short-lived proteins.

To conclude, we found that bufalin increases the phosphorylation of eIF2α, thus leading to the block of translation and decreasing protein levels of Cbl-b in HeLa cells. Our future goal is to test the hypothesis that bufalin activates antitumor immunity through down-regulation of Cbl-b and can thus be used as an antineoplastic agent.

#3032

A novel compound ARN-3236 inhibits SIK2 and sensitizes ovarian cancer to paclitaxel.

Jinhua Zhou,1 Albandri Alfredi,1 Shu Zhang,1 Janice M. Santiago-O'Farrill,1 Ahmed A. Ahmed,2 Hailing Yang,1 Weiqun Mao,1 Yan Wang,1 Hariprasad Vankayalapati,3 Zhen Lu,1 Robert C. Bast Jr1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _Arrien Pharmaceuticals, Salt Lake City, UT_.

Ovarian carcinomas account for 4% of total cancers in women in the United States. Improved outcomes might be attained if sensitivity to primary chemotherapy were enhanced. A recent study discovered that the salt inducible kinase 2 (SIK2) plays a key role in the initiation of mitosis and regulates paclitaxel sensitivity. Here we show that SIK2 is overexpressed in 30% ovarian cancer specimens, associated with a poor prognosis. ARN-3236, a selective, highly potent small molecule SIK2 inhibitor with function similar to SIK2 siRNA, blocks cell proliferation in a panel of ovarian cancer cell lines, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression. ARN-3236 also enhanced response to paclitaxel in ovarian cancer cells (OC316, SKOv3, A2780, HEY, ES2 and UPN251). Similar to the function of SIK2 siRNA, ARN-3236 effectively inhibits AKT phosphorylation at Ser473 and Tyr308, as well as the expression of the downstream effector survivin. ARN-3236 uncouples the centrosome from the nucleus in interphase, and blocks centrosome separation in mitosis, resulting in the accumulation of cells in prometaphase. In addition, ARN-3236 enhances tumor growth inhibition of paclitaxel in ovarian cancer xenograft models. Thus, functional inhibition of SIK2 with ARN-3236 had the same effects on ovarian cancer cell function as knockdown of SIK2 with siRNA. ARN-3236 deserves further study as a SIK2 inhibitor that significantly improves the sensitivity to paclitaxel for treatment of human ovarian cancer cells and xenografts.

#3033

Bumped kinase inhibitors: A novel therapy for castration-resistant prostate cancer.

Cynthia C. Sprenger, Stephen Plymate, Dustin Maly, Wesley Van Voorhis, Kayode K. Ojo. _University of Washington, Seattle, WA_.

BACKGROUND: Prostate cancer (PCa) is the second most common cancer in men and one of the leading causes of cancer death. Resistance to current PCa therapies, including androgen deprivation, occurs in almost all patients leading to development of castration resistant prostate cancer (CRPC). Resistance is associated with expression of splice variants of the androgen receptor (AR-Vs) that are constitutively active. Therapies that indirectly target AR by inhibiting factors important in regulating AR levels and activity may be the most successful against these constitutively active variants. We originally designed bumped kinase inhibitors (BKIs) to inhibit Cryptosporidium sp. Further examination demonstrated that this class of BKIs also inhibits members of the mammalian protein kinase D (PKD) family. In prostate cancer, expression of all PKD isoforms has been shown to increase with cancer progression. Further, PKDs are implicated in PCa replication, migration, invasion, and progression. In regard to AR activity, studies suggest that PKDs in conjunction with heat shock protein 27 (Hsp27) are regulators of AR transport into and out of the nucleus as well as AR transcriptional activity. Such studies, suggest that targeting PKDs would affect AR signaling and thus be a promising anti-prostate cancer therapy.

OBJECTIVE: To demonstrate that BKIs targeting PKD isoforms inhibit CRPC.

RESULTS: Immunohistochemical staining of a tissue microarray (representing 50+ patients) with a PKD antibody demonstrated that PKD expression increased with tumor progression. On a screen of over 80 human protein kinases our BKIs inhibited members of the PKD family at low nM concentrations. Furthermore, several optimized BKI compounds, including BKI-1553, were screened against >360 human protein kinases using Cellzome's™ inhibitor profiling methodology and only PKDs were found to be targeted significantly. This is unique from previously published BKIs, which are PKD inhibitors but also inhibit other human protein kinases such as mutated Src. In vitro our BKIs decreased proliferation of AR positive PCa cell lines but not AR negative lines. We next examined the effects of our lead compound, BKI-1553, on AR transactivation using the LNCaP cell line. BKI-1553 treated cells had decreased ARE-luciferase reporter activity. Interestingly, we also found that BKI-1553 decreased mRNA and protein levels of AR-FL and AR-V7 in these cells. In toxicity studies, BKI-1553 was non-toxic in three species at oral doses that achieved therapeutic levels. In vivo BKI-1553 significantly decreased growth of two PCa xenografts (LNCaP95, p < 0.005 and LuCaP35, p < 0.01).

SUMMARY: Since we found that the PKD inhibitor BKI-1553 selectively inhibits AR positive prostate cancers, and it is thought that the majority of CRPC are AR driven, BKI-1553 could be a novel therapy for cancers that are resistant to currently available therapies.

#3034

Overcoming gefitinib resistance in non-small-cell lung cancer by inducing epidermal growth factor receptor (EGFR) degradation via methionine 790 oxidation.

Elaine Lai-Han Leung,1 Xing-Xing Fan,1 Maria Pik Wong,2 Zhi-Hong Jiang,1 Zhong-Qiu Liu,3 Xiao-Jun Yao,1 Lin-Lin Lu,3 Yan-Ling Zhou,1 Li-Fong Yau,1 Vicky Pui-Chi Tin,2 Liang Liu1. 1 _Macau University of Science and Technology, Macau, Macao;_ 2 _The University of Hong Kong, Hong Kong, Hong Kong;_ 3 _Guangzhou University of Chinese Medicine, Guangzhou, China_.

Personalized therapy is becoming a dominant cancer therapeutic strategy, Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed to treat non-small-cell lung cancer (NSCLC) patients with EGFR mutation but they only inhibit EGFR phosphorylation, acquired-resistance still remains unavoidable without EGFRT790M removal. Here, we report a new treatment strategy to overcome such drug resistance by selectively inducing EGFRT790M degradation via specific stimulation of methionine 790 (M790) oxidation. The basal reactive oxygen species (ROS) levels in EGFRT790M containing gefitinib-resistant NSCLC cell lines were substantially high and 63 clinical lung tumors showed higher NADPH oxidase 2 (NOX2) expression than 61 normal lung tissues which may contribute to high basal ROS in cancer. Interestingly, only NOX3 was upregulated by sanguinarine, a pharmacological agent to elevate ROS, resulted in EGFR over-oxidation and degradation as well as apoptosis. By contrast, such responses were lacking in EGFRWild-type (WT) cells, suggesting a new promising drug development strategy with wide therapeutic window between EGFRT790M and EGFRWT. Selective EGFRT790M degradation was manipulated by REDOX imbalance between NOX3 and methionine reductase (MsrA). When NOX3 was activated, NADPH, the key substrate of MsrA, was depleted to generate excessive ROS, resulting in inhibition of MsrA physiological reduction function on oxidized-methionine, thus localized methionine 790 oxidation

of EGFRT790M occurred. Furthermore, the in vivo tumor suppression effect of sanguinarine, NOX3 upregulation and EGFR degradation were confirmed. These findings indicate that specific inducing EGFRT790M degradation via manipulating REDOX imbalance between NOX3 and MsrA would be promising strategy for treating gefitinib-resistant NSCLC.

#3035

Development and characterization of CCK2R-targeted SMDCs and identification of GIST as a potential therapeutic indication.

Jonathan M. Shillingford, Joseph A. Reddy, Melissa Nelson, Marilynn Vetzel, Garth L. Parham, Ryan Dorton, John Guan, Nikki Parker, Haiyan Chu, Iontcho A. Vlahov, Christopher P. Leamon. _Endocyte, Inc., West Lafayette, IN_.

Cholecystokinin B receptor (CCKBR/CCK2R) is a GPCR of the cholecystokinin receptor family that is primarily expressed in the CNS and the gastrointestinal tract where it binds the ligands cholecystokinin and gastrin, respectively, and mediates their downstream actions. Since it has been reported that CCK2R is overexpressed in certain cancer types, we have explored the potential of utilizing CCK2R as a receptor system to deliver potent therapeutic warheads. To this end, we designed a series of CCK2R-targeted small-molecule drug conjugates (SMDCs) containing either a non-peptidic antagonist (Z-360) or a peptidic agonist (CCK8) as their ligand moiety and explored their activity in in vitro and in vivo models. While EC1812 (Z-360-tubulysin B) failed to elicit either competable cytotoxicity in vitro or robust therapeutic response in vivo (n = 5 ; 1 PR) when tested against a HEK293-CCK2R overexpressing cell line, the addition of a PEG36 linker to generate EC1977 greatly improved therapeutic efficacy in vivo (n = 5 ; 1 CR, 4 cures) despite having no activity in vitro. In contrast, while EC1868 (CCK8-tubulysin B) demonstrated potent, competable cytotoxicity in vitro it had only modest activity in vivo (n = 5 ; 5 CRs). To explore the clinical relevance of a CCK2R-targeted SMDC, we performed mRNA profiling of a human cancer array to identify indications that have high CCK2R mRNA expression. Of all the indications present on the array, those representing gastrointestinal stromal tumor (GIST) were identified as expressing the highest level of CCK2R mRNA. To further elucidate the clinical relevance of this finding, we procured human clinical samples from GIST patients and determined CCK2R-specific radioligand binding and mRNA levels. These data revealed that 50% of GIST samples express high levels of CCK2R mRNA and exhibit functional ligand binding. Interestingly, although 50% of normal stomach samples had similar mRNA levels as the high-expressing GIST samples, they failed to exhibit detectable radioligand binding. In contrast, HCC, colon cancer, and pancreatic cancer samples were all negative for both mRNA expression and ligand binding. Subsequent screening of a series of GIST patient-derived xenograft (PDX) models for functional binding and mRNA levels failed to identify an appropriate model for further in vivo testing. In conclusion, we have developed and characterized a series of CCK2R-targeted SMDCs and tested their activity in vitro and in vivo. Given the expression and functional binding of CCK2R in GIST we propose that GIST may represent an indication for CCK2R-targeted therapies.

#3036

Targeting FACT complex with CBL0137 to overcome acquired resistance to EGFR-TKI in lung adenocarcinoma.

Neelesh Sharma,1 Josephine Dermawan,2 Andrei Purmal,3 Daniel Lindner,4 Gary Wildey,1 Afshin Dowlati,1 Andrei Gudkov,5 Katerina Gurova,5 George R. Stark2. 1 _University Hospitals, Case Western Reserve University, Case Comprehensive Cancer Center, Cleveland, OH;_ 2 _Lerner Research Institute, Cleveland Clinic, OH;_ 3 _Cleveland Biolabs, Buffalo, NY;_ 4 _Cleveland Clinic, Cleveland, OH;_ 5 _Roswell Park Cancer Institute, Buffalo, NY_.

Multiple randomized clinical trials have demonstrated that first line treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) such as afatinib in lung adenocarcinoma patients with EGFR mutations results in better progression free survival, response and quality of life, compared to chemotherapy. Despite excellent initial responses, almost all patients eventually acquire resistance to EGFR-TKI and overall survival for these patients remains dismal. Acquired resistance to EGFR-TKI results from several distinct mechanisms, including second site mutations (such as T790M), Met amplification and NFκB activation. An evolving hypothesis is that most patients achieve an initial incomplete response to EGFR-TKI but have residual disease comprised of cells that have developed an adaptive survival response to EGFR inhibition. Activation of NFkB and acquisition of stem cell like properties are important for this adaptive response. CBL0137 is a curaxin that inhibits NFκB and targets cancer stem cells preferentially by causing chromatin trapping of the FACT (facilitates chromatin transcription) complex. We evaluated the combination of CBL0137 and afatinib in EGFR mutant cell lines and a patient-derived xenograft (PDX) with acquired resistance to EGFR TKI.

Drug synergy experiments were done in the EGFR mutant lung adenocarcinoma cell lines H1975 and H1993 with acquired resistance due to T790M and Met amplification, respectively. Cell viability was measured by MTT assay and synergy was quantified by using ChouTalalay combination indices (CI). The PDX was obtained from a patient with an EGFR mutation and progression after treatment with erlotinib with no detectable T790M mutation. To evaluate the efficacy of the afatinib-CBL0137 combination in mice bearing PDX tumors, afatinib (5 mg/kg orally/days 1-5 every week) alone, CBL0137 (50 or 75 mg/kg IV/once a week) alone, a combination of afatinib and CBL0137, or vehicle alone were administered for 52 days and tumor growth was measured every other day. Ten mice were used for each test.

In the cell lines, the combination of afatinib and CBL0137 was synergistic with CI less than 1. The combination of afatinib and CBL0137 was well tolerated in the mice. In the treatment groups of vehicle alone, afatinib alone, CBL0137 50 mg/kg alone or 75 mg/kg alone, tumor volumes increased by factors of ~35, 17.5, 17.5 and 12.5, respectively. There was no significant tumor growth for 3 weeks in mice treated with the combination of afatinib and CBL0137. After 3 weeks of treatment, the tumors started to grow slowly and increased in volume approximately 5 fold by day 52 when the treatment was stopped.

We conclude that the combination of CBL0137 and afatinib is synergistic and can overcome acquired resistance to an EGFR TKI in an EGFR mutant lung adenocarcinoma. Phase I oral and IV clinical trials of CBL0137 monotherapy in solid tumors are ongoing, and combination studies with afatinib are being planned.

#3037

Tumor cell death triggered by sustained IRES inhibition is a population-wide event involving a quorum-sensing mechanism in which putative tumor stem cells are eliminated along with their progeny.

Christos Vaklavas, William E. Grizzle, Hyoungsoo Choi, Zheng Meng, Kurt R. Zinn, Scott W. Blume. _UAB Comprehensive Cancer Ctr., Birmingham, AL_.

The internal ribosome entry site (IRES) is a feature contained within the 5'-untranslated region of the mRNA which allows the efficiency with which that mRNA is translated to be regulated independently of the general rate of protein synthesis in the cell. It appears that tumor cells rely on IRES-mediated translation to promote their own survival under adverse microenvironmental conditions, ultimately contributing to chemoresistance, relapse, and metastasis. Our lab has identified and characterized a series of compounds (prototype IRES inhibitors) which selectively modulate IRES function. Here we examine the phenotypic consequences of sustained IRES inhibition in triple-negative breast carcinoma and glioblastoma.

72 hours continuous exposure to the IRES inhibitor results in sudden loss of viability affecting >99.9% of the tumor cell population. Cell death is a consequence of the inhibition of synthesis of critical IRES-driven proteins, and not a typical cytotoxic response. The treated cells exhibit prominent features of terminal differentiation, with marked gains in cytoskeletal organization and planar polarity, extensive intercellular networking, and formation of tight junctions or neuronal processes. In addition to depletion of Myc protein, specific changes in IGF1R, connexin 43, BiP, and CHOP also correlate with phenotypic outcome. The highly undifferentiated triple-negative breast tumor and glioblastoma cells apparently cannot tolerate the transition to a fully differentiated state, or the restoration of normal homeostatic mechanisms allows the abnormalities that cause these cells to be malignant to be recognized. Once triggered, cell death is rapid and comprehensive. PARP cleavage is not observed, caspase 3/7 activation is limited, and no protection afforded by Z-VAD-fmk. The cells show no evidence of recovery even following prolonged incubation in the absence of compound. In contrast, normal human mammary epithelial cells (which are already differentiated) are resistant to IRES inhibition.

The synchronized cell death response to IRES inhibition is a highly orchestrated event, with the population of malignant cells becoming physically linked together and responding as a unit, in contrast to typical stochastic processes such as apoptosis, programmed necrosis, and autophagy, where cell death decisions are made at the level of the individual cell. Importantly, tumor stem cells, which typically are resilient to conventional cytotoxic or targeted therapeutic regimens, appear to be vulnerable to IRES inhibition.

Together, these findings support the concept that IRES-mediated translation is critical for maintenance of the undifferentiated phenotype. IRES inhibition potentially represents a new strategy for treatment of undifferentiated tumor types such as triple-negative breast cancer and glioblastoma.

#3038

Investigating novel targeted therapies for double hit diffuse large B-cell lymphoma (DH-DLBCL).

Priyank P. Patel,1 Alison Zeccola,2 Juan Gu,1 Cory Mavis,1 Sheila N. J. Sait,1 Vishala Neppalli,1 Francisco J. Hernandez-Ilizaliturri1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _University at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, NY_.

Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. Previously, our retrospective review from single institution series revealed that 30 out of 611 DLBCL patients had aberrations in c-MYC and BCL2 or BCL6 by FISH. These patients exhibited inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS), suggesting that newer therapies are in dire need. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL.

Materials & Methods: At the pre-clinical level, we studied 3 novel therapeutic agents targeting BCL2 (ABT-199), c-MYC (JQ-1), and various cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT-199 activity (irreversible proteasome inhibitor carfilzomib(CFZ)) using DH lymphoma (DHL) cell lines (Val, DOHH-2, ROS-50). DHL cell lines were exposed to ABT-199 (0-10 uM), JQ-1 (0-100 uM) and carfilzomib (CFZ) (0-50 nM) at 24, 48 and 72 hours. Changes in cell viability were evaluated using Presto Blue assay. Subsequently, DHL cells were exposed to doublet combinations of ABT-199, JQ-1 and CFZ for 48 hours. Coefficient of synergy was calculated using CalcuSyn.

Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1.

Conclusion: ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing.

#3039

Lurbinectedin specifically targets transcription in cancer cells, triggering DNA breaks and degradation of phosphorylated Pol II.

Gema Santamaria-Nunez,1 Carlos M. Genes-Robles,2 Juan F. Martínez-Leal,1 Carlos M. Galmarini,1 Jean Marc Egly2. 1 _PharmaMar S.A., Madrid, Spain;_ 2 _Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France_.

Cancer cells are characterized for their avid demand for active transcription, which reaches the level of a real addiction in solid tumors as small cell lung cancer (SCLC) or triple-negative breast cancer. Pharmacological modulation of transcription may thus provide a therapeutic approach to treat tumor types that depend on deregulated transcription for the maintenance of their oncogenic state. Lurbinectedin, currently under evaluation in a Phase III clinical trial for platinum-resistant ovarian cancer patients, and with very promising activity in combination with doxorubicin in SCLC, inhibits active transcription. Here we demonstrate that, after binding to specific DNA triplets highly represented in the CG-rich region surrounding the promoter of genes, this drug induces a rapid degradation of RNA Polymerase II (Pol II). Our results show that the hyperphosphorylated form of Pol II, already engaged in transcription elongation and likely blocked during this process by the lurbinectedin-DNA adduct, is then specifically submitted to the ubiquitin/proteasome degradation process, which finally removes the majority of the Pol II protein pool in treated cells. Disappearance of Pol II is followed by the formation of DNA breaks, process in which the nucleotide excision repair (NER) machinery, specifically the endonuclease XPF, has an important role. Pol II degradation and subsequent DNA damage were not only abrogated by inhibitors of CDK7 and CDK9 cyclin dependent kinases (DRB and flavopyridol), ubiquitin ligation (PYR-41), or proteasome activity (MG132), but also correlated with the antiproliferative activity of lurbinectedin in different cancer cell line models. In summary, lurbinectedin exemplifies a prototype drug for targeting transcriptional dependency in tumor cells and, thus, it could represent a new therapeutic alternative for solid tumors with this addiction.

#3040

A novel methoxyflavanone from a Chinese medicinal herb (Perilla frutescens) induces G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma cells.

Amer Ali Abd El Hafeez Mohamed,1 Takashi Fujimura,1 Rikiya Kamei,1 Noriko Hirakawa,2 Kenji Baba,2 Miyako Nakano,1 Kazuhisa Ono,3 Seiji Kawamoto1. 1 _Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan;_ 2 _Mishima Food Co. Ltd., Hiroshima, Japan;_ 3 _Department of Food Sciences and Biotechnology, Faculty of Life Sciences, Hiroshima Institute of Technology, Hiroshima, Japan, Hiroshima, Japan_.

Background: Perilla frutescens is a common traditional Chinese medicinal herb used for a variety of diseases, e.g. allergic asthma, cough and intestinal disorders. Flavonoids isolated from P. frutescens such as rosmarinic acid, caffeic acid and apigenin have been reported to inhibit proliferation of a variety of cancer cell lines. We recently identified a novel methoxyflavanone named PDMF (Perilla-derived methoxyflavanone) from P. frutescens as a candidate for anti-allergic drug. The objective of the study is to investigate antitumor potency of PDMF on A549 human lung adenocarcinoma and to elucidate its carcinostatic mechanisms.

Methods: Effect of PDMF on cell proliferation and viability of A549 cells were assessed by BrdU incorporation assay and trypan blue dye exclusion test. Cell cycle and apoptosis analyses were carried out by flow cytometry. To elucidate antitumor mechanisms of PDMF, expression and activation status of cell cycle/apoptosis regulators (p53/p21/BAX/caspases) were thoroughly analyzed by immunoblotting.

Results: Stimulation with PDMF not only triggered G2/M cell cycle arrest but also induced apoptosis of A549 cells in a dose-dependent manner. Mechanistically, PDMF stimulation significantly induced protein levels of p53 and its phosphorylated active forms (pSer15) accompanied by induction of p21 and BAX. We also found that proapoptotic caspase molecules (caspase 3, 8, and 9) were all activated upon stimulation with PDMF.

Conclusion: These results suggest that this Perilla-derived methoxyflavanone represents a useful cancer-preventive phytochemical that triggers G2/M arrest, apoptosis, and activation of p53 pathway.

### Preclinical Radiotherapeutics

#3041

Blood-brain barrier penetrating ATM inhibitor (AZ32) radiosensitises intracranial gliomas in mice.

Steve T. Durant,1 Jeremy Karlin,2 Kurt Pike,1 Nicola Colclough,3 N Mukhopadhyay,2 S F. Ahmad,2 J M. Bekta,2 M Tokarz,2 Catherine Bardelle,3 Gareth Hughes,1 Bhavika Patel,3 Andrew Thomason,3 Elaine Cadogan,1 Ian Barrett,1 Alan Lau,1 Martin Pass,1 Kristoffer Valerie2. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Virginia Commonwealth University, Richmond, VA;_ 3 _AstraZeneca, Manchester, United Kingdom_.

Ataxia-telangiectasia mutated (ATM) kinase is a central DNA damage response (DDR) component signalling the presence of DNA double strand breaks (DSBs) to DNA repair, checkpoint and survival pathways. Clinical doses of fractionated radiotherapy directed at tumours kill cells by inducing single strand breaks and DSBs, the latter being particularly lethal to all cells if not repaired. Poor survival rates of glioblastoma multiforme (GBM) patients is attributed to an inability to excise all invasive tumor tissue (if operable) and an intrinsic tumour chemo/radioresistance, which has been linked to elevated ATM activity in glioma stem cells.

ATM inhibition (ATMi) radiosensitises cancer cell lines in vitro and in vivo. ATMi also acutely radiosensitises patient-derived glioma stem cells more effectively than by inhibiting other DDR or cell cycle checkpoint components (PARP, ATR, Chk1). Checkpoint-defective glioblastoma multiforme (GBM) cell lines seem to be particularly radio-sensitised by ATMi. In addition, several studies suggest that normal brain is radioprotected when ATM activity is deficient, suggesting ATM is required for apoptosis in neurons. ATMi in brain may therefore provide a wide therapeutic margin. Furthermore, ATM's role in signalling DNA damage independent redox stress has been linked to promoting neoangiogenesis in tumour vasculature. Taken together, these studies suggest ATM is an extremely attractive target to inhibit during and potentially following radiotherapy. However, one impediment to preclinical and clinical studies is that current ATMi's have limited CNS bioavailability.

We report AZ32 as the first known selective, orally bioavailable blood-brain barrier (BBB) penetrating ATMi probe. AZ32 inhibits the DDR and radiosensitizes p53/checkpoint-defective GBM cells in vitro. Oral daily dosing providing sufficient mouse free brain pharmacokinetic exposures over AZ32's ATM IC50, during just four daily doses of whole brain irradiation results in significant improvement in median overall survival of syngeneic orthotopic mouse glioma models (log-rank AZ32/radiation vs. radiation p=0.0194). Tumor eradication was confirmed by tumor imaging. This result was recapitulated with a human orthotopic model. These findings support the development of clinical grade BBB-penetrating ATMi as a potential treatment for GBM and potentially other intracranial tumours dosed in combination with fractionated radiotherapy used in standard clinical practice.

#3042

The novel Akt inhibitor AZD5363 following radiotherapy improves tumor control in mouse models of head and neck cancer.

Emma J. Searle,1 Brian Telfer,1 Duncan Forster,1 Kaye J. Williams,1 Barry Davies,2 Timothy Illidge,1 Ian Stratford1. 1 _University of Manchester, Manchester, United Kingdom;_ 2 _AstraZeneca, Macclesfield, United Kingdom_.

Radiation therapy (RT) is an important anti-cancer treatment. Understanding the molecular pathways involved in the response of tumors to RT has led to interest in developing drugs which target these; one such target is Akt. Previous studies support the hypothesis that Akt inhibition might enhance the radiosensitivity of tumors through modification of cellular and micro-environmental factors. AZD5363 is a novel, orally bio-available, ATP competitive inhibitor of Akt and here we have evaluated whether AZD5363 will increase the efficacy of RT in preclinical models of head and neck (H&N) cancer.

The ability of AZD5363 to inhibit the Akt pathway was investigated in vitro in 5 H&N cell lines and in vivo (FaDu xenografts) using western blot and ELISA techniques. The effect of AZD5363 on the radiosensitivity in vitro was assessed using clonogenic assays. The FaDu xenograft model was used to test the effect of AZD5363 on tumor growth when used as a sequential treatment (for 7 days before RT) or adjuvant therapy (continuously after RT). RT was given as a 6Gy single dose. The effect of these treatments on the tumor microenvironment prior to and after irradiation was also assessed using FAZA PET/CT, immunohistochemistry and ELISA techniques.

AZD5363 effectively inhibited the Akt pathway in in vitro and in vivo. There was no effect on radiosensitivity in vitro when drug was administered in a variety of sequences and durations around RT. In vivo, there was no improvement in tumor control when AZD5363 was given for 7 days before RT. But survival was improved (p=0.006) when AZD5363 was administered continuously after RT with reduced tumor growth (p=0.0035, 7 days after RT). There was no effect of drug alone on tumor growth and no difference in the level of tumor hypoxia (measured using FAZA PET/CT or pimonidazole binding) after 7 days AZD5363. Human VEGF and HIF1α levels were reduced (p=0.0104 and p=0.002, respectively) after 7 days AZD5363. There was no difference in microvessel density (MVD) prior to RT, but 7 days after, MVD was significantly reduced if adjuvant AZD5363 had been given (p=0.0317). AZD5363 was also found to effect the rate of proliferation of human umbilical vein endothelial (HUVEC) cells in vitro.

AZD5363 effectively improves long term control of FaDu tumors when combined with RT. However, this only occurs when the drug is used in the adjuvant setting. AZD5363 has no effect on the radiosensitivity of H&N cells in vitro, which suggests that the in vivo effects are dependent on the tumor microenvironment. AZD5363 reduces tumor VEGF and HIF1α in an oxygen independent manner and inhibits the rate of proliferation of vascular endothelial cells, effects that would reduce vascular recovery after RT. This is the first time a specific inhibitor of Akt has been reported to have this sequence dependent ability to enhance the effects of RT and has implications for how AZD5363 and potentially other Akt inhibitors are used in combination with RT in the clinic.

#3043

Survivin-mediated radio-sensitization response in p53 mutant tumor cells.

David J. Grdina,1 Jeffrey S. Murley,1 Richard C. Miller,1 Ralph R. Weichselbaum,1 Alfred W. Rademaker,2 Gayle E. Woloschak,2 Jian Jian Li3. 1 _University of Chicago, Chicago, IL;_ 2 _Northwestern University, Chicago, IL;_ 3 _University of California at Davis, Davis, CA_.

Doses of ionizing radiation ≤100 mGy induce changes in radiation sensitivity expressed by cells exposed to subsequent higher doses. This is referred to as an adaptive effect. We describe a unique survivin-associated adaptive response in which increased radiation resistance or sensitization of cells can be induced by exposure to doses as low as 5 mGy. Experiments were performed using two murine sarcomas, SA-NH and FSa,grown either in culture or as tumors, and two human cancer cell lines, Panc-1 (pancreatic epithelioid carcinoma) and MDA-MB-231 (breast adenocarcinoma). Doses of 5 mGy were used to induce changes in the response of these tumor cells to higher therapy doses using a multi-dosing paradigm. Effects on radiation sensitivity were associated with changes in both survivin concentration and its translocation to the cytoplasm (SA-NH, p53 wild type) or nucleus (FSa, Panc-1 and MDA-MB-231, p53 mutant). In vitro survival assays (2 Gy per fraction, two once daily fractions) and tumor growth delay (TGD) (5 Gy per fraction, five once daily fractions) studies were performed on SA-NH and FSa cells. Intracellular localization of survivin was determined by ELISA and correlated to survival response. 2 Gy alone had no effect on intracellular translocation of survivin. When preceded 15 min earlier by a 5 mGy exposure, survivin increased in the cytoplasm of SA-NH as compared to the nuclei of FSa, Panc-1 and MDA-MB-231 cells. SA-NH tumors were protected by 5 mGy exposures (1.9 day decrease in TGD, P = 0.032) while FSa tumors were sensitized (4.5 day increase in

TGD, P < 0.001). Panc-1 and MDA-MB-231 tumor cells were also sensitized to ionizing radiation following 5 mGy exposures. Exposure of SA-NH to 4 mM of the free radical scavenger N-acetylcysteine inhibited the effect of 5 mGy on inducing this adaptive response. We have identified a very low radiation dose-induced survivin-mediated radio-sensitization response that could enhance therapeutic outcomes associated with the increased usage of imaging procedures in radiation therapy. This work was supported by NIH/NCI grant R01 CA132998 and DOE Low Dose Program/Project Grant DE-SC0001271 (D.J.G.)

#3044

Patient-derived adenoid cystic carcinoma xenografts to study molecular target modulation of tumor radiosensitivity.

Prashanth Prabakaran, Kwangok P. Nickel, David T. Yang, Lauryn R. Werner, Justine Y. Bruce, Aaron M. Wieland, Timothy M. McCulloch, Gregory K. Hartig, Paul M. Harari, Adam D. Swick, Randall J. Kimple. _University of Wisconsin School of Medicine and Public Health, Madison, WI_.

Background: Adenoid cystic carcinoma (ACC) is a relatively rare cancer that typically arises in salivary tissues of the head and neck region. Hallmark characteristics include slow growth rate, peri-neural tumor spread, and a high propensity for late distant metastasis. Surgery and radiation are the mainstays of treatment with no effective systemic agents to date. Due to infrequency, studies of novel therapeutics are not routinely feasible. In addition, whether these tumors can be sensitized to radiation by concurrent chemotherapy is not known. We report here the establishment and examination of ACC patient derived xenografts (PDX) to investigate the efficacy of novel chemotherapies and combinations of chemotherapy and radiation.

Methods: PDXs have been established and maintained in NOD-SCID gamma (NSG) mice from both research biopsies and surgical specimens. Common cancer-associated mutations in both the primary patient tumor and PDX were identified using the Illumina TruSeq Amplicon Cancer panel. Well described immunohistochemical markers of ACC were used to compare histological characteristics between the primary tumor and PDX. The ACC PDX was engrafted into the flanks of nude mice and treated with focal radiotherapy (5 Gy x 8 fractions delivered twice weekly), a panel of chemotherapeutic agents, or combination radiochemotherapy. Tumor size was measured over time and comparisons between treatment groups made by the extra-sum-of-squares f test.

Results: PDXs established from ACC maintain the histologic and physical characteristics of the primary tumor. Targeted mutational analysis of ACC identified expected alterations based on previously reported large scale sequencing of other human tumors including mutations in the receptor tyrosine kinases(RTKs) cKit and KDR/VEGFR2. Based on identified tumor mutations, several targeted therapies were selected including dovitinib, a multi-RTK inhibitor, BEZ235, a PI3K/mTORC inhibitor, and cetuximab, an EGFR mAB. Treatment with each of these compounds showed varying degrees of growth inhibition without evidence of frank tumor regression. However, combining these drugs with radiation demonstrated significantly improved tumor control in comparison to drug alone.

Conclusions: Studies using our PDX model suggest that several molecular targeting agents can significantly augment the impact of radiation on ACC tumor growth. These preliminary data identify the rationale to investigate selected molecular drug/radiation combinations for ACC, particularly when driven by tumor specific genetic biomarkers. Expansion of these ACC studies may be valuable to advance the design of new investigational treatment strategies for this challenging tumor.

#3045

The SMAC-Mimetic LCL161 exhibits strong synergy with radiation in vivo in head and neck squamous cell carcinoma by promoting tumor cell apoptosis.

Linlin Yang, Bhavna Kumar, Tianyun Li, Mitchell Romito, Theodoros N. Teknosa, Amab Chakravarti, Terence M. Williams. _Ohio State University, Columbus, OH_.

Background and purpose: Evasion of apoptosis contributes to radioresistance of head and neck squamous cell carcinoma (HNSCC), calling for novel strategies to overcome apoptotic resistance. Second mitochondria-derived activator of caspase (SMAC) - mimetic is a new class of targeted drugs that specifically induce apoptotic cell death and block pro-survival signaling by antagonizing selected members of the inhibitor of apoptosis protein (IAP) family. The present study was designed to investigate the radiosensitizing effect of a SMAC mimetic, LCL161, in HNSCC and the underlying mechanisms for radiosensitization.

Material and methods: Extent of radiation enhancement of 6 HNSCC cell lines (3 HPV[+], 3 HPV[-]) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Combination effect of LCL161 and ionizing radiation was evaluated by cell cycle analysis, Annexin-V assay, and immunoblotting. Correlation between apoptosis associated molecules and HPV status was examined by analyzing mRNA expression in The Cancer Genome Atlas Database (TCGA) in which 279 HNSCC tumors are included, and screening protein expression in 6 HNSCC cell lines via immunoblotting. Human tumor xenografts in nude mice were generated and treated with LCL161, radiation, or the combination. Target inhibition in vivo was confirmed by xenografts lysate immunoblotting, and tissue section immunomhistochemistry.

Results: LCL161 displayed minimal single-agent cytotoxicity with IC50 values ranging between 32μM - 95μM in 6 HNSCC cells. LCL161 is a potent inhibitor of cIAPs, and caused downregulation of cIAP1 at nanomolar concentrations in less than 1 hour. Interestingly, we found that LCL161 could induce radiosensitization only in HPV[-] HNSCC cell lines, but not in HPV[+] HNSCC cells.TCGA database analysis indicated significantly higher mRNA expression of cIAP1 in HPV[-] compared with HPV[+] HNSCC tumors. Consistent with this finding, immunoblotting confirmed that that protein expression of cIAP1 was elevated in HPV[-] HNSCC cells. LCL161 mediated radiosensitisation was associated with enhanced activation of caspases-3, -7, -8, -9, and PARP. Blockage of caspase activation via a pan-caspase inhibitor, z-VAD-fmk, attenuated LCL161 radiosensitization. Finally, LCL161 monotherapy had minimal activity in attenuating tumor growth in HNSCCxenografts. However, the combination of LCL161 and radiation synergistically reduced xenograft tumor volume, with pharmacodynamic inhibition of cIAP1 and caspase activation confirmed in vivo.

Conclusion:Taken together, these results suggest that LCL161, a SMAC-mimetic, significantly radiosensitizes a subset of HNSCC tumors, via a mechanism involving caspase activation and apoptosis induction both in vitro and in vivo. These data provide support for the combination of LCL161 and radiation in the treatment of HNSCC.

#3046

Combining Chk1/2 inhibition with cetuximab and irradiation enhances in vitro and in vivo cytotoxicity of head and neck cancer models.

Ling Zeng, Reena Beggs, Tiffiny Cooper, Eddy Shih-Hsin Yang. _University of Alabama at Birmingham, Birmingham, AL_.

Background:

Epidermal growth factor receptor (EGFR) overexpression is present in 80-100% of head and neck cancers, and targeting EGFR with cetuximab has improved outcomes when combined with radiotherapy (XRT). However, over half of these patients still succumb to this disease, necessitating novel therapies. Checkpoint kinase 1 and 2 (Chk1/2) are serine/threonine kinases that prevent cell cycle progression and serve as critical regulators of the DNA-damage response. LY2606368 monomesylate monohydrate (CHKi), a potent inhibitor of Chk1/2, exhibits single agent activity in tumors of squamous cell histology, including head and neck. Because of the role of Chk1/2 in DNA damage response, we hypothesized that CHKi may enhance the cytotoxicity induced by cetuximab and XRT in head and neck cancer.

Methods:

The HPV-negative UM-SCC1 and HPV-positive UM-SCC47 head and neck cancer cells were used. Doses of drug compounds are as follows: CHKi (LY2606368, Eli Lilly) 1nM; Cetuximab 0.25μg/mL. Cell proliferation was assessed using a Beckman Coulter Counter. Clonogenic survival was measured with colony formation assays. Apoptosis was analyzed using Annexin V-FITC staining and western blot analysis for cleaved and full length caspase-3 and 9. DNA damage and checkpoint signaling was investigated via western blot analysis for phospho- and total Chk1, Chk2, ATM, ATR, and γ-H2AX. Animal studies were performed using orthotopic tongue injection of UM-SCC1-Luc or heterotopic flank injection of UM-SCC47 cells in athymic nude mice, which were then subjected to 3 cycles of once-weekly CHKi (4mg/kg), cetuximab (0.1mg), and 2Gy XRT.

Results:

The addition of CHKi to cetuximab and XRT resulted in the greatest reduction of cell proliferation and increased cytotoxicity in both HPV positive (HPV+) UM-SCC47 and HPV negative (HPV-) UM-SCC1 cancer cells in vitro. Triple combination treatment effectively reduced cell growth by ~89% in UM-SCC1 and ~63% in UM-SCC47 at 96hrs compared to untreated cells. A robust decrease in clonogenic survival in both cell lines was also observed (approximately 90% at 2Gy). Furthermore, this combination abrogated the DNA damage checkpoints induced by cetuximab and XRT, resulting in persistent DNA damage and activation of apoptosis. Specifically, we observed CHKi-mediated downregulation of total and phospho- Chk1, Chk2, ATR, and ATM, with concomitant increases in cleaved caspase-3 and caspase-9. Importantly, combining CHKi, cetuximab, and XRT led to a significant tumor growth delay in mice bearing orthotopic or heterotopic head and neck tumor xenografts compared to control mice. Additionally, no significant weight loss or toxicity was observed in the treatment.

Conclusions:

CHKi enhanced the in vitro and in vivo cytotoxicity of cetuximab and XRT against human head and neck tumor models. A clinical trial to test this treatment for head and neck cancer patients is underway (NCT02555644).

#3047

Sensitization of prostate cancer to chemo-radiation by ribosomal protein S6 inhibition.

Suleman S. Hussain,1 Carolina B. Livi,2 Nikos Papanikolaou,1 Daniel C. Chan,3 Rita Ghosh,1 Addanki P. Kumar1. 1 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _Agilent Technologies, Inc., Santa Clara, CA;_ 3 _University of Colorado Denver, Denver, CO_.

Radiation Therapy (RT) is curative for most localized prostate cancer (PCA) patients, but is limited by dose-related toxicities and radioresistance. Administration of Nexrutine® (Nx), an inexpensive supplement from Phellodendron amurense bark extract prior to RT inhibited progression to poorly differentiated carcinoma in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. However, the precise molecular mechanism underlying Nx-mediated tumor growth inhibitory effects in combination with RT is unclear. Using global transcriptome profiling coupled with GeneSpring analysis, we have identified significant modulation of number of signaling pathways including genes involved in mTOR and NFκB signaling. Based on these data, we hypothesized that downregulation of ribosomal protein S6 (rpS6) sensitizes prostate tumor to overcome radioresistance. PCA cell lines had higher levels of total and p-rpS6 compared to non-tumorigenic prostate cell lines. Using clonogenic assays, we observed that pretreatment of Nx for 8h could potentiate the effect of low dose RT in LNCaP (AR positive with WT p53), PC-3 (AR negative & p53 null) and PC-3 AR (AR expressing & p53 null) cells. The combinatorial effect depicted strong synergism and had similar effects as high dose RT. Notably, we observed increased activation of Akt/mTOR/NFκB signaling molecules after treatment of PC-3 cells with RT, which was either abrogated (p-rpS6, p-NFκB & p-p70S6K) or decreased (p-Akt) in cells pretreated with Nx prior to RT. Also, Nx pretreatment inhibited the levels of HIF1-α and CyclinD1, which are downstream targets of rpS6. Using immunofluorescence assay, we saw that the combination treatment increased the ɣH2AX foci compared to 2 Gy alone, indicating increased DNA damage. This is associated with increased protein levels of key G2/M regulatory proteins including Wee-1, p-cdc2 levels and decreased Chk1, p-cdc25C resulting in prolonged RT-induced G2/M arrest. The combination treatment also caused a significant increase in apoptosis, which was evidenced by increased cleaved PARP levels, subG1 levels and Annexin/PI staining. Strikingly, knockdown of rpS6 sensitized PC-3 cells to RT and reversed the observed effects of Nx, indicating the importance of rpS6 in mediating these changes. Remarkably, p-mTOR, p70S6K, NFκB, Ki67 & Cyclin D1 were decreased in the prostate tissue of mice receiving the combination compared to RT alone. Recently, a phase 0/1 clinical trial of Nx in PCA patients receiving RT decreased PSA levels and was well-tolerated. Interestingly, tumor tissue from Nx treated patients showed decreased rpS6 staining intensity compared to the normal tissue. Collectively, our data suggests that Nx has radiosensitizing effect in a range of PCA cell lines and could prevent progression to advanced PCA by inhibiting rpS6. Supported by NCCAM (R01 AT-007448) & VA-MERIT Award (I01 BX 000766)

#3048

**Drug repurposing: Validation of sulfasalazine as a radiosensitizer in melanoma by blocking system X** c- **.**

Hilde Elise Førde,1 Linda Sleire,1 Heidi Espedal,2 Jan Ingemann Heggdal,3 Frode Selheim,4 Paal-Henning Pedersen,5 Per Øyvind Enger6. 1 _Oncomatrix Research Lab, Department of Biomedicine, University of Bergen, Bergen, Norway;_ 2 _Department of Biomedicine, Molecular Imaging Center (MIC), University of Bergen, Bergen, Norway;_ 3 _Department of Oncology and Medical Physics, Haukeland University Hospital, Bergen, Norway;_ 4 _Department of Biomedicine, Proteomics Unit (Probe), University of Bergen, Bergen, Norway;_ 5 _Department of Clinical Medicine, K1, University of Bergen, Bergen, Norway; Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway;_ 6 _Oncomatrix Research Lab, Department of Biomedicine, University of Bergen, Bergen, Norway; Department of Neurosurgery, Haukeland University Hospital, Bergen, Norway_.

Melanoma is a cancer that has become increasingly frequent over the last decades in the western world. Initial treatment for primary and locoregional melanoma is surgery. In metastatic disease, systematic treatment and recently immunotherapy has been the mainstay. At this stage however, the prognostic outlook is still bleak. Thus, new treatments are urgently needed.

Melanoma is considered to be a radioresistant cancer. The mechanisms underlying radioresistance are multiple and incompletely characterized. In some tumors, radioresistance is mediated by increased synthesis of anti-oxidants that scavenge reactive oxygen species (ROS), induced by radiotherapy. Glutathione (GSH) is an anti-oxidant synthesized from cystine and constitutes a major defense system against oxidative stress in mammalian cells. Cystine is taken up through system Xc- (SXC), an anti-port transmembrane protein with catalytic subunit xCT. Sulfasalazine, a drug approved in the 1950s for treatment of rheumatoid arthritis and inflammatory bowel disease, has been shown to block SXC. Based on previous reports by others, and our own recent findings, we hypothesize that: I) xCT represents a mechanism for radioresistance and is expressed in radioresistant melanoma cancer and II) xCT inhibitors can act as radiosensitizers to potentiate the efficacy of radiotherapy. Expression of the catalytic subunit of SXC, xCT, was found in tissue micro array of primary and secondary melanoma biopsies. In addition, SAS treatment dramatically reduced cystine-uptake and GSH levels in melanoma cells in vitro, and markedly increased the levels of reactive oxygen species (ROS). Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the anti-oxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and irradiation synergistically reduced subcutaneous melanoma tumor growth in vivo, compared to controls or either treatment alone. Thus, future experimental studies are warranted to validate SAS as a radiosensitizer in the treatment of metastatic melanoma. Future studies will also be aimed at assessing the effect on pulmonary melanoma metastasis.

#3049

Arachidonate 12-lipoxygenase contributes to radiation-induced type II pneumocyte senescence and fibrosis.

Eun Joo Chung, Luca F. Valle, Ayla O. White, Deborah E. Citrin. _National Cancer Institute, Bethesda, MD_.

Introduction: Ionizing radiation (IR) is commonly used in the treatment of thoracic malignancies. Exposure of adjacent normal lung may result in lung injury and fibrosis. Recently, we reported that accelerated senescence of type II pneumocytes, the alveolar stem cell, results in parynchymal depletion and precedes pulmonary fibrosis. Arachidonate 12-lipoxygenase (12-LOX) oxidizes arachidonic acid to form 12-Hydroxyeicosatetraeonic acid (12-HETE), a pro-inflammatory mediator. Increased expression of ALOX-12 has previously been associated with aging. We hypothesized that mice deficient in ALOX-12 would be resistant to IR induced fibrosis and senescence.

Methods: C57/Bl6J (WT) and Alox12 homozygous knockout (Alox12-KO) mice (n>3 per group) were exposed to thoracic IR (0Gy, 5Gy, 17.5Gy, 5x5Gy, or 6x5Gy). Fibrotic foci were identified with aniline blue staining of fixed lung sections. Levels of 12-LOX mRNA and protein were assessed in WT lungs after IR with microarray, quantitative PCR, and western blotting. In vitro studies with primary murine pneumocyte cultures and A549 cells included staining for senescence associated β-galactosidase activity and western blotting for NOX4, p21, and PML following IR and 12-HETE treatment.

Results: A significant increase in 12-LOX mRNA and protein was observed in murine lungs exposed to fibrogenic doses of IR (17.5 Gy, 5x5 Gy and 5x6 Gy) compared to low dose IR (5 Gy) or controls (0 Gy). 12-LOX deficiency significantly reduced fibrotic foci in murine lungs receiving fibrogenic doses of thoracic IR (6x5 Gy) at 18 weeks. Treatment of murine primary pneumocytes with 12-HETE (1 and 150 nM) increased the rate of pneumocyte senescence (2.1 fold). Treatment of murine primary pneumocytes with 12-HETE increased the expression of NOX4 (a mediator of superoxide generation), p21, and PML (senescence markers), paralleling increases observed after IR. Similarly, human type II pneumocyte A549 cells exhibited increased 12-LOX expression in β-galactosidase-stained senescent cells at 3 days after IR.

Conclusion: These studies identify 12-LOX as a critical mediator in radiation lung fibrosis and type II pneumocyte senescence. 12-LOX may serve as a novel therapeutic target in mitigating IR-induced lung fibrosis.

#3050

Proteotoxic isolevuglandins are a central feature of radiation-induced pulmonary injury.

Stacy Mont, Sean S. Davies, L Jackson Roberts, Raymond L. Mernaugh, W Hayes McDonald, Brahm H. Segal, Jonathan A. Kropski, Timothy S. Blackwell, Konjeti R. Sekhar, James J. Galligan, Lawrence J. Marnett, Michael L. Freeman. _Vanderbilt University School of Medicine, Nashville, TN_.

The development of radiation induced lung injury (RiPI) is a major barrier that limits the dose that can be administered for controlling locally advanced lung cancer. Although progress has been made toward identifying the pathophysiological events responsible for RiPI, there is still a substantial gap in knowledge. It is well established that oxidative stress is central to the progression of RiPR. In addition, reoccurring cellular injury appears to be a critical event for promotion of radiation-induced fibrosis. Yet, the exact mechanism by which reactive oxygen species (ROS) promotes injury and the nature of the reoccurring injury remain to be fully elucidated. Isolevuglandins are generated by free radical peroxidation of phospholipid-esterified arachidonic acid. Isoketalation, defined as covalent adduction of isolevuglandins to the ε-amino group of protein lysine residues, is emerging as a novel mechanism by which ROS contributes to the genesis of some diseases. Although isoketalation promotes proteotoxicity and cytotoxicity that can contribute to disease progression, there is currently a lack of knowledge concerning its role in RiPI, the identity of susceptible proteins or whether the process can be genetically regulated. We used a multifaceted approach that included wild type, Nrf2 and p47 null mice, confocal microscopy, and LC-MS/MS analysis of affinity purified proteins to address these questions. Mass spectrometry and gene ontology analysis identified several potential protein targets involved in cytoskeletal regulation, Wnt signaling, integrin signaling, chemokine and cytokine signaling and histone biology. Genetic evidence linking oxidant challenge to protein adduction was provided from the mouse knockout studies where it was shown that Nrf2 expression suppressed, while NADPH oxidase activity promoted isoketalation. We found that when isoketalation exceeded a critical level, cells underwent apoptosis. We identified the cell types in mouse lung that are susceptible to adduction. In a C57BL/6j murine model of radiation induced pulmonary injury we found that ionizing radiation increased the level of adduction concomitant with the development of a chronic apoptotic phenotype. We used human idiopathic pulmonary fibrotic (IPF) tissue as a surrogate for radiation-induced pulmonary fibrotic tissue, which is very hard to obtain. Human IPF and radiation-induced lung injury share a common phenotype that includes slow/chronic development and a prominent ROS component. We found an abundance of adducted protein in human IPF compared to control lung tissue, identified collagen 1α1 as one of the highly adducted proteins and show that adduction impairs collagen degradation by MMP1. In summary, these data support the hypothesis that radiation-induced oxidant stress promotes pulmonary injury, in part, by a hitherto, unrecognized mechanism: isoketalation. Supported in part by NIH/NHLBI Grant RO1HL112286.

#3051

Improving the efficacy of pretargeted radioimmunotherapy in preclinical murine models by utilizing bioorthogonal click chemistry.

Jacob Houghton,1 Rosemery Membreno,2 Dalya Abdel-Atti,1 Wolfgang W. Scholz,3 Brian M. Zeglis,2 Jason S. Lewis1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _City University of New York, New York, NY;_ 3 _MabVax Therapuetics, San Diego, CA_.

Monoclonal antibodies (mAbs) are promising vectors for delivering therapeutic radiation to cancer cells, but the slow pharmacokinetics of immunoglobulins can lead to high radiation doses to healthy organs, which has hampered the advent of targeted radiommunotherapy (RIT) in the clinic. In response, considerable attention has been dedicated to RIT strategies — including pretargeted radioimmunotherapy (PRIT) — that reduce the radiation burden to healthy tissue. PRIT harnesses the tumor-targeting properties of mAbs while avoiding their drawbacks by employing process in which we are able to radioactively label mAbs after they have accumulated at their target site in vivo. Recently, an innovative approach has emerged based on the extraordinarily selective and rapid inverse electron demand Diels-Alder cycloaddition between a tetrazine (Tz) bearing radioligand and transcyclooctene (TCO) modified mAb. This system has yielded PET images with high contrast while delivering only a small fraction of the radiation dose produced by directly labeled mAbs. Given the success of these pretargeted imaging modalities, the logical next step is to leverage this technology for the development of a safe and effective approach to PRIT.

To accomplish this goal, we synthesized two novel 177Lu-labeled Tz radioligands in excellent radiochemical yield (>98%). We compared their uptake in a PRIT system with TCO-modified 5B1 — a human, anti-CA19.9 mAb — using preclinical murine models of pancreatic ductal adenocarcinoma (PDAC). Biodistribution studies in mice bearing subcutaneous xenografts indicated that 177Lu-DOTA-PEG7-Tz—the best ligand tested—had rapid (4.6 ± 0.8 %ID/g at 4h) and persistent (16.8 ± 3.9 %ID/g at 120h) uptake in xenografts while rapidly clearing from blood and non-target tissues. Single-dose therapy studies using 5B1-TCO and varying doses of 177Lu-DOTA-PEG7-Tz (400, 800, and 1200μCi) showed that our system elicits a dose-dependent therapeutic response in mice bearing human xenografts, the higher two doses causing marked reduction in size or complete elimination of the tumors in vivo. Moreover, dosimetry calculations indicated that, as expected, the PRIT approach reduced the effective absorbed dose of radiation relative to directly labeled 177Lu-5B1 by as much as an order of magnitude at doses that elicited an equal therapeutic response. This was particularly true in metabolic organs such as the liver, kidneys, and spleen as well as tissues such as dose-limiting tissues like blood and red marrow.

In the past, several strategies for PRIT have been limited by intrinsic problems (i.e. immunogenicity or radionuclide washout due to noncovalent binding) that prevented clinical translation. We have adapted and optimized a novel PRIT system without those inherent limitations that matches or improves upon their efficacy in preclinical studies. Studies to expedite clinical translation are currently underway.

#3052

Combining α-particle radiopharmaceutical therapy using Actinium-225 and immunotherapy with anti-PD-L1 antibodies in a murine immunocompetent metastatic breast cancer model.

Anders Josefsson, Jessie R. Nedrow, Sunju Park, Sagar Ranka, George Sgouros. _Johns Hopkins University School of Medicine, Baltimore, MD_.

The programmed cell death ligand 1 (PD-L1) plays an essential role in suppressing immune recognition of cancer. PD-L1 is expressed on a variety of cells including tumor cells, tumor associated macrophages (TAMs) and other cells within the microenvironment of the tumor. When PD-L1 binds to the programmed death 1 (PD-1) receptor it inhibits CD8+ T-cell effector function. By upregulating the expression levels of PD-L1, tumor cells and TAMs are capable of avoiding T-cell immune recognition. Immunotherapy using anti-PD-L1 antibody (Ab) has shown promising anti-tumor effect against a number of cancers including breast cancer, and is currently used in several clinical trials. Furthermore, studies have shown that anti-PD-L1 Ab targeted immunotherapy synergizes with radiation therapy. The aim of this study was to investigate a possible gain in therapeutic efficacy when combining targeted α-particle radiopharmaceutical therapy using 225Ac with anti-PD-L1 Ab immunotherapy in a murine immunocompetent metastatic breast cancer model.

6-8 week old healthy female neu-N mice were injected in the left cardiac ventricle (LCV) with 50,000 NT2.5 (endogenously derived) tumor cells to create highly aggressive widespread breast cancer metastases. Groups (n=8) of mice were injected intravenously (i.v.) in the tail vein 72 h after the LCV injection with either 1) a single dose of 300 or 400 nCi 225Ac-DOTA-anti-PD-L1 Ab (0.15 mg/kg), 2) a single dose of 100 times (100x) anti-PD-L1 Ab (15.9 mg/kg) or 3) a 400 nCi single dose 225Ac-DOTA-anti-PD-L1 Ab (0.15 mg/kg) in combination with a single dose of 100x anti-PD-L1 Ab (16.1 mg/kg). The mice in the control group were injected i.v. in the tail vein with 100 µl of saline.

The 225Ac-DOTA-anti-PD-L1 conjugate was radiolabeled having a specific activity of 0.137 µCi/µg with a radiochemical purity >95%. The group receiving the single dose of 100x anti-PD-L1 Ab had the highest median survival of 44 days (p=0.0007) followed by the 400 nCi 225Ac-DOTA-anti-PD-L1 Ab group with 39.5 days (p=0.0413) compared with the control group 31.5 days. The survival for other treatment groups were not significant compared with the control group. Furthermore, the survival from the single dose of 100x anti-PD-L1 Ab treatment was significantly higher than the single dose treatment of 400 nCi 225Ac-DOTA-anti-PD-L1 Ab (p=0.0308).

The highest survival rate was the mice treated with 100x anti-PD-L1 Ab. The combination treatment using 400 nCi 225Ac-DOTA-anti-PD-L1 Ab and 100x anti-PD-L1 Ab showed a significant lower survival compared to each treatment by itself. However, the combination treatment was only performed with one dose and the injection was at the same time, different concentrations and time spaced injections could possibly favor the combined treatment method over the single modality treatments.

#3053

Inhibition of heregulin-mediated ErbB3 signaling as a radiosensitization therapy for head and neck cancers.

Marta Baro,1 Josep Balart,2 Joseph N. Contessa1. 1 _Yale University, New Haven, CT;_ 2 _Hospital de la Santa Creu i Sant Pau, Barcelona, Spain_.

PURPOSE: EGFR signaling confers resistance to radiation therapy (RT) and is a validated target in head and neck squamous cell carcinoma (HNSCC). Inhibition of EGFR function in combination with RT improves local control and overall survival in this patient population, however, the mechanisms of resistance to combined treatment with RT + cetuximab are incompletely understood. We sought to develop cell line models of true cetuximab resistance and to define the molecular characteristics of these tumor cells with respect to radiation sensitivity.

EXPERIMENTAL PROCEDURES: A431 and FaDu cell lines with resistance to cetuximab were generated by two methods: (i) exposure to increasing doses of cetuximab over 14 weeks or (ii) single cell clonal selection prior to identical cetuximab exposure regimens or for untreated controls. Receptor tyrosine kinase activity and dependent downstream signaling were assessed by phospho-blot analysis. Proliferation was determined with MTT. Radiosensitivity was determined through clonogenic survival analysis. The EFM-19 cell line was used as a functional indicator of heregulin family ligand expression. The efficacy of cetuximab was assessed in xenografts grown in nude mice.

RESULTS: Cell line cultures exposed to increasing doses of cetuximab achieved only partial resistance in vivo. The process of clonal selection allowed us to identify distinct cell populations: (a) cetuximab-sensitive clones from untreated controls, (b) partial-cetuximab resistant clones from untreated controls, and (c) cetuximab-resistant clones from cell lines exposed to increasing doses. Partial- and cetuximab-resistant clones showed an increase of phospho-ErbB3, AKT, and Met. These clones were also more resistant to ionizing radiation. We also tested whether KTN3379, a monoclonal antibody that inhibits ligand-dependent and independent ErbB3 activation, affected cell proliferation and survival. After 5 days, KTN3379 + cetuximab reduced proliferation of cetuximab-resistant clones but did not further blocked partial- and sensitive-clones compared to cetuximab alone. This corresponded to a significant reduction of ErbB3/Akt (and Met) mediated by KTN3379. Using the EFM-19 cell model, which lacks endogenous heregulin, we demonstrate that cetuximab resistant cells upregulate heregulin family ligand expression and that KTN3379 effectively blocks this autocrine survival signaling.

CONCLUSION: Tumor cell heterogeneity obscures the identification of operative resistance mechanisms in vitro. Autocrine ligand activation of ErbB3 is a mechanism for therapeutic resistance to cetuximab and may also cause radioresistance. Cross-resistance to both therapeutic modalities identifies HRG and ErbB3 as attractive therapeutic targets for improving delivery of radiation therapy for HNSCC.

#3054

CD47 signaling regulates a DNA damage response pathway by suppressing the expression of Schlafen-11 (SLFN11).

Anthony L. Schwartz, Sukhbir Kaur, Sai-Wen Tang, Yves Pommier, David D. Roberts. _National Institutes of Health, Bethesda, MD_.

Schlafen-11 (SLFN11) expression was recently shown to determine the sensitivity of human cancer cells to DNA damaging agents including topoisomerase inhibitors, DNA alkylating agents, and DNA synthesis inhibitors. High expression of SLFN11 in cancer cell lines results in increased sensitivity to these agents, whereas depletion of SLFN11 induces cellular protection. In addition, high expression of SHFN11 is associated with greater overall survival than low SLFN11 expression in ovarian patients treated with cisplatin. These observations suggest that SLFN11 may be an integral part of a critical DNA repair pathway. However, the pathways that regulate SLFN11 expression have not been reported. CD47 is an integrin-associated protein widely expressed on mammalian cells and frequently elevated in cancers. Its ligand, thrombospondin-1 (TSP1) induces CD47 signaling that regulates some growth factor receptors, cell fate, viability, and responses to cellular stresses such as radiation and chemotherapy. Our laboratory has previously shown that CD47 deficiency results in protection of nontransformed cells, whereas high CD47 expression sensitizes the same cells to ionizing radiation and DNA-damaging chemotherapy. We identified SLFN11 expression as a potential target of CD47 signaling by microarray analysis of a CD47-deficient mutant cell line and found that CD47 expression also correlates with that of SLFN11 in some human cancers. Thus, we sought to investigate the possible regulation of SLFN11 by CD47 by treating various cell lines with the function-modifying CD47 antibody B6H12 or its physiological ligands TSP1 and signal regulatory protein-α (SIRPα). qRT-PCR analysis revealed significant decreases in SLFN11 expression at 3 and 6 hours post-B6H12 treatment. Furthermore, treatment with TSP1 significantly decreased SHFN11 mRNA expression at the same time points, but SIRPα-Fc did not. Conversely, transient re-expression of CD47 in deficient cells elevated SLFN11 expression. These data demonstrate that pharmacological and physiological modulation of CD47 signaling acutely alters SLFN11 expression and suggests a potential mechanism by which CD47 modulates cellular sensitivity to DNA damaging chemotherapy agents. SLFN11 may in turn be a functional target and useful biomarker for the therapeutic CD47 antibodies currently in clinical trials.

#3055

In vitro **evaluation of combination chemotherapy/targeted radiotherapy for translation to an** in vivo **model of prostate cancer.**

Tammy L. Rold, Nicole E. Bernskoetter, Timothy J. Hoffman. _HS Truman VA Hospital, Columbia, MO_.

Introduction: We have previously shown that radiosensitizing chemotherapy when used in combination with targeted radiotherapy agents can result in improved tumor growth inhibition in preclinical xenograft mouse models of prostate cancer (PC). The challenge is determining optimum drug combinations to achieve therapeutic synergy. Herein we performed in vitro studies to determine if synergism existed and then optimize effective drug combination levels to maintain synergism for the treatment of PC.

Methods: The Lu-177 radiolabeled peptide (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was prepared using an Eckert & Ziegler automated system. Cultured PC-3 cells were allowed to adhere overnight in a standard tissue culture environment after plating. Docetaxel chemotherapy was prepared in culture media without supplements and added to wells containing fresh growth media to achieve a final concentration of docetaxel of 0, 1.25, 2.5, and 5 nM. After 24 hrs, all media was removed and replaced with fresh growth media (non-chemo containing) prior to treatment with 0, 5, 10, 20, or 40 uCi/well of Lu-177 labeled peptide receptor targeted radiotherapy (TRT) to each well. At 72hrs post TRT addition, all media was removed and fresh growth media was added. MTT reagent was added to each well at pre-determined time points. Plates were incubated in a standard tissue culture environment for 4 hours prior to removal of all liquid from the wells followed by addition of 200uL of DMSO to each well. Plates were read at 540nm and data was normalized to non-treated controls. Calcusyn software was used to calculate CI values to determine if synergy was achieved. Cell cycle experiments were performed after docetaxel treatment at 0, 2.5, 5, 10, 20, 40, and 80 nM. After 6, 24, 48, and 72 hrs of treatment, cells were collected from each well, fixed in 70% ethanol, and stained with propidium iodide prior to cell cycle analysis (5,000 events).

Results: Cell cycle analysis confirmed a sustained shift (> 50%) of docetaxel treated cells to the radio-sensitive G2/M phase of the cell cycle after 24, 48, and 72hrs of treatment. Synergy was observed when docetaxel at 1.25 and 2.5 nM was combined with 20 and 40 uCi of Lu-177 TRT. At concentrations of docetaxel >2.5nM only additive effects of combination therapy were observed. At concentrations of Lu-177 TRT <10uCi/well in combination with docetaxel only slight synergy or antagonism was observed. Conclusions: Effectively optimizing combination chemotherapy and Lu-177 TRT in an in vivo model of prostate cancer remains challenging. These initial results demonstrate a synergistic window where chemotherapy and Lu-177 TRT can potentially yield optimum results however achieving synergy requires further detailed drug combination optimization both in vitro and in vivo.

#3056

Combined disulfiram and radiation therapy against atypical teratoid/rhabdoid tumor.

Yeoung Eun Lee,1 Seung Ah Choi,1 Pil Ae Kwak,1 Kyu-Chang Wang,1 Ji Hoon Phi,1 Ji Yeoun Lee,1 Sangjoon Chong,1 Youn Joo Moon,1 Anshika Jangra,1 Kyeung Min Joo,2 Seung-Ki Kim1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Samsung Biomedical Research Institute, Seoul, Republic of Korea_.

Disulfiram (DSF) has been studied as an anticancer drug and radiosensitizer. In our previous study, the therapeutic potential of DSF against atypical teratoid/rhabdoid tumor (AT/RT), one of the most aggressive forms of childhood cancer, had been confirmed using in vitro and in vivo studies. To develop more effective and safer therapy in AT/RT, we evaluated the sensitizing potential of DSF on radiation therapy (RT). The effect of DSF in low concentration alone and in combination with RT was investigated in vitro and vivo. Clonogenic assay, western blot analysis, autophagy assay and γ-H2AX foci staining were performed using several primary cultured AT/RT cells and cell lines. Also, in vivo study was performed using AT/RT orthotopic xenograft mouse model. Combination therapy with DSF and RT potently reduced clonogenicity and down-regulated protein expression of survivin and BCL2. The combination treatment strongly promoted the autophagic cell death and produced abundant γ-H2AX foci in all AT/RT cells. Moreover, the combination treatment significantly reduced the tumor growth and prolonged the survival rate compared to single treatment in AT/RT mouse model. Taken together, our results demonstrated that the combination therapy with DSF and RT has a synergistic therapeutic effect on AT/RT, suggesting a potential clinical application for AT/RT patients.

#3057

Synthetic novel Psammaplin A derivatives enhance radiation lethality in human cancer cells.

Jin Ho Kim,1 Chan Woo Wee,1 Hak Jae Kim,1 Soo Youn Suh,1 Eun Sook Ma,2 Boom Soo Shin,2 Il Han Kim1. 1 _Seoul National University, Seoul, Republic of Korea;_ 2 _Catholic University of Daegu, Daegu, Republic of Korea_.

DNA methyltransferase (DNMT) inhibitors show not only anticancer effects but radiosensitization activity. Psammaplin A (PsA), a known DNMT inhibitor, enhances radiation lethality of human cancer cells, but the chemical instability hampers in vivo application of PsA. The purpose of the present study was to synthesize novel radiosensitizers using PsA as the backbone structure. Eight novel PsA-based derivatives (MA2, MA3, MA5, MA6, MA7, MA8, MA9 and MA5M) were synthesized. They were tested for in vitro cytotoxicity and radiosensitizing effects in A549 (lung cancer) and U373MG (glioblastoma) cells using a clonogenic assay. A paired ratio t-test was used to test a statistical significance of sensitizer enhancement ratios of the compounds. All 8 PsA derivatives inhibited in vitro cell survival with 50% inhibitory concentrations ranging 16-150 μM (A549) and 15-50 μM (U373MG). MA7, MA9 and MA5M significantly enhanced radiation lethality in A549 cells, while compounds MA2, MA3 and MA7 showed a significant radiosensitization in U373MG cells (p<0.05). MA3 and MA6 marginally radiosensitized A549 and U373MG cells, respectively (p<0.10). For further investigation, we selected MA3 and MA7, because only these two compounds showed radiosensitization in both A549 and U373MG cell lines. A DNMT activity assay showed that MA3 inhibited DNMT1 activity, but that MA7 showed no inhibitory effect on DNMT1. Pharmacokinetic parameters were determined in mice by a validated LC-MS/MS assay method using a non-compartmental analysis. The average degradation half-life of MA3 and MA7 was under 10 minutes in mouse serum. In the present study, we synthesized two novel PsA-derived compounds that enhanced radiation lethality in two human cancer cell lines. However, they lacked chemical stability to allow for in vivo experiments. Based on the present study, a further study is necessary to improve bioavailability of PsA-derived radiosensitizers. [Supported by 2013M2A2A7043683]

### Small Molecule Inhibitors

#3058

Phenotype-based discovery and development of novel therapies for the treatment of colorectal cancer.

Clare T. Butler,1 Breandán Kennedy,1 William M. Gallagher,1 Jacintha O'Sullivan2. 1 _University College Dublin, Dublin 4, Ireland;_ 2 _Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland_.

Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world. The treatment of late stage CRC using Bevacizumab®, a targeted anti-VEGF therapy, produces insufficient response rates among patients and inevitable acquired tumour resistance. There is a clinical need for improved treatment options.

Methods: Previously, anti-angiogenic screens in Tg[fli1:EGFP] zebrafish identified quininib, a small molecule drug subsequently shown to be as effective as bevacizumab® at reducing tumour volume in vivo and which reduces tumour CD31 expression in xenografted mice. Here, structural analogues of quininib were synthesised to identify novel chemical entities with higher anti-angiogenic and anti-inflammatory efficacy. Analogues were examined for anti-angiogenic effects in the Tg[fli1:EGFP] zebrafish intersegmental-vessel assay. Hits were then progressed for safety analyses in cytotoxic assays, anti-angiogenic efficacy in aortic explants, and effects on cell proliferation and tubule formation using HT29_Luc2 and HMEC cells. Leads were then tested in ex vivo human CRC tumour explants to determine effects on angiogenic and inflammatory factor secretion. Following determination of the maximum tolerated dose (MTD) of the highest ranking analogues, CRC-specific murine xenograft models created by subcutaneous injection of HT29_Luc2 cells were assessed for anti-tumorigenic analysis.

Results: Five out of 12 drug candidates (CC16_HCl, CC12_HCl, Z_HCl, CC8_HCl & OS_1) reduced developmental angiogenesis in Tg[fli1:EGFP] zebrafish significantly more than quininib (***P<0.01). These analogues were well tolerated in human endothelial and CRC cell cytotoxic assays and were more effective than quininib at reducing tubule formation and angiogenic factor secretion in HMEC cells. Two analogues reduced aortic ring vessel sprouting equally or more effectively than quininib. Some analogues can reduce inflammatory and angiogenic factor secretion from ex vivo human CRC explants more significantly than quininib. The analogue with the highest efficacy ranking was identified and its maximum tolerated dose study (MTD) determined using balb/c wild type mice. The anti-tumorigenic effects of the highest ranking analogue is being assessed by i/p injection in an optimised model of HT29_Luc2-CRC tumourigenesis in Balb/c nude mice.

Conclusions: 5 analogues significantly more effective than quininib at reducing inflammation and angiogenesis were identified. The highest ranking analogue is tolerated by i/p injection in mice up to 50 mg/kg with no obvious adverse effects. The highest ranking analogue is currently being tested in murine xenografts with preliminary data suggests it is more effective than quininib at reducing tumour growth.

#3059

Discovery of BET family proteins as cancer targets using phenotypic-based profiling and affinity capture mass spectrometry.

Scott E. Warder, Shaun M. McLoughlin, T. Matthew Hansen, Paul L. Richardson, Denise M. Wilcox, Sadiya N. Addo, Hua Tang, Chaohong Sun, Andrew M. Petros, Sanjay C. Panchal, Chang H. Park, M. Shannon Duggan, Melanie J. Patterson, F. Greg Buchanan, Dong Cheng, Heather M. Davis, David J. Calderwood, Steven W. Elmore, Yu Shen. _AbbVie, North Chicago, IL_.

As part of a multi-year technology integration strategy to identify unprecedented targets, AbbVie has committed to building broad-endpoint profiling assays to enable phenotypic screening campaigns and compound prioritization. Early on, phenotypic cell-based screening employing a panel of protein-fragment complementation assays (PCAs) identified A-1107604 as a hit with potent activity against a subset of endpoints. While its activity profile had some commonalities with known anti-cancer agents, the overall profile of PCAs that were significantly and concomitantly modulated represented a unique signature. Further analysis revealed A-1107604 to have potent and selective activity in a panel of human tumor cell lines. Inhibitor affinity capture from cellular lysates coupled with mass spectrometry identified the BET family of proteins as the putative cellular targets of A-1107604. Binding was localized to the bromodomain of the target proteins using affinity capture-protease digestion and was confirmed by thermal shift assay, solution

binding and X-ray crystallography. This binding was found to be highly selective when A-1107604 was counter-screened against a 150-member kinase panel and an 80-member receptor panel. To correlate target affinity with cellular efficacy, a series of analogs were prepared with affinities spanning 3 orders magnitude. Affinity for BRD4, a BET family member, strongly correlated with efficacy in human tumor cell lines. Finally, A-1107604 was evaluated in human tumor xenograft models where it demonstrated significant tumor growth inhibition. This discovery effort laid the foundation for our BET inhibitor program. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the

publication.

#3060

Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib.

Brian B. Hasinoff, Xing Wu, Daywin Patel. _Univ. of Manitoba, Winnipeg, Manitoba, Canada_.

Bortezomib and carfilzomib are anticancer drugs that target the proteasomal system. Case reports indicate that the use of these drugs can result in cardiotoxicity. These drugs may also be used in combination with doxorubicin, which is itself cardiotoxic. Thus, in order to determine the mechanism of this cardiotoxicity studies were undertaken using a neonatal rat myocyte primary cell model. Using LDH release as a measure of damage in the myocyte model we showed that even brief exposure (6 h) to submicromolar concentrations of bortezomib resulted in significant myocyte damage. Carfilzomib was slightly less toxic and displayed myocyte damage in the low micromolar concentration range. Utilizing a fluorogenic substrate both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the submicromolar concentration range. The inhibition kinetics were consistent with formation of an essentially irreversible covalent complex at the active site. Bortezomib irreversibly inhibited proteasomal activity about four-fold faster than carfilzomib. Thus, the reduced toxicity of carfilzomib may be due to its slower inhibition kinetics. A brief pre-exposure (6 h) of myocytes to low non-toxic nanomolar concentrations of bortezomib greatly increased doxorubicin-mediated damage. The doxorubicin cardioprotective agent dexrazoxane partially protected the myocytes from doxorubicin treatment and from bortezomib plus doxorubicin treatment under these conditions. However, at slightly toxic bortezomib concentrations dexrazoxane offered little protection against bortezomib plus doxorubicin treatment. Bortezomib, at least over short times, did not induce oxidative damage in myocytes as measured with a fluorogenic DCF assay. Likewise, a 6 h bortezomib treatment did not affect the mitochondrial membrane potential of myocytes as measured in a JC-1 ratiometric assay. In conclusion both bortezomib and carfilzomib are potently toxic to myocytes at submicromolar concentrations, likely through their ability to irreversibly inhibit myocyte proteasomal activity. Support: CIHR; a Canada Research Chair in Drug Development.

#3061

AS-10: a new small molecule with promising activity against pancreatic cancer.

Deepkamal Karelia,1 Manoj K. Pandey,1 Daniel Plano,2 Shantu Amin,1 Arun K. Sharma1. 1 _Penn State University College of Medicine, Hershey, PA;_ 2 _University of Navarra, Pamplona, Spain_.

Patients with pancreatic cancer (PC) have a median survival of only 6 months and a five-year survival of less than 5%, hence making PC one of the deadliest cancer. Severity of PC is due to its identification at late stages, rapid local invasion, early metastases, and meager response to current chemotherapeutic agents. Current therapies result in minimal survival advantage and are linked with multiple adverse events and drug resistance. Hence, there is an urgent need for novel agents which are less toxic and offer greater benefits over conventional therapy. There is ample evidences in literature demonstrating that inflammation plays a critical role in PC growth and promotion. Nuclear factor κB (NF-κB) pathway, one of the major inflammatory pathway, is well known for its inflammatory response, cell proliferation, and resistance to apoptosis. It has been demonstrated that activation of NF-κB in PC is also responsible for resistance towards first line chemotherapeutic agent, gemcitabine, in PC. Through extensive structure-activity relationship studies, we have recently identified a novel small molecule, AS-10, which was lethal to PC cells. We sought to evaluate the mechanism of action of AS-10 responsible for inhibiting the growth of PC cells. In vitro, AS-10 reduced PC cell growth with an EC50, in the range of 2.5 to 5 µM. Growth arrest was confirmed by cell cycle studies, which showed that AS-10 induced G1 and G2 cell cycle arrest. The cell cycle arrest was associated with an increase of cell cycle inhibitory markers like p21 and p27. Effect of AS-10 on cell cycle was translated into activation of apoptosis, confirmed by caspase 3/7 activity, PARP cleavage and Annexin V staining. Due to its structural characteristics, we evaluated the effect of AS-10 on inflammatory NF-κB pathway. Our studies, in Panc-1 cells, showed that AS-10 inhibited NF-κB DNA binding activity as well as NF-κB translocation to the nuclei in the presence of inflammatory stimuli (tumor necrosis factor (TNFα)). Since, up regulation of NF-κB activity has been known to be responsible for gemcitabine resistance in PC, we evaluated the activity of our NF-κB inhibitor AS-10 in combination with gemcitabine, and showed that AS-10 synergistically potentiated gemcitabine activity in PC cells. Taken together, these results suggest that AS-10, may represent a potentially promising therapeutic agent for PC. Detailed investigations regarding the efficacy and mechanism of action of these compounds will be presented.

#3062

Silencing the epigenetic silencer KDM4A for TRAIL and DR5 simultaneous induction and antitumor therapy.

Junjian Wang,1 Haibin Wang,2 Lingyu Wang,3 Demin Cai,1 Zhijian Duan,1 Peng Chen,2 June X Zou,1 Jianzhen Xu,4 Xinbin Chen,5 Hsing-Jien Kung,1 Hong-wu Chen1. 1 _Department of Biochemistry and Molecular Medicine, School of Medicine, University of California at Davis, Sacramento, CA;_ 2 _First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China;_ 3 _Department of Urology, School of Medicine, University of California at Davis, Sacramento, CA;_ 4 _Shantou University Medical College, Shantou, China;_ 5 _Schools of Veterinary Medicine, University of California at Davis, Davis, CA_.

Recombinant TRAIL and agonistic antibodies to death receptors (DRs) have been in clinical trial but displayed limited anti-cancer efficacy. Lack of functional DR expression in tumors is a major limiting factor. We report here that chromatin regulator KDM4A/JMJD2A, not KDM4B, plays a pivotal role in silencing tumor cell expression of both TRAIL and its receptor DR5. In TRAIL-sensitive and -resistant cancer cells of lung, breast and prostate, KDM4A small-molecule inhibitor compound 4 (C-4) or genetic silencing strongly induces TRAIL and DR5 expression, and causes TRAIL-dependent apoptotic cell death. KDM4A inhibition also strongly sensitizes cells to TRAIL. C-4 alone potently inhibits tumor growth with marked induction of TRAIL and DR5 expression in the treated tumors and effectively sensitizes them to the newly developed TRAIL-inducer therapeutics ONC201. Mechanistically, C-4 does not appear to act through the Akt-ERK-FOXO3a pathway. Instead, it switches histone modifying enzyme complexes at TRAIL and DR5 transcriptional activator CHOP gene promoters by dissociating KDM4A and NCoR-HDAC corepressor complex, and inducing the recruitment of histone acetylase CBP. Thus, our results reveal KDM4A as a key epigenetic silencer of TRAIL and DR5 in tumors and establish inhibitors of KDM4A as a novel strategy for effectively sensitizing tumors to TRAIL pathway-based therapeutics.

#3063

Discovery of a third-generation EGFR inhibitor, which is highly selective and potent against both resistant and activating EGFR mutations for NSCLC therapy.

Rudi Bao, Peng Gao, Fujun Zhang, Zhaolong Tong, Hongping Yu, Yaochang Xu. _Hansoh Pharmaceuticals, China_.

EGFR T790M mutation which renders resistance to the first-generation EGFR inhibitors,accounts for about 50% of all originally responsive non-small cell lung cancer patients. In order to overcome this type of resistance, we have developed a small molecular drug candidate with unique structural features that offer differentiated pharmacological and safety profiles pre-clinically when compared with other leading third-generation EGFR inhibitors. The compound selectively inhibits T790M as well as activating EGFR mutations, with much less inhibition to the wild type EGFR. Enzymatic inhibition IC50 against EGFR T790M,T790M/Del19 and T790M/L858R is less than 1nM. In addition, enzymatic inhibition IC50 against EGFR activating mutations Del19 and L858R is around 1nM, with much less inhibition to the wild type EGFR, a 10-time selection ratio. In cell based assays, the compound potently inhibits proliferation of EGFR T790M mutated NSCLC cell lines H1975(T790M/L858R)and PC9-GR(T790M/Del19)with an IC50 around 3nM. Similarly, it potently inhibits EGFR activating mutant NSCLC cell lines HCC827(Del19)and PC9(Del19). However, the compound displays about 200-fold less sensitivity to the EGFR wild type cancer cell line A431, with an IC50 around 600nM. When dosed to tumor-bearing animals, the compound dose-dependently induces profound regression of xenografts derived from both H1975 and primary human lung cancer LU1868, both of which carry an EGFR T790M mutation. It also induces regression of HCC827 xenografts potently, similarly to the first- and second-generation EGFR inhibitors; whereas it displays much less inhibition to the EGFR wild type A431 xenografts, demonstrating a higher specificity to the mutated EGFR forms when compared with the EGFR inhibitors on market. The drug candidate displays favorable PK and safety profiles. These properties warrant further development of this compound as first- as well as second-line therapy for NSCLC.

#3064

Newer platinum agent, Triplatin NC, is as effective as Triplatin for metastatic breast cancer and pancreatic cancer carcinomatosis.

Eriko Katsuta, Stephanie C. DeMasi, Samantha J. Katner, Hiroaki Aoki, Erica J. Peterson, Nicholas P. Farrell, Kazuaki Takabe. _Virginia Commonwealth Univ., Richmond, VA_.

Introduction: Since metastatic cancer is the most advanced disease and is associated with the worst outcome, an effective treatment for this Stage is expected to directly improve overall survival. Recent clinical trials demonstrated that Cisplatin is effective in certain metastatic breast cancer, however with severe side effects. Polynuclear platinum analog, Triplatin, was developed to overcome the severe toxicity, however, pharmacokinetics limited its clinical efficacy in Phase II clinical trials. Recent development of Triplatin NC resolved the PK issue, and it is expected to be more clinically useful if it is as effective as Triplatin with less cytotoxic properties.

Methods: HUVEC cells were used for tube formation assays. After orthotopic implantation of Murine breast cancer 4T1-luc2 cells, female Balb/C mice were randomized on Day 1 to Day 1, 5, 9 i.p. administration of Triplatin (0.3mg/kg), Triplatin NC (25mg/kg) or Saline and mice were sacrificed on Day 20 when lung metastasis was evaluated by ex vivo imaging. Pancreatic cancer carcinomatosis, generated by ip injection of 1 million Panc02-luc cells into C57/Blk6 mice. Animals were treated every 4 days beginning on day 1 by i.p. injection with either Triplatin (0.3mg/kg), Triplatin NC (25mg/kg) or Vehicle.

Results: IC50 of 4T1-luc cells at 72h were 9µM and 8µM for Triplatin and Triplatin NC, respectively. 10µM of Triplatin or Triplatin NC demonstrated significant suppression of tube formation compared from no treatment (p=0.005 and p=0.002, respectively). Additionally, 0.1µM of Triplatin NC demonstrated significant suppression of tube formation as well (p=0.003). In 4T1-luc2 implanted model, bioluminescent imaging on Day 7 demonstrated reduced tumor growth in Triplatin and Triplatin NC to 54% and 57% of the control, respectively. By Day 20, Triplatin and Triplatin NC suppressed the tumor size to 20% and 43% of the control, respectively. Triplatin and Triplatin NC significantly reduced the amount of lung metastasis to 14% and 21% of the control, respectively.

In pancreatic cancer peritoneal carcinomatosis model, Triplatin and Triplatin NC reduced total tumor burden to 35% and 39% of the control on Day 9, respectively. Mouse survival was significantly enhanced by Triplatin treatment group in comparison with the control (p < 0.005). All of Triplatin treatment mice survived more than 40 days, whereas all of control group mice died less than 35 days. No mouse developed weight loss more than 25%.

Conclusion: Both Triplatin and Triplatin NC effectively suppressed 4T1-luc2 breast cancer cell growth, and angiogenesis in vitro. Both agents demonstrated similar growth suppression not only of the primary tumor, but also in lung metastasis and panc02-luc carcinomatosis. New platinum compounds with less toxicity and favorable pharmacokinetics warrant further investigation for advanced metastatic cancer.

#3065

Erlotinib derivative, devoid of EGFR kinase inhibiting effect, induced apoptosis of triple-negative breast cancer cells through modulating Elk-1/CIP2A signaling pathway.

Ling-Ming Tseng,1 Yi-Ting Chen,1 Chun-Teng Huang,2 Pei-Yi Chu,3 Wan-Lun Wang,1 Chun-Yu Liu,1 Chung-Wai Shiau,4 Kuen-Feng Chen5. 1 _Taipei Veterans General Hospital, Taipei City, Taiwan;_ 2 _Yang-Ming Branch of Taipei City Hospital, Taipei City, Taiwan;_ 3 _Show Chwan Memorial Hospital, Changhua City, Taiwan;_ 4 _National Yang-Ming University, Taipei City, Taiwan;_ 5 _National Taiwan University Hospital, Taipei City, Taiwan_.

Background:

Triple-negative breast cancer (TNBC), a subgroup of breast cancer, is characterized by its aggressive nature and poor prognosis. Therefore, the discovery and identification of new therapeutic molecule is currently one of urgent issues. Previous studies have suggested an oncoprotein, cancerous inhibitor of protein phosphatase 2A (CIP2A), is a potential therapeutic determinant that mediates the anti-cancer effects of several new agents. We have developed an erlotinib analogue TD-19 that is devoid of EGFR tyrosine kinase inhibition effect, and demonstrated its anti-tumor effects through the suppression of CIP2A-mediated pathway in non-small-cell lung cancer cells (J Pharmacol Exp Ther 2014). In this study, we tested the apoptotic effect of TD-19 in TNBC cells and examined the drug mechanism upstream of CIP2A.

Methods:

TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of TD-19 was tested in nude mice with breast cancer xenografts.

Results:

TD-19 triggered significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-468 and BT-20 cell lines, as well as in non-TNBC cell lines (SK-BR3 and MCF-7). The induced apoptotic effects are associated with CIP2A and p-Akt downregulation in both dose- and time-dependent manners. Overexpression of CIP2A protected MDA-MB-468 from the induced apoptosis of TD-19. In addition, PP2A activity was increased in TD-19 treated MDA-MB-468 cells. Moreover, forskolin, a protein phosphatase 2A (PP2A) activator, enhanced the cell death induced by TD-19, whereas okadaic acid, a PP2A antagonist, attenuated the apoptosis. Mechanistically, we demonstrated that TD-19 downregulated CIP2A mRNA expression through affecting the total amount of Elk-1 in nucleus. Chromatin immunoprecipitation experiments showed TD-19 disturbed the binding of Elk-1 to the proximal CIP2A-promotor. Furthermore, TD-19 showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and downregulated CIP2A as well as p-Akt in these xenografted tumors. Interestingly, combining TD-19 with cisplatin demonstrated enhanced anti-proliferative and apoptotic effects in association with CIP2A downregulation in vitro.

Conclusions:

Our results suggest that the novel erlotinib derivative (TD-19) induced apoptosis through inhibiting Elk1/CIP2A/PP2A/p-Akt signaling in TNBC cells. Pharmacological modulation of Elk-1/CIP2A signaling may be a therapeutic strategy for TNBC.

(Supported by Yen Tjing Ling Medical Foundation (CI-104-07); MOST 104-2628-B-075-001-MY3; Yang-Ming Branch of Taipei City Hospital; and from the Ministry of Health and Welfare, Executive Yuan, Taiwan, MOHW104-TDU-B-211-124-001; and MOHW103TDU-212-114002).

#3066

Anti-angiogenic properties of PM060184.

Carlos M. Galmarini, Maria J. Guillen, Juan F. Martinez-Leal, Marta Martinez-Diez, Pablo Aviles. _PharmaMar S.A., Madrid, Spain_.

Vascular supply of tumors is one of the main targets for cancer therapy. Different studies have shown that most of the microtubule-binding agents present antiangiogenic and vascular-disrupting effects. PM060184 is a new marine-derived drug that binds to a new site in β-tubulin inhibiting tubulin polymerization. The compound is currently being evaluated in phase 1/2 studies in patients with advanced malignancies. The present study describes the antiangiogenic and vascular-disrupting properties of PM060184. Treatment of HUVEC endothelial cells with PM060184 resulted in depolymerization of the microtubule network. Differently from untreated cells, PM060184-treated cells showed a rounded morphology with a well-developed actin filament structure and plasma membrane blebs. Using transwell chambers pre-coated with matrigel™ or collagen matrix, we showed that PM060184 inhibited migration and invasion of HUVEC endothelial cells. We finally found that PM060184 interfered with the correct formation of the HUVEC capillary network grown on matrigel™; moreover, drug treatment also resulted in a significant disruption of the previously formed capillary-like network. This rapid collapse of the endothelial tubular network in vitro occurred in a concentration-dependent manner and was observed at concentrations lower than those affecting cells survival. These in vitro findings were confirmed in nude mice bearing MDA-MB-231 (breast) xenografts: after the administration of a single dose of PM060184 (at its MTD, 16 mg/kg,) a very strong reduction in the number of vessels was quantified in serial tumor sections stained with H&E. Moreover, nude mice xenografted with H-460 (NSCLC) tumors and treated with a single dose of either 2 or 16 mg/kg of PM060184, experienced a strong and dose-dependent decrease of the intratumoral fluorescence after the administration of Angiosense™ 680, a fluorescence vascular imaging probe. Altogether, these data suggest that an anti-vascular mechanism is also contributing to the anti-tumor activities of PM060184.

#3067

A novel tamoxifen analog, GA-11, as potential breast cancer therapeutic.

Gurleen Kaur,1 Mohinder P. Mahajan,1 Manoj K. Pandey,2 Parvesh Singh,3 Srinivasa R. Ramisetti,2 Arun K. Sharma2. 1 _School of Pharmaceutical Sciences, Apeejay Stya University, Gurgaon, India;_ 2 _Penn State University College of Medicine, Hershey, PA;_ 3 _University of Kwa-Zulu Natal (UKZN), Durban, South Africa_.

Tamoxifen is the most commonly used treatment for patients with ER+ breast cancer (BC). Although many patients benefit from tamoxifen in the adjuvant and metastatic settings, resistance is an important clinical problem. We hypothesized that tamoxifen structure can be modulated to generate a more effective and a better drug-like compound. Over the past three years, extensive structure-activity relationship (SAR) studies were carried out in our laboratories to optimize tamoxifen structure. We generated over 50 tamoxifen analogs and evaluated their activity against MCF-7 (ER+) and the triple negative MDA-MB-231 (ER-, PR-, and HER2 -) human breast cancer cell lines following MTT assay. Both ER+ and triple negative cell lines were used to evaluate if the novel analogs were selectively cytotoxic to the ER+ cells similar to tamoxifen. These studies identified a novel compound GA-11 that non-selectively targeted both the ER+ and triple negative cells, interestingly being more cytotoxic to MDA-MB-231 cells. GA-11 exhibited remarkable activity with IC50 values 7 times lower against MDA-MB-231 (triple negative) and 3 times lower against MCF-7 (ER+) than tamoxifen. GA-11 contains a methoxy group in place of N,N-dimethylethyl functionality of tamoxifen, and an additional polar amine group, which makes it more cytotoxic than tamoxifen to both the cancer cell lines (ER+ and triple negative) while being non-toxic to normal mouse embryonic fibroblast (MEF) cells, suggesting that the cytotoxic response of GA-11 is selective for cancer cells. In addition, GA-11 inhibited the expression of MMP-9, c-Myc and Caveolin, the proteins associated with adhesion, migration and metastasis, in a dose-dependent manner. The response of GA-11 in triple negative MDA-MB-231 cells was more dramatic as compared to MCF-7 (ER+) cells, again suggesting its relative selectivity towards triple negative cells and its potential to target triple-negative breast cancer for which currently no effective chemotherapy options exist. Furthermore, GA-11 inhibited the migration and invasion of MDA-MB-231 cells clearly demonstrating its anti-metastatic properties. In silico evaluation showed that GA-11 with cLogP 4.8 and MW 329, fits better into the requirements of Lipinski's Rule-of-Five, formulated to determine the drug-likeness of a small molecule; than tamoxifen (cLogP 7.34). GA-11 is thus expected to have a better oral bioavailability than tamoxifen. These in vitro studies thus indicate GA-11 to be an effective agent that is superior to tamoxifen both in potency and drug-likeness.

#3068

Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model.

Se Hyun Kim,1 Jung Min Kim,2 Tae Soo Kim,2 Won Suk Lee,2 Woo Sun Kwon,2 Seung Tak Lim,3 Joong Bae Ahn,4 Se-Kyu Kim,4 Yeong Woo Jo,5 Sun Young Rha4. 1 _Seoul National University Bundang Hospital, Seongnam, Republic of Korea;_ 2 _Yonsei Cancer Center, Song-Dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 3 _Yonsei Wonju University College of Medicine, Wonju, Republic of Korea;_ 4 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 5 _DAEHWA Pharmaceutical Co., Hoengseong, Republic of Korea_.

Purpose: Paclitaxel is an effective anti-neoplastic agent in many solid tumors including bladder cancer. With the development of new drug delivery strategies, novel formulations of Cremophor EL-free paclitaxel have been developed. Here, we investigated the in vitro and in vivo anti-tumor effect of a mucoadhesive oral formulation of paclitaxel (DHP107) in comparison with iv paclitaxel in a human bladder cancer model.

Methods: Four human bladder cancer cell lines (RT4, T24, J82, HT1376) and one murine bladder cancer cell line (MBT-2) were used in this study. In vitro antitumor activity and underlying mechanisms were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell-cycle analysis and apoptosis assay. In the in vivo study, RT4 cell line was implanted subcutaneously in the flank of nu/nu mice, and treated with iv paclitaxel and 2 doses of DHP107. The effects were assessed by the tumor growth, body weight and Ki-67 staining in tumor tissues.

Results: The IC50 values of DHP107 ranged from 4.15 to 34.72 μg/ml with MTT assay. In RT4 cell line, both drugs induced similar G2-M arrest and apoptosis. The xenograft experiments showed that both DHP107 (per os, 50 mg/kg, equivalent of paclitaxel 10 mg/kg) and iv paclitaxel (intraperitoneal, 10 mg/kg) attenuated the growth of RT4 tumors in mice, equivalently. The higher dose (per os, 100 mg/kg) of DHP107 resulted in increased efficacy compared with the lower dose (50 mg/kg) without weight loss in mice. However, significant weight loss was observed in iv paclitaxel (-20% at Day 11) treated group. We observed significantly decreased proliferation with Ki-67 staining in DHP107 (100 mg/kg) treated group than in DHP107 (50 mg/kg) or iv paclitaxel (10 mg/kg) treated groups.

Conclusion: Our findings provide the evidence that DHP107 showed comparable antitumor effect with iv paclitaxel and is a promising anti-tumor agent in bladder cancer. Clinical studies in patients with bladder cancer warrant further investigation.

#3069

Investigation of pharmacodynamic and predictive biomarkers to define response to proteasome inhibitor marizomib in glioma.

Daniela Bota,1 Annick Desjardins,2 Warren Mason,3 Kaijun Di,1 Ann P. MacLaren,4 Nancy Levin,4 Mohit Trikha4. 1 _University of California, Irvine, Irvine, CA;_ 2 _Duke University Medical Center, Durham, NC;_ 3 _Princess Margaret Hospital, Toronto, Ontario, Canada;_ 4 _Triphase Accelerator, San Diego, CA_.

Proteasome inhibitors (PIs) have been employed with clinical success in multiple myeloma, but have been much less effective in solid tumors, despite the central role of the proteasome in controlling cellular metabolism. Marizomib (MRZ) is a novel second generation proteasome inhibitor which binds irreversibly to and inhibits the enzymatic activity of all three subunits of the proteasome. The unique ability of MRZ among PIs to cross the blood-brain barrier, combined with its pan-proteasome activity, suggest that MRZ may have distinct therapeutic advantages over the approved PIs in the treatment of glioma. Preclinical studies with MRZ have demonstrated anti-tumor activity in intracranial glioma studies, and MRZ is currently being evaluated in a Phase I clinical trial in WHO Grade IV recurrent glioma in combination with bevacizumab (NCT02330562). The aim of this study was to identify pharmacodynamic and predictive biomarkers of response to marizomib in glioma patients.

Analysis of the pharmacodynamic profile of MRZ in packed whole blood from MRZ-treated glioma patients demonstrated >70% inhibition of the chymotrypsin-like (CT-L) activity as early as day 1 of cycle1 at 1 hr post-infusion, with 100% inhibition post-infusion in all patients by the end of cycle 1. Pre-infusion data demonstrate a prolonged effect, with >60% inhibition of CT-L persistent between day 15 of each cycle and day 1 of the next cycle. Trypsin-like (T-L) and caspase-like (C-L ) activities increased after the first 1-2 MRZ doses, presumably due to compensatory hyperactivation of these subunits triggered by CT-L inhibition, which was subsequently overcome by repeated MRZ infusion, resulting in 40-60% inhibition of T-L and 10-30% inhibition of C-L evident through cycle 5.

Analysis of proteasome enzymatic activity in archival glioma tumor tissue revealed that levels of all three proteasome activities are variable between high grade glioma samples, suggesting the potential for differential sensitivity to proteasome inhibition in glioma patients. Further, there is a linear correlation between CT-L activity (the rate limiting enzyme for proteasomal proteolysis) and C-L activity in these samples, suggesting that a PI such as MRZ with pan-proteasome specificity could potentially exhibit more activity in glioma compared to CT-L specific PIs. The data are currently being expanded to evaluate both proteasome enzymatic activity and subunit mRNA levels, to establish whether these endpoints might serve as a proteasome based biomarker.

In conclusion, this study demonstrates that packed whole blood may be suitable as a pharmacodynamic biomarker for proteasome inhibition. This biomarker strategy may be crucial to stratify MRZ responsive patients in glioma.

#3070

Potent and selective inhibition of CDK7 by novel covalent inhibitors.

Leena Khare Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Manoj Kumar Pothuganti, Shilpa S. Nayak, Nandish C, Chandranath Naik, Ravindra MV, Madhu B. Dabbeeru, Nagaraju A, Mahankali B, Thomas Antony, Chetan Pandit, Shekar Chelur, Girish Daginakatte, Susanta Samajdar, Murali Ramachandra. _Aurigene Discovery Technologies Ltd., Bangalore, India_.

Background: Phosphorylation of the RNA polymerase II (RNAPII) in C-terminal domain (CTD) by Cyclin-dependent kinase 7 (CDK7) is an important step in cellular transcription process. Hence pharmacological modulation of CDK7 kinase activity is considered as an interesting approach to treat cancers that critically dependent on transcription to maintain their oncogenic state.

Experimental procedures: Multiple series of novel covalent CDK7 inhibitors were identified by SBDD approach based on the binding mode of known CDK7 inhibitors to find early hits. Iterative medicinal chemistry efforts were performed to identify several lead compounds by optimizing the initial hits to achieve good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile.

Summary: Highly potent ATP competitive covalent inhibitors of CDK7 from two distinct chemical series were identified. They show time-dependent inhibition of CDK7 enzyme activity as a proof of covalent binding and exhibit potent anti-proliferative activity in cell lines derived from various tumor types. CDK7 modulation by these compounds was also confirmed by monitoring cellular pS5RNAPII levels. Representative compounds from each series showed very good selectivity profile in broad kinase (332) panel. Lead molecules were identified based on excellent drug-like properties (solubility, permeability and good oral bioavailability). Tolerability and efficacy studies in rodent xenograft models are ongoing with selected leads to test their impact on tumor growth inhibition and to determine therapeutic window by oral administration.

Conclusion: We have identified novel and selective CDK7 covalent inhibitors from two distinct chemical series with optimized drug-like properties including oral bioavailability. These compounds are being evaluated

for anti-tumor activity in mouse xenograft models.

#3071

Proteasome subunit composition contributes to unusually poor sensitivity of H727 cells to carfilzomib.

Min Jae Lee, Kyung Bo Kim. _University of Kentucky, Lexington, KY_.

Background: While proteasome inhibitors (PIs) have significantly improved the survival of multiple myeloma (MM) patients, significant subsets of MM patients do not respond to the PI- based therapy. Currently, mechanisms of de novo PI resistance in these patients remain completely unknown. In this study, we aim to identify previously unexplored mechanisms of de novo carfilzomib (Cfz) resistance using H727 cancer cells with poor sensitivity to carfilzomib.

Results: Our carfilzomib sensitivity investigation in a panel of 16 cancer cell lines revealed that H727 lung cancer cells are markedly resistant to Cfz compared to other cell lines. H727 cells displayed no detectable P-glycoprotein (P-gp) expression and subsequently were not sensitized by reversin 121 (a P-gp inhibitor) to Cfz, ruling out the involvement of P-gp in the resistance of H727 cells to Cfz. Interestingly, we found that H727 cells are killed by PYR-41 (ubiquitin activating enzyme E1 inhibitor) and WP-1130 (deubiquitinase inhibitor) as effectively as Cfz- sensitive H23 cells, suggesting that ubiquitin-proteasome pathway is essential in H727 cells as in H23 cells. In addition, knock-down of proteasome α7 subunit, a component required for the assembly of a proteasome α-ring and thereby a 20S proteasome complex, resulted in an almost complete cell death, indicating that the proteasome is indispensable for the survival of H727 cells. Intriguingly, despite their poor sensitivity to Cfz, H727 cells were highly sensitive to other structurally distinguished PIs such as bortezomib, epoxomicin, and UK-101. Finally, exposure of H727 cells to interferon-γ (IFN-γ), which has been widely used as a biochemical tool to switch cellular proteasome catalytic subunit composition to the one containing immunoproteasome subunits, sensitized H727 cells to Cfz (Changing IC50 values from 1,355 nM to 185 nM). On the contrary, IFN-γ pretreatment did not affect the sensitivity of H23 cells to Cfz.

Conclusion: We report that H727 cells have a poor sensitivity to carfilzomib (or de novo resistance to Cfz), and that previously reported resistance mechanism involving P-gp upregulation does not play a role. Given our data presented here, we conclude that de novo Cfz resistance of H727 cells may be attributed to distinguished proteasome subunit composition. We believe that our investigation using cancer cell line models may provide important insights into understanding the mechanisms of de novo PI resistance in clinic.

#3072

Combination of ibrutinib and corticosteroids in B-cell non-Hodgkin lymphomas (NHL).

Hsu-Ping Kuo, Sidney Hsieh, Karl J. Schweighofer, Leo WK Cheung, Mutiah Apatira, Mint Sirisawad, Shiquan Wu, Karl Eckert, Yu Liang, Jeff Hsu, Chun-Te Chen, Darrin Beaupre, Betty Y. Chang. _Pharmacyclics LLC, an AbbVie Company, Sunnyvale, CA_.

Introduction: The B-cell receptor (BCR) signaling pathway is a major driver in the pathogenesis of B-cell malignancies. A vast array of BCR-associated kinases have emerged as rational therapeutic targets, including Bruton's tyrosine kinase (BTK), which plays a pivotal role in BCR signaling. Ibrutinib is a first-in-class, oral, covalent BTK inhibitor approved in the US for patients with mantle cell lymphoma and chronic lymphocytic leukemia (CLL) who have received at least 1 prior therapy, CLL patients with 17p deletion, and patients with Waldenström's macroglobulinemia. While ibrutinib is efficacious as a single agent, combinations with other drugs may further enhance efficacy and increase response rates in patients with NHLs. Corticosteroids are included in nearly all combination regimens for NHL treatment, showing promising results when combined with chemotherapy and antibodies (Cunningham, Lancet 2013). We therefore evaluated corticosteroids in combination with ibrutinib in preclinical models of activated B cell-like (ABC) and germinal center B cell-like (GCB) diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma (FL).

Methods: ABC-DLBCL, GCB-DLBCL, and FL cell lines were used in this study. Drug effect on cell growth was evaluated with CellTiter-Glo luminescent cell viability assay (Promega) following treatment with ibrutinib or the combinations for 3 days. Combination index (CI) was determined using CalcuSyn. Synergy score (SS) was calculated using the Chalice Analyzer (Horizon CombinatoRx). Mutation profiles were extracted from the Catalogue of Somatic Mutations in Cancer database.

Results: Synergistic growth suppression of dexamethasone and ibrutinib was identified in 7 of 14 GCB-DLBCL cell lines. Dexamethasone had a stronger effect on cells with lower EC50 on ibrutinib treatment. Intriguingly, 4 of these 7 cell lines that showed synergy of the 2 compounds had BCL-2 nonsynonymous mutations, whereas only 1 of the cell lines with no combination effect had BCL-2 silent mutation. In addition to dexamethasone, other corticosteroids, including hydrocortisone, prednisolone, and mometasone, also showed synergy with ibrutinib in GCB-DLBCL (SU-DHL-4, CI=0.219-0.668, SS=5.08-15.15; SU-DHL-10, CI=0.008-0.224, SS=7.27-11.24), ABC-DLBCL (TMD-8, CI=0.001-0.016, SS=21.1), and FL cell lines (DoHH2, CI=0.020-0.467, SS=12.3-30.86; WSU-FSCCL, CI=0.017-0.022, SS=18.24). The in vivo effect and underlying mechanisms of the combinations are currently under investigation.

Conclusions: We identified synergistic inhibitory effects of ibrutinib and corticosteroids on ABC-DLBCL, GCB-DLBCL, and FL cell growth, providing a rationale for the design of combination clinical trials in these NHL types. Further understanding of the mechanisms contributing to the synergy may help to stratify patient populations and expand treatment to other lymphoid neoplasms.

#3073

Targeting chronic lymphocytic leukemia by interfering glutathione synthesis using a novel therapeutic enzyme cyst(e)inase (AEB3103).

Jinyun Liu,1 Li Feng,1 Everett M. Stone,2 Joseph Tyler,3 Scott W. Rowlinson,3 Michael J. Keating,1 Peng Huang1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _University of Texas at Austin, Austin, TX;_ 3 _Aeglea Bio Therapeutics, Austin, TX_.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western countries. Despite recent advance in new therapeutic agents that have improved treatment outcomes, CLL remains incurable due in part to the inability to completely eradicate the leukemia cells in vivo. Previous studies showed that CLL cells have high intrinsic oxidative stress and are highly dependent on cellular antioxidant glutathione (GSH) to maintain redox balance and cell viability. One logical strategy to impact CLL cells would be to abrogate the glutathione protection of CLL cells in vivo. Recently we discovered that primary leukemia cells isolated from CLL patients were unable to effectively utilize cystine for GSH synthesis due to low expression of the cystine transporter Xc-, and that bone marrow stromal cells highly express Xc- and effectively take up cystine for conversion to cysteine (Zhang et al: Nature Cell Biology, 2012). These findings provide a biochemical basis to develop novel strategies to effectively target leukemia cells in the stromal microenvironment and improve in vivo therapeutic activity. In this study, we tested the depletion of extracellular cystine and cysteine using a novel therapeutic enzyme-cyst(e)inase (AEB3103), as a potential way to block GSH synthesis in CLL cells and abolish the stromal protection of the leukemia cells. Our study showed that AEB3103 was very effective in depleting GSH in CLL cells and caused massive CLL cell death even in the presence of stromal cells. Importantly, AEB3103 could also overcome drug resistance of CLL cells with p53 deficiency both in primary leukemia cells isolated from CLL patients with 17p deletion and mouse leukemic cells isolated from mouse model we recently reported (Liu et al: Leukemia, 2014). In addition, AEB3103 showed very low toxicity to normal cells. These promising in vitro data warrant further animal studies, which are currently on ongoing using the CLL mouse model with TCL1-Tg:p53-/- genotype. Our study suggests that AEB3103 and its combination with standard anti-CLL drugs may potentially be useful for clinical treatment of CLL, even for the more aggressive CLL subtypes with unfavorable cytogenetic alterations such as those with chromosome17p deletion and p53 mutations, and may improve in vivo therapeutic activity.

#3074

Addition of ARN509 and docetaxel, alone or in combination, to castration demonstrates improved efficacy and heterogeneity of molecular response in prostate cancer patient-derived xenografts.

Ryan McMullin,1 Himanshu Gupta,2 Yashoda Rajpurohit,2 Holly Nguyen,3 Lisha Brown,3 Daniel Sondheim,3 Gormley Michael,2 Deborah Ricci,2 Shibu Thomas,2 Corey Eva3. 1 _LabConnect, Seattle, WA;_ 2 _Janssen R &D, Spring House, PA; _3 _University of Washington, Seattle, WA_.

Results from clinical and preclinical investigations suggest potential survival benefits from more aggressive early treatment of some patients. Our objective was to evaluate in vivo the efficacy of adding docetaxel (DOC) or ARN509, alone or in combination, to primary androgen ablation and to investigate the associated molecular response and resistance.

We used hormone-sensitive LuCaP patient-derived prostate cancer xenografts (PDXs) models 35, 77, and 147 that all express AR but differ significantly in responses to castration (CX) and exhibit diverse molecular characteristics. Tumors were grown in intact mice and when established, mice were randomized for CX alone or CX with DOC or ARN-509, or their combination. Tumor volume (TV), and serum PSA responses were monitored. Tumors were harvested 5 days post-treatment (d5) and at end of study (EOS) for analyses.

Our results show differential efficacy of the treatments, mimicking the heterogeneity of clinical response. LuCaP 77 progression was inhibited by CX and the additional treatments (TXs) further inhibited TV, with the most decreased TV and PSA detected in the combination group. When compared to CX, CX combined with ARN509 demonstrated the most significantly improved survival benefit (10w vs 16.5w median survival; HR=0.2, p<0.001), while CX combined with DOC resulted in a smaller survival benefit (10w vs 12.5w, median survival HR=0.5 p=0.036). The combination TX did not result in improved survival due to negative health side effects that resulted in early sacrifice. LuCaP 147 exhibited similar responses to CX as LuCaP 77 but only small additional inhibition of TV with the combination TXs and no significant survival improvements over CX alone. LuCaP 35 was the most responsive to CX and the additional treatments did not enhance the TV inhibition, decreases in serum PSA or survival.

Preliminary IHC analyses of the early response (d5 tumors) did not show significant decrease in AR or GR in any of the PDXs but decrease in PSA indicated the efficacy of androgen suppression. Decreased proliferation (Ki67) was detected in LuCaP 77 and LuCaP 147 after CX and more pronounced decreases in the combination vs CX groups in LuCaP 77 and ARN509 and the combination vs CX in LuCaP 147 with no difference detected in LuCaP 35. Analyses of early gene expression responses at d5 by qPCR revealed changes specifically associated with single and combination treatment with ARN509 in LuCaP 77 for both AR-regulated genes (FKBP5, PCNA, and AKR1C3) and proliferation-associated genes (UBE2C3 and NUSAP1). Further analyses of the responses and alteration of genes and proteins associated with AR signaling, proliferation and drug resistance at d5 and EOS are ongoing.

#3075

Nuclear export inhibitor selinexor (KPT-330) demonstrates anti-tumor efficacy alone and in combination with chemotherapy in multiple breast cancer models.

Natalia Paez Arango, Kurt Evans, Ming Zhao, Erkan Yuca, Stephen Scott, Charissa Kim, Aung Naing, Funda Meric-Bernstam. _University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: The nuclear exporter XPO1 (Exportin1 or CRM1), mediates the transport of over 200 proteins, including several tumor suppressors. For this reason, XPO1 is being pursued as a promising target for cancer therapy options. Selinexor (KPT-330), a selective inhibitor of nuclear export, is an oral agent that has been shown to inhibit XPO1 and is currently in phase 2 trials for hematologic and solid tumors. We sought to determine the antitumor effect of selinexor in breast cancer cells in vitro and in vivo

Methods: We studied the effects of selinexor in vitro using cell proliferation assays; the half maximal inhibitory concentration (IC50) was calculated using isobologram curves after 3 days of treatment. We also tested the effects in combination with chemotherapy and calculated the combination index by the method of Chou and Talalay. In vivo efficacy was tested in triple negative breast cancer (TNBC) patient derived xenografts (PDXs) with varying levels of paclitaxel sensitivity, as single agent and in combination therapy. T/C ratio was calculated using the formula: [(median tumor volume of treated group)/(median tumor volume of control group) x 100]

Results: Selinexor treatment inhibited growth of 12 breast cancer cell lines representing different subtypes (TNBC as well as ER+, HER+) with a median IC50 of 375nM (range 3-750nM). Selinexor significantly reduced tumor growth in vivo in 4 of 5 different TNBC PDX models (with varying status of TP53 and PIK3CA, and gene expression subtypes), with a median T/C score of 48% (range 34-59%) In Multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin and doxorubicin in vitro. In vivo, selinexor in combination with paclitaxel or eribulin had greater antitumor efficacy than either agent alone.

Conclusions: Collectively these findings strongly suggest that selinexor is a promising therapeutic option for breast cancer.

#3076

Characterization of an improved derivative of the Rac/PAK inhibitor EHop-016 in breast cancer.

Linette Castillo-Pichardo,1 Tessa Humphries-Bickley,2 Ingrid Forrestier-Roman,2 Luis Borrero-Garcia,2 Fabiola Pagan Melendez,2 Eliud Hernandez,2 Cornelis Vlaar,2 Luis A. Cubano,1 Surangani Dharmawardhane2. 1 _Universidad Central del Caribe, Bayamon, PR;_ 2 _University of Puerto Rico Med. Sciences Campus, San Juan, PR_.

The expression and activities of the Rho GTPases Rac and Cdc42, and their downstream effector P21-activated kinase (PAK), have been correlated with metastatic cancer. Our published studies with the Rac/PAK inhibitor EHop-016 demonstrate the validity of using Rac inhibitors as anti metastatic cancer therapeutics. However, the relatively high effective concentrations (Rac activity IC50 1μM, tumor inhibition at 25 mg/kg body weight (BW)), and the moderate bioavailability (~30%, t1/2 4.5h) of EHop-016 need improvement. Therefore, we developed EHop-016 derivatives and identified EHop-167 as a Rac inhibitor at nM concentrations. Unlike EHop-016, which does not substantially change breast cancer cell shape, but only reduces cell surface actin based invadopodia, EHop-167 induced a marked decrease in breast cancer cell polarity, cell surface extensions, and cell-extracellular matrix (ECM) attachments (focal adhesions). This phenotype of cell rounding and detachment in response to EHop-167 was demonstrated only by breast cancer cell lines that have undergone epithelial to mesenchymal transition, but not by epithelial breast cancer cells, or MCF-10A mammary epithelial cells. As assessed by pulldown assays and western blotting, Rac and PAK activities were reduced by 80-90% in response to 250 nM EHop-167, in the detached cells. As demonstrated by caspase assays, the cell rounding and detachment from the ECM ultimately resulted in anoikis (cell death due to loss of focal adhesions). Accordingly, Transwell assays of mesenchymal breast cancer cells following 250 nM Ehop-167 showed a ~90% reduction in cell migration in the detached breast cancer cells, and a ~60% inhibition in the attached cells. EHop-167 also reduced the mammosphere formation efficiency of metastatic cancer cells by 50%, indicating an inhibitory effect on cancer stem cells. To determine the in vivo efficacy of EHop-167, athymic nude mice, bearing mammary fatpad tumors of MDA-MB-435 metastatic cancer cells, were treated 3X a week with 0, 1, or 10 mg/kg BW EHop-167 for 50 days. Treatment with 1.0 mg/kg BW EHop-167 resulted in a 50% reduction in tumor growth, while 10.0 mg/kg BW EHop-167 induced an ~95% reduction in tumor growth, compared to controls. Additionally, these mice did not show gross signs of toxicity or significant weight loss. Since the parental compound EHop-016 has no anticancer effects at similar concentrations, we conclude that EHop-167 is an improved Rac/PAK inhibitor that holds promise as an anti metastatic breast cancer therapeutic.

#3077

**Potent inhibition of bromodomain-containing BET family with ABBV-075 induces robust antitumor efficacy in preclinical models of breast cancer and exhibits** in vitro **synergy with doxorubicin.**

Emily J. Faivre, Xiaoyu Lin, Denise M. Wilcox, Xiaoli Huang, Aparna Sarthy, Terry Magoc, Daniel H. Albert, Guowei Fang, Saul H. Rosenberg, Keith F. McDaniel, Warren M. Kati, Yu Shen. _Abbvie, Inc., North Chicago, IL_.

Breast cancer diagnosisis highly prevalent in the US, with an estimated 231,840 new cases occurring in 2015. Targeted therapies, such as tamoxifen or Trastuzumab (herceptin) are available to woman whose tumors are estrogen receptor (ER) positive or HER2 positive, respectively. However, women with triple negative breast cancer or relapsed metastatic disease have limited treatment options. ABBV-075 is a novel BET family bromodomain inhibitor currently under phase I clinical investigation for a wide spectrum of cancer indications. Here we show that ABBV-075 exhibits broad anti-proliferative activity across cell lines representing ER positive, HER2+, and triple negative breast cancer. Notably, ABBV-075 induced G1 arrest and growth inhibition of breast cancer cell lines regardless of ER or RB status. This result suggests that ABBV-075 may provide therapeutic benefit to a broader patient population relative to the recently approved cdk4/6 inhibitor, palbociclib. The G1 arrest mechanism of ABBV-075 was efficacious in breast cancer xenograft studies, where significant tumor growth inhibition was observed. Additionally, ABBV-075 exhibited synergy in ER positive cell lines with doxorubicin, a TOPO2 inhibitor and DNA intercalating agent prescribed to ER positive patients refractory to hormone therapies. Intriguingly, the mechanism by which ABBV-075 and doxorubicin interact was independent of the TOP2-trapping activity of doxorubicin and instead required DNA intercalation. Together, these results demonstrate the potential of ABBV-075 as a treatment option across the different subtypes of breast cancer and in combination with doxorubicin in patients with ER positive disease

#3078

GSK-3 inhibitor 9ING41 potentiates the antitumor effects of CPT-11 in human breast cancer.

Andrey Ugolkov,1 Irina Gaisina,2 Kevin White,3 Alan Kozikowski,4 Thomas O'Halloran,1 Andrew Mazar1. 1 _Northwestern University, Evanston, IL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _University of Chicago, Chicago, IL;_ 4 _University of Illinois in Chicago, Chicago, IL_.

Glycogen Synthase Kinase-3beta (GSK-3beta), a serine/threonine protein kinase, has been demonstrated to be potential therapeutic target in human breast cancer. Our objective was to evaluate novel GSK-3 inhibitors alone and in combination with chemotherapeutic drugs for the targeted therapy of breast cancer. Using a short-term treatment, we found that the GSK-3 inhibitor 9ING41 significantly potentiates the antitumor effects of CPT-11, cisplatin and 5-FU to inhibit breast cancer cell growth. We established hormone-refractory BC-1 and BC-2 patient-derived xenograft (PDX) tumor models from metastatic pleural effusions that were obtained from breast cancer patients. We considered BC-1 and BC-2 PDX tumors as chemoresistant, because metastatic disease progressed in both cases in despite of the fact that patients received many cycles of chemotherapy. Although we found that BC-1 and BC-2 PDX tumors express estrogen receptor (ER), both PDX tumors carried activating mutations in ESR1. These mutations confer ligand-independent activity of the ER in tamoxifen-resistant breast tumors. We found that BC-1 and BC-2 PDX tumors grew without estradiol supplementation in immunodeficient mice, which suggests a hormone-refractory nature of these tumors. We found that GSK-3 inhibitor 9ING41 potentiates the effects of chemotherapeutic drug CPT-11, and leads to the regression of BC-1 and BC-2 breast PDX tumors. Our results demonstrate inhibition of GSK-3 as a promising therapeutic approach to overcome breast cancer chemoresistance, and identify GSK-3 inhibitor 9ING41 as a drug candidate for the targeted therapy of human breast cancer.

#3079

Targeted therapy for EBV-associated B-cell neoplasms.

Siddhartha Ganuguly, Satyanarayana Alleboina, Sudhakiranmayi Kuravi, Joseph McGuirk, Ramesh Balusu. _University of Kansas Medical Center, Kansas City, KS_.

Epstein-Barr virus (EBV) is directly implicated in several B-cell lymphoid malignancies. EBV associated B-cell neoplasms occur in both organ and allogeneic hematopoietic stem cell transplantations due to immunosuppression in these patients to maintain the function of transplant. Treatment of these B-cell neoplasms is limited to reduction in immunosuppression and antineoplastic chemotherapy. EBV associated lymphomas are characterized by prominent activation of Nuclear Factor kappa (NF-kB) pathway and targeting this pathway establishes a rationale for therapeutic approach. The ubiquitin/proteasome signaling pathway plays an important role in regulation of NF-kB pathway. Bortezomib (BZ) is the first FDA approved proteasome inhibitor for treating both newly diagnosed and relapsed/refractory multiple myelomas, and mantle cell lymphomas. BZ acts through inhibition of the 26S proteasome, a large protease complex that degrade ubiquitinated proteins. BZ stabilizes various cellular proteins involved in cell cycle arrest and apoptosis including p21, p27, p53, and IkBα by inhibiting proteasome function. Stabilization of IkBα results in inhibition of NF-kB signaling pathway which promotes tumor cell survival, growth, and angiogenesis. The major limiting factor for long-term administration of BZ through intravenous or subcutaneous is risk of peripheral neuropathy. There is a need to develop bioavailable proteasome inhibitor with low toxicity profile to overcome this conundrum. Ixazomib is an investigational orally bioavailable proteasome inhibitor which inhibits the activity of the 20S catalytic core subunit of the proteasome and currently in clinical trials for patients with relapsed/refractory multiple myeloma. Here we report the first preclinical evaluation of oral proteasome inhibitor ixazomib growth inhibitory effects on EBV-infected B-lymphoblastoid cell lines Raji and Daudi. In these cell lines, treatment with Ixazomib significantly induced apoptosis dose dependently (10-50 nM), in association with poly (ADP-ribose) polymerase cleavage. Cell cycle analysis by flow cytometry showed that Ixazomib treatment induced G2/M arrest dose dependently with concomitant decreases in the percentage of cells in G0/G1 and S phases. Immunoblotting analysis revealed increase in p53 and p27 levels without significant change in the relative levels of p21. Mechanistically, ixazomib treatment resulted in accumulation of polyubiquitinated proteins including accumulation of phosphorylated IkBα as demonstrated by western blot analysis concomitant with significant reduction in NF-kB activity and subunit translocation to the nucleus. Altogether, our pre-clinical data indicate that ixazomib induces apoptosis in EBV-associated lymphoma cells by blocking NF-kB signaling cascade and support the rationale for testing ixazomib in patients with EBV-associated B-cell neoplasms.

#3080

OPB-51602: a novel STAT3 inhibitor that targets mitochondrial respiratory chain and triggers STAT3 dependent ROS production.

Jayshree L. Hirpara,1 Kumi Higuchi,2 Takeshi Tsunoda,3 Boon Cher Goh,1 Shazib Pervaiz4. 1 _Cancer Science Institute, National Univ. of Singapore, Singapore, Singapore;_ 2 _Fujii Memorial Research Institute; Otsuka Pharmaceutical Co., Ltd, Japan;_ 3 _Otsuka Pharmaceutical Co., Ltd, Japan;_ 4 _National Univ. of Singapore, Singapore, Singapore_.

Signal transducer and activator of Transcription (STAT) proteins are activated upon extracellular signals resulting in their phosphorylation and nuclear translocation. Once in the nucleus, STAT proteins, in specific STAT3 activates gene transcription of factors involved in a myriad of cellular processes, including proliferation and survival. By implication, increased expression and activity of STAT3 are associated with human cancers, which argue strongly for the development of STAT inhibitors as a promising chemotherapeutic strategy. We are working with a novel STAT3 inhibitor, OPB-51602, that not only block STAT3 phosphorylation but also inhibits growth and colony formation in a variety of human cancer cell lines. Of note, in addition to targeting STAT3, the compound inhibits mitochondrial respiratory chain and triggers a significant increase in mitochondrial superoxide (O2-) production (within 1 hour of exposure) in NSCLC cell line H1975. Using functional mutants of pY705 and pS727 STAT3, the growth inhibitory activity of the compound could be compromised, however, only S727 mutant rescued the mitochondrial effects. In addition, exposure of cells to OPB-51602 resulted in extracellular acidification (ECAR) together with a complete shutdown of mitochondrial ATP production. Interestingly, these effects could be rescued by 2-deoxyglucose. These data provide evidence that the pharmacological inhibition of STAT3 not only blocks cell proliferation but also induces mitochondrial redox catastrophe and bio-energetic crisis, which could have potential therapeutic implications against cancers that are rendered drug resistant due to the activation of surrogate STAT3 signaling axis.

#3081

Novel cucurbitacin analogue Cuc D exhibits potent anti-cancer activity in cervical cancer.

Mohammed Sikander,1 Bilal Bin Hafeez,1 Fathi T. Halaweish,2 Murali M. Yallapu,1 Meena Jaggi,1 Subhash C. Chauhan1. 1 _University of Tennessee Health Science Centre, Memphis, TN;_ 2 _South Dakota State University, Brookings, SD_.

Background: Cervical cancer is one of the leading cause of mortality among women in US. Naturally occurring dietary compounds have gained increasing attention for their anticancer effects. Cucurbitacins, tetracyclic triterpenoid compound, belong to a family of Cucurbitaceae have shown promising anti-cancer activity. Herein, we investigated the potential anti-cancer effects of a novel analogue of cucurbitacin D (Cuc D) against cervical cancer in in vitro and in a xenograft mouse model.

Methods: In our study, we used human cervical cancer cells (CaSki and SiHa). Cells were treated with Cuc D (0.05 to 1μM) for 48 and 72 hrs. MTS and colony formation assays were performed to investigate the effects of Cuc D on cell viability and proliferation. Western Blot analysis was performed to investigate the effects of Cuc D on cell proliferation and apoptotic markers. To determine the therapeutic effects of Cuc D, we used female athymic nude mice and injected CaSki cells (4 x 106) into the cervix to develop orthotopic xenograft tumors. Cuc D (1 mg/kg body weight) was administered through intratumoral injection four weeks post-tumor cell injection. Tumor volume in these mice were recorded bi-weekly.

Results: Cuc D inhibited cell viability of cervical cancer cells in a dose-dependent manner. IC50 of Cuc D was observed 400 nM and 250 nM in Caski and SiHa cells, respectively. Cuc D treatment effectively inhibited growth of cervical cancer cells which was determined by decreased cell proliferation and colony formation assays. Cuc D treatment induced apoptosis in cervical cancer cells as measured by enhanced Annexin V staining. Western blot result also illustrated cleavage in PARP protein in Cuc D treated cells which further confirms apoptosis induction. Cuc D treatment also inhibited PI3K and c-Myc protein levels and phosphorylation of STAT3 and Rb proteins. In addition, Cuc D treatment induced the cell cycle inhibitory proteins (p21 and p27) and PTEN and the expression of a tumor suppressor microRNA, miR-145, as determined by qRT-PCR. In an orthotopic tumor xenograft mouse model, Cuc D treatment effectively inhibited tumor growth as compared to vehicle control treated mice.

Conclusion: Taken together, our results demonstrate potent anti-cancer efficacy of Cuc D in cervical cancer cells via modulation of key onco/tumor suppressor proteins. Thus, Cuc D could be a useful therapeutic agent for cervical cancer treatment.

#3082

Biomarker-driven inhibition of MET and EGFR pathways in hepatocellular and cholangiocarcinoma models.

Annemilai Tijeras-Raballand,1 Maria Serova,1 Matthieu Martinet,1 Nathalie Colnot,2 Miguel Albuquerque,2 Valérie Paradis,2 Eric Raymond,3 Sandrine Faivre,3 Armand de Gramont4. 1 _AAREC Filia Research, Boulogne-Billancourt, France;_ 2 _Pathology Department, Beaujon University Hospital, France;_ 3 _Hôpitaux Universitaire Paris-Nord Val de Seine, HUPNVS, Paris, France;_ 4 _AAREC Filia Research and Drugs Evaluation Laboratory, Department of Oncology, CHUV, Lausanne, Switzerland_.

Introduction: In hepatocellular carcinoma (HCC) and cholangiocarcinoma (CK), high levels of c-MET and EGFR have been correlated with poor prognosis. Moreover, MET and EGFR has been involved in drug resistance. Our study was aimed at evaluating MET and EGFR inhibition on cell viability, invasion, MET and EGFR signaling as well as on sorafenib sensitivity.

Material and Methods: SU11274 and lapatinib are specific inhibitors of activated MET and EGFR/HER2 respectively. Antiproliferative effects were evaluated in 12 HCC and CK cell lines using MTT assay. Protein levels were assessed by Western blot. Cell motility was investigated by wound-healing and matrigel invasion assays. Ex vivo experiments testing SU11274 were performed on surgical specimens from HCC patients that were cut into 300µM-thick slices and grown in specific media for 48h. Tumor samples were analyzed by IF or IHC.

Results: Expression levels of MET and EGFR defined two subgroups of cells classified as high-MET/low-EGFR or low-MET/high-EGFR cell lines. These subgroups displayed differential sensitivity towards MET and EGFR inhibitors. High-MET/low-EGFR cells displayed high sensitivity to SU11274 (GI50=1,6-3,7µM) but not lapatinib (GI50>20µM), whereas low-MET/high-EGFR cells were more sensitive to lapatinib (GI50=4,8-10,2µM) than SU11274 (GI50>20µM). HGF-dependent invasion was more potently inhibited by SU11274 in high-MET/low EGFR than in low-MET/high-EGFR cells. Cells sensitivity to receptor inhibition could be explained by intrinsic cell responsiveness to MET and EGFR pathways activation. Increased responsiveness of high-MET/low-EGFR cells to HGF was translated into increased sensitivity to SU11274. Similarly, low-MET/high-EGFR cells were more responsive to EGFR and consequently more sensitive to lapatinib. Interestingly, SK-Hep1 cells resistant to sorafenib expressed increased level of MET and were subsequently more sensitive to MET inhibition. Ex vivo results will be detailed on the poster with c-MET, HE, Ki67, and caspase 3 expression.

Conclusion: MET and EGFR expression profiles defined two subgroups of HCC and CK cell lines with differential inhibition sensitivity towards MET and EGFR inhibitors. To maximize the inhibition potential of these molecules, our results suggest that high-MET/low EGFR might be the candidate tumor types to evaluate MET inhibitors, whereas low-MET/high-EGFR tumors may be preferentially tested for EGFR pathway inhibition. Moreover, these results suggest that MET could be an attractive target in sorafenib resistant HCC patients.

#3083

Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis.

Santanu Paul,1 Lisa Stehno-Bittel,2 Animesh Dhar1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _LIKARDA INC, University of Kansas Medical Center, Kansas City, KS_.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) or pancreatic cancer is one of the most lethal malignancies in humans. The survival rate for two years is very limited in most of cases (10%) after diagnosis of this disease. This is because current therapeutic regimens have limited effectiveness. Novel therapeutic targets are needed to treat this disease. GZ17-06.12 contains a nutraceutical for its anti-cancer properties, in several cancers. Crude extracts of the plant Arum palaestinum Boiss used in traditional Israeli and Palestinian medicine, have demonstrated cytostatic and cytotoxic activities in colon, prostrate and lung carcinoma. Therefore, we hypothesize that GZ17-06.12 will inhibit tumor progression and metastasis in PDAC.

Experimental Procedure: In this study, we have determined cell proliferation, pancosphere formation and apoptosis following treatment of different doses oGZ17-06.12 for 24-72 hours using MiaPaCa-2 and S2-007 human pancreatic cancer cells. Cell cycle distribution and apoptosis were measured using flow cytometic analysis. Orthotopic pancreatic cancer model in athymic mice was developed and GZ17-06.12 was given orally for 20 days to those mice. Proliferative markers, pEGFR/pAkt and apoptotic markers, Bax/Bcl-2, were monitored following treatment with GZ17-06.12 in both in vivo and in vitro models. Metastatic markers, MMP-2 and MMP-9 were measured in metastatic tissues in orthotopic models.

Results: GZ17-06.12 inhibited proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. Cell cycle analysis suggested that cells were arrested in S and G2 phase following treatment. In addition, GZ17-06.12 induced apoptosis in both in vitro and in vivo pancreatic cancer. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, GZ17-06.12 decreased the number and size of the pancospheres in S2-007 cells with concomitant inhibition of pancreatic cancer stem cell markers, DCLK1, Lgr5 and EpCam. The effect of GZ17-06.12 suppressed tumor growth and metastatic potential as indicative of MMP-2 and MMP-9 activity in primary and metastatic tumors.

Conclusions: GZ17-06.12 significantly reduces tumorigenesis and metastasis in both in vitro and in vivo pancreatic cancer models, inhibiting proliferation with concomitant increase of apoptosis.

#3084

The effects of NT-1044, a novel AMPK activator, on endometrial cancer cell proliferation, apoptosis, cell stress, and tumor growth.

Dario R. Roque,1 Weiya Z. Wysham,1 Chunxiao Zhou,1 Ken W. Batchelor,2 Wendy R. Brewster,1 Victoria L. Bae-Jump1. 1 _University of North Carolina, Chapel Hill, NC;_ 2 _NovaTarg Therapeutics, Research triangle Park, NC_.

Objectives: Obesity and diabetes are associated with increased risk and worse outcomes in endometrial cancer (EC). Anti-diabetic biguanide drugs such as metformin may have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Metformin requires organic cation transporters (OCTs) for entry into cells, and NT-1044 (NovaTarg Therapeutics) is an AMPK activator designed to have greater affinity for two of these transporters, OCT1 and 3. We sought to compare the effects of NT-1044 on cell proliferation in human EC cell lines and on tumor growth in an endometrioid EC mouse model.

Methods: Cell proliferation was assessed in two EC cell lines, ECC-1 and Ishikawa, by MTT assay after exposure to NT-1044. Apoptosis was analyzed by Annexin V-FITC assay. Cell cycle progression was evaluated by flow cytometry. Reactive oxygen species (ROS) were measured using a DCFH-DA assay. Western immunoblotting was performed to evaluate the effects of NT-1044 on the downstream targets of the AMPK/mTOR pathway and proteins related to cell cycle and cellular stress. For in vivo studies, we utilized the LKB1f/f/p53f/f mouse model of endometrioid endometrial cancer. Mice were treated with placebo or NT-1044 (200 mg/kg/day, oral gavage) following tumor onset for 4 weeks.

Results: NT-1044 significantly inhibited proliferation in a dose-dependent manner in both EC cell lines after 72 hours of exposure (IC50 218 uM for ECC-1; 87 uM for Ishikawa). Treatment with NT-1044 resulted in G1 cell cycle arrest, induced apoptosis and increased ROS production in both cell lines. NT-1044 increased phosphorylation of AMPK and decreased phosphorylation of S6, a key downstream target of the mTOR pathway. Expression of the cell cycle proteins CDK4, CDK6 and cyclin D1 decreased in a dose dependent-fashion while cellular stress protein expression was induced in both cell lines. As compared to placebo, NT-1044 inhibited endometrial tumor weight in the LKB1f/f/p53f/f mice by 56% (p=0.031).

Conclusions: NT-1044 suppressed EC cell growth through G1 cell cycle arrest, induction of apoptosis, cellular stress and inhibition of the AMPK/mTOR pathway. In addition, NT-1044 inhibited EC tumor growth in vivo. More work is needed to determine if this novel biguanide will be beneficial in the treatment of women with EC, a disease strongly impacted by obesity and diabetes.

#3085

The BET family bromodomain inhibitor ABBV-075 targets multiple pathogenesis factors in multiple myeloma and exhibits robust in vivo efficacies as a single agent and in combination with bortezomib.

Tamar Uziel, Xiaoyu Lin, Emily Faivre, Aparna Sarthy, Daniel H. Albert, Leiming Li, Denise Wilcox, Xiaoli Huang, Terry Magoc, Lloyd Lam, Steven W. Elmore, Keith McDaniel, Warren Kati, Yu Shen. _AbbVie, North Chicago, IL_.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins represent a potential new class of cancer therapeutic agents. ABBV-075 is a potent and selective BET family bromodomain inhibitor that was recently advanced to Phase 1 studies. Here we report the in vitro and in vivo characterization of ABBV-075 in preclinical models of multiple myeloma. ABBV-075 exhibited significant anti-proliferative activities and triggered robust apoptosis across many multiple myeloma cell lines, including cell lines that were engineered to become resistant to bortezomib. As reported for other BET inhibitors, ABBV-075 potently inhibited super-enhancer regulated potential cancer drivers such as Myc, IRF8 etc. In addition to directly impacting multiple myeloma cell growth and survival, ABBV-075 also diminished stroma cell derived IL-6 secretion and prevented HUVEC cell proliferation in vitro. Given the importance of angiogenesis and the survival factor IL-6 for multiple myeloma pathogenesis, ABBV-075 could potentially produce significant therapeutic benefits in myeloma by simultaneously targeting multiple critical pathogenesis mechanisms for this disease. ABBV-075 caused significant tumor growth delay in the OPM2 flank tumor model as a monotherapy. Combining ABBV-075 with Bortezomib provided further therapeutic benefit compared with the treatment of either of the two agents individually. Taken together, our results suggest that ABBV-075 could be a promising option to explore for the treatment of multiple myeloma.

#3086

Cyclopamine tartrate, an anti-cancer agent targeting Hedgehog signaling, strongly interferes with mitochondrial function and suppresses aerobic respiration.

Md Maksudul Alam,1 Sagar Sohoni,1 Sarada Preeta Kalainayakan,1 Massoud Garrossian,2 Li Zhang1. 1 _University of Texas at Dallas, Richardson, TX;_ 2 _Logan Natural Products, Plano, TX_.

The Hedgehog (Hh) pathway is a major regulator of many fundamental processes in mammalian embryonic development. These processes include stem cell maintenance, determination of cell fate, tissue polarity, cell survival, cell differentiation and proliferation. Although this pathway is usually silenced in adult tissues, constitutive activation of the Hh pathway can lead to tumorigenesis. This aberrant characteristic can be observed in different types of human cancer including basal cell carcinomas, medulloblastoma, lung cancer, ovarian cancer, gastrointestinal cancer, breast and prostate cancer. This pathway has been shown to regulate proliferation of cancer stem cells and increase tumor invasiveness. Targeted inhibition of Hh signaling pathway is shown to be effective in cancer treatment and prevention. Therefore, the discovery of Hh pathway inhibitors can pave the way of finding effective cancer treatments. The steroidal alkaloid cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. Cyclopamine tartrate (CycT) is an improved analogue of cyclopamine with increased water solubility and bioavailabilty . We performed numerous experiments to examine the effect of CycT in inhibiting Hh signaling on an array of non-small-cell lung cancer (NSCLC) lines. We discovered that CycT has a novel activity in disrupting mitochondrial function and aerobic respiration. Our data show that CycT causes a substantial decrease in aerobic respiration of NSCLC cells, similar to the effect of glutamine deprivation. Mitotracker staining showed that CycT causes mitochondrial fragmentation by increasing mitochondrial fission , which is indicated by the localization of DRP1 at fission sites. Further experiments showed that CycT increases ROS generation, which is presumably a consequence of mitochondrial hyperpolarization. Concomitantly, CycT causes apoptosis and suppress proliferation of NSCLC cells.

#3087

**Combination of ONC201 and bevacizumab significantly impacts colorectal cancer growth and metastasis** in vivo **.**

Jessica Wagner, Wafik S. El-Deiry. _Fox Chase Cancer Center: Temple College of Medicine, Philadelphia, PA_.

ONC201 is a first-in-class small molecule anti-tumor agent (Allen et al., 2013; Wagner et al., 2014; Prabhu et al., 2015; Kline et al., 2015) that upregulates endogenous TRAIL and activates the integrated stress response leading to increased DR5 expression, among other anti-tumor effects. ONC201 recently completed a phase I clinical trial in advanced solid tumors and is currently in multiple phase I/II clinical trials in select advanced cancers (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). Colorectal cancer (CRC) remains one of the leading causes of death from cancer worldwide; predominantly due to its metastatic nature. Increasing clinical evidence including randomized clinical trials has demonstrated that there is an increased efficacy from certain cytotoxic chemotherapeutic agents when placed in combination with molecular agents that target the vascular endothelial growth factor (VEGF) pathway. Bevacizumab (Avastin), a monoclonal antibody that targets VEGF, has become an important therapeutic option for patients with advanced metastatic CRC. The single agent activity of ONC201 in preclinical models of colorectal cancer have been well documented, however a combinatorial regimen for this investigational agent has not been prioritized for this indication. We investigated the efficacy of ONC201 in combination with bevacizumab in preclinical models of colorectal cancer based on the previously reported cooperative efficacy with this combination in preclinical GBM models. While each agent showed efficacy as a single agent, the combination of ONC201 with bevacizumab appeared synergistic in the suppression of CRC xenograft growth including a significant impact on tumor regression and occasional complete tumor regressions. The ONC201 combination with bevacizumab also suppressed metastasis from xenografted human colorectal cancer cells. Mechanistic studies and angiogenesis imaging studies are ongoing to demonstrate the importance of this combination on both tumor growth, migration, and invasion. Our results demonstrate that combining anti-angiogenic bevacizumab with ONC201 enhances anti-tumor efficacy in preclinical models of colorectal cancer and suggest that further testing of the combination is warranted in the clinic. 

## CANCER CHEMISTRY:

### Structural and Chemical Biology

#3088

Transcription factor as target: Novel small molecule inhibits FOXM1 DNA binding and oncogenic gene products.

Michael Gormally,1 Giovanni Marsico,2 Ganesha Rai,1 Christopher Lowe,2 Craig Thomas,1 David Maloney,1 Sam Michael,1 Dijana Matak-Vincovic,2 Ajit Jadhav,1 Anton Simeonov,1 Shankar Balasubramanian2. 1 _National Center for Advancing Translational Sciences (NCATS), Bethesda, MD;_ 2 _University of Cambridge, Cambridge, United Kingdom_.

Forkhead box M1 (FOXM1) is a transcription factor of considerable importance. Aberrant overabundance of FOXM1 through mutations in upstream regulators or gene amplification is now known to be a driving factor of most human cancers. Further, FOXM1 has prognostic value as expression correlates with severity of disease. Thus, chemical inhibition of FOXM1 has become a major goal. To address this need, we designed a novel in vitro assay to detect disruption of FOXM1 DNA binding. We executed a screen of 400,000 compounds from the NIH Molecular Library Small Molecule Repository, consisting of diverse drug-like molecules intended as starting points for medicinal chemistry lead development. After iterative and orthogonal counter screens, we ultimately identified the small molecule FDI-6 as a potent inhibitor of FOXM1. We characterized the perturbation in detail by biophysical analyses and confirmed that FDI-6 binds FOXM1 protein directly. The molecule was cytotoxic to multiple cancer cell lines (GI50 ≈ 10µM) and we demonstrated that the inhibitor displaces FOXM1 protein from promoters of target genes (AURKB, CCNB1, CDC25B) using an MCF-7 breast cancer model. To generalize the effect, we used chromatin immunoprecipitation and next generation sequencing (ChIPseq) to show that the inhibitor physically displaces FOXM1 from consensus binding motifs across the entire genome, reducing FOXM1 peaks by an average of over 60%. Transcriptome-wide expression profiling by RNAseq further confirmed that this displacement by FDI-6 selectively down-regulates the global FOXM1 transcriptional program, suppressing mitotic entry and cell-cycle progression. Importantly, we found that FDI-6 is specific for FOXM1 and has no effect on the expression of genes regulated by related forkhead family factors, which exhibit homology with the DNA binding domain of FOXM1. We are now evaluating the efficacy of this compound in allograft mouse models of FOXM1-driven breast cancer. Our study shows that the genomic interaction of this clinically important transcription factor can now be manipulated with small molecules to regulate the expression of key gene families. This improves our ability to probe transcription factor function, helps establish the oncogenic roles in different disease contexts and demonstrates clear potential for FOXM1 to be pursued as a clinical target in the future.

#3089

A novel G-quadruplex formed in the PDGFR-β promoter that is selectively targeted by a small molecule to repress transcription.

Buket Onel, Prashansa Agrawal, Megan Carver, Robert Brown, Laurence Hurley, Danzhou Yang. _University of Arizona, Tucson, AZ_.

Abnormal expression of PDGFR-β protein contributes to several malignancies. PDGFR-β has become increasingly attractive therapeutic target in the treatments of certain cancers. A G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter has been found to form multiple G-quadruplexes from the overlapping sequences. This G-quadruplex-forming NHE has been shown to regulate approximately 60% of basal promoter activity. Targeting transcriptional control of PDGFR-β provides an attractive target for developing inhibitors for the PDGFR-β signaling pathway, in addition to molecular targeting of the PDGFR-β protein or its cognate ligand. We have previously determined the most stable G-quadruplex formed in the PDGFR-β NHE. Interestingly, the 3′-end G-quadruplex formed in the PDGFR-β promoter NHE appears to be selectively targeted by an ellipticine analog GSA1129, which has been shown to repress PDGFR-β activity in cancer cell lines, and GSA1129 appears to shift the dynamic equilibrium in the full-length sequence to favor this structure. Therefore, characterization of the 3′-end G-quadruplex structure is important for understanding its function and for rational design of small molecules targeting this element. The 3′-end PDGFR-β G-quadruplex appears to adopt an unusual parallel G-quadruplex structure containing an imperfect GGGA tetrad at the 3′-end, as shown by DMS footprinting and CD spectroscopy. We further investigated the stability and structure of the 3′-end G-quadruplex and its interactions with GSA1129 by mutational analysis combined with NMR spectroscopy. The 3′-end G-quadruplex can be stabilized by an A-to-G mutation at position 18. However, the truncated wild-type and mutated 18-mer (Pu18) sequences appear to form predominantly dimer structure as shown by NMR. Using a 19-mer sequence Pu19 with the A-to-G mutation (Pu19A18G), a stable monomeric G-quadruplex can be formed in potassium solution, adopting a parallel-stranded structure as shown by NMR. In the wild-type 3'-end NHE sequences, the formation of a monomeric G-quadruplex in potassium can only be observed in a 20-mer Pu20 sequence with additional 3' G20; the assignment of Pu20 G-quadruplex using site-specific 15N-G-labeled DNA sequences indicated a novel parallel-stranded G-quadruplex structure using G20 instead of A18 in the 3'-end-tetrad. The Pu20 G-quadruplex appears to have high thermo-stability as shown by CD studies; however, its specific formation can only be achieved at low potassium salt. GSA1129 can bind Pu20 and increase the 3′-end G-quadruplex stability by nearly 20 degrees. This study highlighted the dynamic nature of the 3′-end PDGFR-β G-quadruplex and the importance of identifying the proper sequence for the biologically relevant G-quadruplex structure formation. Significantly, the dynamic nature of the 3′-end G-quadruplex may make it an attractive target for drug regulation.

#3090

Molecular structure of the major G-quadruplex formed in the PDGFR-β promoter nuclease hypersensitivity element and its binding with small molecules.

Clement Lin,1 Prashansa Agrawal,1 Yuwei Chen,1 Salil Kalarn,1 Nanjie Deng,2 Laurence Hurley,1 Danzhou Yang1. 1 _University of Arizona, Tucson, AZ;_ 2 _Temple University, Philadelphia, PA_.

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) is associated with multiple cancers, making PDGFR-β an attractive target for anticancer drugs. Few strategies other than molecular targeting of the PDGFR-β protein or its cognate ligand have been reported for developing inhibitors for the PDGFR-β signaling pathway. DNA G-quadruplexes (G4s) formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity, and stabilization of these G4s by small-molecule compounds could serve as a mechanism for cancer therapeutics. We have shown that the major G4 formed in the PDGFR-β promoter is from the central four G-runs, adopting an intramolecular parallel-stranded structure with a broken G-strand and contains three 1-nucleotide (nt) chain-reversal loops and one lateral loop. The novel folding of the PDGFR-β G4 highlights the inherent stability of the 1-nt loops in parallel-stranded G4 structures. Elucidating the structure of the major PDGFR-β promoter G4 is important for designing small-molecule drugs to specifically target this structure to inhibit gene transcription. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the potassium solution structure of this major G4 formed in the PDGFR-β promoter. Our structure showed a unique 3' capping structure involving three guanine nucleotides in the lateral loop. This novel capping structure is determined by the specific loop sequence as well as the broken-stranded PDGFR-β G4 folding pattern. The unique 3' capping structure could be specifically recognized by proteins and small molecule ligands. Unrestrained molecular dynamic calculations showed that this major PDGFR-β G4 and its 3' capping structure are stable in aqueous environment on the ns time-scale. We also investigated the binding of small-molecules to the PDGFR-β G4 using a combination of NMR, circular dichroism (CD), and fluorescence based methods to identify compounds that can selectively bind the PDGFR-β G4 over other classic parallel-stranded G-quadruplexes, such as the c-MYC promoter G4. Our study demonstrates the structural diversity in promoter G4s which may enable specific recognition and the modulation of gene expression by small-molecule drugs.

#3091

Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction.

Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest.

Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z' of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9).

We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors.

#3092

Physiological mimicry to characterize the average topology of the MYC and VEGF promoter G-quadruplexes.

Rhianna K. Morgan, Tracy A. Brooks. _University of Mississippi, University, MS_.

DNA G-quadruplexes (G4s) have become increasingly present in modern drug discovery efforts. The main focus centers on cancer therapeutic development, as G4s are more concentrated in oncogenic promoters than the rest of the genome. Two of the most well described G4s to date are within the MYC and the VEGF promoters; stabilization of either structure fosters transcriptional silencing. Downregulation of MYC decreases cell proliferation and alters metabolism; it is promising for a number of cancers, such lymphomas, leukemia, colorectal cancer and more. VEGF downregulation would modulate angiogenesis, which is of therapeutic benefit for almost all solid tumors. The physiological structure of both of these promoter G4s has been elucidated using plasmid DMS footprinting, which incorporates DNA supercoiling in addition to stabilizing cations or molecules. The current study examines a variety of anhydrous or crowding co-solvents for their ability to recapitulate the physiological structure in single-stranded ex vivo conditions. The co-solvents tested included glucose, acetonitrile, polyethylene glycol, dextran sulfate, sucrose, ficoll, and extracted nucleoplasm, ranging from 0-40%. Significant changes in structure, including loop directionality and number of competing isoforms, were examined by electronic circular dichroism. Electromobility shift assays differentiated inter- and intra-molecular structures, further distinguishing the distribution of isoforms. DMS footprinting with select molecular crowding and dehydrating agents was performed and compared to plasmid footprints, to validate physiological mimicry ex vivo. Ongoing studies examining the physiological conditions regulating G4 stability and function include moving from plasmid to in cell DMS footprinting in order to most completely identify the physiologic isoform, and to using optimized co-solvents to examine the effects of small molecules ex vivo and better predict active hits for in vitro evaluation. Overall the VEGF and MYC promoter G4 structures are promising therapeutic targets for anti-angiogenic and anti-proliferative therapy, respectively. This comprehensive understanding of physiological principles governing the above G4 formation will best inform future drug discovery efforts for these and other oncogenic promoters.

#3093

3D structural report of NRAS isoform 5.

Joseph Markowitz,1 Tapas K. Mal,2 Chunhua Yuan,2 Nicholas B. Courtney,2 Mitra Patel,2 Andrew R. Stiff,2 James Blachly,2 Christopher Walker,2 Ann-Kathrin Eisfeld,2 Albert de la Chapelle,2 William E. Carson2. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

The protein NRAS is part of the NRAS-BRAF-MEK-ERK signaling cascade. Recently, 5 NRAS isoforms were discovered by our group, and it was observed that isoform 5 overexpression in vitro resulted in a more aggressive cell phenotype. NRAS isoform 5 increases phosphorylation of downstream targets (AKT, MEK, and ERK). The structural report is unavailable for NRAS isoform 5. In addition, it was found in the current study that the NRAS isoform 5 does not have GTPase activity due to lack the canonical GTP binding region. Given the importance of this pathway in driving melanoma progression, we studied the structural details of this novel NRAS isoform 5 by two biophysical techniques, NMR and CD spectroscopy.

The circular dichroism spectra of NRAS isoform 5 that was C-acetylated and N-amidated was measured using 11%, 14%, 21%, 45%, 56% and 85% trifluoroethanol (TFE) in a phosphate buffer. The secondary structural elements were induced at ~14-15% TFE. There were no qualitative differences in the CD spectra between 56% and 85% TFE. Therefore 56% deuterated TFE was utilized in the NMR structural studies.

Two dimensional (2D) homonuclear and heteronuclear experiments were acquired at 25°C with Bruker Avance III HD 600 and 800 MHz NMR spectrometers (Campus Chemical Instrument Center NMR facility), each equipped with a triple resonance z axis gradient TXI cryoprobe for amino acid chemical shift assignments and structural determination of NRAS isoform 5. The NMR chemical shifts for the protons were assigned using traditional 2D TOCSY, COSY, and NOESY experiments. The 2D proton-carbon HSQC was utilized to facilitate the assignment of the alpha and side chain aliphatic protons. In the absence of TFE, there were no observable NOEs that could represent ordered secondary structures. Other than intra residue and short range sequential NOEs (i-j ≤ 2), only a weak medium range NOE was observed from the aromatic protons of Tyr 4 to the gamma proton of Val 7/8.

The secondary structure of NRAS isoform 5 was determined via the same NMR techniques in 56% TFE. The secondary structure was defined by 69 sequential NOEs and 39 medium range NOEs. NOEs characteristic of helical structure were observed in the NOESY spectra. For example, NOEs were measured from H(alpha, i) to H(beta, i+3) for residues Tyr 4, Lys 5, and Leu 6. The tertiary fold was determined by 10 key long-range NOEs. The flexible C terminal is brought in close proximity to the N-terminal helix by 4 NOEs between Val 8/9 and Val 14, and 5 NOEs between Tyr 4 and Try 20 to form a helix-turn-coil structure in presence of TFE. In addition, the proton chemical shifts for the aromatic proton chemical shifts for the tyrosine aromatic ring became distinct.

NRAS isoform 5 is highly flexible in aqueous solution, but forms a helix-turn-coil structure in the presence of trifluoroethanol as determined by NMR and CD spectroscopy. This data may be utilized as a starting point to understand the biophysical interactions of this novel NRAS isoform.

#3094

Identification of mimotopes of the epithelial mucin-1 (MUC1) in non-small cell lung cancer.

Yu Qiu,1 Wang Jianfei, Cao Ruoqiong, Zhang Yi, Sun Yangyang, Li Zhong2. 1 _Hebei University College of Life Sciences, Baoding, China;_ 2 _Western University of Health Sciences, Pomona, CA_.

Background

Mimotope analysis has been widely used in mapping epitopes for drug target, and also used in the development of new diagnostics, therapeutics and vaccines. The epithelial mucin-1 (MUC1) is a glycoprotein in human endometrium and has been found highly expressed in tumor tissues of NSCLC patients. Mimotopes of MUC1 has great potentials to be immunotherapy targets for NSCLC. In this study, we use phage display technology to screen a T7 NSCLC phage library to identify mimotope peptides of MUC1 proteins.

Method

A T7 NSCLC phage library was screened by 3-round of biopanning with anti-MUC1 monoclonal antibody acting as a molecular target. Forty positive clones were picked for ELISA confirmation using anti-MUC1 antibody. In addition, these clones were then amplified and coated with ELISA plates. Forty NSCLC patient and 40 control serum samples were used to test the ELISA plates. Statistical analysis was preformed, and the most significant clones were picked and PCR amplified for sequencing.

Result

Two peptide sequences, AAPDFRP and SAPDDRP, were identified and the competitive inhibitory rates of MUC1 were more than 50%. The intensity between MUC1 monoclonal antibody and the positive clones of these two sequences had positive correlation. In addition, in serum binding specificity detection, these two amino acid sequences showed higher tumor binding specificity with lung cancer patients serum than that of healthy human serum(P<0.05).

Conclusion

Peptides of AAPDFRP and SAPDDRP showed a high homology in the mimic epitopes of the epithelial mucin-1 (MUC1) glycoprotein in NSCLC. These may be potential immunotherapy targets which could also act as candidates of tumor vaccines

#3095

Fluorescent probe activation and hNQO1 activity in solid tumor mimics.

Bijeta Prasai, Robin L. McCarley. _Louisiana State University, Baton Rouge, LA_.

Fluorescence molecular imaging is an emerging field with potential to aid in optically guided surgery for cancer treatment. To overcome the drawbacks of always-on fluorescent probes, such as indocyanine green, activatable fluorescent probes are being developed that allow high signal-to-background imaging for better discrimination of cancerous cells from normal cells. The activatable fluorescent probes designed in our research group rely on the presence of two-electron reductase hNQO1 (human NAD(P)H:quinone oxidoreductase isoenzyme 1), which is highly upregulated in tumor cells.

Two-dimensional monolayer cell culture systems lack the features of real tissues, such as complex microenvironment and cellular heterogeneity. To overcome the limitations of two-dimensional (2-D) systems, we expanded our research to three-dimensional (3-D) cell culture systems. Our goal is to understand the dependence of probe activation on the availability and activity of hNQO1 in the heterogeneous regions of solid tumors. Hence, 3-D cell cultures were incorporated in our study using the traditional liquid overlay method for the HT-29 cancer cell line. Following the original approach for monolayer cell imaging, probe activation is observed in the outer proliferating cell regions of 3-D HT-29 colon cancer cell assemblies (multicellular spheroids, MCSs). We are utilizing the in vitro cultured MCSs as mimics of solid tumors to investigate the activity of hNQO1 in the avascular regions of solid tumors with the help of hNQO1 activatable fluorophores, such as previously reported QMeNN.

Paraffin-embedded sectioning is used to determine hNQO1 presence in the avascular, or necrotic regions, of these tumor mimics. Immunofluorescence data obtained from more than 20-day-old HT-29 MCSs exhibited a distribution of hNQO1 throughout the peripheral cellular region and in the fragmented cells and debris of the necrotic center of the tumor mimics. Preliminary results, from wide-field fluorescence microscopy of MCSs that were trypsinized for periods of time to yield released cells that were then incubated with the QMeNN probe, showed minimal probe activation in the inner regions of MCSs, implying diminished hNQO1 activity in the core region. These results counter increased hNQO1 activity, based on literature and our work, that has been observed in the later stages of the monolayer growth cycle of HT-29 cells. Currently, experiments are being performed to determine if hNQO1 expression and its activity are dependent on the age of MCSs, through the use of immunofluorescence, and UV/vis and fluorescence spectrometry methods, with the aid of fluorescent probes. hNQO1 is a potential tumor target, not just for contrast agents, but for bioactivatable prodrugs as well. Understanding the bioavailability of the hNQO1 biomarker in the avascular regions of solid tumors and their activity may answer some aspects of the reasons for chemoresistance.

#3096

Mapping lysine acetyltransferase-metabolite interactions in complex proteomes: a window into the metabolic regulation of epigenetic signaling.

Jordan L. Meier. _National Cancer Institute, Frederick, MD_.

Substantial evidence indicates that chromatin modifiers are sensitive to changes in the cellular concentration of metabolites and cofactors. These findings potentially connect metabolism and gene expression, and have major implications for the regulation of cell signaling in cancer. However, missing from many of these studies has been the direct identification of the specific chromatin modifiers that sense change in metabolic state. Their identification is critical to our basic understanding of how metabolism regulates epigenetically-driven phenotypes, and may inform novel strategies to combat pathologies characterized by aberrant metabolism, including cancer. Towards this goal, here we report a chemical proteomic strategy to globally map chromatin modifier-metabolite interactions in complex proteomes. Our initial efforts have focused on lysine acetyltransferases (KATs), a central class of chromatin modifiers responsible for histone acetylation. First, we validate an optimized chemical proteomic method and demonstrate its ability to globally profile members of the KAT enzyme superfamily at endogenous expression levels. Then we apply this approach to rapidly profile KAT-ligand interactions, and identify novel candidate metabolic regulators of protein acetylation, including acyl-CoAs cofactors and metabolites, as well as new metabolically-sensitive KAT enzymes. By defining the molecular interactions linking metabolism and gene expression, these studies have the potential to illuminate our understanding of biology and inspire new therapeutic strategies targeting acetylation in disease.

#3097

Structural and biophysical characterization of anti-apoptotic protein Bcl-2 and GTPase Rac1 interaction.

Shani Ajumal,1 Jia Kang,1 Thomas Joseph,2 Shazib Pervaiz,1 Kunchithapadam Swaminathan1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _Bioinformatics Institute, Singapore, Singapore_.

We have recently reported a novel mechanism linking the pro-oxidant activity of the anti-apoptotic protein Bcl-2 to its interaction with the small GTPase Rac1. In this current study, we investigate the nature of this interaction using activated (GTP loaded) Rac1 and Bcl-2 peptides (spanning the BH3 domain and its adjacent loop region) using coimmunoprecipitation, isothermal titration calorimetry (ITC), X-ray crystallography, docking/modeling, fluorescence spectroscopy, molecular dynamics and mutational studies. Our results will develop to translational relevance in the treatment of human cancers that are refractory to chemotherapy due to overexpression of Bcl-2 .

#3098

Epigenetic therapy to target neuroendocrine prostate cancer using precision medicine models.

Loredana Puca,1 Wouter R. Karthaus,2 Dong Gao,2 John Wongvipat,2 Andrea Sboner,1 Marcello Gaudiano,1 Chantal Pauli,3 Rema A. Rao,1 Juan Miguel Mosquera,3 Joanna Cyrta,3 Theresa Y. MacDonald,1 Giorgio Ga Inghirami,1 Yu Chen,2 Mark A. Rubin,3 Himisha Beltran3. 1 _Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY;_ 2 _Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Englander Institute for Precision Medicine, New York, NY_.

Background

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer that may either arise de novo or much more commonly after hormonal therapy for prostate adenocarcinoma. Patients diagnosed with NEPC are often treated with platinum chemotherapy able to produce only short duration responses underling the urgent need of identifying novel potential therapeutic targets for this lethal disease.

In the context of our Englander Institute for Precision Medicine we developed patient derived 3D NEPC tumor organoids and patient derived PDXs to test specific inhibitors on molecular targets identified by genomic analysis of native tumors. Emerging data from an integrative molecular analysis of metastatic tumors from a large cohort of castration resistant prostate cancer (CRPC) patients, including NEPC, points to a key role of the Polycomb gene EZH2 and the epigenome in the pathogenesis of NEPC.

Methods

Tumor organoids were developed according to protocols developed by our Englander Institute for Precision Medicine and other Institutes. Briefly the tissue biopsies (liver and bone biopsy) were washed, enzymatically digested and then seeded in a Matrigel (BD) droplet. Organoids were then characterized at both genomic (WES) and protein level (IHC) to confirm the expression of specific markers. Organoids were also subcutaneously injected in NSG mice to generate PDX for drug treatment in vivo.

Results

Based on the significant EZH2 overexpression in NEPC tumors by RNA-Seq and tissue microarray, we checked the expression of EZH2 and H3K273M, the readout of its activity, in NEPC organoids and we found out that both EZH2 and H3K273M were high expressed in NEPC organoids. Therefore we evaluated the effects of the EZH2 inhibitor, GSK343, in NEPC versus CRPC organoids and in the castration resistant line DU145 versus the NEPC cell line NCI-H660. We found out that GSK343 effectively inhibited H3K27me3 and resulted in a significant reduction of NEPC organoids and H660 viability while DU145 as well as CRPC organoids were insensitive to the drug. We extended our studies generating PDXs by subcutaneously injecting NEPC tumor organoids in NSG mouse. The tumor extracted from the PDXs showed a high proliferative phenotype with molecular features characteristic of NEPC as chromogranin A expression and no androgen receptor expression. NEPC PDXs were treated with the EZH2 inhibitor, GSK126, and we observed a significant reduction of tumor size along with the treatment suggesting that EZH2 is a potential therapeutic target for this highly aggressive disease.

Conclusions

In the Englander Institute for Precision Medicine we are generating NEPC patient tumor organoids and PDXs to unveil new targets to facilitate therapeutic decision at this late stage disease. Among the possible hits, EZH2 represents a promising drug target and a potential modulator of the NEPC phenotype.

#3099

KRAS **and clinical context: Differential dynamic signaling output of** KRAS **mutant lung, colorectal and pancreatic cancer cell lines when exposed to targeted anticancer drugs.**

Adam Stewart,1 Elizabeth Coker,1 Anna Minchom,2 Parames Thavasu,1 Alexandros Georgiou,2 Anguraj Sadanandam,1 Timothy A. Yap,2 Johann S. de Bono,2 Bissan Al-Lazikani,1 Udai Banerji2. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Institute of Cancer Research and The Royal Marsden, London, United Kingdom_.

Background

Clinical trials have shown that cancers originating from different tissues driven by the same oncogene respond differently to targeted anticancer drugs. We aimed to understand different signaling patterns in KRAS mutant cells derived from non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic cancer.

Materials and methods

We optimized a 50 phosphoprotein antibody-based assay on the Luminex 200 platform. We then exposed a panel of 15 KRAS mutant cell lines (5 cell lines each originating in the lung, pancreas and colon) to a DMSO control (n=3) and clinically significant concentrations (Cmax achieved in humans adjusted for protein binding in culture medium) of a PI3K (GDC-0941), AKT (AZD5363), m-TOR (everolimus), BRAF (vemurafenib), EGFR (gefitinib), MEK (trametinib) and an HSP90 inhibitor (luminespib) for 1 hr. We quantified the change in phosphorylation of proteins for each drug compared to control. Logistic regression analysis was used to analyse differences between KRAS-driven cell lines originating from different anatomical sites.

Results

There were changes in phosphorylation related to the pharmacodynamic effects of the drug independent of cell line of origin; however, there were interesting differences between KRAS mutant cells originating from different anatomical sites. In NSCLC cell lines, p-EGFR levels changed significantly less when exposed to PI3K, AKT and m-TOR inhibitors (p = 0.047, 0.022 and 0.047, respectively) when compared to cells originating from CRC and pancreatic cancer. CRC cell lines, when compared to NSCLC and pancreatic cancer cell lines, showed significantly less changes in phosphorylation of key cell cycle regulators such as CHK1 when exposed to PI3K, AKT and m-TOR inhibitors, (p = 0.001, 0.047 and 0.047, respectively) and RB when exposed to an AKT and m-TOR inhibitor (p = 0.047 and 0.047, respectively). Interestingly, pancreatic cell lines showed significantly more changes in p-m-TOR compared to CRC and NSCLC cell lines following exposure to PI3K and AKT inhibitors (p = 0.0095 and 0.022, respectively). Of note, drugs not directly targeting the PI3K pathway differentially regulated different nodes in the PI3K pathway, for example, BRAF inhibitors significantly differentially changed levels of phosphorylation at different nodes in the PI3K pathway such as AKT in NSCLC cell lines, p = 0.047, p70S6K in CRC cell lines, p = 0.0472 and PRAS40 in the pancreatic cancer cell lines, p = 0.022.

Conclusion

These results suggest that there are significant differences in signaling patterns caused by PI3K pathway inhibitors in KRAS mutant NSCLC, CRC and pancreatic cancer cell lines. Our findings shed light on the putative use of PI3K pathway inhibitors in KRAS mutant cancers. They also question the universal application of solely using genetic mutations to stratify patients in 'basket' clinical studies.

#3100

Hsp90 inhibition results in GR, AR and JAK protein degradation, decreased triple-negative breast cancer proliferation and increased paclitaxel sensitivity.

Abena S. Agyeman, Wesley J. Jun, Suzanne D. Conzen. _University of Chicago, Chicago, IL_.

Glucocorticoid receptor (GR) activity in triple-negative breast cancer (TNBC) is associated with cell survival and chemotherapy resistance. Targetable molecular drivers for TNBC have been difficult to identify and therefore standard treatment remains limited to chemotherapy. Recently, new-generation small molecule Hsp90 inhibitors (e.g. ganetespib and NVP-AUY922) have proven to be both potent inhibitors of Hsp90 client proteins and far less toxic than previous Hsp90 inhibitors. Moreover, favorable clinical responses have been observed in some TNBC patients enrolled in a phase II clinical trial of ganetespib monotherapy. A ganetespib and paclitaxel clinical trial is ongoing in breast cancer; however, it is not known which Hsp90 client proteins contribute to increased tumor chemotherapy sensitivity.

Recent studies in our lab found that inhibition of glucocorticoid receptor (GR) activity plays an important role in Hsp90 inhibitor-mediated sensitization of TNBC cells to paclitaxel-induced cytotoxicity. However, we also observed that following significant shRNA-mediated GR depletion, ganetespib still provided some sensitization of TNBC cells to paclitaxel. This suggests that inactivation of additional anti-apoptotic Hsp90 client proteins play a role in mediating ganetespib effects. Proteins closely associated with GR function that are also key Hsp90 inhibitor client proteins include the androgen receptor (AR, implicated in TNBC proliferation and apoptosis) and JAK, a component of JAK/STAT signaling pathway implicated in TNBC tumorigenesis and chemoresistance. Here we hypothesize that AR and JAK/STAT signaling have overlapping but distinct roles in Hsp90-mediated chemotherapy sensitization of TNBCs. Western blot analysis of TNBC xenografts treated with ganetespib +/- paclitaxel showed depletion of AR as well as JAK1 and 2. Ganetespib treatment also led to decreased levels of TMPRSS2 gene expression, an AR target gene. Additionally Western blot analysis showed decreased levels of activated phosphorylated STAT3 transcriptional factor, a downstream mediator of JAK, following ganetespib treatment of TNBC cell lines. These results suggest the involvement of AR and JAK in mediating ganetespib mechanism of action. We are currently beginning to determine how AR and JAK/STAT signaling pathways may interact with GR signaling by establishing CRISPR/Cas9 knockouts of GR, AR, and/or JAK in TNBC cells treated with ganetespib +/- paclitaxel. Understanding the relative roles of Hsp90 client proteins in TNBC is predicted to lead to a more rational selection of patients with TNBCs likely to benefit from Hsp90-inhibitor treatment.

#3101

The mutant IDH1 inhibitor prevents growth of glioblastoma with IDH1 mutation in patient-derived xenograft (PDX) model.

Yukino Machida,1 Yoko Ogawara,1 Koichi Ichimura,1 Hironori Matsunaga,2 Seki Takahiko,2 Kazushi Araki,2 Issay Kitabayashi1. 1 _National Cancer Cancer Research Institute, Tokyo, Japan;_ 2 _Daiichi Sankyo, Tokyo, Japan_.

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are expected to have minimum side effects as those of anti-cancer agents.

We established a mouse AML model harboring IDH mutations by co-transducing four mutant genes that frequently occur simultaneously in human AML patients. Conditional deletion of the IDH mutant blocked 2HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH mutations are critical for the development and maintenance of AML stem cells.

Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects on the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment.

To test the effect of the mutant IDH1 inhibitor on glioblastoma, we have established a patient-derived xenograft (PDX) model of glioblastoma carrying IDH1 mutation, and tested the effect of the mutant IDH1 inhibitor on this model. The inhibitor reduced 2HG levels in tumors as well as in plasma. The inhibitor prevented the growth of the tumors with IDH1 mutation. On the other hand, the inhibitor did not affect the growth of the glioblastoma with wild type IDH1. Gene expression analysis identified a set of genes, of which expressions is changed by the IDH1 inhibitor. The most strongly upregulated gene is Glial fibrillary acidic protein (GFAP), which is a differentiation marker for astrocytes. Up-regulation of GFAP is confirmed by immune-staining and quantitative RT-PCR analyses. These results suggest that IDH inhibitor induces differentiation of the glioblastoma with IDH1 mutation. We have found that the glioblastoma is transplantable in mouse brain. We are now looking at effects of the inhibitor on this model.

Taken together, the results indicate that IDH1 mutant inhibitors are effective for the treatment for AML as well as glioblastoma with IDH1 mutations.

#3102

Identify potential therapeutic drugs on head and neck cancer through systemic bioinformatics approach.

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

Head and neck cancer (HNC) is one of the ten most frequent cancers worldwide. In Taiwan, with the the rising incidence in the past decade, HNC has become the fourth male malignant disease since year 2009. The standard therapy methods of HNC are surgery, radiation, chemotherapy or the combination of these modalities. Overall 5-year survival rate in patients with HNC is one of the lowest among common malignant neoplasms treatment of this disease and has not significantly improved over the past decades. To improve the identification of potential therapeutic drug process, integrating the signature of diseases, genes, and drugs was analyzed through bioinformatics method to find therapeutic candidates through comparison of gene signatures between disease and drug treatment. Here, we integrated HNC gene signatures (including 202 oral squamous carcinomas and 69 normal control species) and connectivity map drug database, and after querying, the drug shown with negative correlation may serve as therapeutic candidates to reverse oral cancer signature. Four potential small molecules with antitumor properties including natural product extracts or clinical therapeutic drugs in other diseases were identified. One of the natural product extracts was found it inhibited cell migration ability, cell invasion capacity and cancer stem cell sphere formation capability. Furthermore, the compound was demonstrated that tit regulated the epithelial-mesenchymal transition markers, suppressed the expression of stemness-related transcription factors (SOX2, Oct-4, and NANOG), and increased the expression of cell differentiation marker (CK18). The compound can also increase cell sensitivity to chemodrug- and radiation treatment then then suppress tumor growth in vitro and in vivo. Taking together, our study demonstrates a novel approach in drug development, by a bioinformatics method to effectively identify potential compounds for head and neck cancer therapy.

#3103

The reduced immunogenicity anti-mesothelin immunotoxin RG7787 in combination with nab-paclitaxel has significant anti-tumor efficacy against primary mesothelioma cell lines and patient derived mesothelioma tumor xenografts.

Jingli Zhang, Qun Jiang, Christine Alewine, Swati Khanna, Betsy Morrow, Anish Thomas, Ira Pastan, Raffit Hassan. _National Cancer Institute, Bethesda, MD_.

Mesothelin is a tumor differentiation antigen that is highly expressed in many cancers including malignant mesothelioma. RG7787 is a second-generation anti-mesothelin immunotoxin that is designed to be less immunogenic in patients and has reduced non-specific toxicity in pre-clinical studies. In this study, we evaluated the anti-tumor efficacy of RG7787 alone and in combination with nab-paclitaxel using primary mesothelioma cell lines obtained from patients with pleural or peritoneal mesothelioma. Four early passage malignant mesothelioma cell lines: NCI-Meso16, NCI-Meso19, NCI-Meso21 and NCI-Meso29 were evaluated for mesothelin expression and have 18x103\- 56x103 mesothelin sites/cell. All four cell lines were sensitive to RG7787 with IC50s of 0.3 to 10 ng/ml. Three of the cell lines: NCI-Meso 16, NCI-Meso 21 and NCI-Meso 29 were also sensitive to nab-paclitaxel with IC50s of 4, 40 and 2 ng/ml respectively while, NCI-Meso 19 was resistant with IC50 >100 ng/ml. Synergistic in vitro cell cytotoxicity was observed with both NCI-Meso16 and NCI-Meso21 cells, when treated with the combination of RG7787 and nab-paclitaxel. To evaluate the efficacy of RG7787 with nab-paclitaxel in vivo, athymic nude mice were inoculated subcutaneously with 5x106 NCI-Meso 16 cells. When the tumors reached 100 mm3 in size, mice (n=7 in each group) were treated with 2 cycles (separated by 3 days) of either RG7787 2.5 mg/kg every other day x 3 doses, nab-paclitaxel 100 mg/kg on day 1, a combination of RG7787 and nab-paclitaxel at the same dose and schedule as for single agent therapy, or received no treatment (control). Although treatment with RG7787 and nab-paclitaxel alone resulted in tumor shrinkage compared to control mice, none of the mice had complete tumor regressions. However, in mice treated with RG7787 plus nab-paclitaxel all mice had complete tumor regressions by day 64 that persisted in 7/7 mice when the experiment was terminated 100 days after starting treatment. In the slow growing NCI-Meso 21 in-vivo model, we also observed similar synergistic anti-tumor effects with the combination of RG7787 and nab-paclitaxel. Taken together, our findings show that RG7787 in combination with nab-paclitaxel has significant anti-tumor activity against primary mesothelioma cell lines both in vitro and in vivo, suggesting that this combination treatment could be useful to treat patients with mesothelioma.

#3104

Stapled peptide inhibitors of the estrogen receptor/steroid receptor coactivator interaction.

Terry W. Moore,1 Thomas E. Speltz,1 Sean W. Fanning,2 Christopher G. Mayne,3 Jonna Frasor,4 Geoffrey L. Greene,2 Emad Tajkhorshid3. 1 _University of Illinois at Chicago College of Pharmacy, Chicago, IL;_ 2 _University of Chicago, Chicago, IL;_ 3 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 4 _University of Illinois at Chicago College of Medicine, Chicago, IL_.

Despite undeniable successes, one of the major unsolved problems in estrogen receptor-positive breast cancer therapy is resistance to endocrine therapy. One-third of breast cancer patients who are given tamoxifen will develop recurrent cancer within 15 years. Importantly, the estrogen receptor is still present and active in 80-85% of these recurrent cases, but it is no longer sensitive to current therapies, so new ways of targeting the estrogen receptor are needed. Because selective estrogen receptor modulators indirectly block estrogen receptor/coactivator interactions, our hypothesis is that directly blocking the estrogen receptor/coactivator interaction will provide a more robust blockade that will antagonize forms of estrogen receptor that are active in both wild-type and some endocrine-resistant breast cancers.

As our starting point for inhibiting estrogen receptor/coactivator interactions, we have chosen to use a class of compounds that mimic α-helices: "stapled peptides." These non-natural, synthetic peptides have a key alkene bond that resembles a staple. Typically, peptides make poor drugs because they are rapidly hydrolyzed by proteases and are poorly cell-permeable. Because of their non-natural linkage, stapled peptides are poor substrates for proteases, so they are metabolically stable. Some stapled peptides are also cell-penetrant. This effect is highly sequence-specific, but it seems to be aided when peptides have a relatively high formal positive charge. Stapled peptides have been used to inhibit a variety of important protein-protein interactions in cell culture and animal models. In this work, we have created a library of stapled peptides that inhibit the interaction of the estrogen receptor with the steroid receptor coactivator in vitro. The best peptides show nanomolar IC50 values in a time-resolved fluorescence resonance energy transfer assay. They show high helical content according to circular dichroism studies. We have solved x-ray crystal structures of these molecules bound to the estrogen receptor mutant Y537S, which demonstrate their binding mode, and these studies are in agreement with molecular dynamics simulations of these molecules bound to the estrogen receptor ligand binding domain.

#3105

Probing cytochrome P450 activity with benzofuran-based duocarmycins in a panel of head and neck cancer cell lines and CYP1A1, 1B1 and CYP2W1 transfected cell lines.

Daniela Presa,1 Paul M. Loadman,1 Giuliano Nigro,1 Helen M. Sheldrake,1 Mark Sutherland,1 Steven D. Shnyder,1 Valerie Le Morvan,2 Jacques Robert,2 Magnus Ingelman-Sundberg,3 Laurence H. Patterson,1 Klaus Pors1. 1 _Univ. of Bradford, Bradford, United Kingdom;_ 2 _University of Bordeaux, Bordeaux, France;_ 3 _Karolinska Institute, Stockholm, Sweden_.

Background:

The cytochrome P450 (CYP) isoforms CYP1A1, 1B1 and 2W1 are highly expressed in tumour tissue and surrounding stroma compared to nearby normal tissue, which

provides an opportunity for development of selective cancer therapeutics as previously reported by us.1-2 However, no reagent is available to interrogate CYP2W1 and hence the purpose of this study was to evaluate a series of novel benzofuran-based duocarmycins as chemical probes to study the functional activity of CYP2W1.

Materials and methods:

Standard materials and methods can be found in the references below. Briefly, these include route to synthesis of duocarmycin bioprecursors, use of recombinant CYP bactosomes with LC/MS for metabolite identification, and cell culture MTT proliferation assay. CYP expression was measured by RT-PCR and western blot.

Results:

A panel of head and neck cancer (HNC) cell lines (SCC4, FaDu, Detroit-562, A-253, OSC19, UT-SCC5, UT-SCC10, UT-SCC14) were analysed for the expression of CYP1A1, CYP1B1 and CYP2W1. Significant expression (5-10 fold) was shown at the gene level while protein expression experiments are currently underway and will be reported at the meeting. Furthermore, CYP2W1 functional activity was probed with a novel series of benzofuran-based duocarmycin analogues. The compounds were incubated with recombinant CYP bactosomes or HEK2932W1-transfected cells and their oxidative metabolism was studied using LCMS. CYP2W1 was shown to metabolise one chemical probe, ICT2726, to a unique metabolite not observed for any other CYP isoform. The compounds were investigated for antiproliferative activity in a panel of cell lines (CHO, CHO1A1, RT112, HEK293, HEK2932W1, Cal27, Cal271B1, Cal33, Cal331B1). Obtained IC50 values > 1 μM, which for these ultrapotent (picomolar) duocarmycins, indicated no CYP-bioactivation, consistent with no active duocarmycin metabolite identified from the LCMS studies.

Conclusion:

Our findings support the use of non-toxic ICT2726 as a chemical tool that can be employed in vitro to probe CYP2W1 functional activity.

[1] Sheldrake et al.

Re-engineering of the duocarmycin structural architecture enables bioprecursor

development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem.

2013, 56, 6273-7.

[2] Travica et al. Colon cancer-specific

cytochrome P450 2W1 converts duocarmycin analogues into potent tumor

cytotoxins. Clin Cancer Res. 2013, 19, 2952-61.

#3105A

Titanium(IV) regulation by serum transferrin and citrate sheds new insight into the use of chemical transferrin mimetics for Ti(IV) anticancer drug development.

Arthur D. Tinoco, Sergio A. Loza-Rosas, Alexandra Vazquez, Kennett I. Rivero, Lenny M. Negron, Manoj Saxena, Shweta Sharma, Yamixa Delgado, Annelis Sanchez, Nicole Zambrana, Timothy B. Parks. _University of Puerto Rico-Rio Piedras, San Juan, PR_.

A recently resolved structure of Ti(IV) bound human serum transferrin (sTf) and cell viability studies reveal that the small molecule citrate and the iron(III) transport protein sTf work together to maintain Ti(IV) in a nontoxic speciation in the body. Identification of this biomolecular interaction elucidates important factors that must be considered when developing Ti(IV)-based anticancer agents. Chemical transferrin mimetic (cTfm) ligands are being employed in a Ti(IV) anticancer drug design strategy to take advantage of the structural features of the sTf metal binding site favorable for Ti(IV) coordination to facilitate Ti(IV) cytotoxicity. The very strong Fe(III) binding property of these ligands are exploited to create stable Ti(IV) complexes that are able to deliver Ti(IV) into cells but exhibit lability in the presence of Fe(III). These complexes are intended to operate by the dual function of iron depletion and Ti(IV) attack at intracellular sites. Work with the cTfm ligands N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) and deferasirox reveal the structural features of the cTfm ligands that maximize the stability of the Ti(IV) complex outside the cell but enable sufficient lability to trigger cytotoxicity within the cell. The features are those that minimize induced dissociation due to the presence of citrate and sTf. Preliminary results suggest that while Fe(III) depletion may play an important role in the activity of the Ti(IV) cTfm complexes, the two lead complexes may operate via different mechanisms of action.

## CLINICAL RESEARCH:

### Biomarkers for Gastrointestinal, Hematologic, and Uncommon Cancers

#3106

A blood-based neuroendocrine tumor multi-transcript test predicts and defines PRRT efficacy in neuroendocrine tumors.

Lisa Bodei,1 Mark Kidd,2 Stefano Severi,3 Ignat Drozdov,4 Silvia Nicolini,3 Dik Kwekkeboom,5 Eric Krenning,5 Richard Baum,6 Giovanni Paganelli,3 Irvin Modlin7. 1 _European Institute of Oncology, Milan, Italy;_ 2 _Wren Laboratories, Branford, CT;_ 3 _Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) Srl - IRCCS, Meldola, Italy;_ 4 _Bering Limited, London, United Kingdom;_ 5 _Erasmus Medical Center, Rotterdam, Netherlands;_ 6 _Zentralklinik Bad Berka, Bad Berka, Germany;_ 7 _Yale University School of Medicine, New Haven, CT_.

Background: Peptide receptor radionuclide therapy (PRRT) is an effective treatment for neuroendocrine tumors (NETs). Somatostatin receptor expression defines suitability for therapy. Elevated somatostatin receptor uptake (Krenning scale grade 4) at baseline has ~60% predictive accuracy for efficacy. Increased (>600ng/ml) baseline levels of chromogranin A (CgA) are also considered as predictive. A blood-based 51 multigene NET transcript analysis (NETest) including gene clusters defining cell signaling and metabolism directly correlates with tumor activity. A multialgorithm-derived NETest scale 0-100% (low activity <40%) defines clinical disease activity.

Aim: Assess the effectiveness of the NETest as a predictive biomarker in PRRT-treated NETs.

Methods: 177Lu-based-PRRT treated NETs (n=54) were followed for 33 months. At baseline we evaluated: histological grade, somatostatin receptor imaging (SRI), CgA (ELISA, normal <108ng/ml) and NETest (qRT-PCR with multianalyte algorithmic analyses). A mathematical genomic response index comprising NETest genes regulating metabolism and growth factor signaling integrated with grade was developed as a predictive quotient. RECIST criteria were used to evaluate disease control (responder vs non-responder). Statistical analyses: multiple regression, Kaplan-Meier survival, Chi2 analyses.

Results: PRRT demonstrated a 72% response with median PFS not achieved (median follow-up 16 months). The only baseline clinical characteristic associated with outcome was low grade (G1/G2 [Ki67<20%] - coefficient 0.6±0.2, p<0.005). Although 77% (36/47) low grade and 50% (4/8) high-grade tumors responded, grade alone was not predictive (p=0.12). Neither baseline SRI measurements (p=0.58) nor CgA were predictive (p=0.53). A baseline NETest >40% predicted treatment response and a longer PFS (HR 2.97, p=0.05). NETest accurately (89%, χ2=27.4; p=1.2×10−7) correlated with RECIST-determined treatment response. The baseline NETest decreased in 88% of responders; and increased in 90% of non-responders. Baseline gene expression for metabolism and growth factor signaling had 76% accuracy for predicting PRRT-response. The Predictive Quotient Index (NETest and grade) accurately predicted responders (97%) and non-responders (91%). This offered a significantly better prediction than elevated SRI uptake (94% vs. 38% accuracy: Chi2=31.8, p<0.0001).

Conclusions: The blood-based NETest provided a predictive multi-molecular biomarker for PRRT. Alterations in levels correlate with RECIST responses and assess real time treatment efficacy. A predictive quotient index (NETest and grading) is highly accurate (94%) in predicting efficacy and significantly outperforms (p<0.0001) SRI assessment. NET multigene measurement in blood can be used to predict patients responsive to PRRT.

#3107

A single ddPCR assay to detect KIT exon 11 mutations in tumor and cell free plasma DNA of patients with gastrointestinal stromal tumors.

Pieter A. Boonstra,1 Marco Tibbesma,2 Lisette J. Bosman,2 Ron H.J. Mathijssen,3 Frits van Coevorden,4 Neeltje Steeghs,5 Albert J.H. Suurmeijer,2 Arja ter Elst,2 Jourik A. Gietema,1 Anna K.L. Reyners,1 Ed M.D. Schuuring,2 Dutch GIST Consortium. 1 _Dept. of Medical Oncology, University Medical Center Groningen, Groningen, Netherlands;_ 2 _Dept. of Pathology, University Medical Center Groningen, Groningen, Netherlands;_ 3 _Dept. of Medical Oncology, Erasmus Medical Center Cancer Institute, Rotterdam, Netherlands;_ 4 _Dept. of Surgery, Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital, Amsterdam, Netherlands;_ 5 _Dept. of Medical Oncology, Netherlands Cancer Institute Antoni van Leeuwenhoek Hospital, Amsterdam, Netherlands_.

.

Introduction

Gastrointestinal stromal tumors (GIST) are rare mesenchymal tumors of the gastrointestinal tract. These tumors are characterized by genomic mutations in the c-KIT gene that drive tumor growth and are target for specific inhibitors. Mutations in exon 11 (single nucleotide variations and deletions) are detected in approximately 70% of GIST. Although unique for each patient, most variations cluster in two different mutational hotspots each 25bp in size.

The aim of this study was to develop and test a single quantitative digital droplet PCR (ddPCR) assay to detect exon 11 c-KIT mutations in both formalin fixed paraffin embedded (FFPE) tissue and serial collected cell-free plasma DNA (cfDNA) of GIST patients for diagnostic and treatment response monitoring.

Materials and methods

The drop-off ddPCR assay for detection of exon 11 mutations consists of two fluorescent (FAM and HEX) labeled probes that each cover one of the 2 mutation hotspots. Since double exon 11 mutations are very rare in GIST, one probe will anneal to the non-mutated sequence (reference probe), while the other probe will not anneal. A decrease of one of the 2 fluorescent signals is considered as the presence of a mutation ("drop-off"). For validation, ddPCR results from tumor biopsies were compared with data from next generation sequencing (NGS) using the IonTorrent platform. Pretreatment FFPE tumor material was obtained from 29 (18 exon 11 mutated, 11 non-exon 11 mutated) GIST patients treated in 2014 and 2015. Plasma samples were collected at baseline (before start of treatment with imatinib) and at consecutive time points during treatment.

Results

Using the drop-off ddPCR assay on the same DNA used for NGS, mutations in 17 of the 18 samples were detected. Comparison of the allelic fraction of the mutation shows that ddPCR analysis is very similar to NGS. In 11 control samples, who were wild-type (4 samples) or had mutations other than c-KIT exon 11 (7 samples) no loss of signal was detected.

CfDNA was analyzed of a 72-year old representative patient with metastatic GIST carrying a c-KIT deletion in exon 11 with a fractional abundance (FA) in tissue of 91%. We detected the mutation using the drop-off ddPCR assay in the plasma sample collected before start of imatinib treatment (FA 16%).

Two weeks after initiation of imatinib a rise in mutant allelic frequency was observed (FA 61%) and a decrease after 4 weeks (FA 3%) of therapy.

Conclusion

The detection of multiple exon 11 mutations/deletions using a single ddPCR assay could be used as a rapid prescreening method for the detection of c-KIT mutations in FFPE tissue in GIST patients. Because of the quantitative nature of this assay, ddPCR analysis might be used to monitor response to treatment. The relevance of this drop-off assay for treatment decision making seems promising and is currently validated on a large series of cfDNA samples and a prospective study in GIST patients (NCT02331914).

#3108

PD-L1 expressing circulating tumor cells (CTCs) in patients with head and neck squamous cell carcinoma (HNSCC).

Areti Strati,1 George Koutsodontis,2 Ilias Angelidis,1 Clarence Sasaki,3 Margaritis Avgeris,4 Amanda Psyrri,2 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells, Dept of Chemistry, Univ. of Athens, Athens, Greece;_ 2 _Oncology Unit, 2nd Department of Internal Medicine - Propaedeutic, Attikon University Hospital, Haidari, Greece, Athens, Greece;_ 3 _Department of Surgery, Section of Otolaryngology, Yale University School of Medicine, New Haven, CT;_ 4 _Dept of Biology, Univ. of Athens, Athens, Greece_.

Background: Predictive biomarkers for response to anti-PD1 therapy are lacking. Because therapy with checkpoint inhibitors is cost intensive, noninvasive tools for prediction of responders are of major interest.

Methods: The "Liquid Biopsy''in head and neck squamous cell carcinoma (HNSCC) project involved the isolation of Circulating Tumor Cells (CTC) from patients with HNSCC at baseline, at different time points during treatment and at relapse. Herein, we assessed gene expression of PD-L1 on CTCs in a prospective fashion in a cohort of locally advanced inoperable HNSCC patients treated with curative intent (n=61), in a second cohort of recurrent/metastatic HNSCC patients (n=18) and in 20 healthy individuals, used as normal controls. For the quantification of PD-L1mRNA in CTCs, we developed a highly sensitive, specific, and robust RT-qPCR assay that was firstly analytically validated prior to its application in HNSCC patients.

Results: In patients with locally advanced disease, 54/61(88.5%) samples were evaluable for CTCs at baseline. Twenty four samples were obtained after induction chemotherapy (IC) and 34(55.7%) at the end of concurrent chemo-radiation. At baseline 22/54(40.7%) pts were found to be positive for PD-L1 overexpression, at the post-IC samples 12/24(50%) patients were positive for PD-L1overexpressionand at the end of treatment, 11/34 (32.4%) patients were positive for PD-L1 overexpression. Patients with PD-L1 positive CTCs at the end of treatment had shorter progression-free survival (PFS) (p=0.011) and overall survival (OS) (p=0.004). Multivariate analysis showed that PD-L1 overexpression in patients at the end of treatment was independent prognostic factor of PFS (HR=2422.4; p=0.014) and OS (HR=32.23; p=0.014). Five R/M patients were found to be positive for PD-L1 out of the 18 (27.8%) at baseline.

Conclusions: We demonstrate for the first time that detection of PD-L1+ CTCs at the end of treatment in patients with locally advanced disease is associated with shorter PFS and OS. Serial PD-L1 expression assessment has potential to select and monitor pts for PD-1 checkpoint inhibitors. These data support testing of PD-1 inhibitors in the adjuvant setting in patients with locally advanced HNSCC in whom PD-L1 positive CTCs are detected at the end of treatment.

#3109

The TERT variant rs2736100_AC is associated with reduced risk of upper track urothelial carcinomas.

Xiaotian Yuan,1 Ping Li,2 Magnus Björkholm,1 Dawei Xu1. 1 _Karolinska Institutet, Solna, Sweden;_ 2 _Nursing School of Shandong University, Jinan, China_.

Upper tract urothelial carcinomas (UTUCs) are originated from urothelium, and consist predominantly of renal pelvic carcinomas (RPCs) and ureter carcinomas (UTs). The UTUC incidence has increased over the past two decades, and most UTUCs have become invasive when diagnosed. Therefore, it is urgently needed to identify high-risk populations for the preventive intervention and monitoring. Recent genome-wide association study has provided strong evidence that common single nucleotide polymorphisms (SNPs) are associated with susceptibility of various malignancies. However, little is known about the susceptibility loci to UTUC. Here we genotyped the telomerase reverse transcriptase (TERT) SNPs rs2736100 and rs2736098, two SNPs associated with a risk of other malignancies, in Han Chinese patients with UTUC (n = 217) and evaluated the relationship between the rs2736100 polymorphism and UTUC risk by comparing with that in 289 healthy controls. Genotyping was performed using a TaqMan® SNP Genotyping Assays kit. Neither AA nor CC genotypes differed significantly between cases and control subjects, while the AC carriers were associated with the reduced risk of UTUC (OR = 0.6043; 95% CI: 0.4039 - 0.9042; P = 0.0185). When RPCs (n = 100) and UTs (n = 117) were analyzed separately, such protective effect remained significant in UTs (OR = 0.5728; 95% CI: 0.3540 - 0.9269; P = 0.031). The AC genotype was lower in RPC patients than in controls, but the difference was not statistically significant (OR = 0.6484; 95% CI: 0.3811 - 1.103; P = 0.142). In conclusion, we show for the first time that the rs2736100 AC genotype is protective against UTUC development.

#3110

MASH1 expression in high grade astrocytoma may demonstrate a potential model for prognosis that may lead to a molecular target for future therapy.

David E. Tacha, Jillian Tyrrell, Margaret Lobo. _Biocare Medical, Concord, CA_.

Introduction:

A large U.S. study showed that 25% of patients diagnosed with grade 3 astrocytoma survived for 5 years or more after diagnosis. Unfortunately, the outlook is even worse for patients diagnosed with GBM, as most patients live for less than a year. Achaete-scute complex homolog 1 (ASCL1, MASH1) is a proneural transcription factor that has an essential role in the formation of multiple neuronal lineages. Recent studies have shown that the upregulation of MASH1 and inhibition of the Notch signaling pathway characterize progressive astrocytoma. Proneural proteins have been shown to regulate stem-cell proliferation and differentiation. The convergence with oncogenic pathways combined with the overexpression in glioblastoma has been shown to correlate with poor clinical outcome and has identified these transcription factors as promising therapeutic targets. Recently, a MASH1 mouse monoclonal antibody has been developed. Most studies have shown MASH1 to be a specific marker of neuroendocrine carcinomas; however, only one immunohistochemical study on astrocytoma using a polyclonal antibody has been reported. This study examined MASH1 expression in grade 1-4 astrocytoma cases.

Materials and Methods:

Formalin-fixed paraffin-embedded tissue microarrays consisting of samples of normal brain and astrocytomas [grade 1 (n=22), grade 2 (n=34), grade 3 (n=16) and glioblastoma (n=35)] were evaluated by immunohistochemical analysis using a monoclonal MASH1 antibody. A value of >1% staining was scored as positive.

Results:

MASH1 stained 61.7% (66/107) of astrocytomas (grades 1-4). In grade 1 astrocytoma, MASH1 was expressed in 31.8% versus 85.3% in grade 2, 68.7% in grade 3 and 54.3% in grade 4 (p ~ 0.0028).

Conclusion:

MASH1 is highly expressed in astrocytoma. There was a variable and progressive increase of MASH1 expression in grade 1 versus grade 2-4 astrocytoma; and thus, may demonstrate a potential model for prognosis that may lead to future target therapeutic strategies.

#3111

Elevated expression of hyaluronan synthase 2 associates with poor prognosis in diffusely infiltrating astrocytomas.

Mari J. Poukka,1 Hannu Haapasalo,2 Kirsi J. Rilla,1 Kristiina Tyynelä-Korhonen,3 Ylermi Soini,4 Sanna M. Pasonen-Seppänen1. 1 _University of Eastern Finland, Kuopio, Finland;_ 2 _Department of Pathology, FimLab Laboratories, University of Tampere, Tampere, Finland;_ 3 _Cancer Center, Kuopio University Hospital, Kuopio, Finland;_ 4 _Institute of Clinical Medicine/ Clinical Pathology, University of Eastern Finland, Kuopio, Finland_.

Diffusely infiltrating astrocytomas are central nervous system tumors originating from astrocytic glial cells or their precursor cells. In this retrospective study we investigated the content of hyaluronan and the expression of hyaluronan metabolizing enzymes and its cell surface receptor, CD44, in these aggressive tumors. Hyaluronan is a large extracellular matrix molecule synthesized by three hyaluronan synthases (HAS1-3) on the plasma membrane and degraded by two hyaluronidases (HYAL1-2). In many tumors, aberrant hyaluronan metabolism implicates aggressive disease progression and metastatic potential.

Our material consisted of 165 diffusely infiltrating astrocytomas (WHO grades II-IV). Tumor samples were processed into tissue microarray (TMA) blocks. The TMA sections were stained for hyaluronan, CD44, HAS1, HAS2, HAS3 and HYAL2. The staining intensity of astrocytomas was evaluated with a four-level scoring (0-3; no color, weak, moderate and strong) by two independent observers. The immunostaining results were compared with χ2 -test or with Kruskal-Wallis test for correlation with clinicopathological parameters and survival analyses were done with Kaplan-Meier log rank test and Cox regression.

Hyaluronan and CD44 were strongly expressed in astrocytic gliomas but their expression did not correlate with WHO grade, cell proliferation activity by Ki-67, p53 status, epidermal growth factor receptor (EGFR) amplification or IDH1 mutation. Whereas HAS2 staining intensity showed a significant correlation with IDH1 mutation. Tumors with high HAS2 expression were IDH1 negative (p = 0.003). In addition, in non-parametric tests increased HAS2 staining intensity showed association with increased cell proliferation (p = 0.013) and in log rank test with decreased overall survival of patients (p = 0.001). Variables included in the multivariable Cox regression analysis were HAS2 staining intensity, p53 status, EGFR amplification, IDH1 mutation and WHO tumor grade; in this analysis HAS2 expression turned out to be a significant independent prognostic factor (p =0.008). This study indicates that elevated expression of HAS2 is associated with glioma progression and suggests that HAS2 has a prognostic significance in diffusely infiltrating astrocytomas.

#3112

HMGB1 and its isoform are sensitive and specific biomarkers to detect asbestos exposure and to identify mesothelioma patients.

Andrea Napolitano,1 Daniel J. Antoine,2 Laura Pellegrini,1 Francine Baumann,1 Ian Pagano,1 Sandra Pastorino,1 Chandra M. Goparaju,3 Kirill Prokrym,3 Claudia Canino,3 Harvey I. Pass,3 Michele Carbone,1 Haining Yang1. 1 _University of Hawaii Cancer Center, Honolulu, HI;_ 2 _MRC Centre for Drug Safety Science, Liverpool, United Kingdom;_ 3 _New York University, New York, NY_.

Millions of people have been potentially exposed to asbestos, the primary cause of malignant mesothelioma (MM). Presently, no reliable biomarkers are available to identify among potentially exposed people, those individuals who have actually been exposed and who are at high risk of MM. High Mobility Group Box Protein-1 (HMGB1) is a key mediator of asbestos-induced inflammation and MM pathogenesis. Recently, HMGB1 hyper-acetylation has been functionally associated to its active release by inflammatory cells. Here, we compared the serum levels of total and hyper-acetylated HMGB1 in individuals professionally exposed to asbestos, MM patients and healthy unexposed controls. HMGB1 serum levels reliably distinguished asbestos-exposed individuals and MM patients from unexposed controls. Moreover, the levels of total and hyper-acetylated HMGB1 were significantly higher in MM patients compared to asbestos-exposed individuals, and did not vary with tumor stage, suggesting that early lesions are also associated to increased HMGB1 levels. At a cutoff value of 2.00 ng/mL, the sensitivity and specificity of hyper-acetylated serum HMGB1 in differentiating MM patients from asbestos-exposed individuals was 100%, outperforming, in parallel experiments, other previously proposed biomarkers: osteopontin, fibulin-3, and mesothelin. When comparing MM patients to patients with other non-MM cytologically benign or malignant pleural effusion, the combination of two biomarkers, HMGB1 and fibulin-3, provided the highest sensitivity and specificity in differentiating these two groups. We propose total and hyper-acetylated HMGB1 as valuable biomarkers to differentiate MM patients from individuals professionally exposed to asbestos and from unexposed people.

#3113

SMO mutation identifies a subgroup of malignant pleural mesothelioma (MPM) patients with a worse prognosis.

Monica Ganzinelli,1 Claudia Proto,1 Diego Signorelli,1 Laura Botta,1 Giulia Pasello,2 Marcello Tiseo,3 Annalisa Trama,1 Gemma Gatta,1 Adele Busico,1 Alessandra Fabbri,1 Nadia Zaffaroni,1 Giuseppe Pelosi,1 Ugo Pastorino,1 Filippo De Braud,1 Milena Vitali,1 Marina C. Garassino1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy;_ 2 _Istituto Oncologico Veneto, Padova, Italy;_ 3 _University Hospital of Parma, Parma, Italy_.

Malignant pleural mesothelioma (MPM) is a rare malignant disease with a short prognosis and limited treatment options. However, at population level about 12% of all MPM survive more than three years. The aim of this pivotal study is to investigate whether a different gene profile could divide short versus long survivors.

METHODS:

A cut-off of 36 months of survival was chosen to divide patients with shorter and longer survival. Under this condition we retrospectively collected data on 32 short- and 25 long-survivors from three Italian Institutions. Paraffin-embedded tissue samples were tested for a customized panel of 21 genes (CDKN2, NF2, GSTM1, NAT2, BAP1, TERT, P53, PTCH1, SMO, LATS2, KEAP1, PI3K, KRAS, NRAS, STK11, WT1, FBXW7, CTNNB1, KIT, KDR and REV3). DNA was obtained upon manual microdissection to ensure at least 50% cancer cells. DNA was processed by PGM Ion Torrent. The major prognostic factors and mutations were described. The hazard risk of death was calculated with the Cox Model.

RESULTS:

The main prognostic factors were equally distributed among the two groups (age, sex, histotype, stage, and treatment). The most frequent mutations were BAP-1 (24,5%), NF-2 (17,5%), p53 (14%), SMO (8,7%) PITCH (8,8%), KEAP1 (7%) and TERT (5.3%) considering all 57 patients together. Wild-type patients for this gene panel were 31.6%. Median survival for short survivors was 13 months, while 47 for long survivors. No major differences in gene profile were observed between long and short survivors with the exception of SMO which was mutated only in short survivors (16%). SMO seems strongly associated with a poor prognosis (HR 8.01; CI95% 2.79-22.98 p <0.001). Also when considering only short survivors the negative prognostic effect remained statistically significant (HR 3.67, CI95% 1.28-10.48, p=0.015). The median survival for SMO mutated patients was 7 months.

CONCLUSIONS:

SMO mutation was likely to identify a subset of MPM patients with worse prognosis.

As SMO could be a promising target for specific inhibitors, further researches at clinical level in this subset of patients and also at preclinical level are ongoing.

This study was granted by AIRC.

#3114

Soy isoflavones modulate global methylation in head and neck squamous cell carcinoma (HNSCC).

Laura Rozek,1 Shama Virani,1 Emily Bellile,1 Jeremy Taylor,1 Maureen Sartor,1 Alisha Virani,1 Katie Rentschler,1 Claire Cote,1 Francis Worden,1 Lisa Peterson,1 Douglas Chepeha,2 Mark Prince,1 Scott McLean,1 George Yoo,3 Neil F. Saba,4 Dong M. Shin,4 Omer Kucuk,4 Gregory T. Wolf1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _University of Toronto, Toronto, Ontario, Canada;_ 3 _Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 4 _Winship Cancer Institute, Emory University, Atlanta, GA_.

Soy intake has been associated with improved survival in HNSCC and soy isoflavones have been suggested as potential chemopreventive agents with a favorable therapeutic index and safety profile. Multiple in vivo and in vitro anti-cancer effects have been postulated including modulation of gene methylation. To determine if high dose soy isoflavone treatment changes methylation of genes associated with cancer outcomes, we measured genomic and gene-specific methylation in tumor tissue collected as a part of a multi-institutional neoadjuvant soy isoflavone clinical trial in HNSCC patients undergoing definitive surgical management.

Methods: Thirty-nine patients participated in this clinical trial. Patients were scheduled to receive 2 weeks (range 7-39 days, median = 15.2) of soy isoflavone supplements (300 mg/day G-2535; DCP, NCI, NIH) orally prior to surgery. Levels of methylation of LINE-1 (global methylation), and 6 other candidate genes previously associated with HNSCC (p16, DCC, NDN, CD1a, CCNA1 and Gadd45α) were measured by pyrosequencing in biopsy, resection and whole blood specimens. Twelve patients were stage I/II, 27 were stage III/IV. Thirty one patients had cancer of the oral cavity, 4 had larynx and 4 had oropharynx cancer. Mean age of the patients was 60.1 (sd = 12.5). Changes in methylation were tested using paired t-tests and clinical associations explored using ANOVA.

Results: LINE-1 methylation increased significantly (mean increase 4.9%; range -34.8% to +20.9%) in tumor specimens after soy isoflavone (p<0.005). Amount of change correlated positively with days of isoflavone taken (p= .009). Increases in LINE-1 methylation in tumor were greatest in patients with normal BMI (p<0.03). Similar changes for LINE-1 were not seen in corresponding whole blood samples. No other significant changes in tumor or blood methylation levels were seen in the other candidate genes. Baseline tumor methylation and change in methylation were also studied with respect to nutrition (BMI), drinking, tumor site, tumor stage, nodal status and prior treatment. Baseline CD1a methylation was lowest in current smokers (p<0.04) and increases in CD1a after soy intake were associated with increasing pack years (p<0.02). CD1a methylation was also higher in patients with T1,2 tumors (p<0.03). Pretreatment NDN methylation was lower (p<.008) and LINE-1 higher (p<0.0001) in oral cavity cancer compared to oropharynx or larynx cancers. Toxicity from soy isoflavones was negligible and patient compliance was excellent.

Conclusions: This is the first demonstration of significant increases in tissue-specific global methylation associated with soy isoflavone intake, indicating increased genomic stability. The association of hypomethylation of LINE-1 with genetic instability, carcinogenesis and poor treatment outcomes suggests that soy isoflavones should be studied further as a potential chemopreventive agent in HNSCC.

#3115

Translational clinical utility of a circulating neuroendocrine tumor transcript measurement.

Irvin Modlin,1 Mark Kidd,2 Jaroslaw Cwikla,3 Ignat Drozdov,4 Lisa Bodei5. 1 _Yale University School of Medicine, New Haven, CT;_ 2 _Wren Laboratories, Branford, CT;_ 3 _University of Warmia and Mazury, Warsaw, Poland;_ 4 _Bering Limited, London, United Kingdom;_ 5 _European Institute of Oncology, Milan, Italy_.

Background:

A key issue in the clinical management of gastroenteropancreatic neuroendocrine tumors (GEP-NET) is early identification and the prediction of disease progression or recurrence. Strategies include imaging and biomarker measurement e.g., CgA or NSE. The former is limited by sensitivity and the latter by low specificity and poor reproducibility. We evaluated the role of a blood-based multianalyte gene algorithmic analysis (MAAA) using a 51 NET gene signature (NETest) as an alternative circulating biomarker and assessed its clinical utility in GEP-NET.

Patients and Methods:

We investigated 180 well-differentiated GEP-NET (small intestine: n=93, pancreatic: n=52, colorectal: n=11, stomach: n=3, appendix: n=2 and CUP: n=19; histological grade: G1: n=80, G2: n=86, no data: n=14). Baseline imaging (SRI: n=103, CT/MRI: n=77) were available. Surgery was undertaken in 27 and 28 had somatostatin analog (SSA) therapy. Disease recurrence or progression (RECIST 1.0 criteria) was determined by CT/MRI in treated cohorts. Transcript analysis was by qPCR and multianalyte algorithmic analysis. NETest defines disease activity risk: <14%: negative, <40%: low, >80% high. Transcripts measured by the NETest included genes involved in proliferation e.g., Ki-67, growth factor signaling e.g., RAF pathway and somatostatin receptor expression. Statistical analyses included regression analyses, performance metrics analysis and progression-free survival (PFS: Kaplan-Meier).

Results:

The NETest was positive in 175 (97%) with image-proven GEP-NET. In the surgical samples, matched blood/ tumor sample were significantly correlated (R2=0.7, p<0.0001) and gene expression regulating tumor proliferation, growth factor signaling and somatostatin receptor expression were concordant (R2=0.42-0.8, p<0.05). Surgery significantly decreased NETest levels (85%) and tumor volume decreases partially correlated with the NETest (R2=0.29, p=0.02). Elevated post-resection levels (>40%) predicted recurrence (100%). Baseline disease status (RECIST) was concordant with the NETest in 84% of the 62 evaluable patients; metrics were: sensitivity: 100%, specificity: 70%, PPV: 80% and NPV: 100%. In 28 patients treated with SSAs, NETest at baseline predicted treatment response (median PFS: 245 days NETest >80% vs. undetermined, NETest<40%). Clinically actionable elevations (to >80%) in the NETest occurred 105 days (48-252) prior to image-proven disease progression.

Conclusions:

A blood-based NET MAAA accurately correlated (97%) with image-proven disease. Blood levels were concordant with tissue and correlated with genes regulating tumor activity. NETest levels were decreased by surgery and elevated levels (>40%) were predictive (100%) of recurrence. NETest levels were also correlated (84%) with clinical disease status (RECIST) and values >80% predicted disease progression (100%) on somatostatin analogs.

#3116

MGMT immunohistochemistry (IHC) as a biomarker for response to combination therapy with capecitabine and temozolomide (C/T) in patients (pts) with advanced neuroendocrine carcinomas (aNEC).

Dwight Owen, Andrew J. Alexander, Lai Wei, Jessica Hemminger, Manisha H. Shah. _Ohio State University, Columbus, OH_.

Introduction: Tumor O6-methylguanine-methyltransferase (MGMT) reverses temozolomide-induced DNA injury, and low MGMT tumor expression has been shown as a predictor of response to temozolomide in glioblastoma. C/T therapy induces partial responses in up to 70% of pts with grade 1-2 pancreatic NEC but the role of MGMT expression as a predictor is unclear. We evaluated MGMT expression by IHC as a prognostic and predictive biomarker for pts with aNEC of all grades and primary sites treated with C/T.

Methods: A retrospective review was carried out at Ohio State University of 29 pts with aNEC who received C/T therapy from 2009 to 2013 and who were evaluable for RECIST response. MGMT expression was assessed when available by IHC on pre-treatment tumor samples to test the hypothesis that low MGMT expression (<10%) predicts response to C/T therapy vs high levels (≥10%).

Results: Of 29 pts, primary NEC site was pancreas in 18 pts, and non-pancreas in 11 pts. Objective response, progression-free survival (PFS) and overall survival (OS) data are outlined in the Table. Partial response (PR) rate was 50% in pts with pancreas primary vs 18% for non-pancreas primary. High PRs were observed in pts with grade 3 NEC (57%). Median PFS in the MGMT-low group was 16.6 months vs 9.5 months in the MGMT-high group (p=0.19). Median OS in the MGMT low group was 42.9 months vs 18.1 months in the MGMT-high group (p=0.16). There was a trend toward higher rate of PR (63%) in pts whose tumors had low levels of MGMT expression compared to those with high levels (17%) (p=0.18).

Conclusion: We observed a trend towards increased PR, median PFS, and median OS in aNEC pts whose tumors had low MGMT protein expression by IHC. The small sample size likely limited the statistical significance of the data. Results of this trial serve as strong rationale for future prospective trials to clarify role of MGMT expression in choosing C/T therapy for pts with NEC.

Objective response rate, progression free survival (PFS) and overall survival (OS)

---

|

N | %PR | %SD | %PD | Median PFS | p-value | Median OS | p-value

All patients | 29 | 38 | 52 | 10 | 13.0 | |

29.3

|

Low MGMT by IHC (<10%) | 12 | 62 | 38 | 0 | 16.6 | 0.19 | 42.9 | 0.16

High MGMT by IHC (≥10%) | 8 | 17 | 67 | 17 | 9.5 | |

18.1

|

Well-differentiated tumor grade (Ki-67 <3%) | 7 | 43 | 57 | 0 | 20.0 | 0.34 | NR | 0.027

Moderately differentiated tumor grade (Ki-67 3-20%) | 13 | 31 | 61 | 8 | 9.5 | |

25.9

|

Poorly-differentiated tumor grade (Ki-67 >20%) | 7 | 57 | 14 | 29 | 8.4 | |

13.1

|

#3117

Pleiotrophin is a marker of poor prognosis in Middle Eastern papillary thyroid carcinoma.

Shaham Beg, Maqbool Ahmed, Azhar Hussein, Rong Bu, Zeeshan Qadri, Saif AlSobhi, Fouad Al Dayel, Abdul K. Siraj, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia_.

Pleiotrophin (PTN) is a heparin binding growth factor known to have role in neuronal development. It is highly expressed in embryo but has a very limited expression in adult tissues. PTN is considered a proto-oncogene and has been hypothesized to play role in oncogenesis as its expression is found to be increased in many different cancer subtypes. Its role in cell transformation, cell growth, survival, migration and angiogenesis has also been shown in various different types of cancers. The function of PTN is hypothesized to be carried out by its interaction with cell surface proteoglycans or binding to its selective cell surface receptor, protein tyrosine phosphatase receptor Z1 (PTPRZ1). The significance of role of PTN in pathogenesis of thyroid cancer has not been explored especially with the fact that papillary thyroid carcinoma (PTC) originating in this ethnic population is the second most common female malignancy, after breast. So in search for novel druggable molecular target we sought for PTN expression in a large cohort of Saudi PTC. We analysed PTN alteration in more than 1000 primary papillary thyroid carcinoma in a tissue microarray format with clinical follow up data. We found that PTN was overexpressed in 65.5% (658/1006) of PTC and was significantly associated with aggressive clinical parameters such as tall cell variant histological subtype (p=0.0333), extrathyroidal extension (p=0.0292), lymphovascular invasion (p=0.0182) and large tumour size (p=0.0160). Important significant molecular association was seen with PTPRZ-1 (p=0.0316), Midkine (p=0.0008) and pSTAT-3 (p<0.0001). Finally patients showing PTN overexpression have worse disease free survival compared to patients showing low or absent PTN expression (p=0.0218). On multivariate analysis PTN is found to be an independent marker of poor prognosis taking into consideration parameters such as Age, Stage, Lymph node status and Extrathyroidal extension (p=0.0430). This study highlights the importance of PTN as druggable molecular target which can be therapeutically exploited by its inhibitors in treatment of PTC.

Keywords: Papillary Thyroid Cancer; Pleiotrophin; Pleiotrophin Receptor

#3118

Application of MUM1, CD10 and BCL6 IHC panel to a cohort of a hundred cases of diffuse large b-cell lymphoma on a tissue array.

HAIPING LIU, Sharmini Muralitharan. _Thermo Fisher Scientific, Fremont, CA_.

Diffuse Large B-Cell Lymphoma (DLBCL) is clinically and morphologically heterogeneous and is the most common type of non-Hodgkin lymphoma. DLBCL can be divided into subgroups, namely, germinal center B-cell like (GCB), activated B cell-like (ABC) and type 3 based on gene expression profiles (1,2). Germinal center B-cell like has better survival compared to the ABC and type 3 profiles. It has been demonstrated that immunostaining of a panel of antibodies, CD10, BCL6 and MUM1, can be used to determine the GCB and non-GCB subtypes of DLBCL and predict survival similar to the cDNA microarray (3). The aim of the study is to evaluate several MUM1 clones, select the best clone, combine the clone with CD10 (clone 56C6) and BCL6 (clone LN22) and application of this panel to a cohort of 100 DLBCL cases.

Six clones of MUM1 antibodies, mouse monoclonal antibodies EAU32, MUM1p, and MRQ-8 and rabbit monoclonal antibodies MRQ-43, EP190 and SP114 were evaluated. The clones were first screened for performance on a tonsil sample. All plasma cells and some germinal center B cells, as well as a small number of inter-follicular lymphocytes were expected to stain positive. Optimal dilution for each of the clones was determined independently before the best performing clone was selected. A tissue microarray containing 100 cases of DLBCL was stained with the panel and 97 cases were included in the data analysis (three cases were excluded from analysis due to tissue core loss for one case and due to dubious staining pattern for CD10 of two cases). All IHC assays were performed on the Thermo Scientific Autostainer using the two-step Quanto HRP polymer detection system. Slides were reviewed by a qualified pathologist.

It was found that five of the six clones tested stained as expected except clone MRQ-43, which stained majority of the inter-follicular cells; therefore it was dropped from further evaluation. Clone EAU32 was found to produce the strongest staining intensity and was included with CD10 and BCL6 to constitute the antibody panel for DLBCL. A total of 21 cases were identified as GCB type including 15 CD10+ cases and 5 CD10-/ BCL6+/ MUM1- cases; A total of 76 cases were identified as non-GCB type including 55 CD10-/ BCL6- cases and 21 CD10- /BCL6+/ MUM1+ cases.

The study identified a mouse monoclonal MUM1 antibody that is specific and provided strong immunostaining. The antibody panel of CD10, Bcl6 and MUM1 was effective in classifying the DLBCL into GCB and non-GCB subtypes according to Hans IHC Algorithm (3).

1. Rosenwald A et al, Lymphoma/Leukemia Molecular Profiling Project. N Engl J Med. 2002 Jun 20;346(25):1937-47.

2. Alizadeh AA et al., Nature. 2000 Feb 3;403(6769):503-11.

3. Hans CP et al. Blood. 2004 Jan 1;103(1):275-82

#3119

Expression of OPN isoforms and its potential use on risk stratification in early childhood precursor B-acute lymphoblastic leukemia (pre-B ALL).

Abigail Cristina da Silva Rezende Rezende,1 Mariana Emerenciano Cavalcante de Sa,1 Etel Rodrigues Pereira Gimba2. 1 _Instituto Nacional de Câncer, Rio de Janeiro, Brazil;_ 2 _Universidade Federal Fluminense, Rio das Ostras, Brazil_.

Precursor-B cell acute lymphoblastic leukemia (Pre-B ALL) corresponds to most leukemia cases. Pre-B ALL in children younger than 12 months-old is characterized by a high total leukocytes count, central nervous system (CNS) infiltration and KMT2A rearrangements (KMT2A-r), whereby patients are directed to a high risk treatment protocol. Searching for new additional prognostic biomarkers, especially those that can better predict CNS involvement are needed. It has been reported that osteopontin (OPN) is able to anchor bone-marrow leukemic blasts, protecting these cells from chemotherapeutic cytotoxicity and that circulating OPN has been correlated to blast CNS infiltration. Until now, data regarding OPN in ALL and other non-solid tumors are only correlated to full lenght OPN. However, OPN suffers alternative splicing, generating three splicing isoforms (OPN-SI), named OPNa, OPNb and OPNc. This study aims to evaluate whether OPN-SIs could differently contribute to risk stratification of childhood Pre-B ALL harboring or not the KMT2A-r. This study included 34 childhood Pre-B ALL patients, whose several clinical data, including the status of KMT2A-r are available. Total RNA from bone marrow blast cells have been extracted using the RNeasy (QIAGEN) mini kit and then cDNA has been synthesized. OPN-SI expression has been analyzed by qualitative and quantitative RT-PCR using isoform specific oligonucleotides. Using the qualitative RT-PCR approach, we showed that most patient samples express the 3 OPN-SI (48.64%). Other 10.8% of these cases expressed OPNa and OPNb isoforms, while 13.51% expressed OPNa and OPNc. Only 8.1% of these samples expressed only OPNa or OPNc isoforms. These data showed that OPNa isoform is expressed in most patient samples, followed by OPNc. We also observed that OPNa expression seems to be correlated to the occurrence of KMT2A-r. Considering OPN-SI expression levels, most patient samples (47.05%) overexpress OPNa. Among those patients that harbor the KMT2A-r, 35.29% of them express similar OPN-SI expression levels, while 32.9% overexpress OPNa and 20 % overexpress OPNc. Only 5.88% overexpress OPNb. Among those patient samples that lack KMT2A-r, a higher proportion overexpress OPNa, followed by those that overexpress OPNb, or OPNc or have similar levels of the 3 OPN splice variants. As a whole, our data demonstrate that OPNa isoform is the OPN splice variant expressed in most Pre-B ALL samples, besides being overexpressed in relation to OPNb and OPNc isoforms. Our data also provide evidence that OPNa splice variant expression data could contribute to risk stratification in Pre-B ALL patients, correlated to the occurrence of KMT2A-r, besides being the first study of OPN-SI in non-solid tumors, especially in ALL. Ongoing studies will better better investigate the correlation between OPN-SI expression and additional prognostic data, including KMT2A-r.

#3120

B-Catenin gene expression and its mutations: promising and key component in the pathogenesis of acute myeloid leukemia (AML).

Mohsin Maqbool, Sobuhi Iqbal, Atul Sharma, Sameer Bakhshi, Sreenivas Vishnubhatla, Ashish Datt Upadhyay, Lalit Kumar. _All India Inst. of Medical Sciences, New Delhi, India_.

INTRODUCTION AND AIM: Wnt signaling pathway plays a critical role in stem cell self-renewal and proliferation of leukemic stem cells and its association with the pathogenesis of acute myeloid leukemia (AML) has also been described. Beta-Catenin, a key regulator of Wnt signaling is highly expressed in cancers. Its role has been recently documented in AML however its prognostic significance in AML is not clear. The present study was carried to determine the potential prognostic implications of B-catenin expression and mutations in de novo AML patients.

METHODS: We performed the gene expression of B-Catenin and its mutations analysis in exon 3. We investigated the expression of B-Catenin gene in bone marrow and/or blood samples by quantitative PCR using SYBR-Green chemistry and analysis was done by ΔΔCT Method. 18S RNA and B2M genes were used as reference genes. B-Catenin mutations analysis was by done direct DNA Sanger sequencing.

RESULTS: Total 117 de novo non M3 AML patients and 40 healthy controls were included for the gene expression and analyzed for their clinicopathologic and prognostic significance. Median age for entire cohort (n=117) was 17.0 years (Range 1-60 years) with male:female ratio of (1.8:1). FLT3-ITD mutation was positive in 11 (9.5%) patients. Only 4% of AML patients showed genetic alterations in exon 3 of B-catenin and no significant correlation with base line characteristics. B-catenin showed a significantly increased mRNA expression with a 5 fold rise as compared to healthy controls (p<0.05). B-catenin over expression was significantly associated with poor survival at median followup of 17 months and having less CR rates (p=0.03). There was no significant correlation of B-catenin over expression vs baseline clinical characteristics like Hb, TLC, Platelets, Cyto group (p=0.11), blast percentage (p=0.28), Flt3-itd mutations (p=0.42).

CONCLUSION: These results provide the evidence for WNT signaling activation existence in AML patients and suggest that B-Catenin could be the key component in the pathogenesis and can be prognostically important marker in AML. However B-catenin expression in larger cohort of samples and with longer follow-up is required to confirm our findings.

#3121

Gene expression signature predicts induction treatment response and clinical outcome in adult Colombian patients with acute lymphoblastic leukemia.

Nataly Cruz-Rodriguez,1 Sandra M. Quijano,2 Leonardo J. Enciso,1 Alba L. Combita,1 Jovanny Zabaleta3. 1 _Programa de Investigacion en Innovacion en Leucemias Agudas y Cronicas (PILAC), Instituto Nacional de Cancerologia, Bogota, Colombia;_ 2 _Pontificia Universidad Javeriana, Bogota, Colombia;_ 3 _Louisiana State University Health Sciences Center, New Orleans, LA_.

Background. In Colombia ALL in adults represents a public health problem because its incidence and mortality increase annually. Only 61% of Colombian adult patients with ALL achieve complete remission. The median overall survival to the disease is less than 11.3 months and the event-free survival is 7.34 months. Identification of prognostic factors in patients with ALL is crucial for the proper planning of treatment strategies and the optimal results of therapy. Our goal was to determine gene expression signatures correlated with response to therapy and to evaluate the utility of these expression patterns as predictors of risk prior to therapy of adult Colombian patients with B-ALL.

Methods. This study included 43 adult patients newly diagnosed with B-cell precursor or common B-ALL. Patients were recruited at the Colombian National Cancer Institute and Hospital Universitario San Ignacio, both in Bogota, Colombia. The leukemic blast population from diagnostic samples was separated with magnetic microbeads coated with either anti-CD19 or anti-CD34 antibodies followed by column enrichment using standard procedures and MACS (Miltenyi, Bergisch Gladbach,Germany). Total RNA from purified leukemic cells was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocols. We used microarray analysis to identify genes that distinguish poor from good response to induction treatment using differential gene expression analysis and the response group as reference and the Illumina Custom algorithm embedded in the GenomeStudio software (Illumina). The expression profile was validated by real-time PCR (RT-PCT) using TaqMan probes. The 2-ΔΔCT method was used to estimate the fold induction of each gene using GAPDH and an internal calibrator as controls. Assays were done in triplicate.

Results. We identified 442 genes differentially expressed between 22 leukemia patients who responded and 5 who did not respond to induction chemotherapeutic treatment. Hierarchical analysis with the 99 most differentially expressed genes between the two groups revealed 3 sets of patients that differed in their clinical characteristics giving these genes high prognostic clinical outcome impact capacity. We validated the expression of 7 genes by RT PCR in 43 patients and, in addition to finding a correlation with gene expression profiles, we established correlations with good and poor prognosis from the time of diagnosis.

Conclusions. Our study suggests that the response to induction treatment and clinical outcome of patients can be predicted from the onset of the disease and that gene expression profiles can be used to stratify patient risk adequately and accurately. The present study represents the first showing that gene expression profiling could become a clinically relevant tool for stratification in the early course of disease of Colombian adults B-ALL.

#3122

A strategy for detecting high-risk groups for pancreatic cancer with glycol-biomarkers.

Eiji Miyoshi,1 Tomohiro Maekawa,1 Makiko Ueda,1 Mayuka Shimomura,1 Shinji Takamatsu,1 Kotarosumitomo Nakayama,1 Yoshihiro Kamada,1 Yasuhiko Tomita2. 1 _Osaka Univ. Graduate School of Medicine, Suita, Japan;_ 2 _Osaka Medical Center for Cancer and Cardiovascular Diseases., Osaka, Japan_.

(Aim) Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers, showing a high potential for invasive activity and high recurrence rates. One of the reasons for poor prognosis of PDAC is a difficulty in an early diagnosis. When pancreatic tumors are detected by CT or MRI examinations, most cases are at the advanced stage even if tumor size is within 2 cm. To make an early diagnosis for PDAC truely, we should make a targeting of high-risk groups for developing PDAC like chronic hepatitis followed by hepatocellular carcinoma. Aberrant glycosylation is a promising target for cancer biomarkers. Several kinds of glyco-biomarkers are clinically used for PDAC diagnosis like CA19-9. We have reported that fucosylated haptoglobin (Fuc-Hpt) is a novel type of glyco-biomarker for PDAC. Serum Fuc-Hpt levels are dramatically increased at the stage IV of PDAC as well as a few cases of Stage I-III. In the present study, we performed pathological analysis of surrounding tissues around pancreatic cancer as well as normal pancreas in autopsy cases and investigated the clinical usefulness of glycol-biomarkers for chronic pancreatitis and pancreatic cancer (Subjects and Methods) 88 cases of pancreatic cancer patients and 100 cases of other diseases are enrolled in this study. Inflammation, fibrosis and fatty changes were investigated in these pancreatic tissues by 2 independent researchers in blind way. Serum fucosylated haptoglobin (Fuc-Hpt) levels in patients with chronic pancreatitis and pancreatic cancer were measured with lectin-antibody ELISA, using Aleuria aurantia lectin (AAL) and Pholiota squarrosa lectin (PhoSL). AAL recognizes all types of fucosylation and PhoSL recognizes core fucosylation.

(Results) Pancreatectomy specimens showed a higher ratio of positive change in fibrosis (86% vs. 42%), fatty degeneration (72% vs. 44%), and inflammatory cell infiltration (14% vs. 3%) than control samples. Multivariate analyses demonstrated that each histological change was a significant, independent determinant for PDAC. In contrast, AAL-reactive Fuc-Hpt levels were increased in patients with pancreatic cancer and AAL-reactive Fuc-Hpt was though to be produced in the liver metastasis of pancreatic cancer. In contrast, PhoSL-reactive Fuc-Hpt levels were increased in patients with chronic pancreatitis, compared to pancreatic cancer. Correlations of PhoSL-reactive Fuc-Hpt levels and other biochemical parameters such as serum amylase, lipase, and elastase levels were not observed. The investigation of mechanisms underlying an increase of serum PhoSL-reactive Fuc-Hpt in patients with chronic pancreatitis is underway.

(Conclusion) Cryptogenic pancreatitis might be a premalignant disease for pancreatic cancer and dramatic changes in fucosylation linkage on Fuc-Hpt were observed in pancreatic carcinogenesis. These findings give us a possibility of preventing or diagnosing pancreatic cancer at earlier stage, chronic pancreatitis.

#3123

Aberrant expression of CD33 is associated with poor prognosis in patients with multiple myeloma and tumor progression.

Ki Hong Lee,1 Hee Seoung Seo,2 Ji Yeon Sohn,2 Eunyoung Lee,3 Hyewon Lee,3 Hyeon-Seok Eom,3 Sun-Young Kong1. 1 _Graduate School of Cancer Science and Policy, National Cancer Center, Goyang, Republic of Korea;_ 2 _Department of Laboratory Medicine, Center for Diagnostic Oncology, National Cancer Center, Goyang, Republic of Korea;_ 3 _Hematology-Oncology Clinic, Center for Specific Organs, National Cancer Center, Goyang, Republic of Korea_.

Background: Aberrant antigen expressions of plasma cells in patients with multiple myeloma (MM) have used for diagnosis and monitoring residual disease during treatment. Here, we evaluated prognostic impact of aberrant antigens expression and identify functional roles of CD33 in MM.

Methods: Patient characteristics and immunophenotype from bone marrow aspiration samples of 99 newly diagnosed MM patients in National Cancer Center of Korea were analyzed. Samples were stained with 12 antibodies using 4-color panel for flow cytometry with CD138 gated cells measuring CD38, CD19, CD117, CD45, CD20, CD33, CD13, CD56, CD28, cytoplasmic κ and cytoplasmic λ. Prognostic impact of antigen expressions was evaluated using Kaplan-Meier and log-rank test. To investigate the functional role of CD33 in MM, we evaluated immunophenotype of MM cell lines and performed in vitro experiment on CD33 expression. Transient CD33 knockdown was performed by using small interfering RNA (siRNA) through Amaxa nucleofector device. CD33 knockdown was evaluated using flow cytometry and quantitative real-time PCR. Proliferation was evaluated using IncuCyte Zoom live cell imaging and cell counting kit 8 (CCK-8). The role of CD33 in migration was studied using transwell assay following with IncuCyte Zoom and CCK-8 for quantification of the migrated cells.

Results: The frequencies of antigen expression in newly diagnosed patients with MM were observed as following; expression of CD20, CD19, CD33, CD117, CD13, CD56 and CD38 were 3%, 6%, 15%, 19%, 33%, 66%, and 92%, respectively. CD33 and CD13 expression were associated with lower overall survival (OS; P=0.014 and P=0.036) in Kaplan-Meier analysis. When comparing clinical characteristics according to antigens expression, the level of anemia showed correlation with CD33 expression. Moreover, multivariate analysis showed that CD33 was independently prognostic of shorter progression free survival (PFS; P=0.009) and OS (P=0.024) with correction of clinical prognostic factors. Then we checked CD33 expression level in myeloma cell lines (H929, RPMI-8226, IM-9, and KMS-12-PE) and RPMI-8226 showed high expression of CD33. After knockdown of CD33 in RPMI-8226 by transient transfection, it showed significantly attenuated proliferation and decreased migration.

Conclusion: Clinical and experimental data from our results showed that CD33 might be a prognostic marker, which was associated with tumor progression via increased proliferation and migration in MM.

[This work was supported by a grant from the National Research Foundation (NRF) of Korea funded by the Korean government (MSIP; No. 2014R1A2A2A01002553).]

#3124

Prognostic biomarkers for pancreatic cancer.

Cristina Baciu,1 Robert Grant,1 Hansen He,2 Musaddeque Ahmed,2 Robert E. Denroche,1 Lee Timms,1 Gun Ho Jang,1 Ayelet Borgida,3 Xihui Lin,1 Paul C. Boutros,1 Dianne Chadwich,2 Sheng-Ben Liang,2 Sagedeh Shahabi,2 Michael H.A. Roehrl,2 Sean Cleary,2 Julie M. Wilson,1 John D. McPherson,4 Lincoln Stein,1 Steven Gallinger5. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _University Health Network, Toronto, Ontario, Canada;_ 3 _Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada;_ 4 _UC Davis Comprehensive Cancer Center, Sacramento, CA;_ 5 _Mount Sinai Hospital, Toronto, Ontario, Canada_.

Pancreatic ductal adenocarcinoma (PDAC) has the lowest 5-year-survival rate of common cancers (< 7%), but despite intensive research efforts over the past several decades, its dismal prognosis has barely improved. Recent genome-wide association studies (GWAS) have linked several genetic factors with pancreatic cancer, but there is generally poor overlap in the results from such studies, presumably due to population heterogeneity. In this study, we collected previously published pancreatic risk loci from multiple GWAS studies, and correlated these loci with clinical information gathered on a well-characterized cohort of 148 PDAC patients from the University Health Network in Toronto Canada, and several other North American hospitals, which have undergone whole-genome sequencing. Using this cohort, we attempted to identify prognostic genomic biomarkers by correlating the germline genomic alterations observed in our cohort to overall survival (OS). By understanding the mechanism of germline variants that alter OS, we hope to develop insights that will lead to improved detection and therapy for PDAC patients.

Among the 67 published risk loci we tested using a multivariate Cox proportional hazard model, we found a strong positive association of the single nucleotide polymorphism rs4785367 (RefSNP alleles: C/T on forward strand; MAF=0.474) with overall survival of PDAC donors: HR = 0.426; CI = 0.268 - 0.686; p-value = 0.00029. A more detailed analysis at the genotype level revealed that the presence of the homozygous minor allele has a stronger effect than either the heterozygous or homozygous major allele. The SNP falls within the intergenic region between the ZNF423 and TMEM188 genes, within the exon 2 of lncRNA RP11-305A4.3 and overlaps a CTCF regulatory domain. Preliminary gene expression analysis from RNA sequencing data on a subset of PDAC donors (n=28) shows that patients carrying the minor allele have significant higher TMEM188 expression than of the major type (p-value = 0.012), suggesting that this allele may influence the course of PDAC via TMEM188 activity. A recent study linked the gene product to activation of NK cells, which in turns increases the defense mechanism against the pathogens, infections and transformed tumors. These findings suggest a possible molecular mechanism influencing the course of PDAC. We are also exploring the effect of the presence of the minor allele on the regulatory CTCF region, by applying an integrative pipeline for risk SNP analysis to pancreatic cancer. This will possibly detect the effect on the NANOG motif binding and/or on CTCF looping.

In summary, the present study detected the rs4785367 as a prognostic biomarker for pancreatic cancer, with the novelty of increased TMEM188 gene expression being linked to the presence of the alternate allele in PDAC patients. Further investigations on this and on assessing the effect of the polymorphism on the regulatory CTCF feature are in progress.

#3125

KCNQ1 expression is a strong prognostic biomarker for disease recurrence in stage II and III colon cancer.

Sjoerd H. den Uil,1 Veerle M.H. Coupe,1 Janneke F. Linnekamp,2 Evert van den Broek,3 Jeroen A.C.M. Goos,1 Pien M. Delis-van Diemen,3 Eric J.T. Belt,4 Nicole C.T. van Grieken,1 Patricia M. Scott,5 Louis Vermeulen,2 Jan Paul Medema,2 Herman Bril,6 Hein B.A.C. Stockmann,6 Robert T. Cormier,5 Gerrit A. Meijer,3 Remond J. Fijneman3. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Academic Medical Center, Amsterdam, Netherlands;_ 3 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 4 _Albert Schweitzer Ziekenhuis, Dordrecht, Netherlands;_ 5 _University of Minnesota Medical School, Duluth, MN;_ 6 _Spaarne Gasthuis, Haarlem, Netherlands_.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at high risk of recurrence and stage III patients at low risk of recurrence are key unmet clinical needs. We previously identified KCNQ1 as a tumour suppressor gene of which loss of expression was associated with poor survival in patients with CRC liver metastases. The present study aimed to examine the prognostic value of KCNQ1 in stage II and III colon cancer patients.

Methods: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. KCNQ1 protein expression was evaluated by immuno-histochemistry on tissue microarrays of 386 stage II and III colon cancer patients.

Results: Low KCNQ1 mRNA expression in microsatellite stable (MSS) stage II colon cancers was associated with poor disease free survival (DFS) (HR 3.35; 95% CI 1.16-9.66; p<0.05). Loss of KCNQ1 protein expression from epithelial cells was strongly associated with poor DFS in MSS stage II (HR 3.82; 95% CI 2.04-7.14; p<0.0001), MSS stage III (HR 2.93; 95% CI 1.70-5.02; p=0.0001) and MSI stage III colon cancers (HR 5.06; 95% CI 1.07-23.89; p<0.05). Multivariate analysis demonstrated KCNQ1 to have independent prognostic value in addition to established clinicopathological parameters such as angioinvasion, nodal stage en MSI-status.

Conclusion: We conclude that KCNQ1 is a strong prognostic biomarker for prediction of disease recurrence (HR~4) and may aid stratification of patients with stage II MSS colon cancer and stage III MSI CRC for adjuvant chemotherapy. Because KCNQ1 protein expression is determined by immuno-histochemistry, this biomarker can be implemented in standard clinical care using existing workflows.

#3126

CEMIP, a secreted protein highly induced in colon cancer and associated with poor patient survival.

Stephen P. Fink,1 Lois Myeroff,1 Revital Kariv,1 Petra Platzer,1 Baozhong Xin,1 Debra Mikkola,1 Earl Lawrence,1 Nathan Morris,1 Arman Nosrati,1 James Willson,2 Joseph Willis,1 Martina Veigl,1 Jill Barnholtz-Sloan,1 Zhenghe Wang,1 Sanford Markowitz1. 1 _Case Western Reserve Univ., Cleveland, OH;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX_.

Background: Colorectal cancer is the second leading cause of cancer death among adult Americans. Tumor stage still remains the clinical standard for determining prognosis of colon cancer patients and for selecting individuals for treatment with adjuvant chemotherapy. Genes induced in colon cancer provide novel candidate biomarkers of tumor phenotype and aggressiveness. The goal of this study was to identify genes whose expression is dramatically up-regulated in colon neoplasia even at the early stages of the disease, and with the potential to be new prognostic markers of patient outcome and/or targets for new therapies.

Methods: We used expression microarrays, real-time PCR, Western blot, and immunohistochemistry to identify CEMIP (originally named KIAA1199/CCSP1) induction in colon cancer, and characterized the biological properties of the corresponding protein in cell-line and mouse xenografts. Sandwich ELISA was developed to determine CEMIP plasma levels in patients with colon cancer. Prognostic importance of gene induction was demonstrated by real-time PCR measurement of gene expression in colon cancer cases of known clinical outcome.

Results: We originally identified CEMIP as a novel transcript that is induced an average of 54-fold in colon cancer, with a similar increase in protein level. We find that CEMIP is a secreted protein and that plasma levels of CEMIP in colon cancer patients is increased compared to normal subjects (P=0.05). Knocking out CEMIP in a human colon cancer cell line markedly reduced growth of tumor xenografts implanted in athymic mice. Tumors that did grow had increased deposition of hyaluronan, linking CEMIP participation in hyaluronan degradation to the modulation of tumor phenotype. Human stage III colon cancer cases with greater than average increased tumor CEMIP expression had a median survival time of 37 months, versus greater than 140 months for colon cancer cases with below average CEMIP expression (P=0.004). Similarly, among combined stage II plus III colon cancer cases, median survival decreased by 92 months for CEMIP high versus low expressing tumors (P=0.0003).

Conclusions: CEMIP is highly expressed in colon neoplasia and is a novel member of the colon cancer secreted proteome making it a candidate serological marker of early human colon neoplasia. CEMIP facilitates tumor growth, and high CEMIP correlates with poor outcome in stage III and in stages II plus III combined cohorts. CEMIP may have utility as both a prognostic marker of colon cancer outcome, and as a potential therapeutic target.

#3127

MiR-BART9 is a prognostic biomarker associated with PD-1 expression in colon cancer patients.

Marisa Mariani, Deep Pandya, Mirko Andreoli, Shiquan He, Manuela Spennato, Cristiano Ferlini. _Danbury Hospital, New York, NY_.

Background:

Epstein-Barr Virus (EBV) is widely expressed in the population in a latent stage. During cancer progression, antiviral immunity is compromised by the same mechanisms leading to suppression of anticancer immunity.

This study was aimed at developing the expression of miR-BART as biomarkers to select patients at risk of fast progression for a defect of the antiviral/anticancer immunity.

Methods:

We first analyzed the expression of EBV-miRNAs in a panel of 438 colon cancer (CC) patients of the TCGA dataset. Analysis was conducted using level 1 miRNA-seq and level 3 RNA-seq available on the same patients.

In order to confirm the results, we tested the expression of miR-BART9 in an additional set of 271 CC patients using spectral imaging and quantitative fluorescent immunohistochemistry (PD-1 and PD-L1 antigens at the protein level and miR-BART9 using a specific designed ISH probe).

In order to increase the clinical applicability of our findings, we also developed a qPCR test to analyze the expression of miR-BART9 in serum of colon cancer patients.

Results:

In the analysis of 438 CC patients of the TCGA dataset, expression of miR-BART9 was found in 78 patients (18%). Levels of miR-BART9 were prognostic of poor outcome in a multivariate Cox model including age and stage (HR=2.8, CI 1.6-4.7, p<0.0001). In patients positive for miR-BART9 we also noticed a significant higher expression of PD-1, CD3 but not PD-L1 at the gene level. These results suggest that the expression of miR-BART9 is linked to a T cell infiltrate in the cancer tissue capable to induce a potent suppression of anticancer and antiviral immunity.

In order to confirm this hypothesis, we performed spectral imaging in an additional set of 271 CC patients. Expression of miR-BART9 was found in 32 (12%) patients with a prevalent stromal pattern. Also in this case expression of miR-BART9 was associated with poor outcome in Cox-multivariate analysis including age and stage (HR= 2.5, CI 1.6-3.4, p<0.001). Expression of miR-BART9 was again significantly associated with a PD-1 positive T-cell infiltrate.

Based on these results, we have designed a qPCR test to analyze the expression of miR-BART9 in serum using an EBV-infected B lymphoma cell line (Raji) as positive control. The assay was employed for monitoring miR-BART9 expression in serum from CC patients (n=89) and healthy controls (n=95). Human miR-486 and miR-17 were used as positive controls.MiR-BART9 expression was found in 33 (37%) of CC patients and only in 5 (5%) of the 95 healthy controls.

Conclusions: Our results reveal that expression of miR-BART9 is associated with aggressive CC. Patients featuring reactivation of latent EBV-infection have also expression of PD-1+ T cells, which will impact both anticancer and antiviral immunity. The expression of miR-BART9 can be detected also in serum and may serve to identify the right timing to treat CC patients with PD-1/PD-L1 inhibitors in order to restore an effective anticancer and antiviral immunity.

#3128

Detection of genetic and epigenetic DNA markers in urine for the early detection of primary and recurrent HCC.

Surbhi Jain,1 Hie-Won Hann,2 Sitong Chen,1 Selena Y. Lin,3 Ting-Tsung Chang,4 Chi-Tan Hu,5 Wei Song,1 Ying-Hsiu Su6. 1 _JBS Science, Inc., Doylestown, PA;_ 2 _Liver Disease Prevention Center, Division of Gastroenterology and Hepatology, Thomas Jefferson University Hospital, Philadelphia, PA;_ 3 _Drexel University College of Medicine, Philadelphia, PA;_ 4 _National Cheng Kung University Medical College, Tainan, Taiwan;_ 5 _Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualin, Taiwan;_ 6 _Baruch S. Blumberg Institute, Doylestown, PA_.

The purpose of this study was to explore the potential of urine DNA biomarkers for the early detection of primary and recurrent hepatocellular carcinoma (HCC). HCC is an aggressive disease with a 5-year survival rate of 26% in early-stage cancers, and a mere 2% in later stages, with an approximate 50% recurrence rate in the first 2 years of treatment. The most commonly used screening biomarker for HCC is serum alpha-fetoprotein (AFP), which detects only 40-60% of cases. We have previously shown that urine contains fragmented, circulation-derived, cell-free DNA that can be used for detection of cancer-related DNA markers, if a tumor is present. In order to detect circulation-derived, cell-free DNA markers in urine, we have developed short amplicon (~50 bp) PCR-based assays for the three most frequent hotspot mutations in TP53 (codon 249), TERT (-124, promoter), and CTNNB1 (hotspot in exon 3, codons 32-37), and for aberrant DNA methylation in GSTP1 (mGSTP1) and RASSF1A (mRASSF1A). This five marker panel has an area under the receiver operating curve of 0.94 (92.2% sensitivity at 80% specificity) in distinguishing primary HCC (n=77) from non-HCC (n=91) patients by a logistic regression-based combination algorithm. Furthermore, these 5 DNA markers scored 42 of the 45 (93.3%) AFP-negative (< 20 ng/mL) HCC urine samples "positive" in this study population. To validate the urine DNA test for the early detection of recurrent HCC, an on-going blinded study was conducted. Ten patients with treated HCC were monitored for recurrence of HCC with serum AFP and urine DNA markers. Urine samples were collected from these patients at visits with a hepatologist, barcoded, and tested for the five DNA markers. Of the 10 patients, 4 developed recurrence during the study. The HCC-specific urine DNA markers were detected in 3 out of these 4 patients six months prior to MRI diagnosis, and detected concurrent with MRI diagnosis in the other patient. In conclusion, HCC DNA markers can be detected in urine of patients with HCC by short-amplicon, PCR-based assays. Furthermore, we have demonstrated that the urine DNA test detected at least 40% more HCC in an open-labeled study as compared to serum AFP alone, and has the potential to become the first line of screening for HCC in high risk populations.

#3129

Predictive biomarker identification for response to vantictumab (OMP-18R5; anti-Frizzled) using primary patient-derived human pancreatic tumor xenografts.

CHUN ZHANG, Fiore Cattaruzza, Pete Yeung, Wan-Ching Yen, Marcus Fischer, Claire Guo, Alayne Brunner, Min Wang, Belinda Cancilla, Austin Gurney, Rainer Brachmann, John Lewicki, Tim Hoey, Ann M. Kapoun. _OncoMed Pharmaceuticals Inc., Redwood city, CA_.

Background: The WNT/ β-catenin signaling pathway has been shown to play a key role in both normal development and tumorigenesis (Polakis, 2007; MacDonald et al., 2009). We have developed a monoclonal antibody, vantictumab, that blocks canonical WNT/β-catenin signaling through binding of five FZD receptors (1, 2, 5, 7, 8). This antibody inhibits the growth of several tumor types, including pancreas, breast, colon and lung. Furthermore, our studies showed that vantictumab reduces tumor-initiating cell frequency and exhibits synergistic activity with standard-of-care (SOC) chemotherapeutic agents (Gurney et al., 2012).

Material and methods: We set out to identify a predictive biomarker for the response to vantictumab in pancreatic cancer patients by analyzing mRNA-seq gene expression data from 14 patient-derived xenograft (PDX) models. These 14 minimally passaged pancreatic xenograft tumors were tested in vivo and their responses to vantictumab, in combination with the current SOC gemcitabine and nab-paclitaxel were established. Samples from these experiments were collected for Pharmacodynamic (PD) biomarker analysis. We utilized a two-sample Welch's t-test to identify genes that can distinguish between responders and non-responders and the K-nearest neighbor (KNN, Altman 1992) algorithm for classification. A leave-one-out cross-validation was used to measure area under the ROC curve (Fawcett et al., 2006, AUC), accuracy (ACC), positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the model. Results: PD biomarker analysis confirmed inhibition of genes in Wnt and stem cell pathways by vantictumab in combination with gemcitabine as well as gemcitabine plus nab-paclitaxel. The selected 3-gene signature comprising TGFB3, IGF2 and SMO achieved the best performance (AUC = 0.875, ACC = 0.93, PPV=0.91, NPV=1, sensitivity=1, specificity=0.75) in the 14 PDX pancreatic tumor models. In addition, a strong correlation between the gene signature biomarker and the ratio of tumor inhibition (RTI) in the pancreatic xenograft experiments was observed. The identified 3-gene biomarker was used to predict the response to vantictumab in combination with gemcitabine and nab-paclitaxel in three additional pancreatic PDX tumor models. The efficacy in the three models was successfully predicted by the biomarker. Conclusions: The 3-gene biomarker is being evaluated in a Phase 1b study of vantictumab in combination with gemcitabine and nab-paclitaxel in previously untreated stage IV pancreatic cancer (NCT02005315).

#3130

Adiponectin, Leptin, IGF1 and TNFα serum biomarker as noninvasive diagnosis of colon adenoma.

Hassan Ashktorab,1 Akbar Soleimani,1 Alexandra Nichols,2 Komal Sodhi,2 Lakshmi Kannan,1 Laiyemo Adeyinka,1 Mehdi Nouraie,1 Hassan Brim1. 1 _Howard University, Washington, DC;_ 2 _Marshall University, Huntington, WV_.

Background and Aim: The potential role of Adiponectin, Leptin, IGF1 and TNFα as biomarker in colon adenoma has not been studied. Therefore, we investigated the blood serum levels of these biomarkers in colorectal adenoma.

Method: The case-control study consisted of 198 African American patients with colon adenoma (cases) and 198 healthy individuals (controls) at Howard University Hospital. We used Elisa for biomarkers detection. Statistical analysis was performed by t-test and multivariate logistic regression.

Results: The differences in median leptin, Adiponectin, IGF1 and TNFα levels between control and case groups (6.7 vs.16.4), (11.3 vs.46.0), (4.5 vs.12.9) and (71.4 vs. 130.8) were statistically significant (p<0.05), respectively. In a multivariate model, the odds ratio (ORs) for Adiponectin, TNFα and IGF1 were 2.0 (95% CI=1.6-2.5; P≤0.001), 1.5 (95% CI=1.5- 2.0; P 0.004) and 1.6 (95% CI=1.3-2.0; P≤ 0.001), respectively. There were positive correlations between serum Adiponectin and IGF1 concentrations with age (r=0.17, P≤ 0.001 and r=0.13, P=0.009), also between TNFα, IGF1 and Leptin concentration with Body Mass Index (BMI) (r=0.44, P≤ 0.001 and r=0.11, P=0.03; r=0.48, P≤0.001), respectively. There was a negative correlation between Adiponectin concentration and BMI (r=-0.40, P≤0.001), respectively.

Conclusion: These data support the hypothesis that serum Adiponectin, IGF1 and TNFα are risk biomarkers for noninvasive detection of colorectal adenomas.

#3131

Expression of FGF19/FGFR4 related biomarkers in hepatocellular carcinoma.

Zhong-Zhe Lin, Yung-Ming Jeng, Chiun Hsu, I-Lun Tsai, Kuan-Yu Chen, Fu-Chang Hu, Chih-Hung Hsu, Hey-Chi Hsu, Ann-Lii Cheng. _National Taiwan Univ. Hospital, Taipei, Taiwan_.

Background:

Fibroblast growth factor 19 (FGF19) and fibroblast growth factor receptor 4 (FGFR4) signaling play critical roles in hepatocarcinogenesis. This study explored the expression and clinical significance of FGF19/FGFR4 related signaling molecules in hepatocellular carcinoma (HCC).

Method:

We examined the mRNA expression of FGF19, FGFR4, klotho-beta (KLB), cyclin D1 (CCND1), and FGF4 in 151 surgically resected, primary unifocal HCC using quantitative real-time PCR analysis. The correlation of gene amplification and mRNA overexpression for FGF19 and FGF4 was investigated using a real-time PCR based copy number assay. FGF19 amplification was verified using fluorescence in situ hybridization. Univariate and multivariate analyses were performed to evaluate the prognostic value of these biomarkers for tumor recurrence and survival of patients.

Results:

Overexpression of FGF19, FGFR4, KLB, CCND1, and FGF4 mRNA was detected in 40%, 32%, 26%, 15%, and 35% of 151 tumors, respectively. In multivariate analyses, large tumor size (> 12.7 cm) and advanced tumor stage (stage ≥ II) independently predict worse patient survival. Using generalized additive models, we found mRNA expression of FGFR4 and KLB was significantly associated large tumor size (> 12.7 cm); mRNA expression of FGF19 and KLB was significantly associated advanced tumor stage (stage ≥ II). Furthermore, gene amplification of FGF19 and FGF4 significantly correlated to their mRNA overexpression (P = 0.006 for FGF19; P = 0.039 for FGF4).

Conclusions:

Overexpression of FGF19/FGFR4 related biomarkers is frequently found in HCC. Overexpression of FGF19 and FGF4 mRNA significantly correlates to their gene amplification. Expression status of FGFR4, KLB , and FGF19 may determine patient survival through their impact on tumor size (FGFR4 and KLB) and tumor stage (FGF19 and KLB).

#3132

Genome-wide scan for genetic markers modifying survival of pancreatic cancer patients.

Hongwei Tang. _UT MD Anderson Cancer Center, Houston, TX_.

Background: Genetic factors that are associated with pancreatic cancer survival have previously been examined in Genome-Wide Association Studies (GWAS), but the results are inconsistent due to limited sample size.

Method: We performed a two-stage study on pancreatic cancer survival. The discovery phase took advantage of the existing GWAS data of 868 patients with European descendent from a case-control study conducted at MD Anderson. The validation study on ten top hits was conducted in another 820 white patients with Taqman. The missing genotypes were imputed using IMPUTE2 based on 1000 Genomes Project. Cox regression model was fitted to calculate allele-specific hazard ratios (HR) and 95% confidence intervals (95% CI) with adjustment for demographics, tumor stage, resection, and chemotherapy.

Results: After imputation, 7,738,399 SNPs with a Quality Score >0.03, minor allele frequency >0.01, and Hardy-Weinberg equilibrium were obtained for analysis. Three loci in complete LD at chromosome 2 were associated with overall survival (OS) at genome-wide significance (P = 1 ×): rs113988120 in PAIP2B (poly(A) binding protein interacting protein 2B), rs112493246 and rs138529893 in DYSF (dysferlin). The variant allele was associated with 3.02 fold higher risk of dying (95% CI: 2.07-4.40). SNP rs113988120 and nine others were examined in the validation study and rs113988120 remained nominally significant (P = 2.85 ×; HR: 1.45, 95% CI: 1.04-2.03). In the combined datasets of 1688 patients, this SNP was significantly associated with OS (P = 1.46 × and HR = 1.80 (95% CI: 1.42-2.30).

Conclusion: These observations support our hypothesis that genetic variations to a certain degree influence patient survival in pancreatic cancer.

Key words: Pancreatic cancer, GWAS, Survival

#3133

Glypican-1 is a potential marker of prognosis and involved in chemoresistance of cisplatin in esophageal squamous cell cancer.

Takahiko Nishigaki,1 Tsuyoshi Takahashi,1 Satishi Serada,2 Minoru Fujimoto,2 Hisashi Hara,1 Yasuhiro Miyazaki,1 Tomoki Makino,1 Yukinori Kurokawa,1 Makoto Yamasaki,1 Kiyokazu Nakajima,1 Shuji Takiguchi,1 Masaki Mori,1 Yuichiro Doki,1 Tetsuji Naka2. 1 _Department of Surgery,Osaka University Graduate School of Medicine, Suita, Japan;_ 2 _Laboratory for Immune Signal, Nation Institute of Biomedical Innovation, Ibaraki, Japan_.

[Introduction]Despite recent improvements in multi-treatment approaches, including surgery, radiotherapy and chemotherapy, the prognosis for patients with Esophageal squamous cell cancer (ESCC) remains unsatisfactory. In addition, predicting a prognosis and treatment response for ESCC had been constantly being required but remains difficult, even when using the TNM system. We identified Glypican-1(GPC1) as a novel disease specific antigen of ESCC by quantitative proteomic analysis focused on cell surface membrane proteins. GPC1 is one of the cell-surface heparan sulfate proteoglycans which has been reported as a prognostic factor in pancreatic cancer.

[Objectives]This study aimed to clarify correlations between GPC1 expression and the prognosis in patient with ESCC and also reveal the association of GPC1 with the drug resistance to chemotherapeutic agents in squamous cancer cell lines and esophageal cancer patients treated with chemotherapy.

[Methods]ESCC tissues were obtained from 175 patients who underwent esophagectomy at Osaka University Hospital(Osaka, Japan), from 2001 to 2013 and were subjected to immunohistochemistry. We performed immunohistochemical (IHC) assessment using anti-GPC1 antibody and investigated the association between GPC1 expression and the clinicopathological parameters. We divided the patients into two groups such as high expression group (HG) and low expression group (LG). Furthermore, we investigated associations between GPC1 expression and clinical response of neo-adjuvant chemotherapy for patients with ESCC. We established GPC1 knocked out cells of TE-14 using Crisper-Cas9 system and stable GPC1-transfected cells of LK-2(Lung squamous cell carcinoma cell line) using pcDNA3.1-GPC1 plasmid transfection. To investigate the relationship between GPC1 expression and sensitivity to cisplatin (CDDP), we examined sensitivity to CDDP by the WST-8 assay.

[Results]The patients characteristics as follows, age (median)=64yrs(39-80), gender (Male: Female)= 155:20. By the IHC analysis, 99 patients were classified into HG and 76 were LG. HG patients exhibited a poorer prognosis compared with the LG patients. (p<0.001) In multivariate analyses pT, pN, and GPC1 high expression were became significant prognostic factors (p<0.01).

We clarified there was a significant negative correlation between histological responses and GPC1 expression in patient with neoadjuvant chemo therapy. Furthermore, in vitro experiments showed that the IC50 values for CDDP of GPC1 expressing cells (TE-14 and GPC1 transfected LK-2) were significantly higher than that of GPC1 no-expressing cells (GPC1 knocked out TE-14 and LK-2).

[Conclusion]Our study showed that GPC1 was an independent prognostic factor in esophageal cancer. Furthermore, we showed that GPC1 was a critical molecule for chemoresistance to cisplatin.

#3133A

Circulating tumor DNA as "liquid biopsy" in pediatric osteosarcoma.

Michael Fremed,1 Sajida Piperdi,2 Wendong Zhang,1 Shahina Maqbool,1 Brent Calder,1 Raquel Castellanos,1 Jonathan Gill,3 Michael Roth,3 Bang Hoang,4 David Geller,4 Richard Gorlick,3 Daniel Weiser3. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Montefiore Medical Center, Bronx, NY;_ 3 _Children's Hospital at Montefiore, Albert Einstein College of Medicine, Bronx, NY;_ 4 _Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY_.

BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumor and children and adolescents represent half of new cases diagnosed each year. During and after treatment there is no non-invasive test to assess disease response and early relapse, respectively. Monitoring of circulating tumor material as a "liquid biopsy" has potential to provide this critical information in OS, as it has proven useful in other solid tumors. We hypothesized that circulating tumor DNA (ctDNA) can be extracted from mouse plasma and identified using next generation sequencing. This could ultimately be used to assess tumor burden, evaluate response to treatment, and monitor for recurrence in OS. We overcome the challenge of identifying tumor DNA in a background of host DNA by first using primary tumor material to identify genomic aberrations in a targeted gene region known to be altered in >95% of patients with OS. We then use next generation sequencing to identify in circulation the rare tumor DNA based on known aberrations expected to be present when there is a tumor burden.

MATERIALS AND METHODS: Human osteosarcoma cell lines 143b and M17 were grown in culture and introduced into SCID mice via tail vein, subcutaneous flank, and tibial plateau injections. Terminal bleeds were performed 30 minutes after tail vein injection and at max tumor growth respectively. DNA was extracted from primary tumor cell lines as well as from the plasma of injected mice. We then performed next generation sequencing (NGS) with the Illumina HiSeq 2000 using custom designed probes capturing genes commonly altered in osteosarcoma including TP53, RB1, ATRX, DLG2, MET, PTEN, and SLC19A1. DNA extracted from the plasma of a SCID mouse that did not receive tumor cell injection was used as a negative control.

RESULTS: Gene coverage of approximately 80% was obtained for targeted genes to a depth ranging from 250x to 2000x coverage for tumor cell lines. Cell free circulating tumor DNA was identified in plasma of mice injected with 143b mice via tail vein and flank injections but not from tumor cell free plasma negative control. Over 1000 mutations were identified, most notably c.467G>C p.R156P, a well documented osteosarcoma mutation, which was present in the 143b cell line as well as the DNA extracted from the plasma of tail vein and flank injected mice.

CONCLUSIONS: Circulating cell free tumor DNA can be successfully extracted from SCID mouse plasma and identified using next generation sequencing of target genes. Based on these mouse findings, we anticipate that this will serve as a non-invasive biomarker of disease burden and response to therapy, as well as a biomarker to assess recurrence. The pilot work provides rationale to expand these findings into clinical trials that can prospectively validate our methods and findings. Our approach has the potential to improve outcomes for a childhood cancer frequently associated with poor survival.

### Circulating Biomarkers 2

#3134

Prediction of early recurrence after resection of metastatic liver tumors from colorectal cancer using circulating cell-free DNA.

Takuma Iwai,1 Takeshi Yamada,1 Hayato Kan,1 Michihiro Koizumi,1 Seiichi Shinji,1 Yasuyuki Yokoyama,1 Goro Takahashi,1 Shiro Kitano,2 Masato Nakayama,2 Zenya Naito,1 Keiichiro Ohta,1 Eiji Uchida1. 1 _Nippon Medical School, Tokyo, Japan;_ 2 _Toppan Printing Co. Ltd., Saitama City, Japan_.

Background: We have reported that the amount of total circulating cell-free DNA (ccfDNA) increases with tumor growth and decreases upon tumor shrinkage. However, adverse effects of drugs and surgical stress can increase total ccfDNA because ccfDNA is derived from both normal and cancer cells. Thus, the ability to determine how much ccfDNA is derived from cancer cells is a critical issue. It has been reported that the length of ccfDNA derived from cancer cells is greater than 200 bp while that from normal cells undergoing apoptosis is less than 200 bp. Furthermore, the ratio of ccfDNA to β-globin reflects the amount of mitochondrial DNA derived from normal cells undergoing stress-induced apoptosis. We developed a new biomarker readout, the LINE-1 long fragment (longer than 200 bp) to β-globin ratio (LBR), based on this principle. In this study, we evaluated the clinical utility of the LBR to detect early recurrence of liver metastasis from colorectal cancer after liver resection.

Methods: We enrolled 20 patients who underwent curative liver resection of metastatic liver tumors from colorectal cancer. Total ccfDNA and LBR were measured pre-surgery, and at 1 week and 1 month post-surgery. ccfDNA was purified from 1 mL serum using the QIAamp Circulating Nucleic Acid Kit. Total ccfDNA was measured using Qubit Fluorometer. LINE-1 long fragment and β-globin in ccfDNA were measured using real time PCR. The Ethics Review Committee of our institution approved the study protocol. Written informed consent was obtained from each patient.

Results: We completed 1-year follow-up in 13 of 20 patients. Recurrence was detected in 7 patients and no signs of recurrence were detected in the other 6. Total ccfDNA increased 1 week after surgery in all 13 patients, which could have been caused by surgical stress. Total ccfDNA 1 month after surgery increased in 5 of 7 patients with recurrence. Total ccfDNA 1 month after surgery increased in 3 of 6 patients without recurrence. The 3 patients with increased ccfDNA had post-operative complications or drug-induced liver dysfunction. Notably, LBR increased in the 7 patients with recurrence and decreased in the 6 patients without recurrence 1 month post-surgery.

Conclusion: LBR has potential as a novel biomarker readout for early detection of recurrence after liver resection of metastatic liver tumors from colorectal cancer.

#3135

Prediction of acquired resistance in colorectal cancer patients treated with EGFR blockade by detection of a new KRAS mutation in ccfDNA.

Takeshi Yamada,1 Hayato Kan,1 Takuma Iwai,1 Goro Takahashi,1 Michihiro Koizumi,1 Akihisa Matsuda,1 Seiichi Shinji,1 Yasuyuki Yokoyama,1 Atsushi Tatsguchi,1 Tetsuro Kawagoe,1 Shiro Kitano,2 Masato Nakayama,2 Satoshi Matsumoto,1 Keiichiro Ohta,1 Eiji Uchida1. 1 _Nippon Medical School, Tokyo, Japan;_ 2 _Technical Research Institute, Toppan Printing Co., Tokyo, Japan_.

Background: Oncogenic KRAS mutations can be used to predict a lack of response to epidermal growth factor receptor (EGFR) blockade. KRAS status is usually determined by biopsy from the primary site of colorectal cancer (CRC). However, the genomic profiles of primary tumors and metastases are not always concordant because of intrinsic molecular heterogeneity. Furthermore, chemotherapeutic agents and targeted drugs can alter the tumor molecular landscape. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment involving EGFR blockade. To account for these spatial and temporal changes, the genomic profiles of patients with CRC can be repeatedly evaluated during the course of therapy. In this study, we evaluated the utility of KRAS mutation detection using ctDNA before and during chemotherapy.

Method: Experiment 1: We enrolled 46 metastatic colorectal cancer patients. Before starting chemotherapy, ctDNA was purified from 1 mL serum using the QIAamp circulating nucleic acid kit. We detected nine KRAS (G12A, G12R, G12D, G12C, G12S, G12V, G13D, Q61H, and Q61R) mutations using the Invader method and digital PCR. Experiment 2: Fifteen patients were treated with systemic chemotherapy including EGFR blockade. ctDNA was extracted from these patients every 2 months until disease progression, and the KRAS mutation was detected in nine.

Results: Experiment 1: KRAS mutations in circulating tumor DNA were detected in 88% (14/16) of patients with KRAS mutations in their primary tumor, but in 10% (10/30) of patients without KRAS mutations in their primary tumors. Experiment 2: The response rate was 87% (13/15). In two non-responders, KRAS mutations in ctDNA were detected before chemotherapy. Disease progression was identified in five patients and KRAS mutations in ctDNA were detected in all patients; the five patients with disease progression included the two non-responders in whom KRAS mutations in ctDNA were detected before chemotherapy. However, the genotypes detected after disease progression were different from those detected before chemotherapy (G13D to G12C and Q61R to Q61H). New KRAS mutations occurred in codon 12 in the other three patients in whom no KRAS mutations in ctDNA were detected before chemotherapy. New KRAS mutations in codon 61 disappeared during chemotherapy and disease progression was not detected at that time in two patients.

Discussion: It has been reported in a retrospective study that new KRAS mutations were detected in patients who acquired resistance to EFGR blockade, and many of them were in codon 61. However, new KRAS mutations in codon61 can disappear during treatment involving EGFR blockade. This phenomenon may indicate that new KRAS mutations in ctDNA do not always indicate acquired resistance to chemotherapy involving EGFR blockade.

#3136

Evaluation of HER-2 status by FISH in circulating tumor cells isolated from metastatic gastric cancer patients.

Myoung Shin Kim,1 Eunjoo Hwang,1 Yun Gyu Jeong,1 Hye Seon Lee,1 Ji-hyun Uh,1 Won Suk Lee,2 Woo Sun Kwon,2 Hyun Cheol Chung,2 Cham Han Lee,1 Sung Ho Choi,1 Byung Hee Jeon,1 Sun Young Rha2. 1 _Cytogen, Seoul, Republic of Korea;_ 2 _Yonsei University College of Medicine, Seoul, Republic of Korea_.

Human epidermal growth factor receptor 2 (HER-2) is involved in the pathogenesis and poor outcomes of gastric cancer. Targeted drugs inhibiting HER-2 pathway such as trastuzumab showed benefit in patients with HER2-positive metastatic gastric cancer. Reliable evaluation of HER-2 status is a useful and significant tool for treatment selection in gastric cancer patients. However, tumor biopsy in patients with recurrent and/or metastatic disease is not always possible. Here, we suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative to tumor tissue biopsy. Ten milliliters of blood samples were collected in ACDA tubes from 34 patients with metastatic gastric cancer. The blood samples were divided into two parts: one for immunofluorescent staining and other for culturing. Both samples were processed by size-base filtration using CTC isolation kit (Cytigen, Inc.). CTCs (≥2) were detected in 12 of 35 patients (34.3%, range 2-11). CTC culturing was successful in 30 of 35 cases (85.7%). Cultured CTCs were analyzed for HER-2 amplification by fluorescence in situ hybridization (FISH), and compared to HER-2 status on the primary tumor tissues. The overall concordance of HER2 status was 67% between cultured CTCs and primary tumor tissues. These results suggest that the isolation and culture of CTCs can be a substitute method for tumor tissue biopsy, and may provide clinical applications, including serial blood samplings for the personalized cancer therapy based on their genomic information.

#3137

NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases.

Florence Le Calvez-Kelm,1 Matthieu Foll,1 Magdalena B. Wozniak,1 Geoffroy Durand,1 Priscilia Chopard,1 Maroulio Pertesi,1 Tiffany Delhomme,1 Ivana Holcatova,2 Lenka Foretova,3 Vladimir Janout,4 Eleonora Fabianova,5 Maxime P. Vallée,1 Paul Brennan,1 James D. McKay,1 Graham Byrnes,1 Ghislaine Scélo1. 1 _International Agency for Research on Cancer, LYON, France;_ 2 _Charles University of Prague, First Faculty of Medicine, Institute of Hygiene and Epidemiology, Prague, Czech Republic;_ 3 _Masaryk Memorial Cancer Institute and Medical Faculty of Masaryk University, Brno, Czech Republic;_ 4 _Department of Preventive Medicine, Faculty of Medicine, Palacky University, Olomouc, Czech Republic;_ 5 _Regional Authority of Public Health in Banska Bystrica, Banska Bystrica, Slovakia_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls.

Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (>4ng) and sequencing (>1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value<10-3. We also estimated the detection threshold of our assay using serial dilutions of DNA from SW480 KRAS mutated cell-line (p.G12V) in wild-type DNA.

Results: Sequencing of the serial dilutions of KRAS p.G12V mutated DNA indicated a detection threshold at a minor allele frequency of 0.2%. KRAS mutations in cfDNA were detected in 83 (19.3%) PDAC cases (73 on codon 12; 8 on codon 61; 1 on codon 13; and 1 multiple codons, i.e., similar in proportions as reported in tumor tissue from the International Cancer Genomic Consortium); 3 (2.2%) chronic pancreatitis (all on codon 12); and 8 (2.1%) healthy controls (4 on codon 12 and 4 on codon 61). Stage was significantly associated with the proportion of detected mutations in cancer cases (chi-squared p=0.0005): the proportions of cases with detectable KRAS mutations in plasma were 7.9%, 14.9%, and 31.1% for local, regional, and advanced stages, respectively.

Conclusions: The NGS-based KRAS mutation screening is a sensitive approach to detect low allelic fraction in plasma cfDNA, although its utility for early detection is still limited. However, it has the capacity to identify specific KRAS mutations, which could be useful in a panel of other non-invasive biomarkers.

#3138

Changes in tumor cell-free DNA copy number instability (CNI) predict therapeutic response in metastatic cancers.

Glen J. Weiss,1 Julia Beck,2 Donald P. Braun,3 Kirsten Bornemann-Kolatzki,2 Heather Barilla,1 Rhiannon Cubello,1 Ashish Sangal,1 Robert P. Whitehead,1 Madappa Kundranda,1 Vivek Khemka,1 Howard B. Urnovitz,2 Ekkehard Schutz2. 1 _Western Regional Medical Center, Cancer Treatment Centers of America, Goodyear, AZ;_ 2 _Chronix Biomedical, Gottingen, Germany;_ 3 _Cancer Treatment Centers of America, Zion, IL_.

Background: Tumor cell-free DNA (cfDNA) has the potential to provide minimally invasive patient specific biomarkers to monitor tumor burden. Gains and losses of chromosomal regions - as a hallmark of cancer - have been detected in plasma as copy number aberrations (CNAs), and for several cancers a relation to tumor size has been reported. Longitudinal observations during anti-cancer therapy have been mostly anecdotal. We measured CNAs changes during treatment by computing a genomic copy number instability index (CNI) of cfDNA to evaluate its potential to predict cytotoxic chemotherapy (chemo) response. Methods: 24 patients (pts) with advanced esophageal cancer (EC; n=2), colorectal cancer (CRC; n=3), non-Hodgkin lymphoma (NHL; n=3), pancreatic ductal adenocarcinomas (PDAC; n=4), and non-small cell lung cancer (NSCLC; n=12) were included and assessable for response. DNA was extracted from pre-treatment plasma samples at baseline and sequentially for up to six cycles of chemotherapy. Copy-numbers were called from shotgun sequencing (Illumina) after mapping and quality filtering reads were counted in 5.5MBp windows (sliding) yielding a read coverage of 24,000-fold per bin. The read counts were transformed into log2 ratios and converted into a score based on Gaussian transformations. Concentration of total cfDNA was determined by digital PCR. Treatment response by imaging was recorded by RECIST 1.1 or EORTC PET/CT criteria. For pts with baseline CNI>40 (representing the 99.99% level for non-cancer healthy controls), CNI change was considered predictive of response or stable disease when there was a reduction of CNI ≥50% relative to baseline. Disease progression was demonstrated when: 1) CNI was ≥50% over nadir and <80% reduction relative to baseline; OR, 2) CNI was ≥5 fold the nadir; OR, 3) CNI demonstrated an absolute increase by 1,000 above baseline CNI. For pts with baseline CNI≤40, CNI change was considered predictive of response or stable disease when CNI remained ≤40, and PD when CNI≥2X above the baseline CNI. CNI was an early predictor of response if these results were observed by ~3-8 weeks prior to imaging or a major change in pt's clinical status. Results: CNI was measured in 124 samples. Median baseline cfDNA was 7,238 cp/mL (range 2,319-91,978) and CNI was 171 (range 26-10,170). In general, NHL and tumors with high lymph node disease burden had highest baseline cfDNA and CNI, whereas overall the CNI was not correlated to the cfDNA content. 22 of 24 pts (91.7%) had CNI changes that were predictive of treatment response. For at least 15 pts, CNI change predicted response ~3-8 weeks prior to scan results demonstrating response or PD. Conclusions: CNI change may serve as predictor (potentially early predictor) of therapeutic response to standard chemo for the investigated cancer types. Highest baseline cfDNA and CNI appear to be present in lymph node predominant disease, suggesting more readily shed DNA into the circulation.

#3139

Liquid biopsy (ctDNA) testing in clinical management of solid cancers: 5-years of experience.

Lucie Benesova,1 Barbora Belsanova,2 Tereza Halkova,2 Jiri Pudil,3 Bohus Bunganic,4 Milos Pesek,5 Bretislav Gal,6 Miroslav Zavoral,4 Miroslav Ryska,7 Marek Minarik2. 1 _Genomac Research Institute, Prague, Czech Republic;_ 2 _Genomac Research Institute Prague, Prague, Czech Republic;_ 3 _Surgical Clinic, 2nd Faculty of Medicine, Charles University and Military University Hospital, Prague, Czech Republic;_ 4 _Department of Internal Medicine, 1st Medical Faculty of Charles University, Military University Hospital, Prague, Czech Republic;_ 5 _Department of Pneumooncology, Faculty Hospital Pilsen, Prague, Czech Republic;_ 6 _Department of Otorhinolaryngology and Head and Neck Surgery, St. Anne's University Hospital, Brno, Czech Republic;_ 7 _3Surgical Clinic, 2nd Faculty of Medicine, Charles University and Military University Hospital, Prague, Czech Republic_.

Background: Detection and profiling of circulating tumor DNA (ctDNA) is an attractive tool for management of cancer patients, in particular for early detection of the relapse after surgery or monitoring of response to systemic therapy. The main advantage is minimal invasivity and applicability to a wide range of solid cancers. Methodologies are based on digital PCR with the limit of detection (LOD) below 0.01% mutated alleles, however, these often require significant amounts of input DNA (10s to 100s of ng). In the present work we demonstrate routine detection and clinical utility of ctDNA in a cohort of 423 patients covering a range of 5 different solid tumors.

Methods: ctDNA is detected by somatic mutations found in primary tumor tissue by applying a specific mutation panel targeted to the tumor tape. Detection was done by denaturing capillary electrophoresis (input DNA at concentrations of 5 pg, LOD 0.03 - 1%). The cohort included samples from 257 colorectal cancer patients (CRC), 97 patients with ductal adenocarcinoma of the pancreas (PDAC), 32 patients with non-small cell lung cancer (NSCLC), 12 patients with gastric adenocarcinoma (GA) and 6 patients with head and neck cancers (HNC). A longitudinal monitoring of ctDNA levels prior to surgery, a week after surgery and at three-month follow-up intervals, was performed in 16 CRC patients. Overall ctDNA detection rate, radicality of resection, disease recurrence, tumor dynamics and survival prognosis were evaluated.

Results: ctDNA rates were at 32% for NSCLC, 31% for CRC, 30% for GA, 25% for PDAC and 25% for HNC. When looking at a subgroup of patients in Stage IV of the disease the rates increased to 53% for NSCLC, 77% for CRC, 50% for GA, 46% for PDAC and 50% for HNC. 14 out 16 CRC patients with R0 resection remained ctDNA negative (88%), two patients dropped out of the study. Follow-up monitoring lead to detection of progression in 9 out of 13 patients (69 %). In 3 patients (23 %), ctDNA positivity preceded standard detection by CT scan. In 5 patients with follow-up exceeding 1 year (5 to 10 sample acquisitions over 15 to 28 month period) the ctDNA levels correlated with the clinical course of the disease (progression/stabilization/remission). There was a borderline statistically significance for prognostic role of ctDNA presence in PDAC patients with 140 days vs. 200 days (P=0,0519, long-rank test) for ctDNA positive and negative, respectively.

Conclusion: We have introduced a ctDNA method to routine management of 5 different solid cancers. Our results indicate clinical utility for resection radicality confirmation as well as early detection of disease progression and tumor dynamics in CRC patients. The same is applicable to approx. 50 % of patients with advanced GA, NSCLC and HNC. CtDNA positivity may also indicate a negative prognosis for PDAC patients. Supported by IGA MZ grant no. NT 13660.

#3140

Differential clonal selection in tumor tissue and cell-free DNA from a neratinib-treated refractory breast cancer patient harboring an activating ERBB2 (HER2) mutation.

Lars Joenson,1 Christina W. Yde,1 Olga Østrup,1 Morten Mau-Sørensen,2 Finn C. Nielsen,1 Ulrik Lassen2. 1 _Center for Genomic Medicine, Rigshospitalet, Copenhagen, Denmark;_ 2 _Department of Oncology, Rigshospitalet, Copenhagen, Denmark_.

The ongoing Copenhagen Prospective Personalized Oncology (CoPPO) research program aims to offer patients with exhausted treatment options, targeted therapy against actionable driver mutations identified in freshly obtained biopsies by whole exome sequencing (WES). Mutations are tracked in circulating cell free DNA (cfDNA) to examine the clonal selection of tumor cells evoked by therapy. Here we report the results from a patient with metastasizing HER2 negative and estrogen receptor (ER) positive breast cancer (Luminal A) previously exposed to seven lines of chemotherapy as well as ER antagonists and aromatase inhibitors. After enrollment in the program, examination of liver metastases by whole exome sequencing, RNA-Seq and microarray expression analysis revealed high expression of the estrogen receptor 1 (ESR1) as well as a mutation in the ligand-binding domain of ESR1 (H524L). Moreover, somatic variants in ERBB2 and PIK3CA were identified, including an activating mutation in ERBB2 (S310Y), and consequently the patient treated with neratinib, an irreversible pan-HER (EGFR/ERBB2/ERBB4) tyrosine kinase inhibitor, through a compassionate use program (Puma Biotechnology Inc.). Neratinib caused a rapid decrease in the allelic frequency of ERBB2 (S310Y) cfDNA after 2 days with a continuous decline during the next 7 days. Consistent with this neratinib treatment effect, MRI scans showed regression of the liver metastases. In contrast, the PIK3CA mutation showed an increase in the allele frequency, indicating the existence of a subclone that was insensitive to neratinib. Two months later, the total amount of cfDNA increased and continued to do so. After 5 months on neratinib, the patient progressed with the appearance of brain metastases which were surgically removed and subject to WES. The vast majority of the mutations, including the ERBB2 mutation, observed in the liver metastasis could not be identified in the brain metastases, except for the PIK3CA mutation. More than 300 new variants were exclusively identified in the brain metastases, among these, ERBB3 as well as new PIK3CA, and ESR1 mutations, that were not present in the pre-treatment cfDNA samples. After 4 months of treatment, an increase in the mutation frequency of the liver metastasis specific ESR1 and ERBB2 mutations was observed. In conclusion, neratinib was able to suppress an activating ERBB2 mutation in a heavily pre-treated ER+ breast cancer patient. However, refractory tumor clones harboring ERBB3, PIK3CA and ESR1 mutations developed in brain. These data indicate that combining neratinib with fulvestrant or inhibitors of the HER3/PI3K/AKT/mTOR pathway might prove beneficial to overcome potential resistance mechanisms to therapy.

The two first authors contributed equally.

#3141

Pro-Seq: A novel method to improve sequencing accuracy for liquid biopsy of ctDNA from healthy individuals and cancer patients.

Andre Marziali. _Univ. of British Columbia, Vancouver, British Columbia, Canada_.

A significant barrier to widespread clinical deployment of sensitive circulating tumor DNA (ctDNA) assays (liquid biopsy) is the high assay cost compared to potential reimbursement. Assay cost is currently dominated by the large amount of DNA sequencing required to achieve coverage of a broad gene panel, and by the high read depth required for high clinical sensitivity. Attaching unique molecular barcodes to ctDNA fragments for the purpose of error reduction further increases sequencing requirements, making liquid biopsy commercialization in many clinical applications impractical.

We present a novel library construction process for NGS sequencing that increases the accuracy of the combined library construction and sequencing process by an order of magnitude. Named Proximity-Sequencing (Pro-Seq), the method duplicates the sequence information in each original DNA strand prior to the bulk of library construction in such a way as to provide redundant, linked templates to the sequencer. The redundant templates remain linked through the library construction process, allowing detection of PCR errors as sequence disagreement between the two strands. The linked templates are amplified in a single sequencing reaction, such that base quality and incorporation information can be used to determine which bases of the sequence were corrupted during PCR amplification steps. Since both strands are amplified as part of the same sequencing read, sequencing accuracy is improved without requiring use of additional reads on the sequencer.

A key element of this process is a novel linked-linear amplification in which DNA primers linked by a short molecule amplify a single strand in the same sense, ensuring twin copies in the same sense that remain physically linked. The method is entirely based on novel reagents and can be implemented without additional instrumentation beyond standard NGS equipment.

The method is expected to have significant utility in any applications that require detection of rare sequence variants, including analysis of cell free DNA for liquid biopsy applications. We demonstrate the ability to achieve sequence accuracy similar to barcoded sequencing methods, without the additional sequencing burden required by such methods.

We present the method using an Illumina platform, and present data from sequencing of bacterial and human cell free DNA that demonstrate error rates that are improved by an order of magnitude over the current state of the art NGS chemistries. The addition of Pro-Seq library construction to NGS assays enables lower cost, high sensitivity, and high specificity liquid biopsy tests to be developed, enabling commercialization in applications with limited reimbursement, potentially including early cancer detection.

#3142

Circulating microRNAs can identify cancer-free women at risk for breast cancer.

Nicholas H. Farina, Jon E. Ramsey, Melissa E. Sands, Tiffany J. Rounds, Janet L. Stein, Gary S. Stein, Jane B. Lian, Marie E. Wood. _University of Vermont College of Medicine, Burlington, VT_.

Introduction: MicroRNAs (miRNAs) are well documented to regulate cancer cell activity by modulating signaling pathways to promote disease onset and progression. Recent evidence correlates miRNAs measured in the serum of breast cancer (BCa) patients to expression in breast tumors. Thus, miRNAs are important biomarkers for stages of tumor development. However, no studies have evaluated the potential of circulating miRNAs (c-miRNAs) as risk biomarkers that predict BCa development years before tumor identification.

Objective: Use global miRNA analysis of serum from at-risk but pre-cancerous women who have subsequently been diagnosed with BCa or remain cancer-free to 1) identify expression levels of miRNAs with known BCa association, 2) define cohorts of c-miRNAs differentiating women later developing cancer from cancer-free women with similar clinical risk, and 3) provide insight into c-miRNA mechanisms that promote BCa development.

Methods: Subjects were identified from a cohort of 571 women enrolled in the High Risk Breast Program at the UVM Cancer Center; 35 diagnosed with BCa since enrollment. Enrollment criteria included no prior cancer and increased BCa risk due to family history, benign breast disease, prior irradiation for Hodgkin's disease, known pathogenic abnormality, and/or > 20% lifetime modeled BCa risk score. Clinical data and serum are collected at study enrollment and subsequent cancer-free 4-year interval visits. 28 BCa cases were matched to controls for risk factor and age (+/- 5 yrs) at serum sampling (DCIS, Stage IV BCa, BRCA1/2 excluded). RNA was isolated from 68 serum samples representing 52 individual women following a standardized protocol (Farina et al J Cell Biochem 2014) and over 2500 mature human miRNAs profiled on Affymetrix microRNA v4.0 microarrays. Principal component analyses (PCA), hierarchical clustering, differential expression testing, gene ontology, and biological pathway analyses were performed.

Results: Our analyses show that women who developed BCa (largely hormone receptor positive, HER2 and lymph node negative) share similar patterns of miRNA expression that differ from women who remain cancer-free. PCA, based on a subset of dynamic miRNAs, segregates serum samples into 3 groups: cancer, cancer-free, and overlapped. Several miRNAs consistently cluster together when cases are contrasted against controls suggesting a functional relationship in providing an environment ideal for BCa development. Pathway analyses of miRNA gene targets reveal enriched PDGF, Wnt, Ras, and MAPK signaling pathways.

Conclusion: These data suggest that a cohort of dynamic c-miRNAs discriminates at-risk women who will develop BCa from those who will remain cancer-free. While we consider these findings preliminary until a larger sample set is screened, we conclude that circulating miRNAs will become valuable biomarkers of BCa risk.

#3143

Deciphering mechanisms of circulating tumor cells in breast cancer dormancy.

Debasish Boral,1 Haowen N. Liu,1 Wei Yin,1 Monika Vishnoi,1 Antonio Scamardo,2 Goldy C. George,2 David S. Hong,2 Dario Marchetti1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

More than 67% of deaths in breast cancer patients occur after the initial 5-year survival period while residual disease can be dormant for periods longer than 20 years. Patients are asymptomatic because circulating tumor cells (CTCs) remain dormant and are undetectable by current clinical tools. Dormant CTCs may retain their long-term tumor-initiating (LTI) potential by adhering to their original genome, unlike rapidly cycling cancer cells that are known to have increased genomic instability. We hypothesized that hyperactive mechanisms of DNA repair preserve the genomic make-up of dormant CTCs allowing them to retain their LTI potential, ultimately causing disease relapse.

We isolated and characterized EpCAM-negative breast cancer CTCs by mutiparametric flow cytometry and DEPArrayTM. Individually isolated breast cancer CTCs had a large proportion (>40%) of dormant (Ki67-/PCNA-) cells. Dormant CTCs had a lower incidence of double-strand DNA breaks (DSB) than proliferating cells as assessed by the phosphorylation status of Serine139 on gamma H2AX. This observation was further validated in a panel of eight genetically distinct breast cancer cell lines. Second, to understand whether dormant cells are inherently more resistant to DSB, we induced DSB in breast cancer cells by UV radiation and bleomycin treatment, and measured residual DSB at regular intervals. Results showed that besides being more resistant to DSB de novo, dormant breast cancer cells were also more efficient in repairing their DNA. There are two distinct phases of DSB repair - early [within 2 hours of DSB using Non-Homologous End Joining (NHEJ) methods] and late [evident after 24 hours using Homologous Recombination (HR)]. Unlike proliferating (S-G2M) cells, dormant (G0) cells lack the sister chromatid and repair their DNA exclusively by NHEJ methods. Therefore, and third, we investigated key players of the NHEJ pathway and examined their roles in maintaining genomic integrity. We found that the human telomere-associated protein RIF1, a mediator of alternative NHEJ, was significantly up-regulated in a dormant CTC subset. Dormant sub-populations of breast cancer cells confirmed RIF1 foci formation in areas of DNA damage. Fourth, mis-sense mutation of RIF1 in CAMA-1 cells (ΔRIF1 E1598K) attenuated resistance of the dormant subset to UV and bleomycin treatment.

Collectively, these findings suggest that RIF1 may play functional roles in maintaining the genomic integrity of dormant CTCs. Further investigations are being pursued to assess RIF1 contributions to retain CTC LTI potential leading to CTC-driven metastasis.

#3144

Detection of HER2-HER3 heterodimers in patient circulating tumor cells using proximity ligation assay.

Hatem Soliman, Fatema Khambati. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

BACKGROUND: The HER2 protein is a key oncogene in approximately 20% of breast cancers, and preferentially forms heterodimers with other members of the HER family (such as HER3) to activate its signaling cascade. Dual HER2 signaling blockade by using both trastuzumab and pertuzumab (a HER2 dimerization inhibiting monoclonal antibody) yields superior clinical benefit in HER2+ patients compared to trastuzumab monotherapy. A phase 1/2 clinical trial is ongoing to assess the safety and efficacy of the combination of gemcitabine with trastuzumab and pertuzumab in metastatic HER2+ pretreated patients. (NCT02139358) One of the correlative experiments is looking at the feasibility of detecting HER2-HER3 heterodimers using a proximity ligation assay on circulating tumor cells isolated from patients on treatment as a possible pharmcodynamic marker.

METHODS: Whole blood was collected in two 10cc Cellsave tubes (Veridex, Cat#: 7900005) at baseline, week 4, 7, 10. Samples were drawn before treatment was administered, to avoid contamination of the samples with cytotoxic agents. The blood was subjected to red cell lysis with 1XRBC lysis buffer (Santa Cruz). The lysed cells were fixed with 4% paraformaldehyde followed by negative selection utilizing CD45+ magnetic beads (EasySep CD45 depletion kit, cat#18259). The CD45 depleted cell suspension was re-fixed with 4% PFA on coated glass slides following cytospin. These cells were then stained for PLA to test for HER2-HER3 using Sigma Aldrich DuoLink PLA kit + DAPI fluorescence mounting medium (DUO92013) with anti-HER2 (PA5, Thermo Scientific, diluted 1:50) and anti-HER3 (MA5-12675, Thermo Scientific diluted 1:50) as primary antibodies plus cytokeratin (CK) and CD45 antibodies. A set of HER2+ and HER2- PLA controls using SKBR3 were included in the analysis. Control samples using SKBR3 cells were used to optimize the method.

RESULTS: Using the spiked SKBR3 HER2+ blood samples it was possible to isolate CK+ CD45- DAPI+ CTCs demonstrating HER2-HER3 heterodimers following optimization of the PLA method. Using this method in the first four patients we successfully collected CTCs in 7/11 samples processed. In those 7 CTC+ samples, 4 contained CTCs with positive HER2-HER3 PLA signals.

CONCLUSIONS: We have demonstrated a feasible methodology for the detection of HER2-HER3 heterodimers on the surface of circulating tumor cells from HER2+ metastatic breast cancer patients. This can provide investigators with a non-invasive method to monitor the pharmacodynamics of pertuzumab therapy on tumor cells. The collection of these samples is ongoing and correlation with benefit from therapy will be analyzed in the phase 2 portion of the study.

#3145

Clinical evaluation of streck cell-free DNA blood collection tubes for liquid profiling in oncology.

Inga Medina Diaz,1 Stefan Holdenrieder,2 Annette Nocon,1 Makbule Kobilay,2 Dirk Skowasch,3 Stefanie Held,4 Johanna Weiland,1 Claudia Stamm,1 Frank Diehl,1 Frank Holtrup1. 1 _Sysmex Inostics GmbH, Hamburg, Germany;_ 2 _Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany;_ 3 _Medical Clinic II, Pneumology, University Hospital Bonn, Bonn, Germany;_ 4 _Medical Clinic III, University Hospital Bonn, Bonn, Germany_.

Liquid profiling based on circulating tumor DNA (ctDNA) extracted from blood has become a powerful diagnostic approach in oncology. Making ctDNA analysis broadly available requires shipping whole blood to a clinical laboratory while ensuring cell-free DNA (cfDNA) and blood cell integrity to prevent dilution of ctDNA with cellular genomic DNA. Since standard EDTA blood collection tubes cannot retain this integrity for a prolonged time, several laboratories have proposed Streck Cell-Free DNA blood collection tubes (Streck cfDNA BCTs) as an alternative for sample collection.

Qualification data for the use of Streck cfDNA BCTs in oncology is limited and mainly based on blood collected from healthy individuals as well as data extrapolated from the prenatal field. However, the data generated from these samples may not represent the unique dynamics of clinical oncology specimens and therefore a study of true clinical oncology samples is required to support the use of Streck cfDNA BCT tubes in routine practice.

In this study, we performed a clinical evaluation of cfDNA sample integrity from matched Streck cfDNA BCTs vs standard EDTA tubes from colorectal, pancreatic and non-small cell lung cancer patients (N > 50). Blood drawn into Streck cfDNA BCTs was either processed immediately or 3 days after phlebotomy. DNA quantification was followed by BEAMing digital PCR (OncoBEAM™) on KRAS, NRAS and EGFR mutations and compared to matching specimens collected in EDTA tubes.

Our results suggest that cfDNA yield as well as the genomic DNA background is not affected by prolonged storage of clinical samples in Streck cfDNA BCTs for up to 3 days. In all sample sets containing mutated ctDNA, the detected mutational load was comparable between Streck cfDNA BCTs and EDTA tubes.

In conclusion, this study represents a comprehensive clinical evaluation of Streck cfDNA BCTs vs EDTA tubes for ctDNA profiling. In conjunction with the findings of our previously presented Streck cfDNA BCT shipping condition studies, our data supports the compatibility of clinical oncology specimens collected in Streck cfDNA BCTs with the BEAMing technology.

#3146

Circulating tumor DNA assay performance for detection and monitoring of KRAS mutations in urine from patients with advanced cancers.

Takeo Fujii,1 Cecile Rose T. Vibat,2 Daniel D. Karp,1 Sarina A. Piha-Paul,1 Vivek Subbiah,1 Apostolia M. Tsimberidou,1 Siquing Fu,1 David S. Hong,1 Helen J. Huang,1 Kiran Madwani,1 Debra L. Andrews,1 Saege Hancock,2 Aung Naing,1 Rajyalakshmi Luthra,1 Bryan K. Kee,1 Scott Kopetz,1 Mark G. Erlander,2 Vlada Melnikova,2 Funda Meric-Bernstam,1 Filip Janku1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Trovagen, San Diego, CA_.

Introduction: Non-invasive urinary ctDNA-based liquid biopsy approach can be used to detect and track cancer driver mutations for rapid diagnosis and disease monitoring. Using highly sensitivity ctDNA mutation detection platform, we examined the detection of KRAS G12/13 mutations in urine obtained from advanced cancer patients, assessed urine sample requirements, and compared the results with matched tumor tissue in patients with advanced cancers.

Methods: 41 patients with advanced solid cancer with KRAS mutations on archival tumor from CLIA laboratory testing were prospectively enrolled with informed consent (colorectal cancer, n=29; non-small cell lung cancer, n=6; pancreatic cancer, n=2; ovarian cancer, n=2; other, n=2). Urine was collected before and during experimental therapies. Urinary DNA was isolated using a method that enriches for highly fragmented, systemically derived cell-free DNA. KRAS G12/13 analysis was performed using mutation enrichment PCR coupled with next generation sequencing (MiSeq). Analytical sensitivity of the KRAS G12/13 assay is 0.006% mutant alleles in the background of 60 ng wild-type (wt) DNA and 0.002% mutant alleles in 360 ng wt DNA. Clinical data was collected retrospectively from the electronic medical record.

Results: For 41 patients enrolled on a study, urine volumes in pretreatment samples ranged from 13 to 120 mL (median, 55 mL). Urinary DNA yields were 151 to 23059 ng (median, 1039 ng). Using tissue as the reference, the positive percent agreement (PPA) between urine and tumor KRAS G12/13 test results was 54% (22/41) for urine samples with all volumes (13-120 mL) and any DNA input amount (2-360 ng) and 92% (12/13) for urine samples with volumes ≥50 mL and DNA input amount ≥60 ng. For metastatic CRC patient cohort, the PPA between urine and tumor KRAS G12/13 test result was 60% (18/30) for urine samples with all volumes and any DNA input amount (20-120 mL, 2-360 ng) and 100% (10/10) for urine samples with volumes ≥50 mL and DNA input amount ≥60 ng. Feasibility of longitudinal monitoring KRAS G12/13 mutational burden in urine of patients treated with experimental therapies was demonstrated.

Conclusion: KRAS G12/13 mutational status can be assess in urinary DNA with highest PPA amongst patients with urine volume ≥50 mL and DNA input amount ≥60 ng (92%). KRAS mutation detection from urine should be considered as a viable approach, particularly when tumor tissue is not available.

#3147

Identification of developmental endothelial locus-1 on circulating extracellular vesicles as a novel biomarker for early breast cancer detection.

Pyong-Gon P. Moon,1 Jeong-Eun Lee,1 Young-Eun Cho,1 Soo Jung Lee,2 Jin Hyang Jung,3 Yee Soo Chae,2 Han-Ik Bae,4 Young-Bum Kim,5 In-San Kim,6 Hoyong Park,3 Moon-Chang Baek1. 1 _Kyungpook National University School of Medicine, Daegu, Republic of Korea;_ 2 _Department of Oncology/Hematology, Kyungpook National University Hospital, Daegu, Republic of Korea;_ 3 _Department of Breast & Thyroid Surgery, Kyungpook National University Hospital, Daegu, Republic of Korea; _4 _Department of Pathology, Kyungpook National University Hospital, Daegu, Republic of Korea;_ 5 _Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA;_ 6 _Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea_.

Purpose: Currently, there are no molecular biomarkers for the early detection of breast cancer (BC). This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage BC.

Experimental Design: Circulating EVs, isolated from the plasma of 10 patients with BC (stages I and II), and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different enzyme-linked immunosorbent assays (ELISAs) were used to measure Del-1 in plasma samples from healthy controls (n = 81), patients with BC (n = 269), BC patients after surgical resection (n = 50), patients with benign breast tumors (n = 64), and patients with non-cancerous diseases (n = 98), in two cohorts.

Results: Plasma Del-1 levels were significantly higher (P < 0.0001) in patients with BC than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was area under the curve (AUC), 0.961 [95% CI, 0.924-0.983], sensitivity of 94.70% and specificity of 86.36% in test cohort, and 0.968 [0.933-0.988], 92.31% and 86.62% in validation cohort for early-stage BC by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage BC using the other type of ELISA (0.946 [0.905-0.972], 90.90%, and 77.14% in the test cohort; 0.943 [0.900-0.971], 89.23%, and 80.99% in the validation cohort).

Conclusions: Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage BC and distinguish BC from benign breast tumors and non-cancerous diseases.

#3148

Clinical validation of a next-generation sequencing assay specifically for blood-drop liquid biopsy.

Chen-Hsiung Yeh,1 Jonathan Spurgin,1 Andrew Ford,1 John Athanasuleas,1 Upender Manne2. 1 _Circulogene Theranostics, Homewood, AL;_ 2 _Department of Pathology, University of Alabama at Birmingham, Birmingham, AL_.

Next-generation sequencing (NGS) technology enables rapid analysis and turnaround of multiple genes for clinically actionable somatic variants. Genetic sequencing on tumor biopsy tissue to inform treatment decisions is the hallmark of precision medicine. However, a one-time, single-site tissue sample may not be a true representation of tumor dynamic profile. A liquid biopsy, based on circulating cell-free DNA (cfDNA), can capture the entire heterogeneity of the disease, and offer what tissue biopsies can't, the opportunity to take serial samples in order to monitor tumor genomic evolution in real time. We have recently developed a proprietary cfDNA enrichment process that requires only droplet volumes of blood. Here, we presented clinical validation of the blood-drop liquid biopsy NGS assay interrogating 2855 mutation hotspots in 50 cancer-associated genes using the AmpliSeq cancer panel and Ion Torrent Proton sequencer.

One nanogram of cfDNA prepared from 20 uL of plasma/serum was used as template to amplify 207 mutation hotspot amplicons. Deep sequencing of total 95 clinical specimens (≥ 1000× average coverage across the capture regions) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). The variants at AF of ≥ 10% in most samples (90/95), except 5 cases, were confirmed by another laboratory. Repeated sequencing of 15 samples, and progressively diluted samples from two reference standard DNAs with known mutations and AF demonstrated reliable detection sensitivity at 1% for most single nucleotide variants (SNV) with high within-run and between-run reproducibility. Validation also revealed sensitivity and specificity for detecting variants at 5% AF was 100% (95% CI = 95.7-100.0) using Tru-Q 4 NGS reference standard DNA, and NA19240 or NA12878 HapMap individual, respectively.

Overall, our new-generation liquid biopsy NGS assay, with the capability to multiplex and simultaneously sequence multiple patient samples using finger-stick blood, is a robust and sensitive method for mutation analysis of clinical significance, further expedite treatment decision-making and identify targeted therapies for eligible patients in a time- and cost-efficient manner.

#3149

Enumeration and mutational profiling of CTCs and comparison to ctDNA and colorectal cancer liver metastases.

Evelyn Kidess-Sigal,1 Haiyan E. Liu,2 Melanie Triboulet,1 James Che,3 Georges A. Poultsides,1 Brendan C. Visser,1 Andre Marziali,4 Marc Lee,4 Valentina Vysotskaia,4 Matthew Wiggin,4 Vishnu C. Ramani,1 Ulrich Keilholz,5 Ingeborg Tinhofer,5 Amin Zia,6 John Coller,6 Jeffrey A. Norton,1 Elodie Sollier,2 Stefanie S. Jeffrey1. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _Vortex Biosciences Inc, Menlo Park, CA;_ 3 _UCLA Bioengineering, Los Angeles, CA;_ 4 _Boreal Genomics, Vancouver, British Columbia, Canada;_ 5 _Charite University Hospital, Berlin, Germany;_ 6 _Stanford University, Stanford, CA_.

Background

Colorectal cancer (CRC) is the 3rd most common cancer diagnosed worldwide in both men and women. Only 39% of cancers are diagnosed at a localized stage, and 5-year survival rates decrease rapidly for patients with advanced and metastasized disease (stage III 61%, stage IV 8%). Better markers for detection of disease progression, therapeutic resistance and minimal residual disease are still needed. Liquid biopsies, such as CTCs and ctDNA, are emerging biomarkers shed by the tumor into the blood stream. Both markers currently are attracting growing interest for their use in disease prognosis, early detection of recurrence and are promising candidates for guiding cancer therapy in real-time.

Method

For rapid label-free isolation of CTCs from peripheral blood we used the Vortex technology, a microfluidic device using inertia and laminar microvortices. From 15 patients with metastatic CRC to the liver that underwent liver metastatectomy with curative intent, we collected CTCs preoperatively, at the 5th postoperative day and during follow-up visits. Cells collected were immunostained for EpCAM, CD45 and DAPI, enumerated using standardized classification criteria, and subjected to Sanger sequencing. CTC enumeration and mutational patterns were compared to the primary tumor, liver metastases and ctDNA (detected by a multiplexed PCR and enrichment technology; Kidess E et al., 2015) as well as CEA levels when available.

Results

41 blood samples from 15 patients were collected at different time points prior to and after surgical resection of liver metastases. More CTCs were found in preoperatively collected CRC patient samples (2.4 CTCs/mL, 0.1 - 5.5/mL) than in age-matched healthy controls (0.1 CTCs/mL, 0 - 0.4/mL). 80% of all CRC samples were identified as positive for CTCs (based on a calculated threshold from healthy controls), with varying levels of EpCAM expression (81.4% of CTCs being EpCAM+). The number of CTCs for each patient, showed a close correlation to clinical parameters and ctDNA levels: detection of CTCs, CTC mutational profiles as well as ctDNA revealed minimal residual disease and anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging. For example, for P006, postoperative imaging surveillance revealed progressive disease, which was accompanied by rising levels of CTCs (up to 29 CTCs/mL at the last time point) and PIK3CA mutant DNA in both plasma ctDNA and CTC DNA, while CEA remained in the normal range.

Conclusion

Our data illustrate that CTCs as well as ctDNA can efficiently reveal disease recurrence as well as disease progression earlier than imaging and far more reliable compared to CEA, the currently standard biomarker for CRC. Beyond enumeration, CTC molecular analysis gives additional information and will potentially help to promote the development of tailored therapies for every individual patient.

#3150

Analysis of the extracellular vesicles content present in the plasma of patients with head and neck squamous cell carcinoma for identification of molecular markers for treatment response.

Dorival Mendes Rodrigues-Junior,1 Soon Sim Tan,2 Sai Kiang Lim,2 N Gopalakrishna Iyer,3 Andre Luiz Vettore1. 1 _Federal Univ. of São Paulo, São Paulo, Brazil;_ 2 _Institute of Medical Biology, Singapore, Singapore;_ 3 _National Cancer Centre of Singapore, Singapore_.

Previous data showed that induction chemotherapy (IC) with cisplatin and paclitaxel followed by chemoradiotherapy (CRT) based on cisplatin is safe, well tolerated and improves overall survival for patients with locally advanced head and neck squamous cell carcinoma (HNSCC) (stage III -IV). However, approximately 30% of these patients have incomplete response to IC and CRT and there is no clinical marker able to segregate these patients from those that respond to the treatment. The recent discovery of extracellular vesicles (EVs) in different body fluids carrying proteins and nucleic acids may indicate an alternative route for new specific markers present in the plasma of patients with HNSCC. In this study, Cholera toxin B chain (CTB) and Annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine, were used to isolate EVs from plasma of HNSCC patients that respond and not respond to IC and CRT. The proteins in the vesicles were identified using antibody array through a pool of 6 responders' patients (R) in comparison to 6 patients with incomplete response (IR). A total of 327 proteins were identified into CTB-EVs, from which 54 and 20 proteins were exclusive to R and IR patients, respectively. Beside this, 271 proteins were identified into AV-EVs, from which 20 proteins were present in R patients and other 20 proteins were present only in IR ones. A preliminary analysis of this data revealed that HNSCC resistant patients have elevated levels of TGFβ3 in CTB-EVs while patients with incomplete response have elevated levels of HSP90AB1 in AV-EVs. This study demonstrated the successful use of CTB and AV to isolate circulating plasma EVs for biomarker discovery in HNSCC and represents a novel technology that can be applied to segregate patients that may respond or not to IC or CRT.

#3151

Genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients.

Costanza Paoletti,1 Andi K. Cani,1 Kimberly Aung,1 Elizabeth P. Darga,1 Emily M. Cannell,1 Daniel H. Hovelson,1 Maryam Yazdani,2 Allen R. Blevins,2 Nahomi Tokudome,1 Paul J. Baratta,1 Jose' M. Larios,1 Dafydd G. Thomas,1 Martha E. Brown,1 Christina Gersch,1 Anne F. Schott,1 Daniel Robinson,1 Arul M. Chinnaiyan,1 Farideh Bischoff,2 Daniel F. Hayes,1 James M. Rae,1 Scott A. Tomlins1. 1 _University of Michigan Comprehensive Cancer Center (UM CCC), Ann Arbor, MI;_ 2 _Silicon Biosystems, Inc., San Diego, CA_.

Introduction: Cancer-associated mutations are present in circulating cell free plasma tumor DNA (ptDNA). We have previously reported mutation profiles of DNA extracted from CTC (CTC-DNA) from two patients with MBC (#2 and 24 in table). Here, we report an expanded cohort with an updated gene panel.

Methods: We studied seven patients (two previously reported, along with five additional patients) with MBC who were enrolled in Mi-CTC-ONCOSEQ, had ≥5 CTC/7.5 ml whole blood (WB), and had at least one CTC with high quality DNA determined by the Ampli1™ quality control kit. CTC were enriched from WB with CellSearch© and purified from white blood cells (WBC) (DEPArray™). DNA from individual CTC and WBC was isolated and subjected to whole genomic amplification (Ampli 1™ WGA) and genotyped by multiplexed PCR-based next generation sequencing with the Oncomine Comprehensive Panel (OCP) on the Ion Torrent Proton. Exome sequencing of research biopsies of metastatic tissue was performed using an Illumina HiSeq 2500 platform. Previously reported patients (#2 and 24) sequenced with a beta version of the OCP were re-run, and updated results are provided.

Results: Six of seven patients were ER positive. Patients #2, 12, and 24 had CTC with mutations also found in the research biopsy (table). Novel alterations were found in comparison to research biopsy in five of the seven patients (table). In two patients (#19, 24), two potential actionable mutations (PTCH1 and NOTCH1) were found in CTC-DNA but not in tissue-DNA. No mutations were detected in any WBC.

Conclusions: We demonstrate the ability to purify CTC, and to isolate and amplify DNA of suitable quality for genetic analysis using a comprehensive targeted sequencing panel. Mutations found in tissue as well as novel mutations were found in CTC-DNA. Two potential actionable mutations were identified in CTC, but not in tissue, opening potentially new therapeutic opportunities. We conclude that mutational analysis of CTC-DNA and of tissue may be complementary.

Prioritized mutations in CTCs

---

Pt # | Gene (Mutation) | # CTC

Single (S)

Pooled (P) | # with mutation (variant fraction) | # without mutation | # not evaluable (insufficient coverage) | # WBC (all pooled) | # with mutation | Present in Biopsy?

2 | CDH1 (p.Q641X) | 7 (S) | 5 (1.00) | NA | 2 | 3 | 0 | Y

CDH1 (S70F) | 7 (S) | 5 (1.00) | NA | 2 | 3 | 0 | Y

ESR1 (p.Y537S) | 7 (S) | 4 (0.46) | 2 | 1 | 3 | 0 | Y

ESR1 (unreported mutation) | 7 (S) | 1 (0.56) | 5 | 1 | 3 | 0 | N

8 | NA | 3 (P)* | NA | NA | 3 (P) | 4 | 0 | NA

12 | PIK3CA (H1047R) | 1 (S) | 1 (0.85) | NA | NA | 1 | 0 | Y

TP53 (p.R248Q) | 1 (S) | 1 (0.72) | NA | NA | 1 | 0 | Y

14 | HNF1A (p.W206C) | 3 (P) | \+ (0.17) | NA | NA | 4 | 0 | N

17 | BRCA2 (p.Q1931X) | 4 (P) | \+ (0.10) | NA | NA | 4 | 0 | N

19 | PTCH1 (p.E1242X) | 3 (P) | \+ (0.28) | NA | NA | 3 | 0 | N

24 | CDH1 (p.I584fs) | 5 (P)

4 (P)

4 (P) | \+ (0.79)

\+ (0.68)

\+ (0.77) | NA | NA | 4 | 0 | Y

CDH1 (p.E841X) | 5 (P)

4 (P)

4 (P) | 0

\+ (0.14)

0 | NA | NA | 4 | 0 | N

TP53 (p.152_156del) | 5 (P)

4 (P)

4 (P) | \+ (0.94)

\+ (0.29)

\+ (0.36) | NA | NA | 4 | 0 | Y

NOTCH1 (p.S2492X) | 5 (P)

4 (P)

4 (P) | 0

\+ (0.17)

0 | NA | NA | 4 | 0 | N

Legend: NA = not applicable; + = mutation present in pooled CTC; Y = Yes; N = No; *CTC-DNA from the pool of 3 CTC had low and high quality.

#3152

Breast cancer-derived extracellular vesicles: clinical and prognostic impact.

Lisa König,1 Vera Rebmann,2 Oliver Hoffmann,1 Ann-Kathrin Bittner,1 Bettina Wagner,2 Luis Felipe Santos Manvailer,2 Sabine Schramm,2 Agnes Bankfalvi,3 Bernd Giebel,2 Rainer Kimmig,1 Peter A. Horn,2 Sabine Kasimir-Bauer1. 1 _Department for Gynecology and Obstetrics, University Hospital Essen, Essen, Germany;_ 2 _Institute of Transfusion Medicine, University Hospital Essen, Essen, Germany;_ 3 _Institute for Pathology, University Hospital Essen, Essen, Germany_.

Background: Extracellular vesicles (EVs) are released from cancer cells into the tumor microenvironment, there participating in intercellular communication by altering recipient cell function. Breast cancer (BC) derived EVs are hypothesized to have an impact on tumor growth, immunosuppression or metastatic development, indicating analytical significance of EV concentrations in BC patients. For this reason, we investigated plasmatic EV numbers from locally advanced, neoadjuvant treated (NACT) BC patients in association with clinical parameters and prognostic impact.

Material and methods: Plasmatic extracellular vesicles were isolated using ExoQuick™ precipitation reagent (SBI Inc., Mountain View, CA, USA), according to manufacturer's instructions, from locally advanced BC patients before (n=142) and after (n=156) NACT as well as from healthy female controls (n=16). Subsequently, number and EV particle size were analyzed using ZetaView Laser Scattering Video Microscope (Particle Metrix GmbH, Microtrac, Meerbusch, Germany). Samples were 1:50.000 in PBS pre-diluted to obtain particle concentrations of approx. 1 × 10^6 particles per ml. Circulating tumor cells (CTCs) and stem cell-like circulating tumor cells (slCTCs) were evaluated using the AdnaTests BreastCancer, StemCell and EMT, respectively (QIAGEN Hannover GmbH, Germany).

Results: EV particle concentrations (mean ± SEM in 10^9/ml) were significantly (p < 0.0001) elevated in BC patients (n=104, respectively) before (2370 ± 170) and after (3524 ± 523) NACT compared to healthy females (90 ± 19). Paired analysis before and after NACT revealed higher EV levels after NACT (p = 0.008). In association studies, high EV numbers before NACT were related to less differentiated carcinomas and to lymph node spread. ROC analysis showed optimal cut-off values of EV levels (i) before NACT: 3540 x 10^9/ml (sensitivity: 66.7%; specificity: 78.1%; AUC = 0.706) and (ii) after NACT: 2480 x 10^9/ml (sensitivity: 100%; specificity: 44,3%; AUC = 0.654). In Kaplan-Meier analysis, a decreased 3-year PFS was significantly associated with EV particle concentrations after NACT > 2480 x 10^9/ml (p = 0.005). A reduced OS of BC patients was significantly associated with (i) EV levels before NACT > 3540 x 10^9/ml) (p = 0.001) and (ii) EV levels after NACT > 2480 x 109/ml) (p = 0.003). Decreased EV levels were found in BC patients after NACT with CTCs expressing ERCC1 (p = 0.025) or the stem cell marker ALDH1 (p = 0.004).

Conclusion: In conclusion, BC patients showed elevated plasmatic EV levels compared to healthy females, while increased EV levels after NACT might be due to therapeutic effects. The association between EV particle concentrations and clinical parameters as well as their prognostic impact on clinical outcome in BC indicate the importance of EVs as a mediator in the BC tumor microenvironment. Therefore, determination of plasmatic EV particle amount might serve as a biomarker for BC monitoring.

#3153

A new circulating plasma/serum Epstein-Barr virus (EBV) DNA test by locked nucleic acid (LNA) PCR technique with enhanced sensitivity in diagnosis & monitoring of patients suffering from nasopharyngeal carcinoma (NPC) and other EBV associated malignancies.

Timothy T.C. Yip,1 Hazel Y.Y. Kwok,1 Sharon S.Y. You,2 Dora L.W. Kwong,3 Alvin H.W. Fong,1 W.W. Cheng,4 Christopher Lai,2 Anthony C.C. Shek,2 Victor W.S. Ma,1 Maria LiLung,3 Roger K.C. Ngan1. 1 _Clinical Oncology Dept, Queen Elizabeth Hospital, Kowloon, Hong Kong;_ 2 _Pathology Dept, Queen Elizabeth Hospital, Kowloon, Hong Kong;_ 3 _Clinical Oncology Dept, University of Hong Kong, Pokfulam, Hong Kong;_ 4 _Pathology Dept, Queen Mary Hospital, Pokfulam, Hong Kong_.

Circulating plasma/serum Epstein-Barr virus (EBV) test has been established as routine first line diagnostic test for Nasopharyngeal Carcinoma (NPC). This real time quantitative PCR test is useful in primary diagnosis of NPC as well as monitoring distant metastases of NPC to lung, liver and distant lymph nodes. However, it is still lacking sensitivity in diagnosis of early stage I/II NPC and local relapse in nasopharynx. Detection sensitivity of this PCR test relies on detecting small fragments of circulating EBV DNA shed into the blood stream from NPC tumor lesions which underwent apoptosis/necrosis. Previous literature already showed that the smaller the molecular size of the circulating EBV DNA targets detected, the higher the detection sensitivity. Using Locked Nucleic Acid (LNA) PCR technique, we have designed a new set of LNA primers for PCR amplification of a much smaller EBV gene target (BamH1-W DNA at 46 bp) than the conventional EBV gene target (BamH1-W at 76 bp). Using this LNA marker, we successfully achieved detection sensitivity of 95.7% in 46 NPC patients (44/46 positive) and detection specificity of 98.3% in 59 normal blood screening subjects (58/59 negative). 89.1% (i.e. 41/46) of the NPC patients had higher EBV DNA gene copies per mL in the LNA marker than the conventional marker. Folds of gene copies per mL of increase of LNA marker over those of conventional marker varied from 6 folds up to 3800 folds in 27/46 NPC patients. The results were confirmed in a validation set of 141 NPC patients (113/141) in a multicenter study under the umbrella of NPC Area of Excellence (AoE) program in Hong Kong with 79.6% (113/142) of NPC patients having higher gene copies per mL of LNA marker than the conventional marker initially tested. On longitudinal monitoring of the new LNA marker in 9 NPC patients for a period of 4.9 months to 5.6 months, higher sensitivity of detection was also achieved at disease onset, during the clinical course of disease and at distant metastases (2/9) than the conventional marker. Three patients with Lymphoepithelioma Like Carcinoma of Lung (LELC) and 3 patients with extra nodal nasal NK/T cell lymphoma were monitored from 3 months to 19.3 months. Detection sensitivity was also higher in the new LNA marker in 3/3 LELC and 1/2 NK/T lymphoma with disease progression whereas 1 NK/T lymphoma with remission had LNA marker remained negative. With such enlightening findings, we are expanding the scope of this new test to many more NPC patients as well as other EBV associated malignancies. We are also recruiting patients in embarking on a large scale prospective study.

#3154

Cell-free circulating tumor DNA methylation in high-grade serous ovarian cancer.

Kayleigh R. Davis, Kirsty J. Flower, Jane V. Borley, Charlotte SM Wilhelm-Benartzi, Robert Brown. _Imperial College London, London, United Kingdom_.

Background Ovarian cancer is often diagnosed late leading to poor patient survival. Over two thirds of women with ovarian cancer are diagnosed with Stages III/IV where the relative 5year survival is <20%, compared to >90% for Stage I. New strategies for early detection and patient stratification are urgently required, as well as non-invasive ways of monitoring tumour evolution. Circulating tumour DNA (ctDNA) can be detected in plasma from ovarian cancer patients. Genome-wide methylation analysis of tissue has identified tumour specific changes in DNA methylation which could be present in ctDNA. However, lysis of peripheral blood mononuclear cells (PBMCs) can release DNA into the plasma confounding detection of tumour DNA. If specific methylation changes that differentiate tumour DNA from PBMC DNA are identified, this would allow DNA methylation in ctDNA to be more accurately quantified.

Methods In this study, publically available genome-wide array-based DNA methylation datasets (Cancer Genome Atlas Research, 2011) (Fridley et al., 2014) were interrogated to identify regions that are highly methylated in high-grade serous ovarian carcinoma, (HGSOC), and hypomethylated in PBMCs and vice versa. The methylation status of these loci was then confirmed in an independent dataset of HGSOC and benign

tissue. Sodium heparin blood samples were collected from healthy volunteers and patients suspected of having HGSOC at Hammersmith Hospital, London. Blood samples were separated by low speed followed by high speed centrifugation, DNA extracted and analysed using bisulfite pyrosequencing.

Results Ten loci were identified from the publically available data that had >80% methylation in HGSOC (n=342) as well as <10% methylation in PBMCs (n=276). Conversely, 2 loci identified had <10% methylation in HGSOC and >80% methylation in PBMCs. Seven of these have been independently validated as differentially methylated by bisulphite pyrosequencing in HGSOC versus PBMCs from the Hammersmith patient cohort. Methylation can be detected using pyrosequencing using DNA amounts less than 10ng. For three loci analysed so far, we have shown significant differences in methylation between DNA isolated from plasma of ovarian cancer patients compared to healthy volunteers. One locus was capable of distinguishing ovarian cancer patients from healthy volunteers with 100% sensitivity and specificity.

Conclusions We have identified differentially methylated loci between HGSOC and PBMCs with high sensitivity and specificity. We have shown that these epigenetic changes can be detected in plasma ctDNA from patients with ovarian cancer.

References Cancer Genome Atlas Research Network., Integrated genomic analyses of ovarian carcinoma. Nature 2011, 474:7353 Fridley et al., Methylation of leukocyte DNA and ovarian cancer: relationships with disease status and outcome. BMC Med Genomics 2014, 7:21

#3155

Investigating chemoresistance in small cell lung cancer through the molecular profiling of single circulating tumour cells.

Louise R. Carter,1 Dominic G. Rothwell,1 HuiSun Leong,2 Yaoyong Li,3 Deborah J. Burt,1 Jenny Antonello,1 Cassandra Hodgkinson,1 Karen Morris,1 Lynsey Franklin,1 Crispin J. Miller,2 Fiona Blackhall,4 Caroline Dive,1 Ged Brady1. 1 _Clinical and Experimental Pharmacology, Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 2 _RNA Biology Group/Computational Biology, Cancer Research UK Manchester Institute, Manchester, United Kingdom;_ 3 _The Faculty of Life Science, the University of Manchester, Manchester, United Kingdom;_ 4 _The Institute of Cancer Sciences, The University of Manchester, Manchester, United Kingdom_.

Background

Chemoresistance, both intrinsic and acquired, represents one of the greatest challenges in the management of Small Cell Lung Cancer (SCLC), contributing to the very poor survival seen in this disease. The investigation of SCLC, and particularly serially monitoring the development of changes associated with resistance, is hampered by the limited availability of tumour tissue for research. Circulating tumour cells (CTCs) in contrast, are abundant in this disease and represent a potential minimally invasive 'liquid biopsy' to study the molecular landscape of SCLC.

Methods

To investigate tumour genetics in SCLC CTCs were isolated from chemorefractory patients and chemoresponsive patients' blood samples using the DEPArray©. Blood samples were collected prior to chemotherapy and again at progression with relapsed disease. Following single-cell whole genome amplification low coverage whole genome sequencing and whole exome sequencing (WES) of CTCs were used to investigate mutations and to generate genome-wide patterns of copy number alterations (CNA).

Results

Hallmark SCLC molecular abnormalities such as TP53 mutations and copy number loss in tumour suppressor genes such as RB1 (previously identified in bulk tumour profiling), were noted in the isolated SCLC CTCs. Distinct CNA profiles were found in the CTCs isolated from patients with chemorefractory disease compared to those isolated from patients with chemoresponsive disease. A potential signature based on differential copy number changes in 2183 loci between the two groups of patients' CTCs was identified. When tested in an independent set of CTC-derived explants this signature achieved 100% accuracy in classifying patients with chemoresponsive disease. There were no profound differences in CNA pattern in the CTCs isolated from initially chemoresponsive patients when they had relapsed post chemotherapy when compared to the baseline samples. However, changes in the proportion of C>A transversions were identified by WES between baseline and relapse CTCs.

Conclusions

Our study highlights the utility of molecular profiling CTCs in the research of SCLC. The data presented confirm CTCs represent a novel medium for the investigation of chemoresistance.

#3156

NGS-based screening for TP53 mutations in circulating cell-free DNA: A first step towards early detection of lung cancers.

Patrice H. Avogbe,1 Tiffany Delhomme,1 Noémie Leblay,1 Florence Le Calvez-Kelm,1 Priscilia Chopard,1 Valérie Gaborieau,1 Ghislaine Scelo,1 Behnoush Abedi-Ardekani,1 David Zaridze,2 Anush Mukeria,2 Graham Byrnes,1 Paul Brennan,1 Lynnette Fernandez-Cuesta,1 Matthieu Foll,1 James D. McKay1. 1 _International Agency for Research on Cancer, Lyon, France;_ 2 _Russian N.N.Blokhin Cancer Research Centre, Moscow, Russian Federation_.

Background

The US National Lung Cancer Screening Trial (NLST) demonstrated in 2011 that screening with computed tomography (CT) scans could reduce lung cancer mortality by 20%, but with important financial costs and high number of false positives. The identification of novel biomarkers is a need to obtain the maximum benefit from CT screening. Given its economical and minimally invasive nature, screening for somatic mutations in circulating tumor DNA (ctDNA) using next-generation sequencing may complement existing screening tools. However, for application in early detection, variant detection must also be done agnostically, i.e. without prior knowledge from tumour tissue of the mutations expected and unfortunately, most currently available variant callers are not adapted for this task.

Methods

We performed multiplex PCR on circulating free DNA (cfDNA) extracted from the plasma of 35 lung squamous cell carcinoma (SCC) and 64 small-cell carcinoma (SCLC) patients. We additionally included 133 hospital controls to evaluate the specificity of ctDNA. We applied (>10,000X) Ion torrent targeted sequencing on the full-coding region of TP53 since this gene is known to be mutated in more than 70% and 90% of SCC and SCLC, respectively. Each amplification, library preparation, and sequencing was performed in duplicate to control for amplification and sequencing errors. Detecting mutations on ctDNA raises important statistical and bioinformatics challenges as it represents only a small fraction of cfDNA. We therefore developed and applied a method based on the idea that a data-derived model of sequencing errors has the potential to improve our ability to detect low-allelic fraction (AF) somatic variants.

Results

We detected TP53 non-synonymous coding mutations with AFs between 0.04% and 85% (median 1.7%) in 8 (23%) SCC patients, 28 (44%) SCLC patients, and 8 (6%) controls. We estimated odds ratios of 4.6 (p=0.006) for SCC and 12.0 (p=6.7x10-10) for SCLC. Observations in controls are surprising, but in this instance there was no information regarding a subsequent cancer diagnosis.

Conclusion

We show that it is possible to detect ctDNA in the cfDNA of lung cancer patients. Since only patients with early stage (I-IIA) SCC tumours were included, these results support the potential utility of the approach for early detection. Nevertheless, if such mutations are found prior to diagnosis has not been explored in a prospective study design with pre-diagnostic plasma samples and individuals without a cancer diagnosis through a follow-up period.

#3157

Serial droplet digital PCR (ddPCR) of plasma cell-free DNA (cfDNA) as pharmacodynamic (PD) biomarker in Phase 1 clinical trials for patients (pts) with KRAS mutant non-small cell lung cancer (NSCLC).

Cloud P. Paweletz, Geoffrey R. Oxnard, Nora Feeney, John F. Hilton, Leena Gandhi, Khanh T. Do, Adrienne Anderson, Andrew Wolanski, Alexander Tejeda, Jessie M. English, Paul T. Kirschmeier, Pasi A. Jänne, Geoffrey I. Shapiro. _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Phase 1 clinical trials of novel therapeutics have historically focused on toxicity, but increasingly are doubling as efficacy studies in biomarker-enriched populations. Given the small sample sizes (~3-6 patients per dose), response on imaging may be a coarse marker of therapeutic effect. Here we piloted serial ddPCR of plasma cfDNA as a PD marker in a phase I combination study of a MEK inhibitor and a CDK 4/6 inhibitor in patients with RAS mutated cancers.

Methods / Results: Twenty-five pts with RAS-mutated cancer (incl. 17 patients with KRAS-mutant NSCLC) have been enrolled to date in a phase I dose escalation trial of the MEK inhibitor PD-0325901 with the CDK4/6 inhibitor palbociclib (NCT02022982). Plasma for cfDNA genotyping was collected at baseline prior to therapy and at the beginning of cycle 2. Plasma genotyping for KRAS G12X mutations was performed using a validated and highly quantitative droplet digital PCR assay.

Pts were enrolled in 5 dose level cohorts ranging from 75 mg palbociclib daily (3 weeks on, 1 week off) with 2 mg PD-0325901 BID (3 weeks on 1week off) to 125 mg palbociclib daily with 8 mg PD-0325901 BID (Table). KRAS mutations were detected in 14/24 pts at baseline (59%, median 1402 copies/mL plasma, range: 11-93000), consistent with the previously reported sensitivity of 64%. A second blood draw at cycle 2 was obtained for all 14 pts. A positive plasma response, defined as decrease of KRAS G12X mutants from first to second dose, was observed in 6 pts (range -6% - -100%) with the most plasma responders (n=4 pts) at the maximum administered dose. At lower administered doses, there was a median increase in plasma KRAS mutant levels.

Conclusions: Increasing dose levels resulted in more consistent decreases in KRAS mutation in cfDNA, consistent with a dose-dependent pharmacodynamic effect.These results highlight the potential value of serial plasma ddPCR as a PD marker in early phase clinical trials.

Dose Level | Palbociclib

(QD, 3 wks on

1 wk off) mg | PD-0325901

(BID, 3 wks on

1 wk off) mg | N Enrolled

(N analyzed) | Median Plasma change KRAS mut(%)

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

1 | 75 | 2 | 3 (1) | +24

2 | 75 | 4 | 3 (3) | +60 (range +31 - +150)

3 | 100 | 4 | 8 (4) | +150 (range -6 - +341)

4 | 125 | 4 | 4 (2) | +9 (range -45 - +61)

5 | 125 | 8 | 7 (4) | -27 (range -7 - -43)

#3158

Prostate cancer derived biomarkers within platelets have the ability to predict therapeutic response in castration resistant patients.

Lee-Ann Tjon-Kon-Fat,1 Marie Lundholm,2 Thomas Wurdinger,3 Camilla Thellenberg-Karlsson,1 Anders Widmark,1 Pernilla Wikström,2 Jonas Nilsson1. 1 _Radiation Sciences, Oncology, Umeå, Sweden;_ 2 _Medical Biosciences, Pathology, Umeå, Sweden;_ 3 _Neurosurgery, VU University Medical Center, Netherlands_.

Background: Novel therapies for castration resistant prostate cancer (CRPC) have been introduced in the clinic with possibilities for individualized treatment plans. Best practice of those expensive drugs requires predictive biomarker monitoring. In this article a novel platform for circulating biomarker analysis has been used to follow cancer-derived transcripts implicated in therapy resistance.

Method: The platelet population of blood samples and QRT-PCR were used to identify selected biomarkers in CRPC patient's prior chemo- or androgen synthesis (AS)-directed therapies. The association between biomarker statuses (positive vs. negative) and therapy response, progression-free survival (PFS) and overall survival (OS) was examined.

Results: 40 patients received either docetaxel (n=17) or AS-directed (n=23) therapy, with a therapy response rate of 29% respectively 48%. The cancer-associated biomarkers were present within the platelet fraction. Analyzing these biomarkers in the chemotherapy group were associated with a short OS (p=0.025). In the AS-directed group, the biomarkers were associated with shorter PFS (p=0.015), and with an increased risk of therapy failure (HR: 5,5; p=0,023), as well as short OS (p=0.012).

Conclusions: Analyzing circulating biomarkers in the platelet population enables us to predict who will benefit from AS-directed therapy with high accuracy, and platelet based analysis of cancer derived biomarkers may be used in treatment stratification of patients scheduled for AS-directed therapies.

#3159

Identification of circulating tumor cells from renal cell carcinoma patients by a multi-parameter flow cytometry assay.

Joshua A. Desotelle,1 Chorom Pak,1 Erika Heninger,1 Jennifer L. Schehr,1 Rana R. McKay,2 Benjamin K. Gibbs,1 Craig Norton,2 Toni K. Choueiri,2 Joshua M. Lang1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Harvard Medical School, Boston, MA_.

BACKGROUND: Renal cell carcinoma (RCC) is one of the top ten causes of cancer death in the United States. The last ten years have shown a dramatic increase in the number of available treatment options, however metastatic RCC remains largely incurable. The classes of drugs that have been developed can be divided into agents that target the Vascular Endothelial Growth Factor (VEGF) pathway (Sunitinib, Sorafenib, Pazopanib, Axitinib, Bevacizumab), those that target the mammalian Target of Rapamycin (mTOR) pathway (Everolimus, Temsirolimus) and immune based therapies (IL-2 and PD-1 inhibition). There are currently limited biomarkers to guide clinical decisions, mainly due to the lack of tumor cells for longitudinal molecular analysis. Circulating tumor cells (CTCs) are potential a source of tumor cells that can be identified from a blood draw for serial analysis. CTCs have not been reliably detected in RCC due to the significant heterogeneity and high rate of false positive events in this disease when EpCAM has been used. We sought to identify RCC CTCs with alternative markers, such as carbonic anhydrase IX (CAIX) which is found in greater than 90% of clear cell RCC tumors.

METHODS AND RESULTS: To evaluate for the presence and subtypes of CTCs from patients with RCC, we utilized a multi-parametric flow cytometry assay. We evaluated heterogeneity across subpopulations of putative CTCs with Epithelial Cell Adhesion Molecule (EpCAM), Carbonic Anhydrase IX (CAIX), Carbonic Anhydrase XII (CAXII), PAX8, and Cytokeratin (CK). Negative controls for immune and endothelial events were performed with markers for CD45, CD14, CD34, CD11b and CD61. We tested twenty blood samples from patients with RCC at the Dana Farber Cancer Institute and University of Wisconsin Carbone Cancer Center. CTC frequency in RCC ranges from 0-5410 CAIX+/CK+ events with a median of 24.5 putative CTCs/7.5mL of blood. A subset of patients with radiographic progression had a higher number of CTCs with a median of 295.2 CTCs/7.5 mL. A range of 1-231 EpCAM+/CK+ events were identified with a median of 5.5 CTCs/7.5mL. There was low frequency of events being CAIX+/EpCAM+/CK+, with a median of 1 CTC/7.5mL of blood. Assay specificity was dramatically improved through the combination of multiple positive markers with stains for immune and endothelial cells given frequent non-specific staining for cytokeratin in RCC blood samples.

CONCLUSIONS: CTCs can be identified in patients with RCC using non-traditional markers. CAIX is a more sensitive marker than EpCAM to identify putative CTCs from patients with RCC. Specificity in the assay is critical given the high frequency of false positive events identified if only CD45 is used as a marker for immune cells. Our investigation is ongoing for further molecular characterization of orthogonal endpoints in identified CTCs. Future directions include longitudinal monitoring of CTCs during treatment with VEGF inhibitors.

#3160

Circulating tumor cell enrichment and dielectric manipulation for ultra-pure cell recovery in advanced bladder cancer.

Gareth Morrison, Cory Hugen, Tong Xu, Yucheng Xu, Dorff Tanya, David Quinn, Sarmad Sadeghi, Amir Goldkorn. _University of Southern California, Los Angeles, CA_.

Background: Advanced transitional cell carcinoma (TCC) of the bladder is a lethal malignancy with few available biomarkers to guide therapy. Recent evidence suggests that circulating tumor cells (CTCs) are detectable in TCC and may have a prognostic role; however, most studies to date have focused on CTC enumeration. We leveraged novel enrichment and capture technologies to isolate and recover ultra-pure CTC (upCTC) that can be used for single cell molecular analysis.

Methods: Patients with metastatic bladder cancer were enrolled under an institutional review board approved pilot study. Blood samples (7.5 ml each) were drawn into EDTA or Cell-Free DNA blood collection tubes (Streck), and preliminary CTC enrichment was performed using one of 3 microfluidic platforms: i) EpCAM-based LiquidBiopsy system (Cynvenio); ii) size/deformability-based ClearCell FX system (Clearbridge); or iii) size/deformability-based Parsortix system (Angle). Enriched CTCs were labeled with Cytokeratin (CK), EpCAM, and CD45 immunofluorescent antibodies and Hoechst nuclear stain. Identification and recovery of upCTCs was performed on a DEPArray v2 system (Silicon BioSystems). Additional experiments using cell lines were performed to gauge the recovery of mRNA from rare cancer cells collected in EDTA vs. preservative tubes.

Results: To date, samples were collected from 8 patients of whom 5 (~63%) had detectable CTCs. All 3 enrichment platforms successfully yielded CTC fractions from which Hoechst+/CK+/CD45- upCTCs were subsequently identified and recovered on the DEPArray v2 system. Additional cells that were Hoechst+/CK-/CD45- were identified and collected, and cells also were gated by integral Hoechst intensity and collected for correlation to aneuploidy. Comparison of mRNA recovery from 10 cancer cells collected into EDTA vs. fixative tubes (CellSave, Cynvenio, Streck Cell-free RNA) demonstrated that gene expression readings by qPCR from rare fixed cells was feasible but associated with variable results, likely due to variable proteinase K reversal of mRNA-protein cross-linkages.

Conclusion: We leveraged new techniques to develop and optimize a workflow for identification and collection of upCTCs from bladder TCC patients, and we established that gene expression profiling is also feasible, albeit currently is still best achieved using EDTA collection and not any of the available preservative tubes. Single upCTC molecular profiles (DNA alterations, gene expression) from patient samples generated using these protocols may help to elucidate mechanisms of disease progression and ultimately may advance the management of advanced bladder cancer.

#3161

Development and implementation of a non-invasive NGS-based diagnostic test for precision medicine in prostate cancer.

Pan Du, Amy Wang, Tak Cheng, Winston Patrick Kuo. _Predicine Inc, Palo Alto, CA_.

The growth and survival of prostate cancer tumors relies primarily on the functioning of the androgen receptor (AR) signaling pathway. Recent studies suggested that the presence of AR-V7, a c-terminal truncated form of AR, in circulating tumor cells (CTCs) of patients with castration-resistant prostate cancer (CRPC) is associated with inherent and/or acquired resistance to enzalutamide and abiraterone, the stand of care androgen deprivation therapies in prostate cancer. A series of data also indicate that AR-Vs (eg, including AR-V7, ARv567es, AR-T878A, AR-F876L, and Arv567es) may drive resistance in CRPC. Expression of AR-Vs has been shown to correlate with disease progression and shortened survival and AR-V7 is most abundant in CRPC specimens. Considering that truncated ARs with C-terminal loss (splice variants) lack a functional LBD and are constitutively active, C-terminal AR-directed therapies may not be effective, and novels agents are needed that target mutated ARs including AR-Vs. Circulating tumor cell (CTC)-based AR-V7 tests are currently being tested in the clinic. However, nearly half of the CRPC patients do not have enough CTCs for AR-V7 test, raising the request for a complementary, non-CTC platform to detect AR variants as well as other resistance markers in circulation.

Here we report the successful development of a non-invasive, next generation sequencing-based diagnostics platform that offers comprehensive profiling of genomic alterations in prostate cancer, including splicing variants (such as AR-V7), point mutation, copy number, and translocation using biofluid samples (blood and urine) from CRPC patients. To the best of our knowledge, this is the first non-invasive diagnostic test that measures genetic alterations especially AR-V7 in all patients with prostate cancer, regardless of their status of CTC enumeration. The successful development and clinical validation of this test has potential to enable precision medicine in prostate cancer.

#3162

Blood-based exosomal miRNAs as biomarkers for diagnosis and prognosis of clear cell renal cell cancer.

Joana Heinzelmann, Sophie Baumgart, Sebastian Hoelters, Martin Janssen, Michael Stöckle, Kerstin Junker. _University of the Saarland, Homburg, Germany_.

Introduction & objectives: In previous studies we identified specific miRNA alterations in tumor tissues of clear cell renal cell cancer (ccRCC) with diagnostic and prognostic value relating to the presence of metastasis. For few years it has been known, that miRNAs are actively packed in exosomes which can be released into body fluids. Therefore, we hypothesize that in a simple blood based test we are able to use specific miRNA signatures as minimal invasive biomarkers for confirmation of the diagnosis and evaluation of the metastatic risk in ccRCC.

Materials & methods: Initially, exosomes were isolated from 1 ml serum of 10 ccRCC patients (metastatic and non metastatic tumors) and 10 healthy volunteers using appropriated exosome isolation kit protocol (Thermo Fisher Scientific). Quality of exosomes was proven using exosomal markers (CD63, CD81, Alix, Synthenin) and Nano Particle Tracking Analysis. Exosomal totalRNA was isolated using miRNeasy Mini Kit (Qiagen). For miRNA analyses total RNA was reverse transcribed using TaqMan Reverse Transcription Kit (Thermo Fisher Scientific), cDNA was preamplified using TaqMan® PreAmp Master Mix (Thermo Fisher Scientific) and real time PCR was performed using Gene Expression master mix (Thermo Fisher Scientific). Expression differences were calculated using REST Software and SPSS.

Results: We have shown that 1 ml serum is sufficient to analyze miRNA expression in exosomes. Exosomes isolated from serum exhibit high amount of exosomal markers and possess the typical exosomal size (30-120 nm). CcRCC serum samples are characterized by downregulation of several miRNAs (including miR-10b and miR451). Furthermore, specific miRNAs (including miR-30c) differentiate patients with or without metastases as well as healthy volunteers.

Conclusion: These initial data confirm our hypothesis that the tissue based miRNA signature could be used as biomarkers for detection of aggressive ccRCC analyzing exosomes from liquid biopsy. This minimal invasive blood based test is easy to handle in clinical practice and could be a powerful tool for early detection and monitoring of metastatic disease. To validate these results the expansion of the sample set is ongoing.

#3163

Peripheral blood circulating multiple myeloma cells (CMMCs ) correlate with disease burden and can be used to characterize high-risk cytogenetics in newly diagnosed and smoldering myeloma.

Brad Foulk,1 Mike Schaffer,1 Steve Gross,2 Chandra Rao,1 Denis Smirnov,1 Shalini Chaturvedi,1 Manjula Reddy,1 Madeline Repollet,2 Claudia Rojas,2 Daniel Auclair,3 Mary DeRome,3 The MMRF CoMMpass Network, Brendan Weiss,4 A. Kate Sasser1. 1 _Janssen R &D, Spring House, PA; _2 _Janssen Diagnostics, LLC, Huntingdon Valley, PA;_ 3 _Multiple Myeloma Research Foundation, Norwalk, CT;_ 4 _Abramson Cancer Center and Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA_.

There is an increasing interest in the ability to dynamically track disease burden and perform molecular subtyping of patients with plasma cell disorders without invasive bone marrow sampling. Circulating multiple myeloma cells (CMMC) have been detected in elevated numbers in the peripheral blood of patients with plasma cell disorders using flow cytometry or circulating cell enrichment platforms. We developed an automated CELLSEARCH® assay to enrich, enumerate, and perform a triplex FISH assay for t(4;14), t(14;16), and del 17p on CMMC (CD138+CD38+, CD45-CD19-) isolated from a 4 mL peripheral blood sample (Gross, et.al. Blood 2011; 118(21):1825). Here we present the enumeration and cytogenetic profiling of CMMC from separate cohorts of patients across the spectrum of plasma cell disorders.

The first cohort consisted of newly diagnosed multiple myeloma patients enrolled in the CoMMpass study (ClinicalTrials.gov Identifier: NCT01454297). One or more CMMC per four ml blood were detected in 684/698 (98%) of newly diagnosed myeloma patients with median CMMC count of 413 per 4 mL of blood. CMMC counts decreased significantly from baseline when a remission was achieved due to treatment (p<0.001). CMMC counts <100 at remission were associated with improved PFS and OS compared to those patients whose CMMC counts were > 100 at remission. CMMC FISH results (n=57) showed overall agreement of 85%, 91% and 80% with bone marrow FISH results and 81%, 91%, and 95% agreement with bone marrow CNV/RNAseq results for the t(4;14), t(14;16), and del 17p assays, respectively.

The second cohort of patients consisted of intermediate/high risk smoldering myeloma patients enrolled in a Phase 2 study of Siltuximab (ClinicalTrials.gov Identifier: NCT01484275). One or more CMMC per 4 mL blood was detected at baseline in 74/79 (94%) of intermediate/high risk smoldering myeloma patients with median CMMC count of 100 per 4 mL of blood. Significantly higher CMMC counts were observed between patients in the placebo arm that progressed versus those without progression (n=34, p=0.031). This is in contrast to standard metrics of percentage of bone marrow plasma cells and serum M protein levels where statistically significant differences were not seen between progressors and non-progressors in the placebo arm (p = 0.068 and p= 0.070, respectively).

CMMCs were collected from a third cohort of 35 patients across the plasma cell disease spectrum with an emphasis on MGUS and SMM. CMMC counts were associated with the disease burden of patients within this cohort.

CMMC may be a useful non-invasive tool for disease monitoring and characterization across the plasma cell disorder spectrum. In myeloma, CMMC may be a useful prognostic marker at remission to delineate those patients at risk for relapse. In SMM, CMMC may be useful for predictive patients at risk of progression to MM. 

### Molecular Classification and Genomic Applications

#3164

Molecular determination of the clonal relationship between multiple tumors in BRCA1/2-associated cancer patients has clinical relevance.

Willemina R.R. Geurts-Giele,1 Victorien M.T. van Verschuer,1 Carolien H.M. van Deurzen,1 Paul J. van Diest,2 Rute M. Pedrosa,1 J. Margriet Collee,1 Linetta B. Koppert,1 Caroline Seynaeve,1 Winand N.M. Dinjens1. 1 _Erasmus MC Cancer Institute, Rotterdam, Netherlands;_ 2 _University Medical Center Utrecht, Utrecht, Netherlands_.

PURPOSE

Female BRCA1/2 mutation carriers commonly develop breast cancer and ovarian cancer. It is of utmost importance to know the clonal relationship between multiple tumor localizations, (multiple primaries or metastasized disease), since prognosis and treatment differ between these tumors and their metastases. We evaluated the value of targeted Next Generation Sequencing (NGS) in the diagnostic workup of BRCA1/2 mutation carriers with ≥2 tumor localizations and uncertain tumor origins.

METHODS

Forty-two female BRCA1/2 mutation carriers with ≥2 tumor locations with tumor material available for pathologic revision were selected. Median age at first cancer diagnosis was 48.0 years (range 30-68). Four patients with inconclusive tumor origin after histological and immunohistochemical analyses were 'cases'; 10 patients with certain tumor origin of ≥3 tumors served as 'controls'. Tumors of cases and controls were analyzed by targeted NGS using a panel including CDKN2A, PTEN and TP53, hotspot mutation sites for 27 different genes and 143 single nucleotide polymorphisms for detection of loss of heterozygosity (LOH). Based on prevalence of identical or different mutations and/or LOH patterns, tumors were classified as 'multiple primaries' or 'one entity'.

RESULTS

In 44 tumors, 48 mutations were found; 39 (81%) concerned TP53 mutations. In all 10 controls and all 4 cases, the intrapatient clonal relationships between the tumors could be unequivocally identified by molecular analysis. In all controls, tumor origins based on molecular outcomes matched the conventional histopathological diagnosis.

CONCLUSION

In 90% of BRCA1/2 mutation carriers with multiple tumors routine pathology work-up is sufficient to determine tumor origins and relatedness. In case of inconclusive conventional pathology results, molecular analyses using NGS can reliably determine clonal relationships between tumors, enabling optimal treatment of individual patients.

#3165

An evidence-based software tool for personalized cancer medicine to recommend therapeutic options and avoid toxicity.

Matthias Löhr,1 Katrin Stecker,2 Caroline Huelsewig,2 Sandra Morandell,2 Isin Ertongur,2 Sarah Luke-Glaser,2 Anna Laib,2 Linnéa Malgerud,1 Carlos Fernández Moro,3 Masoud Karimi,4 Johan Permert,5 Rainer Heuchel,6 Johan Lindberg,7 Valtteri Wirta,8 Alexander Picker,2 Marco Del Chiaro,1 Stephan L. Haas,1 Caroline S. Verbek,3 Lars Engstrand,8 Jens Siveke,9 David Jackson,2 Henrik Grönberg,7 Dirk Jäger,10 Stephan Brock2. 1 _Center for Digestive Diseases, Stockholm, Sweden;_ 2 _MolecularHealth, Heidelberg, Germany;_ 3 _Dept. of Pathology, Karolinska University Hospital, Stockholm, Sweden;_ 4 _Dept. of Oncology at Radiumhemmet, Karolinska University Hospital, Stockholm, Sweden;_ 5 _Innovation Office, Karolinska University Hospital, Stockholm, Sweden;_ 6 _Dept of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden;_ 7 _Dept. of Medical Epidemiology & Biostatistics (MEB), Karolinska Institutet, Stockholm, Sweden; _8 _Science for Life Laboratory, Stockholm, Sweden;_ 9 _Rainer Herzog Comprehensive Cancer Center (RHCCC), Technical University, Munich, Germany;_ 10 _National Center for Tumors (NCT), Heidelberg, Germany_.

Background. For many tumors, therapeutic options are sparse beyond the guidelines, especially for pancreatic cancer. At the same time, more and more biomarkers are known, informing about or supporting treatment decisions. Finally, tumor DNA analysis with next generation sequencing (NGS) is quickly becoming a routine test, at least for tumors. However, to analyse the vast amount of data and provide concrete, evidence-based treatment recommendations has been a challenge.

Methods. Applying NGS in a quality controlled set-up together with the newly developed evidence-based software tool EngineusGUIDETM (CE-marked).

Results. During the time from Oct 2013 until Oct 2015, 87 patients were analysed with EngineusGUIDE. In 7 cases, more than one tumor was investigated. NGS was performed (WXS = 48; panel/paired = 16, panel = 23). Sequencing could be performed in all but one patient (Whole Exome; bone metastasis). Sequencing quality was insufficient for analysis in 3 cases (paired panel), sequencing revealed insufficient tumor content in the sample for one patient (Whole Exome) leaving 82 patients. The indication was: Pancreatic Neoplasms = 21 cases; Colorectal Neoplasms = 12; Breast Neoplasms = 5; CUP = 6; NSCLC = 3; 2 cases each for Bronchial Neoplasms, Adenoid Cystic Carcinoma, Common Bile Duct Neoplasms, Glioblastoma, Multiple Myeloma, Prostatic Neoplasms and 1 case each for Adenocarcinoma, Duodenal Neoplasms, Endometrial Neoplasms, Hemangiosarcoma, Hypopharyngeal Neoplasms, Leukemia (Lymphocytic, Chronic, B-Cell), Liposarcoma, Liver Neoplasms, Lung Neoplasms, B-Cell Lymphoma, Mantle-Cell Lymphoma, Meningioma, Oropharyngeal Neoplasms, Ovarian Neoplasms, Paranasal Sinus Neoplasms, Parathyroid Neoplasms, Sarcoma, Sigmoid Neoplasms, Squamous Cell Neoplasms, Stomach Neoplasms, Thyroid Neoplasms, Urinary Bladder Neoplasms, Uveal melanoma.

In 67/82 patients, druggable targets (response biomarkers) could be identified (range 0-8; median 2). In 45/82 patients (range 0-5; median 1) biomarkers indicated lack of efficacy (e.g. KRAS mutation in CRC). In 65/82 patients, biomarkers indicated increased toxicity (range 0-7; median 2), in 19/82 patients FDA-approved biomarkers for toxicity were detected (28 biomarkers).

Of the positive biomarkers, 40 biomarkers in 26 patients indicated drugs approved in the indication, 75 biomarkers in 45 patients indicated approved drugs, and 58 biomarkers in 39 patients indicated experimental drugs (pre-clinical and phase I - III). Six patients have already received drugs recommended by EngineusGUIDE. In 11 patients, toxicity markers may explain observed toxicity during previous treatment.

We are currently collecting the outcome parameters of EngineusGUIDE recommendations.

#3166

Querying, viewing and analyzing colorectal cancer translational research studies in tranSMART.

Mariska Bierkens,1 Wim van der Linden,2 Ward Weistra,3 Kees van Bochove,3 Jeroen A.M. Beliën,4 Remond J.A. Fijneman,1 Rita Azevedo,5 Jan-Willem Boiten,6 Gerrit A. Meijer1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Philips Research, Eindhoven, Netherlands;_ 3 _The Hyve, Utrecht, Netherlands;_ 4 _VU University Medical Center, Amsterdam, Netherlands;_ 5 _TI Pharma, Leiden, Netherlands;_ 6 _Center for Translational Molecular Medicine, Eindhoven, Netherlands_.

All translational research projects basically share the same design, which is correlating variation in disease phenotype to variation in underlying biology. Typical questions to be addressed are: 'Which (out of thousands) biomarkers predict good/bad outcome?' and 'Which (out of thousands) biomarkers predict whether a patient will benefit from a particular therapy?'. In line with this concept, many research teams have generated large amounts of experimental molecular data from patient samples, yet generally this information is inaccessible for examination due to local storage of both (meta)data and the data processing workflows used. Alternatively, data stored in central databases may only be available for exploration and interpretation by data specialists, provided that the processing workflow has been published and is available. Thus, if data is not recorded and easily retrievable, validation of obtained results (e.g. promising biomarkers) may be virtually impossible. Additionally, it will be difficult to query existing data sets to answer new questions, which may lead to experiments being unnecessarily repeated and biological materials being wasted. In the Netherlands, the Translational Research IT (TraIT) project initiated by the Center for Translational Molecular Medicine (CTMM, www.ctmm-trait.nl) aims to provide IT solutions to support translational research from start to end, including sustainable management and analysis of data. These data types involve the clinical, biomedical imaging, biobanking, (molecular) experimental data domains and their associated workflows. In addition to domain-specific solutions to manage these data types, the processed or 'final' data of these different domains will become available in the data-integration platform tranSMART for querying, visualization and analysis. To ensure sustainable data stewardship and provide easy access to existing data for the CTMM colorectal cancer project 'Decrease in Colorectal Cancer Death (DeCoDe)', data has been made available in tranSMART and more datasets are being added. The data encompasses both clinical and (molecular) experimental data from non-high-throughput molecular profiling (NHTMP) assays and array-based profiling techniques.

It is now possible with tranSMART to explore the respective DeCoDe datasets and perform various analyses (survival, Fisher-exact tests, ANOVA, aCGH tests etc.), without needing deep bioinformatics expertise. Through metadata tags it is possible to trace back raw or pre-processed data in other tools (e.g. clinical data in OpenClinica, array-data in GEO, or histological images of tissue microarrays). Data can be examined in more detail by domain experts using tools they are familiar with or other tools provided by CTMM-TraIT. Thus, existing rich data are made findable, accessible, interoperable and reusable (FAIR) and source data can be traced back for customized processing.

#3167

CNV patterns in 822 routine diagnostic cases of NSCLC, melanoma, and colorectal cancer.

Albrecht Stenzinger,1 Roland Penzel,1 Frederick Klauschen,2 Arne Warth,1 Regine Brandt,1 Daniel Heim,2 Peter Schirmacher,1 Wilko Weichert,3 Volker Endris,1 Nicole Pfarr3. 1 _Institute of Pathology, University Hospital, Heidelberg, Germany;_ 2 _Institute of Pathology, Charité University Hospital, Berlin, Germany;_ 3 _Institute of Pathology, Technical University, Munich, Germany_.

Targeted deep massive parallel sequencing (MPS) has been implemented in routine molecular diagnostics for high-throughput genetic profiling of formalin-fixed paraffin-embedded cancer samples. This approach is now widely used to interrogate simple somatic mutations but experience with the analysis of copy number variations (CNV) is still limited. Here, we retrospectively analyzed CNVs in 822 cancer cases (n=135 melanoma, n=468 non-small cell lung cancers (NSCLC), n=219 colorectal cancers (CRC)) that were sent to our institution for routine molecular profiling using a semiconductor based sequencing platform. Amplifications and deletions inferred by MPS coverage data were independently validated by a qPCR assay. We observed a decreasing frequency of CNV in clinically actionable genes from melanoma to NSCLC to colorectal cancer.

Of 56 melanomas with genetic aberrations in BRAF, 31 showed co-occurring CNV in other genes, mainly affecting CDKN2A. Some tumors (5 cases each) revealed clustered deletions affecting either ABL1, NOTCH1, and RET or STK11, GNA11, and JAK3. 8.1% of the cases had amplifications in clinically actionable genes. In the group of NRAS mutant tumors (n=39), 26 showed co-occurring CNVs in other genes, such as CDKN2A and FGFR3, and 9 NRAS mutant cases were additionally mutated in BRAF. 19.1% had amplifications in clinically actionable genes. In contrast to BRAF mutant tumors, we did not see any specific CNV clusters. In the group of BRAF/NRASwt tumors (n=11), we observed 5 cases with co-amplification of KDR, KIT, PDGFRA and another 6 cases with KIT mutations. While co-amplified cases had many gene deletions, KIT mutated tumors harbored only very few genetic aberrations in other genes.

Across both NSCLC data sets, we identified 14 cases with amplified EGFR (10 of them harboring co-occurring EGFR mutations) and detected 8 NSCLC with KRAS amplifications (of which 7 had co-occurring mutations of KRAS). KRAS mutated tumors displayed frequent amplifications in MYC (n=10) and MDM2 (n=5). Of the 22 BRAF mutant tumors, two harbored mutated KRAS. In contrast to melanoma, we observed no clustering of CNVs in BRAFmut NSCLCs. Within the group of KRAS/EGFR/BRAFwt tumors, we identified 15 cases harboring genetic aberrations in MET (n=8 mutations, n=7 amplifications).

Compared to melanoma and NSCLC, the number of CNV in CRC was rather low. IGF2 amplifications were most prevalent (n=13) followed by MYC (n=9). Two cases showed amplified wild-type alleles of KRAS. Two KRAS mutant tumors showed concomitant amplification of NRAS and three cases harbored amplified EGFR.

In conclusion we demonstrate that i) detection of CNVs by targeted MPS data obtained from FFPE material is feasible and ii) could be validated independently, iii) this approach enables detection of known CNV patterns, and iv) uncovers new CNV patterns in clinically actionable targets across cancers.

#3168

Your targeted population might not be what you predict: Changes in tumor genetic landscapes post standard of care.

Danielle Greenawalt,1 Austin Dulak,1 Manasa Ramakrishna,1 Zhongwu Lai,1 Justin Johnson,1 Hongyue Dai,2 Melissa Mitchell,2 Carl Barrett,1 Stephen Fawell,1 Jonathan Dry1. 1 _AstraZeneca, Waltham, MA;_ 2 _M2Gen, Tampa, FL_.

Our current understanding of the mutation spectrum of relapse/refractory patients is limited. Public sequencing efforts have focused by design, on primary tumors, while re-biopsy of patients who are refractory to first line therapy or who relapse on treatment is not standard protocol. We have performed whole exome sequencing on post standard of care biopsies from 3 patient cohorts, 47 DLBCL patients post-relapse following R-CHOP, 49 triple negative breast (TNBC) following taxol alone or in combination and 41 lung adenocarcinoma (LUAD) following platinum alone or in combination. Comparative analysis of the mutation spectrum of these cohorts was performed against available clinically matched primary tumor cohorts. Comparison of the mutational signatures, somatic variant frequencies and copy number variation between matched pre- and post-treatment samples, and treated-primary tumor cohorts were performed. Significant differences were identified in the frequency of oncogenic drivers including PIK3CA in TNBC and KRAS in LUAD. We also observed mutational signature differences between cohorts that could impact treatment regimens post standard of care. Collectively these data further highlight the value of re-assessing the tumor genetic landscape at the time of treatment rather than relying on primary tumor samples, which may not be representative of the cohort to be treated.

#3169

The benefits and burdens of assaying matched normal tissue when sequencing cancer genomes.

Elena Helman, Michael J. Clark, Ravi Alla, Sean M. Boyle, Shujun Luo, Selene Virk, Deanna Church, Parin Sripakdeevong, Jason Harris, Mirian karbelashvili, Christian Haudenschild, John West, Richard Chen. _Personalis, Menlo Park, CA_.

Targeted sequencing assays are increasingly used to identify tumor mutations that guide therapeutic decisions. Interpretation of a cancer variant's origin and therapeutic impact poses analytical challenges. Recent studies have indicated that jointly analyzing a tumor with its matched normal can accurately discriminate between tumor-specific (somatic) and inherited (germline) mutations. Moreover, a NHGRI/NCI Clinical Sequencing Exploratory Research Consortium Tumor Working Group just released a set of guidelines recommending that laboratories performing cancer sequencing tests should include germline variants. However, procurement of a matched sample is often logistically impractical. In the absence of a matched normal, large databases and analytical techniques are currently used to identify cancer variants in tumor sequencing data. Whether the benefits outweigh the additional burden of sequencing the matched normal for accurate detection of cancer-relevant mutations remains an open question.

To compare tumor-only and tumor/normal analysis of cancer samples, we collected a set of >100 formalin-fixed (FFPE) and fresh frozen cancer samples of various tumor types, where matched normal blood or adjacent tissue was available. We performed augmented target enrichment sequencing (exome and large cancer gene panel) of both DNA and RNA. The data was analyzed using cancer bioinformatics pipelines that detect base substitutions, small insertions/deletions, copy number alterations, and gene fusions in both tumor-only and tumor/normal modes. Variants were annotated using described clinical actionability filtering strategies. Analysis of germline variants for secondary findings was performed.

We find that 67% of mutations detected in tumor-only mode are reclassified as germline variants when analyzed together with the matched normal sample. These include mutations in hereditary cancer predisposition genes, such as BRCA1, VHL, and other genes with ACMG guidelines that warrant germline variant classification and appropriate management. Clinically actionable mutations may be miscalled as somatic when a matched normal is not available; however, we find the definition of 'actionable' can greatly impact the results of this analysis. Finally, the use of newly available large datasets, such as ExAC, substantially decreases the number of miscalled somatic variants in the absence of a matched normal.

The effects of administering targeted therapies to patients with germline mutations in the relevant gene are largely unknown. Mutations of putative germline origin may be important for hereditary cancer knowledge and tumor treatment, and should be reported as such. For NGS-based cancer interpretation to guide clinical decisions in a practical and cost-effective manner, highly optimized tumor-only and tumor/normal analyses must be available with proper attention to germline consent, classification and education.

#3170

Analysis of copy number variations in meningioma samples using microarrays revealed 22q deletions more frequent in higher grade tumors.

Josef Srovnal,1 Vladimir Balik,2 Jiri Ehrmann,1 Miroslav Vaverka,2 Lumir Hrabalek,2 Katerina Staffova,1 Marian Hajduch1. 1 _Palacky University, Olomouc, Czech Republic;_ 2 _University Hospital Olomouc, Olomouc, Czech Republic_.

Background: Meningiomas represent one of the most common intracranial tumors. They are generally thought to progress from low to high-grade lesions. However, the molecular mechanisms underlying their pathogenesis remain still uncertain. We suppose the existence of a correlation between the parameters that will help to predict more precisely their biological behavior and response to therapy. Identification of meningioma molecular subgroups may have significant potential to improve clinical management, through molecular disease risk stratification strategies and the identification of patients who could benefit from targeted molecular therapeutics.

Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 30 patients (10 patients in each meningioma grade) and 5 healthy controls (dura mater). Comprehensive clinical-pathological data were mined. There were 7 males and 23 females; mean age was 56.7 years, range 33 - 100 years. Total DNA was purified from FFPE samples after pathological verification using proteinase K treatment followed by QIAmp DNA FFPE Tissue Kit (Qiagen). Microarray analysis was performed using the OncoScan FFPE Assay Kit (Affymetrix), raw data were obtained using Chromosome Analysis Suite (Affymetrix) in default manner. Subsequently, the data were analyzed using Nexus Express software (Biodiscovery). 10 samples were excluded from analyses because of poor quality of results.

Results: In total, 35 OncoScan arrays were performed for copy number variants (CNV) analyses in meningioma and control samples. Our results confirm that del(22q) and del(1p) are the most common (44%, resp. 24% of cases) deletions in meningiomas. Del(22q) was present in 75% grade 2 menigiomas cases in contrast to 25% in grade 1 patients. Additionally, monosomy of chromosome 6 (12%), 8 (8%), 14 (20%) and 18 (12%) were observed. Surprisingly, chromosomal gains and LOH were found only in small portion of cases (8%). Finally, mutation in TP53 (c.817C>T/A, c.637C>T) and PIK3CA (c.3140A>G) genes were found in 12% of cases. Only common CNV variants were found in healthy control samples.

Conclusion: We have identified the CNV profiles in meningioma patients allowing for better knowledge of pathological pathways and tumor progression. Del(22q) is frequently present in higher grade tumors probably altering NF2 a TP53 pathways. However, it will require further validation.

Acknowledgment: This work was financially supported by Ministry of Health of the Czech Republic, grant nr. 15-29021A, IGA UP LF 2015_010 and NPU LO1304.

#3171

Powerful target capture/NGS approach reveals extensive genetic changes including SNVs, CNVs and gross chromosomal rearrangements in colorectal cancers.

Hongzheng Dai,1 Xia Tian,1 Stella Chen,1 Yue Wang,2 Tzu-Wei Yang,3 Chun-Che Lin,3 Ming-Chang Tsai,3 Chih-Hong Wang,4 Chi-Chou Huang,3 Chin-Ying Shih,4 Guang-Yuh Chiou,4 Yuh-Jyh Jong,4 Lee-Jun Wong2. 1 _Baylor Miraca Genetics Laboratories, Houston, TX;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _Chung Shan Medical University Hospital, Taichung, Taiwan;_ 4 _National Chiao Tung University, Hsinchun, Taiwan_.

Introduction: Accurate and comprehensive molecular analyses of genetic changes in cancer tissues are critical for cancer management, treatment, and genetic counseling. Study of the complete molecular profiles in patient's blood, cacinoma tissue and surrounding pathological normal tissues will help us understand the etiology of colorectal cancers (CRC) as either hereditary or sporadic. In addition, this study will also reveal somatic changes and possible targets for therapy. Multigene target capture followed by deep next generation sequencing (NGS) provides a powerful approach to simultaneously detect both single nucleotide variants (SNVs) and exonic copy number variants (CNVs) in a time and cost effective fashion.

Method: DNA samples were prepared from blood, freshly frozen normal surrounding and carcinoma or polyp tissues from 20 patients with CRC and 10 patients with colorectal polyps. A custom probe library containing 183 cancer related genes was used to capture the target sequences followed by NGS with deep coverage (average depth per base is ~1000X). Both SNVs and CNVs were analyzed based on in-house developed analytical algorithm and bioinformatics pipeline.

Results: This study revealed extensive genetic changes in colon carcinoma tissues. At least 40% of the colon carcinoma samples harbor loss-of-function pathogenic germline mutations in cancer related genes. However, loss of function germline mutations were not found in the polyps. Germline missense variants of unknown significance were identified in 60% of the polyps. Multiple deleterious somatic mutations are identified in all carcinoma tissues but only 1-2 somatic mutations per sample are found in the polyps. Almost every carcinoma tissue harbor gross chromosomal rearrangement, which is only found in about 50% of the polyp tissues. . NGS based CNV analysis revealed 2 germline events (FANCD2 E15-16 del; whole ALK gene dup). Somatic CNV events are more frequently observed in carcinoma tissues. Gross changes at chromosomal levels were also detected by using the NGS CNV analytical algorithm. Recurrent somatic pathogenic variants in genes associated with CRC, such as APC, TP53, and KRAS were identified while novel likely pathogenic variants in novel genes possibly linked to CRC were also discovered for further investigation. Heterogeneity of carcinoma tissues within the same individual was suggested by comparing allele frequency of different somatic mutations. Numerous loss of heterozygosity was observed in both carcinoma and polyp tissues.

Conclusion: Target capture/NGS approach allows simultaneous analyses of SNVs and CNVs with quantitative data to distinguish between germline and somatic mutations. These analyses reveal abundant and valuable molecular information for target therapy, patient management, genetic counseling, and new insights of possible pathogenic mechanism

#3172

Comprehensive analysis of intrahepatic cholangiocarcinoma based on viral infections and mutational status in the IDH1/2 and KRAS genes.

Kazuya Yasui,1 Takeshi Nagasaka,1 Yuzo Umeda,1 Tomokazu Fuji,1 Fumitaka Taniguchi,1 Toshiaki Toshima,1 Keisuke Kimura,1 Takashi Kawai,1 Yoshiko Mori,1 Hiroshi Tazawa,1 Takahito Yagi,1 Ajay Goel,2 Toshiyoshi Fujiwara1. 1 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan;_ 2 _Center for Gastrointestinal Cancer Research; Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

Background: Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with poor prognosis and limited therapeutic options. Recently comprehensive genetic profiles have elucidated novel fusion genes and somatic mutations. Although somatic mutations in the isocitrate dehydrogenase (IDH) and KRAS gene were frequently found in ICC, the clinical features of this malignancy with mutations in these genes remain unclear.

Materials and Methods: A cohort of 49 patients with ICC who underwent curative resection from 2000 to 2013 at Okayama University Hospital were enrolled and analyzed. KRAS (exon2), IDH1 (codon132), and IDH2 (codon172 and codon140) mutations were confirmed by Sanger sequencing. Associations between the mutational profiles of these genes and clinic-pathological features were investigated. Kaplan-Meier curves were plotted, and survival rates were compared using the log-rank analysis.

Results: KRAS mutations were observed in twelve ICC patients (24.5%, KRAS-mutant). IDH mutations were found in eight patients (16.3%); five patients harbored IDH1 mutations and three patients harbored IDH2 mutations (IDH1/2-mutant). Among three patients with IDH2 mutations, a rare IDH2 R140L was identified. One patient harbored both KRAS G12A and IDH2 R172K mutations (classified as KRAS-mutant). The rest of 30 ICCs harbored no mutation in either KRAS or IDH1/2 gene, thus categorized as Wild-type. Infection of Hepatitis B (HBV) or C (HCV) was observed in 10 ICCs (6 ICCs were HBV-positive and 4 ICCs were HCV-positive). Of 10 ICCs with viral infection, only two ICCs harbored KRAS mutations (categorized as virus-infected) and eight ICCs with viral infection harbored no mutation in KRAS and IDH1/2 genes. Finally, ICCs were divided into four subsets; viral-infected, KRAS-mutant, IDH1/2-mutant, and 'others'). With regard to clinic-pathological features, lymph node metastasis was more frequently observed in KRAS-mutant (p=0.039) compared with the others. IDH1/2 -mutant were more frequently observed in mass-forming type (p=0.047). By survival analysis, 3-years survival rate was 85.7% in IDH1/2-mutant, 58.3% in the others, 36.3% in viral-infected, and 22.2% in KRAS-mutant, respectively (p=0.05). Thus, our cohort suggests that IDH1/2, as well as KRAS mutations, have the potential to serve as prognostic biomarkers in ICC.

Conclusion: We conclude that not only viral infections, but also IDH1/2 and KRAS mutations could be potential predictive markers for the identification of good and worse prognosis in ICCs. Hence the mutational profiles of these genes provide an attractive rationale for developing a molecular signature for the development of non-invasive screening for ICCs in future.

#3173

Next generation sequencing (NGS) of dihydropyrimidine dehydrogenase (DPYD) gene in Mexican patients with gastrointestinal (GI) carcinomas.

Erika Ruiz-Garcia,1 Alicia Lopez-Yanez,1 Alette Ortega,1 Jorge Guadarrama-Orozco,1 Gisela Hernandez-Luis,1 Fabiola De la Rosa,1 Abelardo Meneses-Garcia,1 Horacio Astudillo-de la Vega2. 1 _National Cancer Institute, Mexico City, Mexico;_ 2 _Hospital de Oncologia, CMN SXXI, Mexico City, Mexico_.

Background: Fluoropyrimidines (5-fluorouracil (5-FU), capecitabine and tegafur) are the mainstay chemotherapeutic drugs for gastrointestinal cancer. Patients with deficiency of dihydropyrimidine dehydrogenase enzyme (DPD), which interfere with the pyrimidine metabolism, could be at risk toxicity (mild, severe or lethal) because >80% of the administered 5-FU is catabolized by DPD. Previously, our group studied DPYD single-nucleotide polymorphism (SNPs) by real-time PCR assay, in 72 Mexicans. We found that frequency of SNP 85T>C and 85T>T was similar as Oriental race (2-3%), but contrast with Caucasians (0.19%), meanwhile, for SNP 496A>G which Caucasian prevalence was reported 0.8%, in our Mexican sample was very high (14%). Even though, none of our patients with toxicity grade 3-4, were positive to the SNP associated to higher toxicity (IVS14 + 1G>A). Because of this heterogeneity, we decide to sequence the complete gen in our population.

Methods: We included prospectively, 19 patients of the GI Oncology Department of the National Cancer Institute of Mexico, who were treated with fluoropyrimidines. We follow them during the whole treatment. DNA was extracted from whole blood using the DNeasy blood and tissue kit (Qiagen Ltd, Crawley, UK). We use the AmpliSeq™ Designer for the full coverage of all 23 exons of DPYD gene; 25 μg of DNA was used for library construction (Ion Ampliseq Library kit 2.0), we follow the manufacturer's instructions for the target sequencing of DPYD gene with the Ion Torrent PGM platform. The data management was performed with the Ion Reporter Software of Thermo Fisher.

Results: We found that 89.5% of the patients had SNPs (c.85C>T), 44.1% (c.1627A>G) and 5.2% (c.496A>G). Mean while SNPs: c.1896T>C, c.451A>G, c.771C>T, c.2384G>A, c.2605G>A, c.1462A>G, c.1422_1422delA were present in a lower frequencies. These results were associated to clinical toxicity (mucositis, neurotoxicity, hand foot syndrome, hematological toxicity and diarrhea grade >2) but there wasn't any association (chi2 test). Not a single patient were positive for IVS14 + 1G>A.

Conclusion: These are the first results using NGS for DPYD gene in Mexican people. We confirm that our population had a different molecular profile that Caucasians. We need to improve the sample size, to see if we could find an association between SNPs and clinical toxicity.

#3174

Colon cancer molecular subtypes: Concordance, effect on survival and selection of most representative preclinical models.

Zsófia Sztupinszki,1 Balázs Győrffy2. 1 _Semmelweis University, Budapest, Hungary;_ 2 _Hungarian Academy of Sciences, Budapest, Hungary_.

Introduction: Colon cancer is a genetically heterogeneous disease and there is an increasing need for more detailed classification of stage II and III patients. The NCCN guideline currently lists three FDA approved gene-expression based multi-gene prognostic tests for stage II-III colorectal cancer. However, it is undecided how many molecular subtype would most accurately describe these tumours. Here, we cross-validated known molecular subtypes for their concordance and capability to predict prognosis free survival. We utilized gene expression signatures to identify the most representative cell line model for each subtype.

Materials and methods: Using publicly available datasets from the GEO repository we established a database containing clinical and microarray data for 2,166 colon cancer patients. Gene expression measured on Affymetrix HG-U133A and HG-U133 Plus2.0 arrays was processed according to authors' description for each classifier in R. We conducted a systematic review of the literature in order to identify gene-expression based classifiers. We assigned each sample for these classificators and then compared their potential to predict recurrence free survival.

Results: All together 22 different molecular subtypes were re-classified including the CCHS, CIN25, CMS, ColoGuideEx, ColoGuidePro, CRCassigner, MDA114, Meta163, ODXcolon, Oncodefender, TCA19, V7RHS classifiers as well as subtypes established by Budinska, Chang, DeSousa, Marisa, Merlos, Popovici, Schetter, Yuen, and Watanabe (first authors). When comparing the methods the most concordant classifiers were MDA114 and DeSousa (Cramer's V =0.711). The highest efficacy to predict progression free survival in stage II-III patients was achieved by Yuen (p=3.9e-05, HR=2.9), Marisa (p=2.6e-05, HR=2.6) and Chang (p=9e-09, HR=2.35). We also assigned 61 colon cancer cell lines from four independent studies to the closest molecular subtype.

Conclusion: In summary, here we present a comprehensive analysis of human colon cancer molecular subtypes by cross-validation using independent transcriptomic datasets.

#3175

Identification of a unique subpopulation of mesenchymal-like colorectal cancers associated with better survival.

Mingli Yang,1 Michael J. Schell,2 Michael V. Nebozhyn,3 Andrey Loboda,3 Timothy J. Yeatman1. 1 _Gibbs Cancer Center & Research Institute, Spartanburg, SC; _2 _Moffitt Cancer Center & Research Institute, Tampa, FL; _3 _Merck, Sharp and Dohme, West Point, PA_.

The epithelial-mesenchymal transition (EMT) has been well-recognized as an important mechanism promoting cancer cell invasion and "stemness", metastasis and therapeutic resistance. Various types of cancers with high-EMT (mesenchymal-like) features have been correlated with poorer survival. For example, recently, an international consortium (Guinney et al, Nature Medicine, 2015) has coalesced six independent classification systems of colorectal cancers into four consensus molecular subtypes (CMSs), among which only CMS4 (mesenchymal, 23%) was associated with worse overall survival and worse relapse free survival.

Here we report an analysis of 458 colorectal tumors that identified a subpopulation (n=31, 6.8%) of mesenchymal-like colorectal cancers with unique molecular features. Surprisingly, these tumors were significantly associated with better survival. All samples underwent targeted exome sequencing of 1,321 cancer-related genes, global gene expression profiling and microsatellite instability (MSI) analysis, and were evaluated for the Kaplan-Meier survival and various additional correlation analyses. We explored gene expression signatures to measure EMT, RAS/MAPK, growth factor signaling, and mucinous characteristics.

While we recently found that colorectal tumors with wild-type APC (n=151, 33.0%) were associated with worse outcome, we identified a mucinous subpopulation of these tumors (n=31, 20.7%) which were associated with better overall survival (p=0.004). This subpopulation of wt APC tumors were mesenchymal-like, as indicated by its significantly higher mRNA expression of the mesenchymal marker VIM (p=0.0001) along with a robustly higher EMT signature scores (p<0.0001). This is in contrast to another mucinous colorectal subpopulation, MSI tumors that are epithelial-like, also with a good survival. Moreover, unlike hyper-mutated MSI tumors, the subpopulation identified appears to be correlated with the least-mutated tumors that lack common drivers (e.g. TP53, KRAS and BRAF). Compared to other wt APC tumors, this subpopulation had lower mRNA expression of a number of "canonical" Wnt/beta-catenin targeted genes including MYC (p=0.009), MET (p<0.0001), CDKN2A (p=0.03), LGR5 (p=0.0015), HNF1A (p=0.003), and SOX9 (p=0.01), while expression of other targeted genes TCF4 (p<0.0001), TNC (p=0.04) and NOTCH2 (p=0.0006) were upregulated. Furthermore, these tumors were also characterized by attenuated activation of RAS/MAPK and growth factor-mediated signaling.

Taken together, we have identified a specific subpopulation of colorectal cancers with distinct biological and prognostic features, indicating that a "fine-tune" classification of colorectal cancers is necessary to improve resolution of appropriate therapy.

#3176

Improved colorectal cancer screening by new stool-based protein markers.

Linda J. Bosch,1 Meike De Wit,1 Annemieke C. Hiemstra,1 Sander R. Piersma,2 Thang V. Pham,2 Gideon Oudgenoeg,2 Veerle Coupe,2 George Scheffer,2 Sandra Mongera,2 Jochim Terhaar Sive droste,2 Frank Oort,2 Sietze van Turenhout,2 Ilhame Ben Larbi,2 Chris Mulder,2 Beatriz Carvalho,1 Remond JA Fijneman,1 Connie R. Jimenez,2 Gerrit A. Meijer1. 1 _Netherlands cancer institute, Amsterdam, Netherlands;_ 2 _VU University medical center, Amsterdam, Netherlands_.

Background

The fecal immunochemical test (FIT) is used in many countries for non-invasive screening for colorectal cancer (CRC), but its characteristics leave room for improvement. We aimed to identify novel stool-based protein markers in stool that outperform or complement hemoglobin in detecting CRC.

Materials and methods

A series of large scale proteomic discovery and validation was conducted on a total of 313 human stool specimens. A discovery set consisted of 22 stool specimens (12 CRC and 10 controls), and isolated proteins were analyzed by an in-dept mass spectrometry GeLC-MS/MS workflow. An external validation set consisted of 291 independent stool specimens (79 CRC, 40 advanced adenoma (AA), 43 nonadvanced adenoma (A) and 129 controls), and isolated proteins were analyzed by single-shot mass spectrometry (Q-Exactive). As proof of concept, commercially available meso scale discovery (MSD) assays were performed on an independent validation set of 72 FIT fluid samples set including 14 CRC, 16 AA, 18 A and 24 controls.

Results

Of the 468 human proteins quantified in the discovery set, 93 were significantly enriched in CRC vs controls (p<0.05). Of these, 29 were significantly found enriched in CRC vs controls in the validation set (q<0.05). CART analysis and exhaustive search revealed a combination of 4 proteins as the most optimal combination to differentiate CRC from controls. A logistic regression model of this combination of proteins detected CRC with a sensitivity of 73% as compared to 43% for hemoglobin (HBA1) alone at a fixed specificity of 95% (p=0.00003). Another combination of 4 proteins was the most optimal combination to differentiate AA from controls, which showed a sensitivity of 48% vs 8% for hemoglobin (p = 0.0002) at fixed specificity of 95%. Evaluation of one of the markers on FIT fluid samples provided proof of concept that these proteins can be detected in FIT fluid and can significantly discriminate CRC from healthy controls (p < 0.001).

Conclusion

Proteome profiling on stool samples revealed 29 validated proteins significantly enriched in CRC samples compared to controls. A panel of 4 complementary protein markers outperformed hemoglobin for detection of CRC as well as AA. Proof of concept for detecting the proteins in FIT fluids confirmed the high potential of these markers for screening purposes in a non-invasive and cost-effective manner.

#3177

Clinical characteristics and choice of therapy that are associated with altered bladder cancer genes.

Michael L. Nickerson,1 Kate M. Im,2 Sevilay Turan,3 Lee E. Moore,1 James C. Costello,4 Michael Dean,1 Dan Theodorescu4. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Data Science for Genomics, Ellicott City, MD;_ 3 _Trinity Washington University, Washington, DC;_ 4 _University of Colorado, Aurora, CO_.

The identification of altered genes in bladder cancer (BCa) that are associated with clinical characteristics of disease and the choice of therapy is likely to improve cancer diagnosis and treatment. We used next generation sequencing (NGS) and PCR-Sanger sequencing to examine genomic alterations in DNA from muscle invasive and non-muscle invasive tumors from Chinese and U.S. patients, and in 25 bladder tumor-derived cell lines. We show cancer genes are altered by multiple mechanisms, approximately 4-5 tumor suppressors are altered for each oncogene, and alterations are highly predictive of canonical features of disease. Telomerase (TERT) was most frequently altered by somatic sequence changes in the promoter in 37/54 (69%) tumors and by germline variants in 30/54 (56%) tumors. Most germline variants in U.S. patients were novel (19/20 variants) and three were confirmed as somatic in distinct tumors, including the most frequent variant, c.-245 T>C (rs2853669). Variants created or altered transcription factor binding sites and promoter CpGs, telomere length was significantly shorter in tumor versus adjacent normal tissue from the same patient (p=0.004), and somatic alterations were not associated with stage, grade, or other altered BCa genes. Epigenetic genes were frequently altered, including the chromosome X histone 3 lysine 27-specific demethylase (KDM6A) and stromal antigen 2 (STAG2). We identified somatic sequence and copy number alterations in the breast cancer 1-associated protein (BAP1) in U.S. patient tumors that were not observed in 99 Chinese patients (p = 0.003), which remained significant after accounting for differences in tumor grade and stage (p = 0.037). BAP1 mutations were associated with papillary features in a subset of tumors and co-occurred with KDM6A mutations (p = 0.017), indicating the altered cancer genes may provide complementary survival advantages. The cancer gene alterations in the cell lines were similar to those observed in the tumors and we observed homozygous alterations unique to each line. We conclude that altered X chromosome cancer genes (STAG2 and KDM6A) which are single copy in men contribute to the BCa gender bias and speculate that TERT is altered as an early event in BCa. Characterizing cancer gene mutations in cell lines allows more effective utilization of these reagents for molecular and therapeutic analyses. Somatic alterations in BAP1 contributed to frequent BRCA pathway alterations in BCa and indicate treatment with poly(ADP ribose) polymerase inhibitors may be effective.

#3178

Metastatic urothelial cancer 10 years after transplanting a filial kidney: genomically investigating the perpetrator.

David C. Müller,1 Maarit Raemoe,2 Christian Wetterauer,2 Valeria Perrina,1 Luca Quagliata,1 Tatjana Vlajnic,1 Christian Ruiz,1 Beate Balitzki,3 Hans H. Hirsch,4 Rainer Grobholz,5 Lukas Bubendorf,1 Cyrill A. Rentsch2. 1 _Institute for Pathology, University Hospital Basel, Basel, Switzerland;_ 2 _Department of Urology, University Hospital Basel, Basel, Switzerland;_ 3 _Institute of Forensic Medicine, University of Basel, Basel, Switzerland;_ 4 _Transplantation & Clinical Virology, Department of Biomedicine, University of Basel, Basel, Switzerland; _5 _Institute for Pathology, Kantonsspital Aarau, Aarau, Switzerland_.

BACKGROUND

Polyomavirus (BKV) infection has been associated with development of urothelial carcinoma (UC). We report on a patient with conventional UC of the bladder (Ta, G3) 8 years after receiving a kidney graft from a 4 year old male donor. After a recurrence (T1, G3) 9 years after transplantation (NTx), he was diagnosed with a SV-40 positive metastatic micropapillary and muscle-invasive UC 10 years after NTx with involvement of the graft kidney pelvis (pT3), the bladder (pT3) and a pelvic lymphnode metastasis. The aim of this study was to investigate the genealogy and clonal relationship between these cancer locations.

METHODS

Cancer cell nuclei derived from formalin fixed tumor specimens were processed and sorted based on DNA content with a BD Influx FACS. DNA from sorted aneuploid populations was analyzed by whole genome high resolution CGH microarrays (aCGH). In addition, short tandem repeat (STR) analyses were done with the PowerPlex® ESI Kit commercial testing kit. Each run was performed on an Applied Biosystems Genetic Analyzer 3500 and analyzed with the GeneMapper ID-X Software. Integration of PV-genom was investigated by RT-PCR. The IonTorrent platform by LifeTech (PGM, IonAmpliseq Cancer Hotspot Panel v2) was used for next-generation-sequencing (NGS).

RESULTS

Array CGH clarified that the primary two non-muscle invasive bladder tumors differed completely from each other and from the subsequent cancer tissues. The tumor manifestations 10 years after NTx shared the same genomic profile, except for a private amplification at 6p12.3 and a private deletion at Xp22.33 - Xp22.11, both in the bladder tumor. STR identified all three tumors diagnosed 10 years after NTx to originate from the allograft donor. NGS identified two shared mutations of unknown relevance (KDR / InDel chr4:55962545; Akt1 / c.138C>A) in the 10 year after NTx UC sites. No known somatic mutation was found. RT-PCR identified the PV to be integrated in the human cancer genome. Further studies are currently ongoing to define the exact integration sites of BKV and the potentially influenced human genes.

CONCLUSIONS

This case is an impressive example of a, donor-derived, metastatic micropapillary UC developed under immuno-suppression and subsequent BKV-reactivation in a presumably healthy transplanted kidney of a four years old donor.

#3179

Germline deletion of FH in hereditary leiomyomatosis and renal cell carcinoma (HLRCC).

Cathy D. Vocke, Christopher J. Ricketts, Lindsay A. Middelton, Youfeng Yang, W. Marston Linehan. _National Cancer Institute, Bethesda, MD_.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome characterized by germline mutations in the fumarate hydratase (FH) gene that encodes the enzyme responsible for conversion of fumarate to malate in the Kreb's Cycle. HLRCC is associated with the development of cutaneous and uterine leiomyomas, and a highly aggressive form of type 2 papillary kidney cancer. HLRCC renal tumors can be extremely aggressive with patients demonstrating locally advanced or disseminated disease even when associated with small tumors 1-2 cm in size. Therefore, accurate identification of the "at risk" individuals is very important. In our experience, FH point mutations are identified in 90% of individuals that are clinically diagnosed with HLRCC, while in the remainder, FH may be inactivated by deletion or other causes. Several studies have reported complete deletions of the FH gene and a few have estimated the extent of these deletions and identified the additional genes that would be lost in conjunction with FH. Interestingly, families with complete FH deletion have demonstrated cutaneous and uterine leiomyomas but no families have yet been reported with evidence of kidney cancer. A single family has been identified that demonstrates a partial deletion resulting in the loss of exon 1, and in this case kidney cancer has identified within the family. In this study, patients with suspected HLRCC hereditary kidney cancer but no germline point mutation were enrolled on an NCI-IRB approved protocol and an Agilent custom high-definition comparative genomic hybridization (CGH) array was used to identify FH gene deletions. The assay identified complete germline deletion of a single copy of FH within seven suspected HLRCC patients deletion ranging from 249kb to 4.74Mb that in all cases resulted in a minimum addition loss of the KMO, OPN3, CHML, and WDR6 genes. All results were confirmed by CLIA approved tests and a further five new patients were identified by direct CLIA testing, including a novel partial deletion. In contrast to previous reports, seven out of twelve (58.3%) patients/families in our cohort demonstrated a personal or family history of HLRCC associated Type 2 papillary kidney cancer. In conclusion, complete or partial deletion of the FH gene is an infrequent but important cause of HLRCC and has no protective effect on the incidence of HLRCC associated type 2 papillary kidney cancer.

#3180

Deletions of 18q characterizes a subset of aggressive prostate cancers with metastatic potential.

Claudia Hube-Magg, Martina Kluth, Maximilian Graunke, Christina Koop, Steuber Thomas, Markus Graefen, Stefan Steurer, Thorsten Schlomm, Guido Sauter, Ronald Simon. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

Large chromosomal deletions and rearrangements of the TMPRSS:ERG locus are hallmarks of prostate cancer. Deletion of 18q is recurrently found in prostate cancer, but the prevalence and clinical significance of this alteration have not been studied in detail as to yet. We took advantage of our large prostate cancer prognosis tissue microarray (TMA) containing more than 12,000 prostate cancers with clinical follow-up data and performed fluorescence in situ hybridization (FISH) analysis using an 18q21 probe. A total of 6,881 prostate cancers were interpretable by FISH. Deletion of 18q was found in 7.5% of cancers, and was linked to advanced tumor stage (p<0.0001), high Gleason grade (p=0.0013) and biochemical recurrence (BCR, p<0.0001). Comparison with data from TMPRSS:ERG fusion and ERG protein expression revealed that deletions of 18q were only marginally related to an ERG-fusion negative phenotype (p=0.0063). Multivariate analysis showed that the prognostic value of 18q deletion was independent from the established prognostic factors in all cancers (p=0.0004) as well as in the subsets of ERG-fusion negative (p=0.0148) and ERG-fusion positive cancers (p=0.0264). From the clinical data attached to our TMA, we defined additional clinical endpoints beyond BCR, which represent the four hallmarks (local, local invasive, occult systemic and metastatic) of tumor growth and dissemination in prostate cancer. Deletion of 18q was linked to unfavorable outcome also according to these alternative clinical endpoints. For example, the fraction of cancers with occult systemic or metastatic dissemination increased from 33.6% in patients whose tumors lacked 18q deletion to 41.8% in patients with tumors harboring 18q deletion (p=0.0458). In summary, the results of our study demonstrate a pivotal role of 18q deletions for prognosis in prostate cancer independent of the established prognosticators.

#3181

Expression changes of the chromatin modifier PBRM1 in human renal cell carcinomas in relation to histopathological features.

Hans Krause,1 Anica Högner,1 Burkhard Jandrig,2 Ergin Kilic,1 Carsten Kempkensteffen1. 1 _Charité - Universitätsmedizin Berlin, Berlin, Germany;_ 2 _Universitätsklinikum Magdeburg A.ö.R., Magdeburg, Germany_.

Recent next-generation sequencing studies of clear cell renal cell carcinoma (ccRCC) have identified point mutations in chromatin-modifying genes, among them PBRM1 which is now the second most frequently mutated gene in RCCs (in up to 41% ccRCCs cases) and belongs to the m1 TCGA (The Cancer Genome Atlas) subset. The occasional presence of PBRM1 mutations in the absence of VHL mutations indicates, that PBRM1 might be another driver tumor suppressor gene in ccRCCs. We were interested in examining PBRM1 expression at mRNA and protein levels and correlated these findings to histopathological features of RCC patients.

Relative gene expression (RGE) data were obtained by Q-PCR from matched tumor and adjacent normal fresh frozen ccRCC tissues from 57 patients who underwent radical nephrectomy at our clinic. Papillary (pRCC) as well as chromophobic cases (chRCC) were added to analyze the expression of splice variants of PBRM1. Comparative Western blot analyses as well as semi-quantitative immuno-histochemical staining (IHC) for PBRM1 and VHL were performed on all ccRCC cases.

In 78.9% of cases (45/57) PBRM1 mRNA was downregulated at least 1.5-fold, 7% (4/57) showed PBRM1 upregulation, and 14% (8/57) displayed no obvious expression changes between tumor and corresponding tumor tissue. Interestingly, 21 of 45 ccRCCs tumor tissues expressed splice variant 4 more abundantly when compared to normal tissue, whereas the normal tissue preferentially expressed splice variant 1 that includes an additional exon. Remarkably, this differential expression picture is completely reversed in pRCC and chRCC cases.

The majority of 57 ccRCCs displayed weak nuclear PBRM1 staining (52.6%), whereas 31.6% showed moderate and 15.8% strong staining. However, we were not able to demonstrate a significant correlation of IHC expression levels, neither to tumor staging nor to Fuhrman grading.

The observed high frequency of decreased expression of the chromatin-remodeling gene PBRM1 on mRNA (78.9%) and protein levels (52.6%), respectively, as wells as a high mutation rate (about 15% in our data set) indicate a substantial role of PBRM1 in the tumorigenesis of ccRCCs. Preferences in the expression of different PBRM1 splice variants warrant further investigation with regard to renal cell carcinoma development.

#3182

Tumor associated macrophages in undifferentiated uterine sarcoma: association with angiogenesis and therapeutic implications.

Joanna Przybyl,1 Magdalena Kowalewska,2 Anna Quattrone,3 Barbara Dewaele,3 Vanessa Vanspauwen,3 Sushama Varma,1 Robert T. Sweeney,1 Michal Swierniak,4 Elwira Bakula-Zalewska,2 Janusz A. Siedlecki,2 Mariusz Bidzinski,5 Jan Cools,6 Matt van de Rijn,1 Maria Debiec-Rychter3. 1 _Stanford University, Stanford, CA;_ 2 _Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland;_ 3 _KU Leuven and University Hospitals Leuven, Leuven, Belgium;_ 4 _Human Cancer Genetics, Center of New Technologies, CENT, University of Warsaw, Warsaw, Poland;_ 5 _Holycross Cancer Center, Kielce, Poland;_ 6 _KU Leuven and Flanders Interuniversity Institute for Biotechnology (VIB), Leuven, Belgium_.

Sarcomas of the uterus are derived from uterine smooth muscle (leiomyosarcomas) or from endometrial stromal cells. The latter group includes three subtypes: the low-grade (LG) and high-grade (HG) endometrial stromal sarcomas (ESS), each defined by characteristic chromosomal translocations, and undifferentiated uterine sarcoma (UUS), the most aggressive form. The majority of UUS patients present with high-stage disease and even patients with stage I tumors often die within 2 years from diagnosis. Adjuvant radio- and chemotherapy do not improve the clinical outcome. No prior comprehensive molecular analysis of all three types of endometrial stromal tumors has been reported.

We performed genomic analysis (whole exome sequencing and aCGH), gene expression profiling (RNA-seq and microarrays), immunohistochemistry (IHC) and FISH on 31 endometrial stromal tumors, including 17 UUS and 14 LG and HG ESS. All ESS cases carried characteristic JAZF1, PHF1, MBTD1 or YWHAE rearrangements. Selected findings were validated by IHC, qRT-PCR and Sanger sequencing.

Genomic analysis revealed that UUS are characterized by complex chromosomal aberrations. The most frequent somatic mutations (in TP53, KRAS, FBXW7, PIK3CA and ERBB3) and copy number changes (e.g. gains of 19q, 8q11.1-q24.3 and 3q26.2-q29) in UUS resemble those seen in uterine carcinosarcomas and carcinomas.

UUS over-expressed numerous genes encoding M2 macrophage-specific markers, including CD163, CCL18, mannose receptor, stabilin-1, and macrophage galactose-type C-type lectin 2. Immunohistochemistry for CD163 and CD68 confirmed high tumor-associated macrophages (TAM) counts in UUS tissues compared to the ESS. In UUS, we also observed significantly higher mRNA expression for genes implicated in angiogenesis (CD34, MMP9), immunosuppression and tumor invasion (e.g. CCR2, IL10RA, IRAK3, CCL13, CXCL9, CXCL10).

TAMs are associated with progression, resistance to cytotoxic therapy and metastatic spread in most human tumor types. In animal models, TAMs were shown to induce increased angiogenesis. CSF1 is a major regulator of macrophage function and is implicated in these tumor-promoting effects. Clinical trials have shown an effect of inhibitors of the CSF1 pathway in tenosynovial giant cell tumors and our data indicate that these inhibitors may be considered for the treatment of UUS. Our study also showed significantly elevated expression in UUS of two additional therapeutic targets involving macrophage pathways: CCR2 (C-C chemokine receptor type 2) that can be targeted by monoclonal antibodies, and BTK (Bruton's tyrosine kinase) that can be targeted by ibrutinib.

In conclusion, our findings demonstrate abundant presence of TAMs and over-expression of TAM-associated markers in UUS compared to the other tumors derived from endometrial stroma. Moreover, our results indicate novel potential therapeutic targets for UUS patients management.

#3183

Delivery of point-of-care management to patients with gynecologic malignancies using comprehensive genomic profiling.

Lorna Rodriguez-Rodriguez,1 Kim M. Hirshfield,1 Veronica Rojas,2 Siraj Ali,3 Robert S. DiPaola,1 Darlene Gibbon,1 Sara Isani,1 Aliza Leiser,1 Gregory M. Riedlinger,1 Shridar Ganesan1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 3 _Foundation Medicine, Inc., Cambridge, MA_.

INTRODUCTION: Gynecologic cancers were estimated to occur in 98,280 women in 2015 in the United States and result in 30,440 deaths (Siegel 2015). The most common forms of gynecologic cancers occur in the ovary and uterine corpus (Siegel, 2015). Various reasons for poor outcome in these cancers include the late stage of the disease at the time of diagnosis and therapy resistance. We sought to elucidate alternate treatment avenues based on the genomic profiles of recalcitrant or rare gynecologic tumors. In the process, we aim to determine both the clinical utility and the feasibility of using comprehensive genomic profiling (CGP) during clinical care to identify clinically relevant tumor genomic alterations for these patients while enabling point-of-care management.

METHODS: As part of our ongoing prospective clinical trial, we report on the initial 69 patients with gynecologic cancers that were refractory to standard therapy. CGP was performed by Foundation Medicine, Inc. Genomic mutations and alterations were reviewed by members of an expert, multidisciplinary institutional molecular tumor board (MTB). Consensus recommendations by the MTB were provided to the treating physician on genomically targeted, clinically relevant, FDA-approved, on- and off-label therapies and clinical trials. The outcomes were assessed.

RESULTS: Study outcomes were available for 64 patients. On average, patients had 4.97 genomic alterations per tumor (range 1-46), while the time from the testing laboratory report to the formulation of recommendations by the MTB was approximately 3-4 weeks. Targeted

treatment options were recommended by the MTB for 93% of patients; 39% of these patients had the MTB-based recommendations implemented by the treating physician. A response or clinical benefit was seen in 56% of the patients receiving MTB-based targeted therapy.

CONCLUSION: These data suggest that alternatives to standard therapies for recalcitrant gynecologic cancers can be found though CGP and subsequent analysis from members of an expert, multidisciplinary institutional MTB. The formulation of clinical recommendations by an MTB based on tumor genomic profiling results is a feasible option. Further research is needed to understand how this approach shapes clinical decision making.

#3184

Genomic analyses reveal two distinct biologic entities of endometrial endometrioid carcinoma.

Yuko Sugiyama,1 Nobuyuki Fukui,2 Osamu Gotoh,2 Katsuhiko Hasumi,1 Yutaka Takazawa,2 Tetsuo Noda,2 Seiichi Mori2. 1 _Cancer Institute Hospital, Tokyo, Japan;_ 2 _Cancer Institute, Tokyo, Japan_.

Endometrial carcinoma (EC) is classified into two types: type 1 (endometrioid type) and type 2 (non-endometrioid type). Type 1 endometrioid adenocarcinoma (EA) has been generally considered a single disease entity and was developed from endometrial hyperplasia as a precursor lesion. Recently we identified two distinct subgroups in type 1 EA; groups 1 and 2 as tumors with and without endometrial hyperplasia, respectively. The purpose of this study is to identify genes involved those differentially expressed by groups 1 and 2 EA. Genomic analyses including transcriptome and exome sequencing and immunohistochemistry were performed. For the validation, these data were projected to the TCGA (The Cancer Genome Atlas Research Network) data. In the results of transcriptome, group 1 and 2 EAs identified differential regulation of multiple genes and pathways. As an example, estrogen receptor (ER) related pathways were up-regulated in group 1 EA. Immunohistochemical analyses further confirmed overexpression of ER and PgR in the protein level. In the result of exome sequencing, mutation was observed more frequently in group 2 and sixteen driver genes were more frequently mutated in group 2.

#3185

Value of next generation sequencing in a diagnostically challenging case of hematologic malignancy.

Ja Min Byun,1 Youngil Koh,1 Sung-Soo Yoon,1 Daeyoon Kim2. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Cancer Research Institute, Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Traditionally, the diagnosis of hematologic malignancies relied on morphological, immunological along with cytogenetic evaluation of bone marrow and peripheral blood. However, these methods sometimes yield non-diagnosis, diagnostic ambiguity or discordance. The advent of next generation sequencing (NGS) can be of help in such settings. Here, we describe our experience of using NGS data in guidance to diagnose and treat an indeterminate case of hematologic malignancy.

Case: A 77-year old female presented to the clinic with worsening dyspnea on exertion with pancytopenia (white blood cells 1.25x109/L – hemoglobin 7.6g/dL – platelet 22x109/L). Her bone marrow examination showed hypercellular marrow morphologically suggesting acute myeloid leukemia (AML) M4 by French-American British classification, but blast count of only 6.9%. The conventional karyotyping showed 46, XX [20]. Fluorescence in situ panel, including PML-RARA, RUNX1T1-RUNX1, CBFB, KMT2A, MECOM, were all negative, and so were the results of HemaVision®. Direct sequencing and PCR for FLT-3 ITD and TLD and KIT sequencing for exon 9, 11, 13, 17 results were all negative. In order to reconcile the discrepancies between morphological and cytogenetic diagnosis, NGS was performed.

Method: The DNA from bone marrow collected at the time of diagnosis was analyzed using whole exome sequencing (WES). Saliva DNA collected at the time of remission was used matched normal sample. Somatic mutations were determined by MuTect. For structure variation analysis, insertion size was calculated by CollectInsertSizeMetrics module of Picard package and Pindel to call SVs. Gene to gene network analysis was done using STRING database.

Discussion: We identified 13 somatically mutated non-synonymous small nucleotide variants with WES. These included NBPF1/8/9, PBRM1, DDX60L, VPS52, TIMM23B, KIF26A, MYO1E, KRTAP9-8, CRELD2, FOXO4 and TNMD. Through gene to gene networking, we recognized FOXO gene in relation to AKT pathway and JUN signaling. Since FOXO4 amplification appears in 0.5% of AML cases and AKT/FOXO and JNK/c-JUN plays a pivotal role in myeloid leukemia, we recognized this as possible driver mutation. Moreover, fragment analysis with direct sequencing recognized NPM1 gene exon12 c.860_863dupTCTG. NGS data revealed NPM1 1 copy repeat (2 bp insertion). Based on the NGS data, the patient received low dose cytarabine (20mg/m2/day for 14 days), as in elderly AML patients. She responded surprisingly well, and after 3 cycles of chemotherapy, her hemogram was almost normalized (WBC 3.02x109/L – Hb 10.4g/dL – platelet 180x109/L). She is currently being followed up without any further interventions.

Conclusion: Our data highlights the importance of molecular diagnosis based on NGS when confronted with diagnostic challenges in hematology. Efforts to effectively incorporate NGS data in real-world clinical settings should be encouraged to realize the goals of tailored medicine.

#3186

Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR.

Daeyoon Kim,1 Yoojin Hong,2 Youngil Koh,1 Sung-Soo Yoon,1 Choong-Hyun Sun,2 Kwang-Sung Ahn,3 Seungmook Lee,2 Hongseok Yun,2 Suyeon Lee2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Bioinformatics Group, Platform Development Center, CSP R &D, Samsung SDS, Seoul, Republic of Korea; _3 _Functional Genome Institute, PDXen Biosystem Inc., Seoul, Republic of Korea_.

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods.

First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped.

Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially.

In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83).

Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p=0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful.

Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients.

#3187

Concordance of immunohistochemistry (IHC) assay results with gene expression profiling (GEP) methods for diffuse large-B-cell lymphoma (DLBCL) subtype identification.

Michael Schaffer,1 Shalini Chaturvedi,1 Sandy Frans,2 Regina Aquino,1 Jaehong Park,1 Brett Hall,1 Sriram Balasubramanian1. 1 _Janssen, Spring House, PA;_ 2 _Janssen, Beerse, Belgium_.

Introduction: The survival of DLBCL patients who receive immunochemotherapy is correlated with the molecular signature of their tumors. The two most common subtypes which are defined by cell of origin (COO) are the germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes which have 3-year OS rates of 80% and 45%, respectively when treated with R-CHOP. Several methods have been developed to categorize DLBCL into these sub-types as the treatment they receive directly affects their outcome. Microarray GEP was first used to classify DLBCL into ABC or GCB, with a minority unclassified (UNC), however this is difficult to implement widely and has been replaced by more recent technologies. The most commonly used IHC-based Hans method profiles patients into GCB and non-GCB. The primary focus of this study was to evaluate concordance between current GEP and IHC methods to designate DLBCL subtypes.

Methods: For this study, the IHC assay used was a standardized IHC assay based on the Hans algorithm and for the GEP-based assay; the NanoString Technologies Lymphoma Subtyping Test (LST) was used. Commercially available DLBCL samples (N = 138) were confirmed for tumor content and sent to a central laboratory for evaluation by the IHC assay. RNA was extracted from each sample and sent to NanoString for GEP evaluations. To compare methods homogeneously, calls for each method were converted to either "GCB" or "non-GCB". All "ABC" and "UNC" subtype calls from the NanoString platform was converted to "non-GCB" and any "did not pass" (DNP) calls were converted to not available (NA). All NA values were excluded from the tables and concordance calculations.

Results: The Positive Percent Agreement [95% CI] in calling non-GCB between IHC and LST was 87% [78% - 96%] (47/54). Overall concordance between IHC and LST was 80% [73% - 87%] (N = 125, 12 LST samples DNP, 1 IHC sample DNP). The Negative Percent Agreement between IHC and LST (i.e., in calling the GCB sub-type) was 75% [65% - 85%] using the LST result as a non-reference standard (53/71).

Conclusions: Given that the success of targeted therapy for the treatment of DLBCL is contingent on the correct identification of sub-type of DLBCL, it is important to adopt a method that accurately and consistently identifies the sub-type in the majority of patients. Given the good concordance between the GEP and IHC methods, DLBCL can be stratified into COO sub-types using either platform. The results show that an optimized Hans IHC assay standardized in a central laboratory offers a robust, relatively inexpensive and accessible platform for cell of origin sub-typing in DLBCL and, with clinical validation, may offer an excellent tool for the selection of optimal therapy.

#3188

Targeted semiconductor sequencing of 409 cancer-related genes for somatic mutations and copy number variations in multiple myeloma.

Hiroshi Ikeda, Yasushi Sasaki, Kazuya Ishiguro, Tadao Ishida, Yuka Aoki, Takashi Tokino. _Sapporo Medical University, Sapporo, Japan_.

Background:

2016's NCCN guideline recommended that induction therapy used conventional chemotherapy such as proteasome inhibitors and Imids in MM. Overall survival is improving by these Novel agents. However, we don't know how to choose these new agents. In the future, we can use next generation sequencer as a tool of drug choosing system. So we reviewed newly diagnosed 11 patients with MM who had received novel agents in our institution.The comprehensive analysis of genetic alterations in tumor by next generation sequencing can allow for the prediction of drug resistance and facilitate improvements in the treatment of MM patients.

Method:

DNA was extracted from magnetic bead-enriched bone marrow CD138-positive malignant plasma cells from 11 cases of MM, and CD138-negative cells were used as matched non-tumor cells. Forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel that offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. (covered regions: 95.4% of total). We sequenced 15,992 regions which obtained more than 1.5 megabases of target sequence.

Result:

Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 539x, and >95% of targeted bases were represented by at least 20 reads. The average number of non-synonymous mutations detected per patient was 5.8 (range 0-11). We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of MM patients. Pathway assessment has shown that somatic aberrations within MM genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB.

Conclusion:

We performed targeted next-generation sequencing for rapid (2 days), standardized, and cost-effective gene analysis of malignant plasma cells from patients with MM. We can use targeted next-generation sequencing as tool of drug choosing system.

#3189

The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome.

Marina Martello, Daniel Remondini, Enrica Borsi, Mauro Procacci, Barbara Santacroce, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Serena Rocchi, Katia Mancuso, Beatrice Anna Zannetti, Giovanni Martinelli, Michele Cavo, Carolina Terragna. _DIMES-Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy_.

The iperactivation of Hedgehog (Hh) pathway, which controls the refuel of the tumor clone, might be critical to Multiple Myeloma (MM) recurrence. In order to dissect the role played by Hh pathway in different MM cells compartments, and to evaluate its clinical impact on patient outcomes, here we explore the transcriptomic and genomic profiles in both CD138+ plasma cells and CD138-19+ B progenitors cells obtained from newly diagnosed MM patients (pts),

The study included a cohort of 126 pts, homogenously treated with bortezomib-based regimens and ASCT. DNA and RNA were obtained from both CD138+ plasma cell fraction and CD19+ B cells. Gene expression profiling (GEP) (HG U133 Plus 2.0) and genomic analysis (SNP 6.0) were performed on Affymetrix platform. Data were analysed by employing several software: dChip (GEP clustering), Ingenuity Pathway Analysis (GEP Enrichment) and Nexus (Copy number).

By unsupervised hierarchical clustering, an Hh signature of 10 genes - SHH, IHH, DHH, SMO, PTCH1, PTCH2, SUFU, GLI1, GLI2 and GLI3 - was identified, and it was able to significantly cluster pts in two subgroups: cluster 1 (70 pts) and cluster 2 (56 pts. An overall significant activation of Hh pathway was shown in cluster 2, as compared to cluster 1. Of note, the Hh pathway was down regulated in CD19+ B cells obtained from pts included in cluster 2, while it was overexpressed in cluster 1 pts. Western blots on both cell fractions confirmed this opposite Hh genes behavior. A higher genomic instability (e.g. higher frequencies of both t(4;14) and del(17p)) was demonstrated in CD138+ cells from cluster 2 pts and, at least 5 known tumor suppressor genes, such as RB1, BRCA2, PDX1, FOXO1 and TP53 were affected. Conversely, cluster 1 pts were mainly characterized by hyperdiploid karyotypes. The more aggressive phenotype of cluster 2 pts was confirmed by an overall deregulation of cell adhesion processes (CD44, LIMS1, COL4A2, CTGF, COL1A1, FN1), increased proliferation (MYCBP, IL22, SDPR, SOX2, SOX6) and DNA repair mechanisms (SP1, SMARCD3, FOXA3). Hh pathway activation significantly influenced pts' outcome, since those included in cluster 2 had a shorter PFS and OS compared to cluster 1. In fact, the 5-year PFS estimates were 31% vs 56% (p=0.0062), whereas the OS probabilities were 66% and 83%, respectively (p = 0.0071). Of note, both hazard ratios for PFS and OS were doubled in pts included in cluster 2, as compared to pts included in cluster 1. Finally, multivariate analyses confirmed that being part of cluster 2 was an independent prognostic factor for both PFS and OS, along with del(17p) and ISS 3.

Two alternate Hh-driven subtypes of MM might be identified at diagnosis, which correlated with pts outcomes. Stratification of pts according to their molecular background might help the fine-tuning of future clinical studies.

Acknowledgements: FP7 NGS-PTL project, Progetto Regione-Università 2010/2012 L. Bolondi.

#3190

Genomic profiling of salivary gland tumors identifies novel and targetable alterations.

Hanna Majewska,1 Judith Müller,2 Sotirios Lakis,2 Piotr Czapiewski,1 Adam Gorczyński,1 Mariola Iliszko,3 Alena Skalova,4 Rafal Dziadziuszko,5 Jacek Jassem,5 Wojciech Biernat,1 Roopika Menon,2 Johannes M. Heuckmann,2 Lukas C. Heukamp2. 1 _Department of Pathomorphology, Medical University of Gdansk, Gdansk, Poland;_ 2 _NEO New Oncology AG, Cologne, Germany;_ 3 _Department of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland;_ 4 _Department of Pathology, Charles University, Faculty of Medicine in Pilsen, Pilsen, Czech Republic;_ 5 _Dept. of Oncology and Radiotherapy, Medical University of Gdansk, Gdanks, Poland_.

Background

Salivary gland neoplasms represent a heterogeneous group of benign and malignant tumors that comprise 6% of all head and neck cancers. Due to their low incidence, these tumors are poorly understood and remain a diagnostic and therapeutic challenge.

Methods

Out of 182 analyzed salivary gland tumors with various histologies, eight were positive for ALK immunohistochemistry. The cut-off was 15% positive cells in either membranous, nuclear or cytoplasmic compartments. The ALK positive tumors were first subjected to FISH analysis. Subsequently, these tumors were analyzed using hybrid capture based next generation sequencing to confirm possible ALK gene fusions or copy number alterations, and to detect additional genomic alterations of clinical relevance.

Results

An in-depth genomic analysis of the samples resulted in the detection of inactivating mutations in BRAF (p.D594N) and TP53 (p.C238S), as well as amplifications of ERBB2 and ALK. Strikingly, a novel MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion was detected in a patient with adenocarcinoma not otherwise specified. This tumor was FISH positive and 100% of cells showed strong membranous staining for ALK. MYO18A (Exon1-40)-ALK (exon 20-29) gene fusion resulted in the retention of the kinase domain of ALK and the coiled-coil domain of MYO18A. Similarly to other ALK fusions, we hypothesize that the coiled-coil domain of MYO18A mediates the dimerization and activation of MYO18A-ALK, thereby resulting in an overexpression of constantly activated ALK.

Conclusion

Using hybrid capture based next generation sequencing, we identified in salivary gland tumors numerous genomic alterations in therapeutically relevant genes and a novel gene fusion (MYO18A-ALK). Patients harboring this fusion may potentially benefit from treatment with ALK inhibitors.

#3191

Molecular testing in lung cancer: Patient and caregiver experiences.

Jennifer C. King, Maureen Rigney. _Lung Cancer Alliance, Washington, DC_.

Increasingly over the past decade, new targeted therapeutics and immunotherapies with associated companion diagnostics have been approved for the treatment of lung cancer. Lung cancer is often associated with elderly and lower socio-economic populations that may not have a high level of health literacy. As part of a comprehensive needs assessment survey of the lung cancer community, lung cancer patients and their caregivers were asked whether they or their loved ones had undergone molecular testing/biomarker testing as well as asked to report the history of oncologic drugs they had taken. Demographic information and additional cancer treatment history were also collected. Over 300 survey responses were collected. Interim analyses indicated that the majority (68%) of patients currently undergoing active treatment had received molecular testing to help determine their treatment plan. There was a clear improvement in testing rates over time with less than 30% of patients who had completed treatment reporting use of a molecular test. Importantly, there is still an educational gap that needs to be addressed as greater than 20% of patients and greater than 30% of loved ones, including the group who self-identified as the primary caregiver, reported that they did not know whether molecular testing had been performed. In addition, patients reported that they had not received EGFR mutation testing but had taken erlotinib, gefinitib, and/or afatinib. Final results will be presented as well as an analysis of factors that associate with lack of molecular testing. Taken together, these data indicate that there is a critical need for enhanced patient and caregiver education regarding molecular testing.

#3192

Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples.

Jordi Remon,1 Jean Charles Soria,1 David Planchard,1 Cecile Jovelet,1 Chloe Pannet,1 Ludovic Lacroix,1 Annas Gazzah,1 Andrew Lawson,2 Sarah Smalley,2 Kenth Howarth,2 David Gale,2 Emma Green,2 Vincent Plagnol,2 Nitzan Rosenfeld,2 Ken Oulassen,1 Nathalie Chaput,1 Benjamin Besse1. 1 _Gustave Roussy, Villejuif, France;_ 2 _Inivata Limited, United Kingdom_.

Introduction: Approximately 30% of patients with an adenocarcinoma of the lung have an actionable driver mutation. Further understanding the molecular mechanisms of acquired resistance to targeted therapies provides key information for determining subsequent treatment options. Access to tumor tissue to perform either the initial molecular profile or at the point of acquired resistance, however, is often limited. Circulating tumor DNA (ctDNA) can be used as a minimally invasive method for the detection and quantification of molecular abnormalities. We performed a prospective study to assess molecular alterations in the ctDNA of NSCLC patients in whom the initial molecular profile or profile at acquired resistance was unknown due to lack of tumor tissue biopsy or insufficient cellularity in the biopsy.

Methods: Plasma samples were collected from 52 pre-treated advanced NSCLC patients at the Gustave Roussy. DNA was extracted from < 5 ml of plasma and analysed using Inivata's enhanced TAm-SeqTM assay covering regions from 35 cancer-related genes. Sequences were generated using Illumina sequencing. We also analysed plasma taken following treatments prescribed after the original molecular profile detected using plasma ctDNA.

Results: From July 2015 to October 2015, 52 patients were included (63% female, 37% never-smoker, 95% diagnosed with an adenocarcinoma subtype, 95% with stage IV disease, and 54% had EGFR mutant tumors of which 68% had mutations in exon 19 and 32% had mutations in exon 21). ctDNA profiling was successfully performed for all patients, and mutations were detected in 38 of 52 patients. The median number of mutations detected in plasma samples was 1. Within the EGFR mutant subpopulation, T790M mutations were identified including 8 acquired cases (with a concomitant C797S mutation in 1 case) and 1 primary T790M mutation. Of these patients, 5 started personalised treatment with AZD9291 based on the results of ctDNA analysis. In the other 18 patients with EGFR mutant tumors, no acquired mutations associated with resistance were detected. Other results encompassed: 2 plasma samples with EGFR mutation exon 18 (G719A, G719C) leading to initiation of afatinib in one case, 1 case with EGFR mutation exon 21 (L861Q), 1 patients with ERBB2 exon 20 insertion, 3 KRAS mutant detected in plasma (G12C, G12S, G12F), 2 STK11 mutant samples, and 1 patient with a MET mutation (exon 14) who subsequently started crizotinib.

Conclusions: ctDNA analysis with Inivata's enhanced TAm-Seq™ provides an alternative method of 'liquid biopsy' for obtaining molecular profile of mutations present in NSCLC patients in the absence of an invasive tissue biopsy. Liquid biopsy identified cancer mutations in 73% of the study population, and 18% of those patients subsequently received treatment tailored to the plasma ctDNA detected mutations. An update on the analysis of 75 patients will be presented during the conference.

#3193

Development of a colon cancer model system reveals epithelial contribution to poor-prognosis gene signatures.

Pramudita R. Prasetyanti,1 Sander R. van Hooff,1 Hans M. Rodermond,1 Tessa van Herwaarden,1 Kieshen Kalloe,1 Nathalie R. de Vries,1 Ronals van Leersum,1 Joan H. de jong,1 Evelyn Fessler,1 Nicolo Baldi,1 Giorgio Stassi,2 Jan Paul Medema1. 1 _Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands;_ 2 _Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy_.

Background: Recent consensus on molecular classification categorizes colorectal cancer (CRC) into 4 robust subtypes: CMS1 (epithelial-MSI), CMS2 (epithelial-canonical), CMS3 (epithelial-metabolic) and CMS4 (mesenchymal)1. CMS4 is linked to poor cancer prognosis and characterized by mesenchymal and epithelial-to-mesenchymal transition (EMT) gene expression2,3. Recent attempts to deconvolute the transcriptome from CRC tumors have suggested that the mesenchymal gene expression results from a large stromal compartment and is not due to epithelial cells with EMT-like features4,5. This challenges the classic notion that tumor cells activate the EMT program to undergo trans-differentiation into mesenchymal cells as observed in a number of in vitro and in vivo studies6. To resolve these conflicting views a detailed investigation of the tumor or stromal cell contributions to the mesenchymal signature is important.

Material & methods: We developed patient-derived xenografts (PDX) from multiple subtypes of CRCs. Transcriptome analysis was performed to assign each tumor and PDX to a CMS subtype, and further dissect the contribution of both epithelial and stromal gene expression to the overall expression phenotype. Additionally, we established multiple cultures from primary human CRCs to further assess subtypes-differences.

Results: We developed a panel of human CRC PDXs from all subtypes (CMS1-4). The expression profiles of the patient's tumor and PDXs remain stable for multiple serial passages in vivo. Evaluating the gene expression in the PDXs, which are free of human stroma, our analyses identified tumor specific expression markers that were differentially expressed in the mesenchymal subtype (CMS4). This finding suggests the presence of an epithelial tumor derived signal in mesenchymal tumors which contributes to CMS4 specific gene expression. This set of epithelial specific CMS4 tumor markers has been validated in other CRC expression datasets and associated with disease relapse. Our panel of primary cell lines representing various subtypes showed differential sensitivity to certain therapeutic agents thus indicating a subtype-specific drug response.

Conclusions: The identification of diverse subtypes in our panel of CRCs and corresponding PDXs, shows the presence of distinct tumor specific gene expression in mesenchymal CMS4 tumors. Furthermore, the primary CRC cell line panel comprising all subtypes, provides great insight into CRC heterogeneity and serves as a valuable model system to accelerate the development of effective treatment.

References:

1. Guinney, J. et al. Nat. Med. 21, 1350–1356 (2015).

2. De Sousa E Melo, F. et al. Nat. Med. 19, 614–618 (2013).

3. Sadanandam, A. et al. Nat. Med. 19, 619–625 (2013).

4. Isella, C. et al. Nat. Genet. 47, 312–319 (2015).

5. Calon, A. et al. Nat. Genet. 47, 320–329 (2015).

6. Kalluri, R. & Weinberg, R. A. J. Clin. Invest. 119, 1420–1428 (2009). 

### Novel Approaches to Pediatric Cancers

#3194

Cisplatin-DNA adduct formation in patients receiving cisplatin +/- sodium thiosulphate (STS) in the SIOPEL 6 randomized phase III trial.

Gareth J. Veal,1 Milind Ronghe,2 Bruce Morland,3 Edward Neuwelt,4 Rudolf Maibach,5 Penelope Brock6. 1 _Northern Inst. for Cancer Research, Newcastle-upon-Tyne, United Kingdom;_ 2 _Royal Hospital for Sick Children, Glasgow, United Kingdom;_ 3 _Birmingham Children's Hospital, Birmingham, United Kingdom;_ 4 _Oregon Health & Science University, Portland, OR; _5 _International Breast Cancer Study Group, Bern, Switzerland;_ 6 _Great Ormond Street Hospital, London, United Kingdom_.

Background: The SIOPEL 6 randomised phase III trial was designed to investigate the efficacy of sodium thiosulphate (STS) in reducing ototoxicity in patients receiving cisplatin monotherapy for standard-risk hepatoblastoma. Preliminary results from the trial recently presented at ASCO indicated comparable response rates between the two study arms. The trial has now completed patient recruitment, with final evaluation of hearing loss at ≥3.5 years of age awaited as the primary endpoint of the trial. As part of the clinical study, blood samples were collected pre-treatment and 24 hours following cisplatin administration, to investigate the potential impact of STS on the formation of platinum-DNA adduct levels in leucocytes.

Patients and methods: Whole blood samples (5-10ml) were taken prior to cisplatin treatment and 24 hours following the start of a 6 hour cisplatin infusion, with STS administered 6 hours after the end of cisplatin administration in patients randomised to receive STS. Samples were collected in EDTA blood tubes and frozen at -20°C or -80°C prior to transport to Newcastle for analysis. DNA was isolated from whole blood using Qiagen DNA blood Maxi kits and the concentration of DNA in each sample quantified by UV absorption. DNA samples were diluted in 3.5% nitric acid and hydrolyzed overnight at 70°C. Platinum-DNA adduct levels were determined by ICP-MS analysis, with results expressed in units of nmoles platinum/g DNA.

Results: Blood samples were collected from a total of 37 patients, with 22/37 (59%) patients having received cisplatin followed by STS and 15/37 (41%) patients cisplatin alone. A significant difference in median platinum-DNA adduct levels was observed in patients receiving cisplatin followed by STS (median: 6.7 nmol/g DNA; range: 4.2 - 14.0), as compared to patients receiving cisplatin alone (median: 13.5 nmol/g DNA; range: 4.3 - 165.7) (P = 0.0011).

Conclusion: Results indicate that the formation and/or longevity of platinum-DNA adducts determined in whole blood samples collected 24 hours following cisplatin administration are influenced by the co-administration of STS, with significantly higher levels observed in patients receiving cisplatin alone. Bearing in mind the comparable response data reported between the two randomisation study arms, these data would indicate that platinum-DNA adduct formation in leucocytes may act as a surrogate marker, providing novel insights into the mechanism of the otoprotective effects of STS when used in combination with cisplatin.

Acknowledgements: Work supported by Cancer Research UK, the North of England Children's Cancer Research Fund and the Experimental Cancer Medicine Centre Network.

#3195

Synergistic effects of combined treatment with hAT-MSC.sTRAIL and panobinostat in brainstem glioma.

Seung Ah Choi, Chanhee Lee, Pil Ae Kwak, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Sangjoon Chong, Seung-Ki Kim. _Seoul National University Hospital, Seoul, Republic of Korea_.

Human adipose-derived mesenchymal stem cells expressing secreted form of the tumor necrosis factor-related apoptosis-inducing ligand (hAT-MSC.sTRAIL) have demonstrated therapeutic activity against various tumors. However, sTRAIL resistance remains a challenge in developing anticancer strategies. To solve this problem, many studies have tried to combine drugs to produce synergism or sensitize resistant cancer cells. Histone deacetylase inhibitors (HDACi) have been known to induce expression of TRAIL death receptors 4 and 5 (DR4/DR5). Herein, we evaluated the use of combined therapy of hAT-MSC.sTRAIL with HDAC inhibitor, panobinostat, in enhancing sensitivity to hAT-MSC.sTRAIL mediated apoptosis against the brainstem glioma.

A sTRAIL was introduced into the characterized hAT-MSCs using electroporation. To confirm appropriate treatment concentration of panobinostat to the glioblastoma cells, dose titration was tested using cell viability assay. The therapeutic effect of single or combination treatment against glioblastoma was evaluated using primary cultured glioblastoma cells and cell lines. Orthotopic xenograft brainstem glioma mouse model was established using engineered firefly luciferase expressing tumor cells for bioluminescence in vivo imaging.

Panobinostat induced anti-proliferative effects in dose and time-dependent manner (IC50 range, 0.05-0.2 μM) and effectively enhanced the expression of TRAIL DR4 and DR5, but not decoy receptors. Combined hAT-MSC.sTRAIL and panobinostat significantly decreased the tumor cell growth compared to each alone. Intriguingly, the combination treatment not only induced apoptosis but also autophagy. Using a preclinical brainstem mouse model, we confirmed that the combination of hAT-MSC.sTRAIL and panobinostat was safe and induced regression of tumor volume. Furthermore, the combination therapy prolonged the survival of brainstem glioma-bearing mice.

Our results suggested that combination therapy of panobinostat enhanced the anti-cancer effect of hAT-MSC.sTRAIL and represent potential therapeutic approach to the brainstem glioma.

#3196

STAT3 inhibitor WP1066 as a novel therapeutic for medulloblastoma.

Laura K. Metrock, Jingbo Liu, Liangping Yuan, Hongying Zhang, Abhinav Dey, Tobey MacDonald. _Emory University, Atlanta, GA_.

The purpose of this study is to determine the efficacy of the STAT3 inhibitor WP1066 on the sonic-hedgehog (SHH) subtype of medulloblastoma (MB).

STAT3, or signal transducer and activator of transcription 3, is a potential novel target for the treatment of SHH-MB. We have generated substantial preliminary data showing that STAT3 activation promotes the survival of SHH-MB cancer stem cells (CSCs) that are critical for treatment resistance and tumor recurrence.

Smoothened (SMO) antagonists have shown efficacy in treating subtypes of SHH-MB, however all tumors develop resistance and SMO inhibitors are ineffective for patients with SHH-MB harboring mutations downstream of SMO. Our preliminary in vitro data suggests that combined SMO and STAT3 inhibition has synergistic activity, and that constitutively activated STAT3 is protective against SHH-MB cell death mediated by SMO inhibition.

Methods:

The effect of increasing concentrations of WP1066 on Shh-responsive MB cells was assessed by Western blot analysis for STAT3 phosphorylation, WST assay for cell proliferation, and soft agar colony formation. The effect of WP1066 on apoptosis and the cell cycle was assessed by flow cytometry.

An ex vivo murine SmoA1 SHH-MB brain slice culture method was used to assess the effects of WP1066 alone and in combination with radiation. In vivo studies consist of mice treated with 1) WP1066, 2) SMO inhibitor LDE225 3) WP1066 in combination with LDE225 and 4) vehicle alone. All mice are imaged by MRI prior to treatment and every 3 weeks after treatment. Tumor volume is calculated and rate of tumor growth and survival are measured. The mice are followed until death or the end of the experiment at 36 weeks.

Results:

In human Shh-responsive MB cells in vitro, WP1066 blocked IL6-induced STAT3 activation (phosphorylation), decreased cell proliferation and abrogated tumor colony formation. Results regarding the mechanism of inhibition of cell proliferation/viability by WP1066 (i.e. apoptosis and cell cycle analysis) are pending.

In the SHH-MB brain tumor slice cultures, WP1066 treatment induced potent tumor cell kill, especially of the CSCs within the perivascular niche. WP1066 also showed synergy with radiation, but did not add toxicity to normal brain cells.

Preliminary in vivo data indicate a statistically significant decrease in the rate of tumor growth and prolonged survival in mice treated with WP1066 compared to vehicle control. Results of the efficacy of WP1066 with LDE225 are pending.

Conclusions:

WP1066 has potent efficacy against Shh-responsive MB cells in vitro and SHH-MB tumors ex vivo, without toxicity to normal brain cells. Preliminary data is promising for WP1066 activity against SHH-MB in vivo, suggesting WP1066 is a novel therapeutic for SHH-MB.

#3197

BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase pathway.

Ping Zhao,1 Jacob Hall,2 Austin Voydanoff,3 Mary Durston,1 Elizabeth VanSickle,1 Abhinav B. Nagulapally,1 Jeffery Bond,1 Giselle Saulnier Sholler1. 1 _Helen DeVos Children's Hospital, Grand Rapids, MI;_ 2 _Calvin College, Grand Rapids, MI;_ 3 _Kalamazoo College, Grand Rapids, MI_.

Background: Medulloblastoma (MB) is the most common malignant brain tumor in children with poor survival outcome. New treatment strategies are needed for control of MB. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) intracellular signaling pathway plays a key role in cellular metabolism, proliferation, survival and angiogenesis. This pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. BKM120 (Buparlisib) is an oral pan-class I PI3K inhibitor that targets all 4 isoforms of class I PI3K. BKM120 is currently being clinically evaluated for the treatment of different adult cancers including breast cancer, glioblastoma, prostate cancer, advanced non-small cell lung cancer, and colorectal cancer. In this study, we screened our MB established and patient primary cell lines by genomic profiling analysis, and validated the targeted therapy both in vitro and in vivo in xenograft mouse model.

Methods: RNA expression profiling analysis was performed with Affymetrix GeneChip U133 Plus 2.0 genome wide expression cDNA microarray. Analysis was done using R/Bioconductor packages and Partek Genomics Suite. Eleven MB cell lines were treated with increasing concentrations (0-4 µM) of BKM120 for 48 hours. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. Western blot analysis was used to measure phospho-Akt, phospho-mTOR, and cleaved caspase 3 protein levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with CellTiter-Glo luminescent cell viability assay. Xenograft study was performed with DAOY cells implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily.

Results: BKM120 exhibited cytotoxicity in MB cells in a dose-dependent manner by inhibiting activation of downstream signaling molecules Akt and mTOR, and activating apoptotic pathways and inducing cell death in the eleven cell lines tested. IC50s of BKM120 in the MB cell lines ranged from 0.456 to 2.9 µM determined by cell viability assay. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner. In MB xenograft mouse study, BKM120 significantly suppressed tumor growth and prolonged mouse survival at 30 mg/kg and 60 mg/kg.

Conclusion: This study indicates that BKM120 promotes apoptosis and suppresses medulloblastoma tumor growth both in vitro and in vivo. Additional investigation of BKM120

for the treatment of pediatric medulloblastoma is warranted.

#3198

HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma.

Amanda B. Witte,1 Mary Durston,2 Ping Zhao,2 Abhinav B. Nagulapally,2 Jeffrey Bond,3 Giselle L. Saulnier Sholler1. 1 _Michigan State University College of Human Medicine and Helen DeVos Children's Hospital, Grand Rapids, MI;_ 2 _Helen DeVos Children's Hospital, Grand Rapids, MI;_ 3 _University of Vermont, Burlington, VT_.

Background: Embryonal tumors of the central nervous system continue to present therapeutic challenges with high rates of relapse and poor prognosis at advanced stages. Medulloblastoma (MB) accounts for over 15% of pediatric brain tumors, with a five-year survival rate of 62%. Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for 15% of pediatric cancer deaths.

Among many pathways that contribute to MB and NB potency, several histone deacetylases (HDACs) have been shown to prevent cell differentiation. In cells expressing wt-p53, HDAC inhibitors induce nuclear relocalization of p53 to the nucleus to induce expression of the cell cycle inhibitor p21/Waf1/Cip1. As most NB tumors express wt-p53, HDAC inhibitors are promising candidates for therapy. Panobinostat has been shown to induce differentiation, cell cycle arrest, and apoptosis in NB cells. This effect is partially due to down-regulation of CHK1, a pathway by which cancer cells can develop resistance to conventional chemotherapeutic drugs.

This study proposes panobinostat coupled with conventional chemotherapeutics such as doxorubicin, etoposide, and velcade, as an effective target against the HDAC pathway in MB and NB.

Methods: Established MB and NB cell lines and patient derived cell lines (MB: DAOY, D283, D341, 384MED, 458MED, 487MED, 556MED, 581MED, 721MED; NB: BE(2)-C, CHLA-90, SMS-KCNR, SH-SY5Y, MGT8-117-08, BIO-036-08) were used to quantify panobinostat's effects. Cell viability was measured using Calcein AM fluorescent assay at doses of 0.39-50 nM. Isobologram analysis of panobinostat in combination with doxorubicin, etoposide, and velcade were generated using Calcein AM. Western blot was used to measure HIF1 alpha, CHK1, and acetyl H4, Caspase 3 and PARP levels. ATP level per cell was measured using CyQuant fluorescent DNA assay with CellTiter-Glo luminescent cell viability assay.

Results: Cell viability assays show cytotoxicity of panobinostat in MB and NB cell lines, with IC50 values from 2-10 nM in MB cells, 5-20 nM in established NB cell lines and 23-91 nM in patient derived NB lines. ATP/cell activity was inhibited in MB and NB cells following treatment with panobinostat alone. Isobologram experiments suggest synergistic cytotoxicity of panobinostat in combination with doxorubicin, etoposide, and velcade in NB cell lines BE(2)-C and SMS-KCNR. Western blot analysis indicates caspase-mediated apoptosis occurs, with inhibition of overexpressed HDAC proteins among multiple classes in MB and NB cells.

Conclusions: This study indicates that panobinostat targets the HDAC inhibition pathway of MB and NB cells. Additionally, panobinostat synergizes with doxorubicin, etoposide, and velcade in inducing apoptosis in MB and NB cells. This study provides rationale for initiation of a clinical trial in treating MB and NB patients with panobinostat in combination with conventional chemotherapeutics.

#3199

BKM120 is cytotoxic in neuroblastoma targeting the PI3K pathway.

Monica M. Pomaville,1 Ping Zhao,2 Sarah DeCou,2 Abhinav B. Nagulapally,2 Jeffrey Bond,3 Giselle L. Saulnier Sholler1. 1 _Helen DeVos Children's Hospital and Michigan State University College of Human Medicine, Grand Rapids, MI;_ 2 _Helen DeVos Children's Hospital, Grand Rapids, MI;_ 3 _University of Vermont, Burlington, VT_.

Background: Neuroblastoma is the most common extracranial solid tumor found in children, accounting for approximately 15% of cancer-related deaths. Many cellular processes have been discovered to play a role in neuroblastoma's potency, including a family of lipid kinases within the phosphoinositide 3-kinase (PI3K) signaling pathway that contribute to cell survival, proliferation, and differentiation. Targeting this pathway could unveil new treatment strategies that work to specifically treat each child's unique disease. BKM120 is a novel cancer therapeutic that targets the PI3K/Akt/mTOR signaling pathway and has recently been shown to have great potential in the clinic by acting on Class IA PI3Ks. Though Class IA PI3Ks hold multiple types, BKM120 has been shown to act preferentially on PIK3CA mutant isoforms. In this study we show the inhibitory effect of BKM120 on neuroblastoma cell lines that over-express PI3KCA, suggesting a promising role in the clinic for children with this expression profile. It has also been suggested that inhibitors of the PI3K pathway exert their inhibitory effects on cancer cells by destabilizing MYCN, a protein found over-expressed in approximately one third of neuroblastoma patients that encourages malignant progression of the disease.

Methods: Neuroblastoma (NB) cell lines BE(2)-C and SMS-KCNR cells and patient lines MGT-002-13, MGT-003-08, MGT-014-11, and MGT-015-08 were used in these studies. Cell viability was measured using Calcein AM fluorescent assay at BKM120 doses 0.2uM, 0.5uM, and 1.0uM. Western blot analysis was used to measure PI3K pathway members including pAKT, p-mTOR, mTOR, and apoptosis markers including Cleaved Caspase 3, Caspase 3, PARP, and cPARP. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. IncuCyte imaging of sytox and kinetic caspase-3 reagent was used to measure apoptosis in NB cells treated with BKM120.

Results: BKM120 is cytotoxic to NB cell lines with IC50's ranging from 0.9-5.5 uM. BKM120 increases protein expression levels of Cleaved Caspase 3 and cleaved PARP by inducing apoptosis and decreases MTOR, pAKT and MYCN at increasing drug doses. BKM120 decreases ATP/cell related to glycolytic metabolism activity in NB cell lines. Increased expression of MTOR and PI3KCD correlate with increased resistance to BKM120.

Conclusion: This study indicates that BKM120 targets the MTOR/PI3K/AKT pathway and may play a role in MYCN driven tumors. In addition, BKM120 inhibits NB cell proliferation and induces caspase-mediated apoptosis in vitro in NB. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, further assessment in clinical trial setting is needed to assess BKM120's therapeutic activity for children with NB.

#3200

New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma.

Simon Jacques-Ricard, Noel Raynal, Gregory Armaos, Elodie Da Costa, Annie Beaudry. _Ste-Justine Hospital, Montreal, Quebec, Canada_.

Pediatric neuroblastoma is one of the most common extra cranial cancers in children. Despite an improvement in survival with the currently available therapies, neuroblastoma with an amplification of the transcription factor MYCN have a very poor prognosis. New therapeutic approaches must be developed to increase the survival of patients. One such approach is epigenetic drug therapy. Neuroblastoma, like many other pediatric cancers, contains several epigenetic alterations at the level of DNA methylation and histone modifications. In a screening of FDA approved drugs, we discovered some molecules having characteristics of epigenetic drugs that were unknown until now. Our study seeks to demonstrate the efficacy of these molecules in the treatment of neuroblastoma cell lines. Following preliminary tests, one of the molecules approved by the FDA stood out: disulfiram, a medication approved for the treatment of chronic alcoholism. We treated neuroblastoma cell lines (MYCN amplified: IMR-32, N91 and SK-N-DZ; MYCN non-amplified: SK-N-AS and SK-N-SH) for 48 hours with disulfiram at clinically relevant concentrations (from 10 nM to 50 µM). Our results demonstrate a 50% growth inhibition (IC50) of 50nM for the cell lines tested. In addition, after analysis by flow cytometry, we found a cell cycle block in G2/M. We also observed a decrease in the transcription factor MYCN and a reduction in acetylation of several histone marks by Western blots analysis. Further studies are underway to determine the mechanism of action of disulfiram and we also plan on doing RNA sequencing and ChIP sequencing. This study will evaluate the efficacy of disulfiram alone as well as in combination with other epigenetic drugs, such as DNA methyltransferase inhibitor decitabine, for the treatment of neuroblastoma.

#3201

Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 predicts poor prognosis and promotes cell proliferation in Ewing sarcoma.

Caterina Mancarella,1 Linda Calzolari,2 Stefano Ferrari,1 Davide Donati,1 Piero Picci,1 Katia Scotlandi1. 1 _Rizzoli Orthopaedic Institute, Bologna, Italy;_ 2 _Fondazione IRCCS Istituto Nazionale Tumori, Milano, Italy_.

INTRODUCTION: Insulin-like growth factor 2 (IGF-2) mRNA binding protein 3 (IGF2BP3, also known as IMP3) is an oncofetal protein which binds RNA thus influencing the transcript target fate. IGF2BP3 is de novo synthesized in malignancies where it promotes proliferation, drug resistance and metastases. Moreover, IGF2BP3 represents a promising indicator of outcome in several cancers. Ewing sarcoma (EWS) is a rare bone and soft tissue tumor where the IGF system plays a pivotal role. This study aimed to investigate the relevance of IGF2BP3 in EWS and verify its value as a prognostic biomarker.

MATERIAL AND METHODS: Total RNA was extracted from two independent cohorts of 30 and 109 snap-frozen tissues from localized primary EWS samples with more than 5 years follow-up. Microarray analysis (Affymetrix) was performed in the cohort of 30 patients while IGF2BP3 expression was analysed by qRT-PCR (Applied Biosystem) in the cohort of 109 cases. Prognostic value of IGF2BP3 was evaluated both in univariate and multivariate analysis. IGF2BP3 expression was assessed by qRT-PCR and western blot in a panel of 14 EWS cell lines. Plasmids for IGF2BP3 silencing or over-expression were transfected in A673 or LAP-35 and IOR/CAR EWS cells, respectively. Soft-agar growth of cell lines with differential IGF2BP3 expression was evaluated. catRAPID express human web server was employed for prediction of novel IGF2BP3/mRNAs interactions. IGF-2 and ABCF1 expression levels were analysed in vitro and in clinical specimens.

RESULTS: Microarray results evidenced that IGF2BP3 higher expression correlated with a worse outcome (p-value 0.0002). This data was confirmed in the cohort analysed by qRT-PCR both considering univariate (p-value 0.001) and multivariate analysis (HR:2.83. IC95% [1.22-6.53].p=0.015). In vitro, higher IGF2BP3 expression correlated with an increased capability of growth in anchorage-independent conditions and IGF2BP3 knockdown significantly decreased soft-agar growth of EWS cells. Oncogenic potential of IGF2BP3 was not found to be mediated through IGF-2-dependent mechanism but ABCF1, an ABC family member with a key role in translation initiation, was predicted as a target of IGF2BP3 by in silico analysis. Accordingly, IGF2BP3 loss- or gain-of-functions approaches induced down- or up-regulation of ABCF1 protein levels, respectively. In addition, a direct correlation between IGF2BP3 and ABCF1 was found in 109 EWS patients (p-value minor than 0.0001). Functional studies on the role of ABCF1 in EWS will follow.

CONCLUSIONS: IGF2BP3 represents an indicator of prognosis in EWS therefore its detection may be of help to stratify patients according to risk. We also provided evidences for IGF2BP3 in the regulation of EWS aggressiveness and identified ABCF1 as a new putative mediator of IGF2BP3 malignant effects. (Grants: FIRB RBAP11884M_005 and AIRC IG2013_14049 to KS)

#3202

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity.

David Schirmer,1 Richard Klar,2 Oxana Schmidt,1 Dirk Wohlleber,3 Wolfgang Uckert,4 Uwe Thiel,1 Felix Bohne,5 Dirk H. Busch,6 Angela M. Krackhardt,2 Stefan Burdach,1 Günther H. Richter1. 1 _Children's Cancer Research Center and Dept of Pediatrics, Technische Universität München, CCCM Munich - Comprehensive Cancer Center and German Translational Cancer Research Consortium (DKTK), München, Germany;_ 2 _Medical Department III, Hematology and Oncology, Technische Universität München, CCCM Munich - Comprehensive Cancer Center and German Translational Cancer Research Consortium (DKTK), München, Germany;_ 3 _Institute of Molecular Immunology/Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, München, Germany;_ 4 _Max Delbrück Center for Molecular Medicine, Berlin, Germany;_ 5 _Institute of Virology, Technische Universität München, Helmholtz Zentrum München, München, Germany;_ 6 _Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, München, Germany_.

Background: Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology targetable tumor associated antigens (TAA) are identified and exploited for engineered T cell therapy. Here, the specific recognition and lytic potential of transgenic, allo-restricted CD8+ T cells directed against the ES-associated antigen STEAP1 was examined.

Methods: Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells, allo-restricted HLA-A*02:01- CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, TCR α- and β-chains were identified. They were cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01- primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2-/-γc-/- mouse model.

Results: Initially generated and transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific IFNγ release. They lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01- ES lines. In vivo tumor growth was inhibited more effective with transgenic STEAP1130-specific T cells than with unspecific T cells.

Conclusion: Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1 expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1 expressing tumors.

#3203

Phase I results of mitoxantrone in combination with clofarabine in children with refractory/relapsed acute leukemia.

Jessica Hochberg,1 Javier Oesterheld,2 Olga Militano,1 Liana Klejmont,1 Lauren Harrison,1 Berkley Nickerson,1 Mitchell S. Cairo1. 1 _New York Medical College, Hawthorne, NY;_ 2 _Levine Children's Hospital, Charlotte, NC_.

BACKGROUND: Despite excellent outcomes in pediatric ALL, multiply relapsed patients have response rates ~40%, with <10% OS in CR3.(Gaynon, BJH 2005) The prognosis for relapsed or refractory AML is ~20% OS.(Wells, JCO 2003) Novel combinations with improved outcomes are needed. Clofarabine and Mitoxantrone have proven efficacy in children with leukemia and offer possible synergistic activity in vivo with less drug resistance.(Chow, Leukemia & Lymphoma 2000)

OBJECTIVES: To determine the maximal tolerated dose and overall response rate of clofarabine in combination with mitoxantrone as reinduction therapy for refractory/relapsed acute leukemia. To determine the percent of minimal residual disease (MRD) following reinduction.

DESIGN: Prospective, open label, uncontrolled, safety and efficacy study. Patients 0-30.99yr old with ALL, AML or NHL in 1st, 2nd or 3rd relapse OR primary induction failure were given 1 to 3 cycles of clofarabine (escalating doses 20, 30, 35 and 40mg/m2/day) Day 1-5, in combination with mitoxantrone 12mg/m2/day on Day 3-6. Dexrazoxane was given prior to Mitoxantrone. Dose escalation was planned every 3 patients pending dose limiting toxicities. CNS prophylaxis was achieved with intrathecal liposomal cytarabine. Patients were allowed subsequent cycles pending response and anthracycline exposure.

RESULTS: To date 17 patients have been enrolled on the Phase I safety portion of this study. Median Age is 13yrs (8months-23yrs); 11 ALL (3=Induction Failure, 6=Relapse1, 2=Relapse2), 5 AML (4=Induction Failure, 1=Relapse2), 1 NHL (=Progressive Disease). There have been 2 prolonged Grade III/IV toxicities at Dose Level 4 (Clofarabine 40mg/m2) (1 hepatic toxicity, 1 prolonged myelosuppression) hence additional patients were enrolled at Dose Level 3 (Clofarabine 35mg/m2). Median time to neutrophil recovery was 24 days. Fourteen of 16 (88%) evaluable leukemia patients achieved a CR after 1 cycle of therapy. Of these patients, 94% achieved MRD negativity (<0.1%). Two patients with relapsed ALL had no response. One patient with relapsed/refractory NHL had progressive disease after 2 cycles. Thirteen of 14 patients achieving CR went on to receive an allogeneic HSCT with continued remission at a median follow up time of 275 days (range 40-634). Overall 1 year survival for leukemia patients is 78.7% (CI95: 47.2-92.5).

CONCLUSION: The combination of clofarabine and mitoxantrone reinduction therapy for relapsed or refractory acute leukemia appears to be safe and well tolerated in children, adolescents and young adults with poor risk hematologic malignancies. The Phase I MTD of this combination was found to be 35mg/m2 Clofarabine. Hematopoietic recovery appears to be rapid and complete. Initial data from the first 17 patients enrolled is encouraging with an 88% CR rate in leukemic patients. An extended multicenter Phase II Study is ongoing.

## IMMUNOLOGY:

### Immune Checkpoints 2

#3204

INCAGN01949: an anti-OX40 agonist antibody with the potential to enhance tumor-specific T-cell responsiveness, while selectively depleting intratumoral regulatory T cells.

Ana Maria Gonzalez,1 Mariana L. Manrique,1 Ekaterina Breous,2 David Savitsky,1 Jeremy Waight,1 Randi Gombos,1 Yuqi Liu,1 Shiwen Lin,1 Taha Merghoub,3 Daniel Hirschhorn-Cymerman,3 Gerd Ritter,4 Jedd Wolchok,4 Peggy Scherle,5 Gregory Hollis,5 Reid Huber,5 Marc Van Dijk,2 Robert Stein,1 Nicholas S. Wilson1. 1 _Agenus Inc, Lexington, MA;_ 2 _Agenus Inc / 4-Antibody, Basel, Switzerland;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _The Ludwig Institute for Cancer Research Inc, New York, NY;_ 5 _Incyte Corp, Wilmington, DE_.

OX40 (CD134, TNFRSF4) is a T cell co-stimulatory receptor that potentiates T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In preclinical mouse tumor models, surrogate anti-OX40 agonist antibodies have shown remarkable single agent anti-tumor efficacy, as well as the ability to combine effectively with other immunomodulatory antibodies and immune education strategies, such as therapeutic cancer vaccines. Agonistic antibodies targeting OX40 are predicted to counteract the immunosuppressive tumor microenvironment and promote tumor-specific T cell immunity via two primary mechanisms: 1) binding and activating OX40 signaling in tumor-specific effector and memory T cells, thereby enhancing their responsiveness to tumor-associated antigens, and 2) co-engaging Fcγ receptors expressed by tumor-associated effector cells, and facilitating the selective depletion of intratumoral regulatory T cells.

INCAGN01949 is a novel fully human IgG1 monoclonal antibody identified using the Retrocyte Display™ platform that is being developed for the treatment of advanced malignancies. INCAGN01949 recognizes human and cynomolgus monkey OX40 with comparable binding affinity. INCAGN01949 has been optimized to potently mediate receptor forward signaling under conditions of suboptimal TCR stimulation, leading to features like enhanced production of TNFα and IFNγ, and concomitant suppression of IL-10. INCAGN01949 achieves this functionality through OX40 clustering and downstream activation of the NFκB pathway in T cells, which is sustained across a broad range of antibody concentrations. Consistent with mouse preclinical tumor models, OX40 was found to be selectively overexpressed by intratumoral regulatory T cells from a variety of primary human tumor samples. Commensurate with its human IgG1 Fc region, INCAGN01949 can effectively co-engage activating Fcγ receptors on immune effector cells, including natural killer cells and macrophages. Therefore INCAGN01949 has the potential to mediate selective effector cell activity toward intratumoral populations of regulatory T cells.

The biophysical and functional attributes of INCAGN01949 make it suited for clinical development, both as a single agent and in combination with other immunomodulatory antibodies or immune education strategies.

#3205

Defining the immune checkpoint landscape of acute myeloid leukemia (AML).

Naval Daver, Sreyashi Basu, Guillermo Garcia-Manero, Jorge Cortes, Farhad Ravandi, Steven Kornblau, Marina Konopleva, Michael Andreeff, Gautam Borthakur, Nitin Jain, William Wierda, Srdan Verstovsek, Peter Ruvolo, Tapan Kadia, Jairo Matthews, Wilmer Flores, Hui Yang, Carlos Bueso-Ramos, Narmeen Somani, Jorge Blando, James Allison, Hagop Kantarjian, Padmanee Sharma. _MD Anderson Cancer Center, Houston, TX_.

Introduction: The expression of co-stimulatory (costim) receptors/ligands in the bone marrow (BM) and peripheral blood (PB) in patients (pts) with AML has not been defined. Identification of immune-checkpoint pathways that dominate in AML will guide the rational selection of specific antibodies for clinical trials.

Methods: Between March, 2015 and October 2015 we performed 17-color multi-parameter flow-cytometry (MFC) on 15 untreated AML and 25 relapsed AML to assess expression of costim ligands (4-1BBL, B7-1, B7-2, ICOSL, PDL-1, PDL-2, OX40L) on leukemic blasts and costim receptors (4-1BB, CTLA-4, ICOS, PD-1, OX40, GITR, LAG-3, TIM-3) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells: CD3+CD8+. Four healthy human BMs were used as control. Expression is denoted by percentage of specific T-cell subset or gated AML blasts positive for the marker indicated. PB mononuclear cells (PBMCs) and blasts were evaluated at the same time-point.

Results: OX40+ Teffs and OX40+ CD8 cells were higher in untreated AML BM as compared to healthy donor BM (median [med]: 7.2% versus [vs] 0.26%; P=0.0005 and med: 2.03% vs 0.06%; P=0.07, respectively). PD1+ Tregs and OX40+ Tregs were also higher in untreated AML BM as compared to healthy donor BM (med: 19.7% vs 7.5%; P=0.03 and med: 10.3% vs 0.5%; P=0.02, respectively). PD1+ Teffs, OX40+ Teffs, and ICOS+ Teffs were higher in relapsed AML BM as compared to healthy donor BM (med: 17.7% vs 6.7%; P= 0.047, med: 12% vs 0.27%, P=0.002, and med: 13.3% vs 1.1%; P=0.07, respectively). OX40+ CD8 cells, ICOS+ CD8 cells, TIM3+ CD8 cells (med: 5.0% vs 0.07%; P=0.04, med: 18.5% vs 2.6%; P=0.09, and med: 2.7% vs 0.7%; P=0.01, respectively) and OX40+ Tregs (med: 15.5% vs 0.5%; P<0.0001) were also higher in relapsed AML BM as compared to healthy donor BM. GITR+ Teffs, PD1+ CD 8 cells, and LAG3+ Tregs were higher in relapsed AML BM as compared to new AML BM (med: 10.9% vs 1.7%; P=0.08, med: 36.2% vs 21.3%; P=0.03, and med: 46.7% vs 16.9%; P=0.07, respectively). No other noteworthy differences were noted for costim receptor expression. There were no noteworthy differences in ligand expression patterns between relapsed AML and new AML. There was significant variability in BM expression of costim receptors and ligands between individual pts. The expression of costim receptors and ligands differed significantly between BM and PB from the same time-point in the same pt. A larger sample size is needed to confirm these data and find additional associations and this is currently underway at our institution.

Conclusions: Clinically targetable checkpoint receptors including PD1, OX40, and ICOS appear to be overexpressed in the BM of pts with AML as compared to healthy donor BM. Relapsed AML had higher expression of costim receptors than untreated AML. Three anti-PD1 based clinical trials for pts with AML are enrolling at our institution (NCT02397720, NCT02464657, NCT02532231).

#3206

Programmed death receptor-1/programmed death receptor ligand-1 blockade improves priming of antitumor effector T cells after cytotoxic therapies.

Miho Takahashi, Satoshi Watanabe, Ko Sato, Tomohiro Tanaka, Yu Saida, Junko Baba, Aya Ohtsubo, Miyuki Sato, Rie Kondo, Masaaki Okajima, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Toshiaki Kikuchi. _Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan_.

Cytotoxic lymphodepletion therapies, such as chemotherapy and radiotherapy, have been established to augment antitumor immunity. Naïve T cells elicit effector-like phenotypes and functions during recovery from lymphopenia. We and others have repeatedly demonstrated that transfer of naïve T cells into lymphopenic-tumor bearing mice delayed tumor growth. This enhancement of naïve T cells is required an activation through T-cell receptor (TCR).

Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has been demonstrated to augment antitumor immunity. Previous studies have shown that PD-1/PD-L1 blockade therapy stimulates or restores the function of antitumor T cells in the effector phase. Because engagement of programmed death receptor-1 (PD-1) by ligand suppresses TCR signaling and inhibits T cell activation and function, there is a possibility that PD-1 regulates activation of T cells during recovery from lymphopenia after cytotoxic therapies.

In the current study, we transferred naïve T cells into tumor-bearing mice following whole-body irradiation for lymphodepletion. These mice were further treated with anti-PD-1 antibodies. PD-1/PD-L1 blockade therapy after lymphodepletion significantly suppressed tumor progression. Next, we tested several kinds of cytotoxic agents to induce lymphopenia in mice. We found that the kind of cytotoxic agents affected the augmentation of antitumor efficacies of PD-1/PD-L1 blockade. Analyses of tumor-draining lymph-nodes (TDLNs) revealed that the number of tumor-specific effector T cells was significantly increased in mice treated with anti-PD-1 antibodies. By contrast, the number of effector T cells in spleens was not increased by PD-1/PD-L1 blockade therapy. These results indicate that PD-1 regulates a priming of antitumor effector T cells in TDLNs after cytotoxic lymphodepletion therapies. PD-1/PD-L1 blockade therapy is able to enhance antitumor T cell responses not only in the effector phase but also in the priming phase.

#3207

Influence of tumor mutation burden on response to anti-PD-1 treatment in murine models of melanoma.

Elena Galvani, Kate Hogan, Gabriela Gremel, Amaya Viros, Amit K. Mandal, Matthew Smith, Jacqueline Swan, Antonia Banyard, Garry Ashton, Nathalie Dhomen, Richard Marais. _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

Therapeutic antibodies directed against immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1), have recently shown remarkable clinical benefits, particularly in metastatic melanoma and non-small cell lung cancer (NSCLC), cancers largely caused by chronic exposure to mutagenic agents. Recent studies have focused on identifying genomic and immune predictors of response, but consistent, robust biomarkers have yet to be fully characterized. However, some data suggests a high non-synonymous tumour mutation burden is associated with clinical benefit in melanoma patients who received anti-CTLA-4 therapy. A similar correlation was also found in NSCLC patients treated with anti-PD-1.

Using mouse models of BRAFV600E-driven melanoma, we examined the relationship between tumour mutation burden and response to the immune checkpoint inhibitor anti-PD-1. Chronic exposure to ultraviolet radiation (UVR) was used to increase the number of tumour non-synonymous single nucleotide variants (SNVs), and thus mutational load. Responses to PD-1 blockade were compared between tumours having low (~10 SNVs) and high (>100 SNVs) mutation burden and tumours were subsequently subjected to comprehensive molecular and immune profiling using whole exome sequencing, immunohistochemistry, and flow cytometry.

Our initial data suggest there is no significant correlation between tumour mutation burden and response in our model of BRAFV600E-driven melanoma, and we will present a detailed analysis of low- and high-mutation burden tumours in light of their response to PD-1 blockade. Consistent with previous studies, our results highlight that integrated molecular characterization of tumour tissue and associated microenvironment in larger cohorts is needed to identify robust determinants of response to immune checkpoint inhibitors.

#3208

**Chemo-induced biology of PD-L1 and** in vivo **combination immune therapy for ovarian cancer.**

Shannon Grabosch,1 Feitianzhi Zeng,2 Lixin Zhang,2 Tianzhou Ma,3 George Tseng,3 Robert P. Edwards,2 Anda M. Vlad2. 1 _Magee Womens Research Institute, Pittsburgh, PA;_ 2 _University of Pittsburgh, School of Medicine, Pittsburgh, PA;_ 3 _University of Pittsburgh, Graduate School of Public Health, Pittsburgh, PA_.

Rationale: Ovarian cancer patients receive platinum/taxane-based chemotherapy as standard of care. Five year survival has remained unchanged for several decades, at less than 45%, pointing to the need for new and improved therapies. We recently reported in vivo anti-tumor efficacy of PD-L1 immune checkpoint blockade in a new transplantable ovarian cancer mouse model. However, chemo-induced effects on ovarian tumor PD-L1 expression have not been addressed. We studied here the effect of platinum/taxane on tumor PD-L1 expression in vitro and in vivo and tested the efficacy of PD-L1 blockade in combination with cisplatin, using several treatment regimens.

Methods and Results: Our results demonstrate that in vitro exposure of several human ovarian cancer cell lines, with various baseline susceptibilities to cisplatin and taxol, triggers PD-L1 upregulation. Similar effects were observed when newly tumor-derived, platinum sensitive (2F8) and platinum resistant (2F8cis) murine ovarian cancer cell lines were exposed to chemo drugs in vitro. Based on these findings we postulated that PD-L1 immune checkpoint blockade in combination with cisplatin may provide therapeutic benefit in ovarian cancer. In vivo, we have challenged n=37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs or isotype control every two weeks for three doses starting at day 14 post-inoculation. Study endpoint was overall survival. Secondary endpoints were tumor CD8 infiltration, changes in Th1/cytotoxic immunity. Tumors and ascites-derived cancer cells were analyzed with flow cytometry. RNA was extracted from splenocytes and analyzed with Nanostring using probes for n=511 immune genes.

In line with in vitro results, tumor cells isolated ex vivo from cisplatin-treated mice expressed increased PD-L1. Compared to control treated mice, both Cisplatin alone and anti-PD-L1 alone increase overall survival (p=0.002 and p=0.02, respectively). No increase in survival was observed in anti-PD-L1/Cisplatin-treated mice, due to an over-responsive immune cascade overwhelming the animal. Addition of celecoxib, a cyclooxygenase-2 inhibitor, to the anti-PD-L1/cisplatin combination was well tolerated and led to improved overall survival.

Conclusion: Ovarian cancer cells upregulate PD-L1 in response to chemotherapy exposure in vivo and in vitro. Though effective independently, combination cisplatin and anti-PD-L1 blockade did not improve survival, likely due to cytokine release syndrome. Celecoxib added to cisplatin and anti-PD-L1 improves overall survival. Anti-tumor immunity in responder mice revealed a cytotoxic T cell mediated gene signature. These findings reveal benefits and pitfalls of chemotherapy in combination with immune checkpoint blockade and have high translational potential for ovarian cancer treatment.

#3209

CRLX101, an investigational nanoparticle-drug conjugate of camptothecin, demonstrates synergy with immunotherapy agents in preclinical models.

Douglas Lazarus, Christian Peters, Adam Stockmann, Scott Eliasof, Lata Jayaraman. _Cerulean Pharma, Inc., Waltham, MA_.

CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin, is currently being clinically evaluated in multiple treatment-refractory solid tumors. CRLX101 has been shown preclinically to be active in many different tumor types as a dual inhibitor of topoisomerase 1 and hypoxia-inducible factor 1α (HIF-1α). It has a long circulation half-life and has been shown pre-clinically to release camptothecin in a slow and prolonged manner in tumors. Camptothecin itself was identified as an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not shown significant toxicity in over 300 patients to date, offers a unique opportunity to improve cancer treatment in a meaningful way.

Recent publications have suggested that tumor expression of the immune-suppressive molecule PD-L1 is controlled by HIF-1α. Since CRLX101 is a potent inhibitor of HIF-1α, it is possible that CRLX101 behaves as an inhibitor of the PD-1/PD-L1 axis in vivo. We therefore hypothesized that a combination of CRLX101 with agents that are being investigated in combination with the anti PD-1 antibody would lead to increased efficacy. Indole diamine oxygenase (IDO) inhibitors are a new class of drugs that decrease tumor-induced immune suppression and are currently being evaluated in the clinic with anti-PD-1 antibodies. Using syngeneic tumor models, we tested the combination of CRLX101 with three different IDO inhibitors. Treatment of the B16.F10 melanoma model with IDO inhibitors NLG919, INCB024360 or indoximod had no effect on tumor growth while CRLX101 as monotherapy showed moderate anti-tumor activity. When CRLX101 was combined with any of the three IDO inhibitors, the anti-tumor activity was greatly improved compared to monotherapy. Similar results were noted in other syngeneic tumor models. This improved combination response was not observed in IDO inhibitor combinations with the clinically approved topoisomerase inhibitor irinotecan, suggesting that CRLX101 provides a unique advantage in this context. Interestingly, CRLX101/IDO inhibitor combination was also superior to the combination of anti-PD-1 antibody and IDO inhibitor. These data suggest that CRLX101 in combination with IDO inhibitors can successfully block host-mediated immune-suppression to enhance anti-tumor immunity, and this combination may therefore show improved therapeutic activity in the clinic. We therefore plan to explore the immune-specific effects of CRLX101 as well as the mechanistic basis for the observed combination response, and use that information to guide CRLX101 clinical strategy in the setting of chemo-immunotherapy regimens.

#3210

An in vitro approach to study T cell exhaustion.

William Hastings, Glenn Dranoff, Alexander Cao. _Novartis Institute for Biomedical Research, Cambridge, MA_.

Immune checkpoint inhibition is now a validated modality in the treatment of multiple cancers. In vitro immune cell assays are a key tool in the study of pharmacological agents for immune modulation. Assays often involve isolating lymphocytes from human peripheral blood followed by a T cell stimulus such as anti-CD3, staph enterotoxin B, or allogeneic APCs. While this approach is useful to probe the events of immune stimulation, it likely reflects a primary or secondary immune activation, rather than a state of exhaustion. As intra-tumoral T cells are thought to be exhausted by chronic/repeated exposure to self-antigen, we sought to define experimental conditions that would reflect this state in vitro. We reasoned that repeated stimulation of T cells using anti-CD3/anti-CD28 coated microbeads would induce an exhausted phenotype. We found that, using PBMC from normal healthy donors, frequent and repeated stimuli resulted in a loss of T cells' ability to produce IL-2, TNFa, and IFNg as measured by intracellular cytokine staining. Interestingly, only one restimulation was necessary to confer this phenotype, as repeated stimuli over a longer time course did not result in further suppression. We also found that repeat stimulation conferred a surface marker phenotype suggestive of exhausted cells. Finally, we used cell death inhibitors z-7AAD-cmk and EGTA and found no effect on cytokine loss, supporting the hypothesis that cells are exhausted and not deleted by activation induced cell death. Thus we have defined experimental conditions that possibly reflect an exhausted T cell phenotype in vitro. This may be useful for the pharmacological evaluation of different therapeutic agents for their immune modulatory activities in cancer.

#3211

PD-1-targeted Probody therapeutics provide anti-tumor efficacy and a 10-fold dose protection against systemic autoimmunity in preclinical studies.

Kimberly A. Tipton, Chanty Chan, Kenneth R. Wong, Victoria Singson, Jennifer H. Richardson, W Michael Kavanaugh, Bryan A. Irving, James W. West. _CytomX Therapeutics, South San Francisco, CA_.

Immuno-oncology approaches have transformed the treatment of advanced melanoma, NSCLC and an increasing number of other cancers. Antibodies targeting the T cell checkpoint molecules CTLA-4 and PD-1 can reverse tumor suppression of T cell activation that prevents effective anti-tumor immunity. However, blockade of checkpoint molecules, particularly in combination, in non-tumor tissues can result in autoimmune side effects that can limit therapeutic utility. Combination of PD-1 and CTLA-4 blockade provides increased response rates but with concomitant increases in immune-related adverse events. Here we demonstrate that Probody™ therapeutics targeting PD-1 synergize with a CTLA-4 checkpoint inhibitor to eradicate established tumors in a mouse model while protecting against PD1-mediated systemic autoimmunity.

Probody therapeutics are recombinant, proteolytically-activated antibody prodrugs that have the potential to meaningfully widen therapeutic index by minimizing interactions with normal tissue while retaining anti-tumor activity. Probody therapeutics take advantage of protease activities that are abundant in tumors but suppressed in normal tissues. An extension of the antibody light chain with a masking peptide blocks binding of the antibody to antigen in normal tissue. Tumor-associated proteases cleave and release the mask, enabling the active antibody to bind antigen preferentially in the tumor. The feasibility of the Probody approach to checkpoint targets is supported by preclinical and clinical studies in which intratumoral delivery of low dose immune modulators enables anti-tumor responses despite negligible systemic exposure.

For preclinical assessment of PD-1 as a Probody target, a family of Probody therapeutics derived from the anti-mouse PD-1 antibody J43 was developed. The intact Probody therapeutics demonstrated reduced binding to mouse PD-1 relative to the parental antibody that was completely restored following proteolytic activation. The anti-tumor efficacy of the J43 antibody and Probody therapeutics was assessed following systemic administration as single agents or in combination with an anti-CTLA-4 antibody to mice bearing established MC38 syngeneic tumors. As single agents, the PD-1 antibody and Probody therapeutics reduced tumor growth relative to an isotype antibody control and synergized with CTLA-4 blockade. Consistent with their reduced ability to bind target in the absence of proteolytic activation, the PD-1 Probody therapeutics were up to 10 times less potent than the parental anti-PD-1 antibody in inducing autoimmune diabetes in NOD mice. Our preclinical findings demonstrate that PD-1 Probody therapeutics retain anti-tumor efficacy with improved safety profiles and therefore have the promise to enable better tolerated PD-1 combination immunotherapies.

PROBODY is a trademark of CytomX Therapeutics, Inc.

#3212

Combining Toca 511 and 5-fluorocytosine with αPD-1 or αCTLA-4 antibody significantly reduces tumor burden compared to either checkpoint inhibitor alone or in combination in a subcutaneous mouse glioma model.

Leah Mitchell, Fernando Lopez Espinoza, Kader Yagiz, Daniel Mendoza, Maria Rodriguez-Aguirre, Sean Mitchell, Douglas J. Jolly, Joan M. Robbins. _Tocagen, San Diego, CA_.

Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD) protein, selectively replicates and spreads in malignant cells. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an oral anti-fungal drug) to 5-FU, leading to both direct tumor cytotoxicity and extended immunotherapeutic effects. As of 9/25/2015, 116 patients with recurrent high grade glioma have been treated with Toca 511 & Toca FC in 3 ongoing Phase 1 clinical trials (NCT01156584, NCT01470794, NCT01985256), in which potential benefits were found, including extended overall survival and a favorable safety profile. The purpose of this study was to determine if combinations of Toca 511+5-FC with αPD-1, αCTLA-4, or both, are therapeutically useful in a syngeneic mouse model, and for which combinations we could see meaningful variations in tumor infiltrating immune cells.

2x10^6 Tu-2449SC glioma cells (2% pretransduced with Toca 511 to provide a uniform starting inoculum) were implanted on the right flanks of B6C3F1 mice and treatment initiated when tumors reached ≈100mm3. 5-FC treated mice received 500 mg/kg/day IP (5 days per week). αPD-1 was administered at 300µg/mouse IP followed by 3 maintenance doses every 3 days of 200µg/mouse. αCTLA-4 (100µg/mouse, IP) was administered every 3 days (total of 6 treatments). Isotype antibodies were administered to control groups. The study was terminated after 3 cycles of 5-FC. Tumors and spleens were collected and analyzed by flow cytometry for 26 unique immune cell populations.

Tumor burden was significantly reduced with the addition of Toca 511+5-FC to either antibody treatment alone or in combination compared to antibody treatment groups. Treatment with Toca 511 and 5-FC alone was highly effective at controlling tumor growth, therefore, additive treatment with αPD-1, αCTLA-4, or αPD-1 + αCTLA-4 did not significantly further reduce tumor burden. The combination of αPD-1 and αCTLA-4, but not either antibody alone, significantly reduced tumor burden. Treatment with 5-FC significantly reduced tumor associated macrophages as well as myeloid derived suppressor cells and increased CD4+ T cell populations. Of the T cell populations that were increased in the tumors, a greater percentage of T cells were producing IFNγ.

Combining Toca 511+5-FC with αPD-1, αCTLA-4 or the combination of antibodies significantly reduced tumor growth compared to antibody treatments alone. Tumor associated immune cell population analysis also revealed that Toca 511+5-FC altered the immune cell populations in the tumor to generate a more permissive environment for immune activation and anti-tumor immune response. Toca 511 & Toca FC may be broadly applicable in combination with checkpoint inhibitors to help activate the immune system.

#3213

Hypomethylation therapy leads to immune polarization and improved efficacy of immunotherapy in transgenic model of pancreatic cancer.

Tamas Gonda, Jarwei Fang, Catheine Do, Anna Zhukovskaya, Martha Salas, Benjamin Tycko. _Columbia University, NC, NY_.

BACKGROUND

Despite significant activity of immunotherapies in several malignancies, their efficacy in pancreatic cancer has been limited. One possible explanation may be the absence of a tumor microenvironment and immune cells that are necessary. We have demonstrated that 5-aza-2-deoxycitidine (DAC) had a profound effect on pancreatic cancer associated stroma and up-regulated many immune pathways. Therefore we proposed that hypomethylating therapy may alter the microenvironment to allow immue checkpoint inhibitors greater therapeutic efficacy.

METHODS

Transgenic mice that spontaneously develop pancreatic cancer (Kras, Trp53, Pdx-1 Cre [KPC]) underwent weekly transabdominal ultrasound to detect mice initially with larger (6-8 mm) and subsequently with smaller (3-5 mm) tumors. PBS or DAC (5 mg/kg) and anti -PD1H or isotype control antibody were administered every 72 hours intraperitoneally for 3 doses. Tumor size was monitored biweekly and 3D tumor volume reconstruction was used to estimate size. The immune cell infiltrate was characterized by quantitative immunohistochemistry or by FACS.

RESULTS

Mice treated with DAC with 6-8 mm tumors demonstrated a significantly increased survival and tumor necrosis as well as polarization of the infiltrating macrophages towards an M1 (CD86, CD163) phenotype and a trend towards an increase in infiltrating CD8 cells compared with controls. The shift in the T cell population and the macrophage phenotypes was not seen in the spleen or peripheral blood of either wild type or tumor bearing animals treated with DAC compared with controls. We subsequently treated mice with 3 doses of DAC followed by 3 doses of anti -PD1H and compared them to anti-PD1H alone in KPC mice with smaller (3-5 mm) tumors. In DAC + anti-PD1H we noted a decrease in the growth rate of tumors, an increased survival and a significant increase in necrosis and CD8 infiltrates, as well as a significant decrease in Ki67 and increase in Tunel staining compared with anti-PD1H alone.

CONCLUSIONS

Our results suggest that DAC pre-treatment prior to checkpoint inhibitors may favorably polarize the tumor infiltrating immune cell population, lead to increased recruitment of CD8 positive T cells, result in marked tumor necrosis and slow tumor growth. These effects ultimately contribute to increased therapeutic efficacy of checkpoint inhibitors in pancreatic cancer. We plan to optimize the efficacy of epigenetic therapy and checkpoint inhibitors by using other monoclonal antibodies (anti-PD1, PD1L)and different dosing regimens.

#3214

A fully human anti-TIM3 antibody with co-stimulatory activity.

J Dixon Gray, Irina Krapf, Heyue Zhou, Gunnar Kaufmann. _Sorrento Therapeutics, San Diego, CA_.

Targeting immune checkpoint molecules has been demonstrated to have significant benefit to patients with cancer. One such molecule is TIM3 whose expression is associated with a phenomenon referred to as T cell exhaustion. While it has been suggested that ligation of TIM3 provides a negative signal to the T cell, there is no obvious inhibitory signaling motif associated with the molecule. We have developed a fully human antibody to TIM3 which demonstrates potent co-stimulatory activity. T cells stimulated with anti-CD3 plus either anti-CD28 or anti-TIM3 show comparable levels of cell activation. Similar to co-stimulation with anti-CD28, the anti-TIM3 antibody promotes expression of CD25, interferon gamma, Tbet and granzyme B, amongst others. Thus, anti-TIM3 co-stimulation promotes T cells with a pro-inflammitory phenotype. With TIM3 being associated with exhausted T cells, co-stimulation with this antibody would be expected to specifically re-activate these exhausted T cells at the tumor site. Moreover, since some NK cells constitutively express TIM3, it would be expected that this antibody would activate these cells as well. Altogether, we have identified a novel anti-TIM3 antibody that is capable of targeting T cells at the tumor site and activating them.

#3215

GITRL-Fc can significantly reduce tumor growth by stimulating innate and adaptive immunity.

Hyun-Bae Jie, Minu K. Srivastava, Erin Mayes, Rui Yun, Fumiko Axelrod, Jorge Monteon, Austin Gurney, Angie In-Kyung Park. _OncoMed Pharmaceuticals Inc, Redwood City, CA_.

Although the interaction between GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) and GITRL (its ligand) is important for the development of immune responses, the cellular mechanisms underlying anti-tumor immunity including both innate and adaptive immunity is not fully understood. We generated a novel single-gene linkerless GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc), and investigated its effect on controlling tumor growth and immune responses in preclinical tumor models. Treatment with GITRL-Fc significantly reduced tumor growth in several preclinical tumor models by inducing Th1 biased anti-tumor immunity and reducing Treg-mediated immune suppression. Immune cell depletion studies showed that anti-tumor immunity induced by GITRL-Fc depended on CD8+ T cells as well as NK cells. Furthermore, the combination of GITRL-Fc with PD-1 blockade significantly reduced the tumor growth in the Renca murine kidney adenocarcinoma tumor model. Taken together, these results suggest that GITRL-Fc can improve cancer treatment as a single agent or in combination therapy by enhancing innate and adaptive cellular immunity.

#3216

Anti-PD-1, Anti-PD-L1 and Anti-CTLA-4 checkpoint inhibitor treatment leads to different responses in syngeneic tumor models.

Peter Jantscheff, Cynthia Schaefer-Obodozie, Sandra Moor, Holger Weber. _ProQinase GmbH, Freiburg, Germany_.

Checkpoint inhibitor treatment has already become a common therapy of various cancer types. Still there is a growing need for well-characterized preclinical mouse models, as clinical data indicate that patients only partially respond to this regiment. We examined the efficacy of anti-CTLA-4, anti-PD-1 and anti-PD-L1 therapy on 4T1, B16.F10, Clone M-3, CT26wt, LL/2, MC38-CEA and RENCA syngeneic tumor models. The outcome demonstrates a large variation in the response to the immune checkpoint therapy among the analyzed tumor models. Poorly immunogenic models like the LL/2 tumor did not respond to any given therapy, whereas highly immunogenic tumor models like CT26wt or MC38-CEA tumors were inhibited by all tested immunotherapies.

The CT26wt tumor model was further characterized in a re-challenge experiment. Growth retardation was observed in CT26wt-tumor bearing mice treated with anti-PD-L1 antibody. A fraction of mice responded with a complete regression of the tumor. These mice were repeatedly challenged subcutaneously with a high number of fresh CT26wt tumor cells. No re-growth of CT26 tumors was observed, whereas challenging with fresh 4T1 tumor cells resulted in a rapid 4T1 growth in these mice, demonstrating a specific immune protection against CT26wt tumors.

In addition all syngeneic tumor models were analyzed for the distribution of immune cells such as Treg cells, M1/M2 macrophages and M/PMN-MDSCs in tumor tissue based on multi-color flow-cytometric analysis.

These data may help to select a suitable model for testing new drug candidates and define a sensitive combination therapy to support the anti-tumor immune defense in addition to checkpoint inhibitor treatment.

#3217

MGD013, a bispecific PD-1 x LAG-3 Dual-Affinity Re-Targeting (DART®) protein with T-cell immunomodulatory activity for cancer treatment.

Ross LaMotte-Mohs,1 Kalpana Shah,1 Doug Smith,2 Sergey Gorlatov,1 Valentina Ciccarone,1 James Tamura,1 Hua Li,1 Jill Rillema,2 Monica Licea,2 Leilei He,1 Farha Vasanwala,1 Wei Chen,1 Xiao-Tao Yao,1 Francine Chen,2 Jennifer Brown,1 Jeffrey Nordstrom,1 Scott Koenig,1 Ezio Bonvini,1 Syd Johnson,1 Paul Moore1. 1 _MacroGenics, Inc., Rockville, MD;_ 2 _MacroGenics, Inc., San Francisco, CA_.

Introduction: The combination of monoclonal antibodies (mAbs) that targets the immune checkpoint molecules CTLA-4 and PD-1 has shown clinical benefit beyond that observed with either mAb alone. This finding has prompted exploring whether such an approach could be applied within the context of additional combinations of checkpoint molecules, such as PD-1 and lymphocyte activation gene-3 (LAG-3). Animal tumor models have validated combining anti-PD-1 with anti-LAG-3 mAbs in eliciting synergistic tumor-eradicating immunity; expression of PD-1 and LAG-3 on exhausted T cells and tumor infiltrating lymphocytes (TILs) further supports their dual-targeting. We have developed a bispecific DART® protein that targets PD-1 and LAG-3, aimed at inducing potent antitumor immunity through simultaneous blockade of non-redundant checkpoint pathways intrinsic to exhausted T cells.

Methods: mAbs against PD-1 and LAG-3 were generated and selected for DART conversion based on binding, biophysical and functional blocking against their respective receptor/ligand axes and functional activity in re-activation of prior superantigen-stimulated T cells or in antigen-specific recall assays.

Results: Lead PD-1 and LAG-3 mAbs demonstrating favorable functional properties were selected for humanization. Immunohistochemistry confirmed that the lead LAG-3 and PD-1 mAbs display restricted lymphocyte expression in human tissues and overlapping expression in TILs. The humanized mAbs were assembled into MGD013, an Fc-bearing PD-1 x LAG-3 DART protein that demonstrated favorable biophysical and manufacturability properties. MGD013 bound specifically with high affinity to PD-1 and LAG-3 as well as to target-expressing cell lines and chronically-activated T cells. MGD013 blocked PD-1/PD-L1, PD-1/PD-L2 and LAG-3/HLA (MHC-II) interactions and PD-1 signaling. Further functional characterization of MGD013 revealed enhanced cytokine secretion in response to antigenic re-challenge of previously stimulated T cells compared to that observed upon independent blockade of either the PD-1 or LAG-3 pathways alone. Furthermore, under the above experimental conditions, MGD013 mediated greater cytokine secretion than that observed with the combination of equivalent (equimolar) levels of replicas of the approved PD-1 mAb, nivolumab, and the LAG-3 mAb, 25F7, which is currently undergoing clinical testing. Finally, cynomolgus monkey pharmacokinetic studies demonstrated a prolonged circulating half-life consistent with that of an Fc-bearing molecule.

Conclusions: MGD013 blocks both PD-1 and LAG-3 pathways, resulting in enhanced T-cell responses compared to single or combination mAb blockade. Together with favorable cynomolgus monkey PK, these studies support further clinical development of MGD013.

#3218

Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models.

Marion Lapierre,1 Cécile Dejou,1 Carine Paturel,2 Henri-Alexandre Michaud,3 Laurent Gros,3 Armand Bensussan,4 Jean-François Eliaou,3 Jeremy Bastid,1 Nathalie Bonnefoy3. 1 _OREGA Biotech, Ecully, France;_ 2 _Innate Pharma, Marseille, France;_ 3 _INSERM U1194, IRCM, Montpellier, France;_ 4 _INSERM UMR-S 976, Paris, France_.

The CD39-CD73-adenosine pathway is an emerging regulator of the immune antitumor response. CD39 is expressed within tumors and the tumor microenvironment by several cell population including immune and cancer cells. In tumor tissues, the pathway leads to the accumulation of immunosuppressive adenosine together with decreased levels of immunoactivating peritumoral ATP. We reported previously that CD39 blockade increased T cell and NK cell-mediated cytotoxic activity in vitro and disclose, during this meeting, the development of the first human-CD39-blocking humanized antibody (S. Augier et al., Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy). Here we demonstrated that this pathway is involved in tumor-induced resistance to various cancer therapies in syngeneic mouse melanoma, colon cancer and fibrosarcoma models. We used therapy-resistant mouse models or inefficacious treatment regimens in the CD39 knockout mice to assess the capacity of CD39 to affect the response to chemotherapies, tumor associated antigen (TAA)-targeting antibodies and immunomodulators such as anti-PD1 antibodies. We achieved increased response rates, increased response duration and some complete and long lasting tumor regressions in the CD39 deficient context. These preclinical proof-of-concept studies highlight the role of the CD39 immune checkpoint pathway in limiting the efficacy of various anticancer therapies in syngeneic mouse models and thereby support the potential clinical value of the humanized CD39-neutralizing antibody under development.

#3219

Prior vaccination is critical to PD1/PDL-1 treatment efficacy in a mouse breast cancer allograft.

Zhun Wang, Xiaoyu An, Jinping Liu, Xuesong Ouyang, Jiagui Qu, Likun Zhang, Jie Cai, Jean-Pierre Wery, Henry Li. _Crown Biosciences, Santa Clara, CA_.

We previously generated the MuPrime® mBR6004 model, by engrafting the breast adenocarcinoma derived from MMTV-PyVT transgenic mice (GEMM)1 into the syngeneic FVB/N mice2,3. The allograft grows robustly, maintains the histopathology features of the original GEMM, and metastasizes to the lungs in all examined cases when implanted orthotopically2. Additionally, histopathology shows that the models is HER2++, but ER-/PR-. We profiled drug responses to standard of care (SoC)2 and checkpoint inhibitors3. Interestingly, among all checkpoints inhibitors tested, our model is insensitive to PD1/PDL-1 inhibitors, but responds to CTLA-4 inhibitors3. To further explore the underlying mechanism of response, we performed extensive tumor immuno-profiling for the presence of infiltrating immune cells, e.g. TIL, CTL, Treg, immune-suppressive macrophages, NK, etc3. We observed a good pharmacodynamic (PD) correlation between the presence of tumor infiltrating T-cells, particularly CD8+ TIL, and NK and a positive response to therapy, regardless the treatment type. Given this preliminary observation, we attempted animal vaccination with tumor lysates prior to tumor engraftment or treatments. Interestingly, while vaccination had minimal effects on the engraftment take rate (100% take for all animals) and on the growth kinetics of the engraftments, it had profound effects on the tumor response to anti-PD1/anti-PDL-1 antibody treatments, significantly enhancing tumor response to these agents in a model that was otherwise unresponsive. Our results suggest that the immunological status of the animal, at least with regard to the specific anti-tumor immunity, is critically important to determine the response to checkpoint inhibitors. Our observations suggest that vaccination is critical for the overall success of immunotherapy (I/O) treatments utilizing checkpoint inhibitors as well other treatment strategies.

References

1. Guy, C.T., Cardiff, R.D. & Muller, W.J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Molecular and cellular biology 12, 954-961 (1992).

2. Annie Xiaoyu An1, Jinping Liu1, Jie Cai1, Jean Pierre Wery1, and Henry Q.X. Li1,. Building mouse tumor derived allogragfts for immune-oncology research. (2015).

3. Zhun Wang1, A.X.A., 2*, Jinping Liu1, Gavin Jiagui Qu1, Likun Zhang, Jie Cai1, Bin Chen1, Davy Xuesong Ouyang1, Jean Pierre Wery1, and Henry Q.X. Li1,2. Response to checkpoint inhibition by GEMM breast cancer allograft EORTC-AACR-NCI Abstract (2015).

#3220

A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR.

Ana Maria Gonzalez,1 Ekaterina Breous,2 Mariana L. Manrique,1 David Savitsky,1 Jeremy Waight,1 Randi Gombos,1 Yuqi Liu,1 Shiwen Lin,1 Olivier Leger,3 Volker Seibert,2 Takemasa Tsuji,4 Taha Merghoub,4 Sadna Budha,4 Roberta Zappasodi,4 Gerd Ritter,5 Jedd Wolchok,4 Peggy Scherle,6 Gregory Hollis,6 Reid Huber,6 Marc Van Dijk,2 Robert Stein,1 Nicholas Wilson1. 1 _Agenus Inc, Lexington, MA;_ 2 _Agenus Inc / 4-Antibody, Basel, Switzerland;_ 3 _Leger Consulting, Chambéry Area, France;_ 4 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 5 _The Ludwig Institute for Cancer Research Inc, New York, NY;_ 6 _Incyte Corp, Delaware, DE_.

Activation of costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily in T cells is considered a promising alternative approach to potentiate anti-tumor immunity that may complement strategies focused on the blockade of co-inhibitory pathways such PD-1/PDL1. Glucocorticoid-induced TNFR-related protein (GITR, CD357 or TNFRSF18) is an important T cell costimulatory receptor that can potentiate T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In humans GITR expression is generally restricted to subsets of T cells responding to TCR stimulation, and is co-expressed with OX40. Like other TNFR family members, GITR co-stimulation can enhance T cell responsiveness to suboptimal TCR signaling by activating the NFκB pathway, leading to enhanced cytokine responses and survival. GITR signaling in T cells may also promote resistance to the immune suppressive effects of regulatory T cells, thereby enhancing T cell responsiveness to weakly immunogenic tumor-associated antigens. INCAGN01876 is a humanized IgG1 monoclonal antibody being developed for the treatment of advanced malignancies. INCAGN01876 potently binds to human and non-human primate GITR but does not cross-react with related TNFR family members. INCAGN01876 has been optimized to mediate receptor forward signaling under suboptimal TCR stimulatory conditions, leading to increased production of TNFα and IFNγ by both CD4+ and CD8+ T cells. INCAGN01876 achieves this functionality by virtue of its ability to facilitate GITR clustering in TCR-stimulated T lymphocytes. In mouse preclinical tumor models, GITR was found to be selectively overexpressed by intratumoral regulatory T cells, a finding that was also observed in primary human tumor samples from diverse tumor types. In mouse models, this feature enabled a surrogate anti-GITR antibody to co-engage activating Fcγ receptors expressed by tumor-associated effector cells, and mediate the selective depletion of intratumoral regulatory T cells. Consistent with this, INCAGN01876 was designed to co-engage activating Fcγ receptors and was shown to efficiently mediate immune effector cell mechanisms, including ADCC and ADCP. Taken together, the biophysical and functional attributes of INCAGN01876 make it ideally suited for clinical development, both as a single agent and in combination with other immunomodulatory agents.

#3221

Oncolytic virotherapy combined with T-cell co-signaling antibodies for intracranial gliomas.

Zineb Belcaid,1 Cor Berrevoets,1 Jenneke Kloezeman,1 Marielle van der Kaaij,1 Dimitrios Mathios,2 Michael Lim,2 Clemens Dirven,1 Reno Debets,1 Martine Lamfers1. 1 _Erasmus MC, Rotterdam, Netherlands;_ 2 _Johns Hopkins University, Baltimore, MD_.

Introduction: The tumor-selective human adenovirus Delta24-RGD (DNX-2401) is currently under investigation in phase II clinical trials for recurrent glioblastoma. In recent preclinical studies, our group demonstrated that the immune system, and CD8+ T cells in particular, plays an important role in the establishment of a protective anti-glioma immune memory (Kleijn, PLoS ONE, 2014). In order to improve anti-tumor efficacy of Delta24-RGD therapy with antibody immunotherapy, we are currently investigating the role of T cell co-signaling molecules in our model and their contribution toward an anti-glioma T cell response.

Methods: Immune-competent C57bl/6 mice were intracranially injected with GL261 glioma tumor cells and treated on day 5 with an intra-tumoral injection of Delta24-RGD virus. Single cell suspensions of brain tumors and spleens were stained for flow cytometry with antibodies against CD3, CD4, CD8 as well as ten selected T cell co-signaling molecules: ICOS, 4-1BB, CD28, OX-40, CD40L, CTLA-4, PD-1, LAG-3, TIM-3, BTLA. Tumor cell lysis and IFNy production was assessed in ex vivo cultures of brain tumor cells and immune cells. In addition, IFNy production was assessed from co-cultures with splenocytes and GL261 glioma tumor cells treated with either a PD-1 blocking antibody or an agonist ICOS antibody.

Results: In brain tumors, we observed an increase in numbers of CD8+, but not CD4+ T cells, on day 9 and 14 after virus-treatment. Expression analysis of the co-signaling molecules in infiltrating brain T cells revealed higher expression levels of the co-signaling molecules ICOS, CD28 and PD-1. In the ex vivo tumor cell and immune cell cultures derived from Delta24-RGD treated mice, there is a significant positive correlation between the observed tumor cell lysis and IFNy production ex vivo (Spearman r = 0,9524; p < 0.01). Interestingly, the amount of tumor cell lysis inversely correlated with a low density of CD3+ PD-1+ T cells in vivo (Spearman r = -0,7619; p < 0.05). Co-cultures of splenocytes from virus-treated mice and GL261 glioma tumor cells showed a significant increase in IFNy production when treated with a PD-1 blocking antibody (p < 0.01), but not when treated with an ICOS agonistic antibody.

Conclusion: The inverse relationship between the presence of a low density of PD-1+ TILs in vivo and a higher T cell functionality ex vivo provides rationale for inhibiting the PD-1 receptor with antibody immunotherapy in combination with Delta24-RGD therapy in order to improve therapeutic efficacy. In vivo studies combining these therapies are underway.

#3222

Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy.

Severine Augier,1 Ivan Perrot,1 Cecile Dejou,2 Rachel Joly,1 Stephane Delahaye,1 Helene Rispaud Blanc,1 Caroline Denis,1 Laurent Gauthier,1 Armand Bensussan,2 Jean-francois Eliaou,2 Yannis Morel,1 Nathalie Bonnefoy,2 Jeremy Bastid,2 Carine Paturel1. 1 _Innate Pharma, Marseille, France;_ 2 _Orega Biotech, Ecully, France_.

CD39 (ENTPD1) is a cell membrane ectonucleotidase that hydrolyzes extracellular immunoactivating ATP and ADP into AMP, which can be further hydrolyzed by ectonucleotidase CD73 into immunosuppressive adenosine. Within the tumor microenvironment, adenosine accumulation causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. The role of CD39 expression on both Tregs and on tumor cells in promoting immunosuppression has been demonstrated in several reports. Blockade of CD39 may promote anti-tumor immunity by directly accumulating immunostimulating ATP and indirectly by reducing adenosine accumulation. Here, we describe the discovery and preclinical development of an anti-huCD39 blocking antibody for cancer immunotherapy. Parental anti-huCD39 mouse monoclonal antibody was humanized. The humanized mAb specifically binds huCD39 protein, but not CD39-like proteins. Nanomolar affinities for human CD39 were measured in SPR studies on recombinant CD39 protein and in flow cytometry titration studies on CD39 expressing transfectants and tumor cell lines. The humanized mAb blocked human CD39 ATPase activity in vitro in the nanomolar range, as demonstrated using transfected cells, CD39-expressing tumor cell lines, as well as human PBMC and ex-vivo isolated fresh tumor samples. The humanized mAb cross-reacted on cynomolgus CD39 and blocked ATPase activity on cynomolgus PBMC with similar efficacy as on human PBMC. Finally, treatment with blocking anti-CD39 mAb inhibited tumor growth in vivo in mouse tumor models. Taken together, these data support the clinical development of anti-CD39 neutralizing mAb for cancer immunotherapy.

#3223

Androgen receptor stimulation decreases PD-L1 expression in androgen-responsive thyroid cancer cells.

Timmy O'Connell,1 Melanie Jones,2 Anvita Gupta,1 Augustine Moscatello,1 Raj K. Tiwari,1 Jan Geliebter1. 1 _New York Medical College, Valhalla, NY;_ 2 _United States Military Academy Preparatory School, West Point, NY_.

Women develop thyroid cancer more often than men; however, when men develop thyroid cancer, the disease course is more lethal, with a mortality rate that is two to three fold higher than in females. Immune elimination of nascent tumor cells may explain the differences in disease occurrence/course between men and women. To address this possibility, we examined the effect of androgen on the expression of immune checkpoint molecules in an androgen responsive-thyroid cancer cell line. The undifferentiated thyroid cancer cell line, 8505C, does not express a functional androgen receptor (AR). 84E7 is a clone of 8505C that was transfected with an AR containing plasmid resulting in constitutively expressed AR. Transcriptome analysis was performed on 8505C and 84E7, with and without 5α-dihydrotestosterone (DHT) treatment. Raw sequencing reads were aligned to the UCSC hg19 human reference genome with Tophat, and Cufflinks was used to measure transcript abundances in Reads Per Kilobase of exon model per Million mapped reads (RPKM) as well as to find genes with statistically significant changes in expression as per Trapnell et al (2012). RNASeq data indicated that DHT treatment of 8505C cells does not result in a significant change in gene expression (only 14 of 23,285 genes), consistent with the lack of a functional androgen receptor. In contrast, DHT treatment of 84E7 resulted in >2 fold expression changes in 1,552 genes. Examination of the immune checkpoint molecules CD28, CD80, CD86, CTLA-4, PD-1, PD-L1, PD-L2, TIM-3, TIM-3L, 4-1BB, 4-1BBL, OX40, and OX40L revealed that in DHT-treated 84E7 cells, PD-L1 was the sole immune checkpoint molecule that exhibited a significant expression change with a 1.8 log2 fold decrease (p=0, q=0), or 72% reduction in mRNA content. This is potentially significant as PD-L1 is produced by tumor cells as a strategy for subverting and evading the immune response, specifically T cells, allowing for continued tumor growth and metastases. In the thyroid, the presence of androgen-activated androgen receptors could perhaps lead to an environment that is more favorable for the immune system and may help eliminate nascent thyroid cancer cells. Thus, men may experience a decreased incidence of thyroid cancer due to an enhanced (less inhibited) anti-tumor environment. However, failure of this system and a subsequent increase in PD-L1 expression may help to explain the onset and severity of thyroid cancer in males. This finding opens the possibility for the use of immune checkpoint inhibitors in the treatment of thyroid cancer.

#3224

Blocking FSTL1 reprograms cancer-caused abnormal immunity.

Chie Kudo-Saito,1 Masayoshi Toyoura,2 Yuji Shoya,2 Akiko Ishida,2 Ryoko Kon2. 1 _National Cancer Center, Tokyo, Japan;_ 2 _Pharma Foods International Co., Ltd, Kyoto, Japan_.

Purposes:

The close connection between imbalanced immunity and tumor progression has been increasingly recognized, and a number of inhibitors for blocking immune inhibitory checkpoints have been established to improve the anti-tumor immunity in cancer patients. However, clinical responses are limited to a part of the treated patients. We previously identified FSTL1, which is significantly secreted from Snail+ metastatic tumor cells, as a key effector molecule in the cancer-caused immune suppression and dysfunction. FSTL1 generates and expands pluripotent mesenchymal stem cells (MSCs), which are able to generate various immunoregulatory cells and functionally impaired CD8-low T cells, and also to induce de novo tumor dissemination into bone marrow. FSTL1 also directly confers higher invasiveness on tumor cells (EMT induction). To prevent the FSTL1-caused vicious circulation for tumor progression, we attempted to establish anti-FSTL1 blocking mAbs.

Results:

We generated chicken/mouse chimeric anti-FSTL1 mAbs, which specifically recognized human and mouse FSTL1. The anti-FSTL1 mAb significantly suppressed tumor proliferation and invasion, MSC induction in mouse bone marrow cells, and Treg induction in human PBMCs in vitro as compared to the control group. When bone metastatic melanoma models (C57BL/6 mice implanted with snail-transduced B16-F10 melanoma cells both subcutaneously and intravenously) were intraperitoneally injected with the anti-FSTL1 mAb (10 mg/kg) on Day 5 (when tumor metastasis was already observed in bone marrow) and Day 10 after tumor implantation, the FSTL1-caused events were inhibited, and tumor-specific CD8+ T cells with higher CTL activities were generated, resulting in significant suppression of subcutaneous tumor growth and bone metastasis in the treated mice as compared to those of the control mice. At least in the same models, conventional immune checkpoint inhibitors including anti-CTLA4, anti-PD1, and anti-PDL1 mAbs failed to improve the anti-tumor immunity, and rather promoted bone metastasis because of no reduction of regulatory cells, particularly the MSCs in the treated mice.

Conclusions:

These results suggest that blocking FSTL1 reprograms and properly induces anti-tumor immunity by switching the tumor-supportive immune directivity to the active mode against cancer. Our anti-FSTL1 mAb having totally different molecular mechanisms from the conventional ones may be a promising therapeutics as a next generation of "immune checkpoint inhibitors" for radically correcting cancer-manipulated immunity in clinical settings.

#3225

Development of a proximity-based immunoassay to measure the PD1:PD-L1 complex in fixed samples.

Gerald Wallweber, Roy Ravanera, Ahmed Chenna, David Stathas, Weidong Huang, John Winslow, Christos Petropoulos. _Monogram Biosciences, South San Francisco, CA_.

Introduction: The binding of tumor cells expressing PD-L1 to PD1 receptors on activated T-cells inhibits T-cell activation, thereby evading an immune response against tumor progression. Currently, therapeutic monoclonal antibodies directed against either PD1 or PD-L1 block this interaction and restore T-cell function, leading to tumor lysis. PD-L1 immunohistochemistry (IHC) is the most common biomarker assay for these therapies, but disagreement exists on the antibodies used for IHC, which cell types to score, and the criteria for positivity. Quantitative immunofluorescence identified a significant correlation between the proximity of PD1 and PD-L1 protein expression and response to anti-PD1 therapy (Tumeh et al., Nature. 2014; 515:568-71). We have developed a direct quantitative measurement of the PD1:PD-L1 complex for the stratification of patients for anti-PD1 or anti-PD-L1 therapy. The VeraTag PD1:PD-L1 complex immunoassay utilizes two antibody pairs for the proximity-dependent release of a fluorescent reporter (VeraTag), which is measured with high sensitivity via capillary electrophoresis to accurately quantify the amount of complex in fixed samples.

Methods: Anti-PD-L1 rabbit mAb E1L3N was selected from our previously established PD-L1 VeraTag immunoassay. Mouse anti-PD1 mAbs were screened against FFPE human tonsil and PBMCs for development of a PD1 VeraTag assay. The VeraTag assay for PD1:PD-L1 complex combined the anti-PD-L1 rabbit mAb E1L3N and the anti-PD1 mouse mAb NAT105 together with a goat anti-rabbit secondary Ab conjugated to the VeraTag reporter and a goat anti-mouse secondary Ab conjugated to biotin. Fixed cell preparations used in the VeraTag PD1:PDL1 complex assay were prepared by co-culturing MB453, MB231 or RKO cancer cell lines expressing varying amounts of PD-L1 with Jurkat T cells. After 48-hours, non-adherent cells were removed by washing and remaining cells were fixed overnight in formalin.

Results: Following phytohemagglutinin (PHA) stimulation of human PMBCs and Jurkat cells, VeraTag measurements of PD1 protein expression increased 8- to 10-fold, whereas PD-L1 protein expression varied <2-fold under the same conditions. VeraTag measurements for the PD1:PD-L1 complex increased approximately 4-fold with the addition of PHA to co-cultures of PD-L1+ cancer cell line and Jurkat T cells. The level of PD1:PD-L1 complex varied proportionally with the number of Jurkat T cells added to the co-culture. We are currently utilizing VeraTag assays to quantify the amounts of PD1, PD-L1 protein and the PD1:PD-L1 complex with the VeraTag assays across a panel of formalin fixed samples of breast, lung and squamous cell carcinoma of the head and neck (SCCHN) cancer samples.

Conclusions: We have developed sensitive and quantitative assays for PD1, PD-L1 and the PD1:PD-L1 complex, which may provide a more direct measurement of PD1/PD-L1 pathway interaction.

#3226

Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models.

Anais Lagrange,1 Romain Boidot,2 Marc Hillairet de Boisferon,3 Olivier Duchamp,3 Jean-François Mirjolet,3 François Ghiringhelli4. 1 _INSERM 866, Dijon, France;_ 2 _Centre Georges François Leclerc, Dijon, France;_ 3 _Oncodesign S.A., Dijon Cedex, France;_ 4 _INSERM 866 / Centre Georges François Leclerc, Dijon, France_.

PD-1 - PD-L1 pathway disruptors (e.g. nivolumab, pembrolizumab) are now being approved for treatment of patients with locally evolved or metastatic melanoma and advanced non-small cell lung cancer (NSCLC) who have progressed after first line platinum-based chemotherapy. These drugs have also antitumor efficacy in other tumor types (renal cell carcinoma, bladder, Hodgkin lymphoma, colorectal carcinoma with mismatch-repair deficiency ⋯). However, there is still needs to identify predictive biomarkers of response in order to select patients who will benefit from treatments. PD-L1 expression, mutation numbers, or mismatch repair (MMR) deficiencies were already demonstrated to influence response to PD-1 targeting therapies. However, they did not completely discriminate responders from non-responders or cut off levels are not yet precisely defined.

Syngenic models are one of the only experimental models to evaluate immune system targeting therapies. Eight mouse models (4T1, A20, B16-F10, CT-26, EMT-6, MBT-2, LLC and Renca) were tested for response to PD-1 or PD-L1 targeting antibodies. From them, 3 models were characterized as non-responder (i.e. 4T1, LLC and RenCa) while other 7 models were showing to be sensitive to PD-1 inhibitors. PD-L1 expression (using RT-qPCR, WB and flow cytometry) was analyzed on cell lines at baseline and after 24h incubation with IFNg. At baseline, there is no significant difference between responders and non-responders whatever the level of analysis (mRNA, total PD-L1 protein expression or cell surface PD-L1 expression). However, after incubation with IFNg, there is a trend to segregate between responders and non-responders. Increased level of PD-L1 at the cell surface was evidenced for responders. IFNg signaling pathway used interferon regulatory factors (IRFs). IRF1 have been demonstrated to be responsible for both constitutive PD-L1 expression and early induction of PD-L1 after IFNg stimulation. mRNA expression of IRF1, IRF3, IRF7 and IRF9 were analyzed at baseline and after 24h incubation with IFNg. At baseline or after IFNg stimulation, there is no significant difference for IRF3, IRF7 and IRF9 mRNA expression between responders and non-responders. However, IRF1 mRNA is less expressed at baseline in responders as compared to non-responders. Moreover, increase in IRF1 expression after IFNg stimulation is higher in responders when compared to non-responders, which correlates well with changes in PD-L1 cell surface expression after IFNg stimulation. The total number of genomic variations was also analyzed using whole exome sequencing. Responder cell lines have highest number of variations in comparison to non-responder cell lines.

In depth characterization of these syngeneic models will increase the understanding of how translatable are the results acquired from preclinical mouse studies.

#3227

Intratumor diversity of surface marker expressions on tissue infiltrating lymphocytes in renal cell carcinoma patients.

Atsunari Kawashima, Takayuki Kanazawa, Kayoko Maekawa Kato, Kumiko Goto, Mitsunobu Matsumoto, Akiko Morimoto, Kota Iwahori, Takeshi Ujike, Akira Nagahara, Kazutoshi Fujita, Motohide Uemura, Norio Nonomura, Hisashi Wada. _Osaka University, Suita, Japan_.

Introduction and Objectives:

Renal cell carcinoma (RCC) is considered as an immunogenic tumor and several immune checkpoint inhibitors including anti PD-1 antibody has been reported to have impressive antitumor responses. Immunotherapy is believed to overcome tumor heterogeneity as previously reported in RCC patients. However, there were no reports whether there was intra-tumor diversity of surface marker expressions which could combine the new immune checkpoint inhibitors though the existence of polyclonal expansion of T lymphocytes in RCC were reported. Here, we investigated the surface marker expressions of tumor tissue infiltrating lymphocytes (TIL) and classified them based on their functional populations. Moreover, we explored the presence of intra-tumor diversity of surface marker expressions of TILs in each individual.

Material and Methods:

We extracted 70 sites 46 patients underwent surgical resection (clear cell RCC; 40, non-clear cell RCC; 6). Each TIL was characterized based on functional T cell populations (naïve T cell, effector T cell, regulatory T cell, Activated T cell, Exhausted T cell) using seven surface marker expressions (CD4, CD8, CD45RA, PD-1, Tim3, ICOS, CD25) measured by flow cytometry. Then we classified and examined the presence of intra-tumor diversity using 36 TILs from 12 patients by clustering analysis. Finally, all of TILs were classified and we examined both the validity and the clinical importance of its classification. Statistical analysis was performed using SPSS ver. 20.0.

Results:

Firstly, 36 TILs could be classified into three groups. Although 28 TILs of 10 patients were classified into the same group within each individual, 8 TILs of 2 patients (16.7%) were classified into different groups within each individual and those patients had intra-tumor diversity of surface marker expressions of TILs. Secondly, 70 TILs including previous ones were classified into three groups and 2 patients were consistently classified into different groups within each individual and this classification could be validated. Finally, we analyzed the correlation between this classification and clinical features to show the clinical importance of this classification. The presence of lymphovascular invasion was significantly correlated with this classification by both univariate (p=0.002) and multivariate analysis (OR: 0.093, 95%CI: 0.016 - 0.533, p=0.008).

Conclusions:

This study showed the presence of intra-tumor diversity of surface marker expressions of TILs in some patients and the reactivity of the new immune checkpoint inhibitors could differ such as other drugs already used in general practice. Also this classification based on functional populations of TILs might help to understand the comprehensive immune conditions in tumor tissues.

### Innate Immune System, Myeloid Cells, and Tumorigenesis

#3228

High salt induces anti-inflammatory MΦ2-like phenotype in peripheral macrophages.

Suneetha Amara,1 Toral R. Mehta,2 Margaret Whalen,2 Venkataswarup Tiriveedhi2. 1 _Mercy Hospital, St Louis, MO;_ 2 _Tennessee State University, Nashville, TN_.

Macrophages play a critical role in inflammation and antigen-presentation. Abnormal macrophage function has been attributed in autoimmune diseases and cancer progression. Recent evidence suggests that high salt tissue micro-environment causes changes in macrophage activation. In our current report, we studied the role of extracellular sodium chloride (NaCl) on phenotype changes in peripheral circulating monocyte/macrophages collected from healthy donors. High salt (0.2 M NaCl) treatment resulted in a decrease in MΦ1 macrophage phenotype (CD11b+CD14highCD16low) from 77.4±6.2% (0.1 M) to 29.3±5.7% (0.2 M, p<0.05), while there was an increase in MΦ2 macrophage phenotype (CD11b+ CD14lowCD16high) from 17.2±5.9% (0.1 M) to 67.4±9.4% (0.2 M, p<0.05). ELISA-based cytokine analysis demonstrated that high salt treatment induced decreased expression of in the MΦ1 phenotype specific pro-inflammatory cytokine, TNFα (3.3 fold), IL-12 (2.3 fold), CCL-10 (2 fold) and CCL-5 (3.8 fold), but conversely induced an enhanced expression MΦ2 phenotype specific anti-inflammatory cytokine, IL-10, TGFβ, CCL-17 (3.7 fold) and CCR-2 (4.3 fold). Further high salt treatment significantly decreased phagocytic efficiency of macrophages and inducible nitric oxide synthetase expression. Taken together, these data suggest that high salt extracellular environment induces an anti-inflammatory MΦ2 macrophage phenotype with poor phagocytic and potentially reduced antigen presentation capacity commonly found in tumor microenvironment.

#3229

Microglia/macrophages activation status in diffuse gliomas.

Thais F. Galatro,1 Paula Sola,1 Sueli M. Oba-Shinjo,1 Bart J. Eggen,2 Suely K. Marie1. 1 _University of São Paulo, São Paulo, Brazil;_ 2 _University of Groningen, Groningen, Netherlands_.

Diffuse gliomas are primary brain tumors characterized by infiltrative growth and high heterogeneity, which renders the disease mostly incurable. Advances in genetic analysis have characterized molecular and epigenetic alterations leading to impact on patients' overall survival and clinical outcome, particularly in glioblastoma (GBM). However, glioma tumorigenicity is not controlled uniquely by its genetic alterations. The crosstalk between tumor cells and the surrounding microenvironment plays a crucial role in modulating glioma growth and aggressiveness. The most abundant non-neoplastic cells in this microenvironment belong to the myeloid lineage, comprising resident microglia, with central nervous system (CNS)-tailored functions, and infiltrating monocytes/macrophages from the bone marrow. Understanding the dynamics between tumor and myeloid cells and the correlation between oncogenic molecular alterations in the tumor and the inflammatory activation status of innate immunity cells would elucidate potential treatment alternatives. Our large cohort of human glioma consisted of both astrocytoma and oligodendroglioma tumors, ranging from grades II to IV. Using high-throughput DNA sequencing and immunohistochemistry, we have classified GBM samples in proneural, classical and mesenchymal. Next, we evaluated the activation status of microglia/macrophages within these samples. Despite the great heterogeneity, we observed higher levels of myeloid markers (IBA1, CD11b and CD68) in astrocytic tumors compared to oligodendrocytic ones and to non-neoplastic (NN) tissue. Anti-inflammation markers, such as CD163, are also more abundant in astrocytomas, as well as in the mesenchymal and classical GBM subtypes, while pro-inflammation markers, such as IL1-beta, show a more widespread expression throughout samples. Our preliminary data suggests an immune-suppressive and growth supportive characteristic for tumors with worse clinical outcome, linked to an activation profile of myeloid cells. Further exploring the characteristics and dynamics of innate immune cells within the microenvironment of different grades and subtypes of glioma is crucial for the better understanding of the disease progression.

#3230

The role of TRF2 on tumor progression in non-small cell lung cancer: potential modulating effect on myeloid cells.

Linda Bouhlel,1 Marius Ilie,2 Laurie Signetti,3 Julien Cherfils-Vicini,3 Eric Selva,4 Patrick Brest,3 Charles Hugo Marquette,1 Eric Gilson,3 Paul Hofman2. 1 _CHU Nice, Pneumology Department and IRCAN CNRS UMR 7284/INSERM U1081, University of Nice Sophia Antipolis, Nice, France;_ 2 _CHU Nice, Laboratory of Clinical and Experimental Pathology and Institut for Research on Cancer and Aging; Nice (IRCAN); CNRS UMR7284/INSERM U1081; Faculty of Medicine; Nice University, Nice, France;_ 3 _Institut for Research on Cancer and Aging; Nice (IRCAN); CNRS UMR7284/INSERM U1081; Faculty of Medicine; Nice University, Nice, France;_ 4 _CHU Nice, Hospital-Integrated Biobank BB-0033-00025, Nice, France_.

Background: TRF2 is a key factor involved in telomere protection and has been found to be upregulated in various human cancers. The recent discovery by which TRF2 inhibits recruitment of NK cells and prevents tumor cells from NK-mediated elimination established new strategies for cancer support. Oncogenic KRAS is found in approximately 30% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC). Activating mutations in KRAS gene generates a robust inflammatory response in lung adenocarcinomas, characterized by an abundant infiltration of leukocytes including neutrophils. Thus, the concepts of solid (at the tissue level) and liquid (at the blood level, with the involvement of circulating tumor cells-CTCs) microenvironment, are a key element to be taken into account in understanding the mechanisms of carcinogenesis and tumor progression. This study was undertaken to, 1) define the impact of TRF2 expressed by lung carcinoma cells on myeloid cells (both at tissue and circulating level), and, 2) to analyze whether this effect may vary according to cancer cells KRAS mutational status.

Materials and Methods: Normal human bronchial epithelial cells (Beas2B) were stably transfected with different variants of KRAS mutations or wild-type gene. We evaluated TRF2 expression and neutrophil recruitment according to the KRAS mutational status in an in vitro Transwell model. In addition, we investigated the presence of CTCs in patients with metastatic NSCLC, as well as the TRF2 expression on CTCs (by using a cytomorphological assessement), and the circulating myeloid cells populations, neutrophils and myeloid-derived suppressor cells (MDSC), by flow cytometry analysis.

Results: The expression of TRF2 was differentially modulated based on the mutational status of KRAS, being decreased in KRASG12V cells. We found an increased recruitment of human neutrophils upon induction of KRASG12D expression. In addition, we found a significant correlation between the PMN-MDSC phenotype and TRF2-expressing CTCs in patients with metastatic NSCLC.

Conclusion: TRF2 expression in CTCs modulates the phenotypic orientation of MDSC. KRAS mutational status affects the expression of TRF2 and neutrophil recruitment. The complexity of the interactions between these different actors highlights mechanisms requiring an accurate understanding and may represent potential new therapeutic targets.

#3231

Identification of CD245 as myosin 18A, a receptor for surfactant A: A novel pathway for activating human NK lymphocytes effector function.

Jérôme Giustiniani,1 Adèle de Masson,2 Anne Marie-Cardine,3 Jean-David Bouaziz,4 Christian Garbar,1 Martine Bagot,5 Yacine Merrouche,1 Armand Bensussan3. 1 _Institut Jean Godinot/Unicancer, Reims, France;_ 2 _Harvard Skin Disease Research Center, Boston, MA;_ 3 _INSERM U976, Université Paris VII Paris Diderot, Paris, France;_ 4 _INSERM U976, Service de Dermatologie, Hôpital Saint-Louis, Paris, France;_ 5 _INSERM U976, Service de Dermatologie, Hôpital Saint-Louis, Paris, France_.

Natural Killer (NK) cells were identified over 40 years ago as a subset of lymphocytes able to spontaneously kill tumor cells in the absence of pre-stimulation. Present in most mammalian and avian species, NK cells play a critical role in the anti-tumor and anti-infectious immunity and in reproduction. NK cell cytotoxicity is tightly controlled by a balance between signals delivered through the engagement of activating and inhibitory receptors. Beside these receptors, costimulatory molecules co-exist at the surface of activated NK cells to potentiate NK cells functions. 4-1BB (CD137) is a costimulatory receptor expressed on T, B and NK cells whose expression is triggered by engagement of the Fc receptors on the NK cell surface, as during antibody-dependent cell cytotoxicity (ADCC). Consequently the engagement of CD137 increases cetuximab-, rituximab-, and trastuzumab-dependent NK cell cytotoxic function in different cancer models. We previously described CD245 as an unidentified structure on the surface of human peripheral blood lymphocytes that was recognized by the monoclonal antibodies DY12 and DY35. We also reported that CD245 on NK lymphocytes was associated with tyrosine phosphatase activities. We found that CD245 is the unconventional myosin 18A, which is a receptor for the surfactant protein A (SP-A). We characterized the molecular and functional properties of CD245 and identified a novel co-activation pathway that potentiates the NK cell cytotoxicity. Stimulation by specific antibodies or its physiological ligand SP-A is able to increase NK cell degranulation and lymphokine-activated killer activity towards tumor B cells and EBV-infected B cells, respectively. We also found that myosin 18A stimulation is able to induce CD137 expression at the NK cell surface and that the myosin 18A-induced NK cell cytotoxicity toward CD137L-expressing tumor cells is dependent on the CD137/CD137L interaction. Thus, CD245 could represent a novel and promising target in cancer immunotherapy.

#3232

NF-κB regulates suppression of macrophage activity during tumor initiation.

Nivedita Ratnam, David Wang, Denis Guttridge. _The Ohio State University, Columbus, OH_.

Constitutive activation of NF-κB is characteristic of most cancer cells. However, how NF-κB functions during tumor initiation is not well established. Recent findings from our laboratory showed that one mechanism by which NF-κB functions to promote tumor initiation is by suppressing immune surveillance. Here we investigated how this occurs. Initiating tumors are characterized by a massive infiltration of immune cells, especially macrophages that exhibit anti-tumor activity through the secretion of pro-apoptotic factors like tumor necrosis factor (TNF) and nitric oxide (NO). Using co-culture assays with conditioned media from Ras transformed tumor cells in the presence of NF-κB, we find that the tumor cells are able to protect themselves from the pro-apoptotic activity of the macrophages by secreting a neutralizing factor. RNA sequencing analysis identified this factor as macrophage inhibitory cytokine-1 (MIC-1)/ growth and differentiation factor-15 (GDF-15). Our results show that MIC-1/GDF-15 is a direct transcriptional target of NF-κB. Furthermore, xenografts in SCID mice show delayed tumor initiation in p65+/+ Ras tumor cells that are depleted for MIC-1/GDF-15 compared to wild type p65+/+ Ras tumor cells. Moreover, we find that MIC-1/GDF-15 inactivates macrophages directly by suppressing their ability to produce TNF and NO. This suppression occurs through the inhibition of NF-κB signaling in the macrophages. Interestingly, expression of MIC-1/GDF-15 is increased in patients with pancreatic ductal adenocarcinoma. In the pancreatic cancer cell line, Panc02, we show that knocking down MIC-1/GDF-15 prevents macrophage inhibition thereby increasing their sensitivity to macrophage-mediated killing both in vitro and in vivo. Thus, NF-κB functions during pancreatic cancer initiation to suppress the anti-tumor activity of infiltrating macrophages by regulating the expression of MIC-1/GDF-15.

#3233

Macrophages orchestrate early dissemination of HER2+ cancer cells.

Nina Linde,1 Arthur Mortha,1 Nicole Saenger,2 Maria Soledad Sosa,1 Kathryn Harper,1 Ethan Tardio,1 Tanja Fehm,3 Thomas Karn,4 Miriam Merad,1 Julio A. Aguirre-Ghiso1. 1 _Mt. Sinai Icahn School of Medicine, New York, NY;_ 2 _University Hospital, Frankfurt/Main, Germany;_ 3 _Heinrich Heine University, Duesseldorf, Germany;_ 4 _University Hospital, Frankfurt, Germany_.

Metastatic dissemination was proposed to occur only in invasive cancer. However, detection of cancer cells in the bone marrow of patients with pre-invasive breast ductal carcinoma in situ (DCIS) showed that dissemination could, through unknown mechanisms, take place earlier. Late evolved invasive breast cancer cells disseminated by recruiting circulating monocyte-derived tumor associated macrophages (TAMs). We therefore investigated whether macrophages might actively participate in early dissemination. We show that pre-malignant cancer cells in MMTV-HER2 mice produced CCL2 in an NFkB dependent manner and thereby recruited resident mammary tissue macrophages into the duct, months before monocyte-derived TAMs arrived. Intra-epithelial mammary tissue macrophages induced an epithelial-to-mesenchymal transition in early HER2+ cells in a Wnt-dependent manner and thereby drove early dissemination. Consequently, depletion of macrophages from pre-malignant MMTV-HER2 mice significantly reduced early dissemination of pre-malignant cancer cells as measured by reduction in levels of circulating cancer cells and disseminated cancer cells in lungs. Interestingly, macrophage depletion during pre-malignant stages alone was sufficient to reduce the onset of late metastasis in MMTV-HER2 mice, indicating that early disseminating cancer cells contributed to late metastasis formation. In humans DCIS samples we frequently found intra-epithelial macrophages inside of ducts and their presence correlated with reduced E-Cadherin levels. Finally, a first pilot study indicated that those patients whose DCIS lesions contained macrophage +/E-Cadherin low microenvironments frequently had DCCs in the bone marrow. Taken together, we reveal that resident mammary tissue macrophages can promote early dissemination, and unravel a mechanism how early cancer spread might proceed in breast cancer patients and in other cancers where metastasis are diagnosed and the primary tumor is never detected (cancer of unknown primary). We also propose that early DCCs play a long-term causal role in metastasis development when patients are diagnosed in late stages, implying that the heterogeneity of metastases is higher than we might anticipate.

#3234

Genetic deletion of macrophage migration inhibitory factor reduces oral carcinogenesis.

Steve Oghumu, Thomas Knobloch, Cesar Terrazas, Sanjay Varikuti, Claire Bollinger, Hans Iwenofu, Christopher Weghorst, Abhay Satoskar. _Ohio State University, Columbus, OH_.

Oral cancer kills about 1 person every hour, each day in the United States and is the 6th most prevalent cancer worldwide. The pro-inflammatory cytokine 'macrophage migration inhibitory factor' (MIF) has been shown to be expressed in oral cancer patients, yet its precise role in oral carcinogenesis is not clear. In this study, we examined the impact of global Mif deletion on the cellular and molecular process occurring during oral carcinogenesis using a well-established mouse model of oral cancer with the carcinogen 4-nitroquinoline-1-oxide (4NQO). C57BL/6 Wild-type (WT) and Mif knock-out mice were administered with 4NQO in drinking water for 16 weeks, then regular drinking water for 8 weeks. Mif knock-out mice displayed fewer oral tumor incidence and multiplicity, accompanied by a significant reduction in the expression of pro-inflammatory cytokines Il-1β, Tnf-α, chemokines Cxcl1, Cxcl6, Cxcl8 and Ccl3 and other molecular biomarkers of oral carcinogenesis Mmp1 and Ptgs2. Further, recruitment of myeloid-derived tumor promoting immune cells was inhibited in Mif knock-out mice. Our results demonstrate that genetic Mif deletion reduces the incidence and severity of oral carcinogenesis, by inhibiting the expression inflammatory cytokines and infiltration of tumor promoting immune cells. Thus, targeting MIF is a promising strategy for the prevention or therapy of oral cancer.

#3235

IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligands.

Aparna Rao,1 Xiaoran Zhang,1 Christopher Deibert,1 Paola Sette,1 Tim Chan,2 Paola Grandi,1 Nduka Amankulor1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Background: Diffuse gliomas are fatal primary brain tumors that are poorly immunogenic. The basis for insufficient anti-tumor immunity in diffuse gliomas is not understood. Gain of function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. Methods: We analyzed the TCGA database for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligands expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and in genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza- 2'deoxycytodine (Decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. Results: All IDH mutant glioma stem-like cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes were resistant to NK cell- mediated lysis. The hypomethylating agent Decitabine increased NKG2D ligand expression, and restored NK-mediated lysis of IDH mutant cells in an NKG2D- dependent manner. Conclusions: IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULPB3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell- mediated immune surveillance in patients with IDH mutated diffuse gliomas.

#3236

Macrophages increase the expression of RhoC in inflammatory breast cancer leading to increased migration.

Julie Madden, Steve Allen, Yu-Chi Shen, Chelsea Fournier, ZhiFen Wu, Liwei Bao, Sofia Merajver. _University of Michigan, Ann Arbor, MI_.

Inflammatory breast cancer (IBC) is considered the most lethal form of breast cancer due to its ability to progress quickly and the frequent presence of metastasis at diagnosis. African Americas are disproportionally diagnosed with IBC and often have worse outcomes than Caucasians. By investigating IBC in both African American and Caucasian cell lines we seek to understand the differences in IBC progression and help address disparities by providing new anti-IBC strategies. RhoC GTPase is overexpressed in 90% of IBC tumors and is known to increase cell motility. Sites of inflammation, as seen in IBC, attract tumor associated macrophages (TAMs), which have been found to facilitate the movement and invasion of many breast cancers. We hypothesize that TAMs play a role in increasing RhoC expression in IBC cell lines, consequently leading to IBC's severe migratory and metastatic potential.

A novel microfluidic device created by our team was used to measure the migratory phenotype of IBC cell lines in response to macrophage conditioned media (CM) and cytokine stimulation. IBC cell lines were treated with CM, cytokines, or pathway inhibitors then Western blotting was used to determine protein expression and phosphorylation to identify important signaling pathways.

We found the expression of RhoC significantly increased in two different IBC cell lines, SUM149 (African American) and SUM190 (Caucasian), after culturing with conditioned media from the macrophage-differentiated U937 monocytic cell line. This increase was not detected in either the normal-like MCF-10A breast epithelial cell line or the non-IBC MDA-MB-231 triple negative breast cancer cell line. CM caused a significant increase in the migration distance and frequency of both SUM149 and SUM190 cell lines. Analysis of the CM determined CCL2, CCL5, and IL-8 to be the key mediators in the macrophage CM. Western blotting proposes that CCL2, CCL5, and IL-8 stimulation causes twice as much RhoC expression compared to the control. Further analysis suggests a role for the MAPK pathway in controlling RhoC expression and migration.

Macrophage conditioned media causes an increase in RhoC expression in IBC cell lines and stimulates migration. Individual cytokines can lead to an increase in RhoC possibly through the MAPK pathway. Studies involving RhoC inhibitors are ongoing and could yield promising therapies for the prevention of metastasis in IBC. By understanding the specific mechanism of TAMs' effects on IBC, we hope to learn how to control the lethal metastatic nature of IBC and improve outcomes for patients of all ethnicities.

#3237

Assessment and targeting of M2-tumor associated macrophages (M2-TAMs) in prostate cancer.

Jelani C. Zarif, James R. Henandez, Kenneth J. Pienta. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression and metastasis. The central hypothesis of this study is to determine if targeting of M2-TAMs using specific surface antigens will be an effective therapy for lethal prostate cancer and potentially, other cancers. Our study elucidated M2-Macrophage biology including creating homogenous populations of both M1 and M2 macrophages using a new strategy as to allow for identification of novel surface antigen markers. While using our new method for polarization of human monocyte polarization into macrophages, we found that M2 macrophages polarization was stymied in vitro after glutamine deprivation. We then assessed novel markers of macrophage populations, cytokine and chemokine production, T-Cell inactivation and we also assessed whether these surface antigens that are expressed on M2-macrophages are were found in metastatic human prostate cancers indicative of myeloid infiltration. Validation studies using metastatic prostate cancer tissues from rapid autopsies and surgical specimens supplied by the Department of Urology at Johns Hopkins University School of Medicine were also be performed. Complementary studies will be executed to target these markers for anti-tumor cell efficacy using these relevant model systems. By identifying and targeting specific markers on M2-TAMs, we predict that this targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer.

#3238

Imbalance of circulating monocyte subsets in patients with squamous cell carcinoma of the head and neck.

Koichi Sakakura, Hideyuki Takahashi, Sei-ichiro Motegi, Kazuaki Chikamatsu. _Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan_.

Introduction: Human circulating monocyte is classified as three populations according to CD14 and CD16 expression: CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes and CD14dimCD16+ nonclassical monocytes. Recent studies have unveiled the specific surface molecules and functions of each subset; however their dynamics in patients with cancer still remains unclear. In this study, we evaluated the proportions and the expressions of immunological/angiogenic molecules of the monocyte subsets in peripheral circulation in patients with squamous cell carcinoma of the head and neck (SCCHN).

Materials and Methods: Peripheral blood mononuclear cells were isolated from 9 patients with SCCHN and age-matched 4 healthy donors. The 3 monocyte subsets were identified according to CD14/CD16 expressions using multi-color flow cytometry. Mean fluorescence intensities of various HLA molecules and angiogenic markers were analyzed.

Results: Percentage of CD14+CD16+ intermediate monocyte among total monocyte tended to decrease in SCCHN patients than that in normal donors (p=0.079). Higher surface expression of immunosuppressive HLA-G molecule on circulating classical (p=0.045) and intermediate (p=0.052) monocyte was observed in patients with SCCHN. An immunosuppressive receptor ILT4 showed tendency to increase on nonclassical monocyte (p=0.061). Moreover, intracytoplasmic level of milk fat globule-EGF factor 8 (MFG-E8) in association with phagocytosis and angiogenesis tended to increase in intermediate (p=0.064) and nonclassical (p=0.052) monocytes of SCCHN patients.

Discussion and Conclusion: CD16+ intermediate and nonclassical monocytes have been reported as mature, proinflammatory and angiogenic population in periphery. In the present study, we showed possible alternations of these subsets in patients with SCCHN. Furthermore, increasing surface expressions of immunosuppressive molecules HLA-G and ILT4 on circulating monocytes implied immunological deterioration in cancer patients. However the sample size is still small, our preliminary data strongly suggests proportional and functional changes of peripheral monocyte subsets in SCCHN patients. Further analyses in more patients are currently underway.

#3239

Cancer-associated fibroblasts induce immunosuppressive macrophages in head and neck squamous cell carcinoma.

Hideyuki Takahashi, Koichi Sakakura, Kazuaki Chikamatsu. _Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan_.

Background

Cancer-associated fibroblasts (CAFs) are one of main elements in the tumor microenvironment (TME). Among the innate and adaptive immune cells in the TME, tumor-associated macrophages (TAMs) are particularly abundant and play a protumoral role. However, an interaction between CAFs and TAMs in head and neck squamous cell carcinoma (HNSCC) still remains unclear. In the present study, we investigated whether CAFs skewed macrophage function toward the immune suppressive phenotype.

Materials and Methods

In vitro assay: CAFs were prepared from surgical tissue of HNSCC patients, and culture supernatants of CAFs were collected. Using this supernatants and peripheral blood mononuclear cells collected from healthy donors, immunological interactions between CAFs and TAMs was investigated.

In vivo assay: The immunohistochemistry (IHC) of 73 patients with HNSCC was performed. Anti-α-smooth muscle actin, CD68, and CD163 antibodies were used to identify CAFs and TAMs, respectively. The relationship between immunohistochemical parameters and clinical parameters was evaluated.

Results

The expression levels of CD68, CD163, CD14, CD80, CD86, and HLA-G were up-regulated in CD14+ cells co-cultured with the supernatants of CAFs (CAF-educated cells) compared with control cells. Moreover, the gene expressions of ARG1, IL6, IL10 and TGFB1 were also increased in CAF-educated cells. The CAF-educated cells suppressed T-cell proliferation more intensively than control cells, and neutralization of TGF-β and IL-10 led to significant restoration of T-cell proliferation. In IHC, the intensity of CAFs correlated with the number of CD68-positive macrophages, especially with CD163-positive macrophages. It also correlated with several clinical parameters such as lymphatic invasion, vascular invasion, nodal status, tumor stages and recurrence rate. Moreover, the high intensity of CAFs correlated with poor PFS and OS. The high intensity of CAFs and M2 macrophage infiltration were independent prognostic factors of PFS and OS in HNSCC.

Conclusion

The present study suggests that CAFs can induce an immunosuppressive phenotype of macrophages and contribute to establishing the immunosuppressive networks in TME, resulting poor prognosis in HNSCC patients.

#3240

Prostate cancer exosomes and their effects on macrophage engulfment and polarization.

Evita G. Weagel, Wei Meng, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The purpose of this study is to explore the effects of prostate cancer-derived exosomes on macrophage engulfment and polarization. Exosomes are small vesicles secreted by virtually all cells, which contain nucleic acids, lipids, proteins, and carbohydrates. Exosomes can be abundantly found in cancer patient secretions (serum, urine, etc.) and contain a high amount of RNA molecules, some of them being miRNA, and parts of the DNA of the cell of origin. One of the functions of exosomes is to mediate cellular communication. This occurs as they are secreted from one cell and then distribute their contents into other cells. Some exosomes specifically target immune cells. The miRNA the exosomes carry can silence genes involved in the activation of these immune cells, leading to immune suppression and tumor growth. Macrophages are often found surrounding the tumor microenvironment, as they are recruited by tumor signals to promote angiogenesis and metastasis. These macrophages, which are often called tumor associated macrophages (TAMs), are exposed to various signals derived from the tumor microenvironment, including exosomes. Previous data generated in our lab suggests that the co-culture of macrophages with PC3 and DU145 cell lines results in a decrease in macrophage phagocytosis and an M2-like polarization state. For this study, we exposed U937 cells to phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow for differentiation. Then, we co-cultured these macrophages with isolated exosomes from spent media from both PC3 and DU145 cell lines at 1, 2, 3, 4, 5, 6, 12, and 24 hours. We then allowed the macrophages to phagocytose fluorescent microbeads for 1 hour. Flow cytometry analysis of these macrophages revealed that the total bead phagocytosis starts decreasing at 3 hours of exosome co-culture compared to control. Interestingly, total bead phagocytosis resumes to normal at 12 and 24 hours of exosome co-culture. Gene expression analysis of these macrophages suggests an M2 phenotype from hours 2-24. These results suggest that prostate cancer-derived exosomes can target macrophages and affect their phagocytosis. Further research is required to elucidate the specific genes these exosomes target.

#3241

M1 macrophages promote ovarian cancer metastasis through NF-κB/TWIST axis signaling pathway.

Un-Tek Jo, YongSang Song. _Seoul National University, Seoul, Republic of Korea_.

The importance of tumor microenvironment in cancer progression has been recognized recently. However, factors associated with the tumor microenvironment are diverse and the molecular mechanisms are needed to be further defined for therapeutic intervention. Here we focused on the interaction between obesity related inflammatory cell, macrophage type 1 (MФ-1), and ovarian cancer cells in relation to metastasis. Two types of ovarian cancer cell lines; PA-1 and SKOV3, and monocyte-derived macrophages were used in this study. In the treatment with MФ-1 conditioned media, only SKOV3 cells were affected in proliferation activity. However migration and invasion were promoted by treatment with MФ-1 conditioned media in both ovarian cancer cells. In parallel, MФ-1 conditioned media treatment increased TWIST, EMT related gene. Interestingly, TWIST expression was correlated with protein expression levels of nuclear NF-κB (p65 and p50). Upon MФ-1 conditioned media treatment, nuclear translocation level and transcriptional activity of the NF-κB increased. Also, TPCK, NF-κB inhibitor suppressed invasion activity by MФ-1 conditioned media, in parallel with down-regulation of TWIST expression. Taken together, MФ-1 promoted EMT related metastatic potential in ovarian cancer cells through NF-κB signaling pathway. These results provide a new insight into the obesity related tumor microenvironment in ovarian cancer cell progression.

#3242

In depth myeloid cell characterization in the murine syngeneic CT26 colon carcinoma model by 10 color flow cytometry.

Matt Thayer, David Draper, Daniel Saims, Maryland Rosenfeld-Franklin, Scott C. Wise. _Molecular Imaging Inc, Ann Arbor, MI_.

The efficacy of immune-modulating anti-cancer therapeutic antibodies that have been FDA-approved in recent years, such as anti-CTLA-4 and anti-PD-1, has driven growing interest in methods that provide a mechanistic understanding of drug function. Development of new mono- and combination therapies with immune-modulatory effects requires more powerful immunophenotyping techniques capable of in depth cell characterization. To this end, using the CT26 murine syngeneic colorectal cancer model we have developed a 10 color flow cytometry antibody panel that focuses on the identification of tumor-infiltrating immune cell subsets derived from myeloid lineage precursors utilizing the high-throughput-capable 4-laser, 14-color Attune NxT Flow Cytometer with autosampler. The panel includes a combination of antibodies against CD45, CD3, CD19, CD49b, CD335, CD11b, CD11c, Ly-6G, Ly-6C, F4/80, and CD115. By excluding cells of lymphoid lineage, we show that this panel facilitates analysis of myeloid derived cells including natural killer (NK) cells, macrophages, neutrophils, dendritic cells (DCs), and monocytic or granulocytic myeloid-derived suppressor cell (mMDSCs and gMDSCs) subsets in tumor and peripheral blood. In addition, this combination of antibodies allows for a more complete analysis of MDSCs which can differentially express several disease-relevant myeloid specific markers including Ly-6G, Ly-6C, F4/80, CD11c, and CD115. In the tumor, the Ly-6G-high population demonstrates differential expression of Ly-6C, 21% Ly-6C-high (granulocytes) and 77% Ly-6C-low (mMDSCs). The majority of the granulocytic population was identified as gMDSCs and the remainder as neutrophils based on CD115 expression. Macrophages constitute 27% of Ly-6G-low cells. In blood, 98% of the Ly-6G-high population was also Ly-6C-high, and this population is predominantly neutrophils. No macrophages (Ly6G-low and F4/80+) were identified in the peripheral blood. These data confirm the expected distribution of myeloid lineages in the tissue types investigated. Finally, we show that the accuracy of analysis is enhanced by the use of fluorescence minus-one (FMO) controls to identify those markers that generate dim signals, as well as a viability dye used to exclude dead cells from analysis. Identification of additional potentially responsive immune compartments will facilitate identification and development of potential combination therapies otherwise overlooked by looking primarily at T-cells. This panel allows for a significant expansion of our ability to provide a complex description of the myeloid subset.

#3243

Neutrophil elastase regulates PD-L1 expression in breast cancer.

Victor Gall, Anne V. Philips, Akhil Chawla, Na Qiao, Lisa S. St. John, Pariya Sukhumalchandra, Mao Zhang, Jeffrey J. Molldrem, Gheath Alatrash, Elizabeth A. Mittendorf. _MD Anderson Cancer Center, Houston, TX_.

Introduction:

We have previously reported that breast cancer cells take up the inflammatory mediator neutrophil elastase (NE) from tumor-associated neutrophils (TANs). NE uptake leads to: 1) increased cleavage of cyclin E (CCNE) to its low molecular weight isoforms and generation of the CCNE-derived immunogenic epitope CCNE144-152 , 2) cross-presentation of the NE-derived epitope PR1, and 3) increased human leukocyte antigen (HLA) class I expression. NE is therefore a link between innate and adaptive immune responses in breast cancer. The current study was undertaken to investigate the effects of NE uptake on PD-L1 expression as another mechanism impacting adaptive immune responses in breast cancer.

Methods:

The breast cancer cell line MDA-MB-231 was maintained in standard media or media supplemented with NE. The effect of NE on cell surface expression of PD-L1 was determined using flow cytometry. Total PD-L1 protein expression was determined using western blot analysis performed on whole cell lysates. NE was inhibited with either elafin or alpha-1-antitrypsin to assess the requirement of enzymatic activity for the observed effects. Transcriptional regulation of PD-L1 expression by NE was assessed by qPCR. To evaluate specific transcription factors (TFs) involved in PD-L1 regulation, nuclear protein was isolated and analyzed for the activity of 16 TFs using a commercial plate array. The impact of NE uptake on TFs identified on the array was confirmed by western blot analysis. The functional effects of changes in PD-L1 expression were evaluated using an annexin V assay to assess T-cell apoptosis.

Results:

Addition of NE to breast cancer cells resulted in a reduction in PD-L1 surface expression as determined by flow cytometry and in total PD-L1 expression as shown by western blot analysis. The effect was reversed after removal of the cells from NE-supplemented media. The addition of elafin to inhibit NE enzymatic activity led to increased NE uptake by MDA-MB-231 cells with no change in PD-L1 cell surface expression. Treatment with alpha-1-antitrypsin prevented NE uptake and also abrogated its effect on PD-L1 expression. Together, these results suggest that uptake of enzymatically active NE is required to decrease PD-L1 expression. qPCR showed a decrease in the PD-L1 transcript suggesting that the effect of NE on PD-L1 is transcriptionally regulated. NE treatment resulted in decreased expression of TFs known to be involved in PD-L1 regulation, including STAT1, STAT3, and JUN. NE-treated breast cancer cells induced less apoptosis of T-cells compared with untreated breast cancer cells.

Conclusions:

Uptake of enzymatically active NE by breast cancer results in decreased cell surface and total PD-L1 expression, an effect that is in part transcriptionally regulated. These findings suggest another mechanism whereby the innate inflammatory mediator NE may impact adaptive immune responses in breast cancer.

#3244

Tumor infiltrating (TINKs) and tumor-associated (TANKs) natural killer cells (TINKs): A new paradigm in colorectal cancer.

Antonino Bruno,1 Barbara Bassani,1 Davide Giuseppe D'Urso,1 Silvia Zanellato,2 Elisabetta Gini,3 Elisa Cassinotti,4 Luigi Boni,5 Lorenzo Mortara,3 Adriana Albini,1 Douglas M. Noonan3. 1 _IRCCS MultiMedica Milan, Milan, Italy;_ 2 _2Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy;_ 3 _Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy;_ 4 _Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy, Varese, Italy;_ 5 _Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy_.

Tumor infiltrating immune cells often show a skewed phenotype that reflects attenuation of anti-tumor activity and enhancement of pro-tumor activities, including angiogenesis. Natural Killer (NK) cells are effectors lymphocytes of innate immunity, primarily involved in immunosurvelliance against tumors through their cytotoxic activity. We previously reported that NKs from Non Small Cell Lung Cancer (NSCLC) show the decidual-like CD56brightCD16-VEGFhighPlGFhighIL-8+IFNγlow phenotype [Bruno et al, Neoplasia, 2014; Bruno et al, JNCI, 2014].

Here we investigated whether tumor associated (TANKs) and tumor infiltrating (TINKs) NK cells undergo an angiogenic-switch in colorectal cancer (CRC).

NK subset distribution and cytokine profiling were performed by multicolor flow cytometry, using peripheral blood and tissue samples from CRC patients. Conditioned media (CM) from FACS-sorted NKs were used either for secretomic profiling, by antibody membrane array or angiogenesis functional assay on human umbilical endothelial vein cells (HUVECs)

We found that CD56brightCD16- NK cells predominate in CRC adjacent and tumor tissues, produce VEGF, PlGF, IL-8 and show impaired cytotoxicity. Further, TINK/TANKs from CRC patients express the decidual NK markers CD9 and CD49a. Secretomic analysis on CRC peripheral blood NKs revealed up regulation for several pro angiogenic factors, including Angiogenin, Angiopoietin-1/2, TIMP-1/2, Tie-2, MMP1, MMP9. Media conditioned by FACS sorted NK cells from peripheral blood and tumor tissue of CRC patients were able to induce HUVEC proliferation, migration and the formation of capillary like structures.

Our data demonstrate that TINK/TANKS from CRC patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. We propose that TINK/TANKs could represent the hallmark for a new paradigm in CRC inflammation.

1. Bruno A, Focaccetti C, Pagani A, Imperatori AS, Spagnoletti M, Rotolo N, Cantelmo AR, Franzi F, Capella C, Ferlazzo G, Mortara L, Albini A, Noonan DM. The proangiogenic phenotype of natural killer cells in patients with non-small cell lung cancer. Neopla sia. 2013 Feb;15(2):133-42.

2. Bruno A, Ferlazzo G, Albini A, Noonan DM. A think tank of TINK/TANKs: tumor infiltrating/tumor-associated natural killer cells in tumor progression and angiogenesis. J Natl Cancer Inst. 2014

Aug;106(8):dju200

#3245

IL-15 superagonist/IL-15RαSushi-Fc fusion complex (IL-15SA/IL-15RαSu-Fc; ALT-803) markedly enhances specific subpopulations of NK and memory CD8+ T cells, and mediates potent anti-tumor activity of murine breast and colon carcinomas.

Peter S. Kim,1 Anna R. Kwilas,1 Wenxin Xu,2 Sarah Alter,2 Emily K. Jeng,2 Hing C. Wong,2 Jeffrey Schlom,1 James W. Hodge1. 1 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 2 _Altor BioScience Corporation, Miramar, FL_.

Interleukin (IL)-15-N72D superagonist-complexed with IL-15RαSushi-Fc fusion protein (IL-15SA/IL-15RαSu-Fc; ALT-803) has been reported to exhibit significant anti-tumor activity in murine myeloma, rat bladder cancer, and murine glioblastoma models. In this study, we examined the immunomodulatory and anti-tumor effects of IL-15SA/IL-15RαSu-Fc in tumor-free and highly metastatic tumor-bearing mice. Here, IL-15SA/IL-15RαSu-Fc significantly expanded NK and CD8+ T cells. In examining natural killer (NK) cell subsets, the greatest significant increase was in highly cytotoxic and migrating (CD11b+, CD27hi; high effector) NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that IL-15SA/IL-15RαSu-Fc significantly increased IL-15 responding memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 breast tumor-bearing mice, IL-15SA/IL-15RαSu-Fc induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when IL-15SA/IL-15RαSu-Fc was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. Altogether, these studies showed for the first time that IL-15SA/IL-15RαSu-Fc (1) promoted the development of high effector NK cells, (2) enhanced function of NK cells, and (3) played a vital role in reducing tumor metastasis and ultimately survival, especially in combination with checkpoint inhibitors.

#3246

Phytotherapy boosts innate immunity to maximize cancer survival.

Benjamin H. Lau. _Loma Linda University Medical School, Loma Linda, CA_.

We studied surgery, radiation, and chemotherapy in animals and found all these modalities suppressed cancer growth but they also impaired the immune functions of the animal host. We also studied phytotherapy (use of plant chemicals) and found it to have no side effects and with more lasting benefits. Using in vitro cell culture system with both malignant and non-malignant cells, and immune cells (macrophages and natural killer cells), we compared crude extract of Pacific yew and the chemotherapeutic drug Paclitaxel derived from Pacific yew. The crude extract or the drug was incubated separately with: human malignant ovarian cells, nonmalignant ovarian cells, macrophages, and natural killer cells. Cell viability was determined by MTT (methylthiazol tetrazolium) assay. Results show the drug damaged all these four types of cells while the crude plant extract only damaged the cancer cells showing its selective toxicity for cancer cells. Study with other phytochemicals also shows selective toxicity for cancer cells while enhancing the activities of immune cells. Phytotherapy is thus a non-toxic chemotherapy and immunotherapy. In our clinical observation, 160 men and women diagnosed with five types of cancer (72 with breast cancer, 28 with prostate cancer, 18 with colon cancer, 14 with lung cancer, 28 with ovarian cancer) chose to use phytotherapy (a plant-based diet) plus lifestyle modification. The five year survival of these patients were 90%, 89%, 83%, 86%, and 82%, respectively. Compared with five year survival data from National Cancer Institute (www.seer.cancer.gov) for these five types of cancer: 82%, 50%, 62%, 15%, and 45%, respectively, the phytotherapy group exhibits greater cancer survival. Since numerous studies have now revealed that cancer is attributable to our lifestyle habits -- the way we eat, and the way we live, our data suggest that regardless of treatment choices, a change in diet and lifestyle is likely to improve cancer survival.

#3247

Granulocytic MDSCs (CD11b+CD15+CD14-HLADR-) in peripheral blood of mesothelioma patients are elevated and suppress T cell proliferation and function via reactive oxygen species.

Swati Khanna,1 Francis Mussai,2 Anish Thomas,1 Gary Middleton,3 Constance Yuan,1 Betsy Morrow,1 Jingli Zhang,1 Ira Pastan,1 Maryalice Stetler-Stevenson,1 Raffit Hassan1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Birmingham Children's Hospital and the University of Birmingham, UK, Birmingham, United Kingdom;_ 3 _University of Birmingham, Birmingham, United Kingdom_.

The role of myeloid derived suppressor cells (MDSCs) in mediating tumor immunosuppression in patients with malignant mesothelioma has not been well characterized. The goal of our study was to analyze for the presence of both monocytic (Mo) and granulocytic (Gr) MDSCs in blood of mesothelioma patients, and characterize their mechanism of suppression of T cells responses. We evaluated the peripheral blood of patients (n=23) for the presence of Gr-MDSC (CD11b+CD15+CD14-HLADR-) and Mo-MDSC (CD11b+CD14+HLADR-). Gr-MDSC (62.3±13.9% vs. 47.8±17.8%; p=0.005) as well as monocytic MDSC (0.2±0.4% vs. 0.1±0.3%; p=0.01) were significantly elevated in peripheral blood of mesothelioma patients as compared to healthy donors (n=21). The frequency of Gr-MDSC was higher than Mo-MDSC. Thus, Gr-MDSCs appeared to be the main immunosuppressive population and were investigated further.Gr-MDSC from mesothelioma patients were found to inhibit the proliferation of both autologous CD4 and CD8 T cells (mean inhibition of proliferation of 61.9% for CD4 and 75.5% for CD8 at 1:2 T: Gr-MDSC ratio) and was accompanied by a decrease in IFN-γ released by T cells in the co-culture supernatants (92.4 pg/mL at 1:2 ratio). To determine the mechanism by which Gr-MDSC inhibit T cells, we tested the levels of reactive oxygen species (ROS), and nitrite oxide species and arginase activity in Gr-MDSC sorted from peripheral blood. Both arginase (0.3±0.3 vs. 0.2±0.3 μM) and nitrite levels (1.8±2.5 vs. 0 μM) in Gr-MDSC were below detection limit in both patients and donors and were marginally affected by the addition of their respective inhibitors (nor-NOHA and L-NMMA). However, the levels of ROS in Gr-MDSCs derived from mesothelioma patients were on average 3 folds higher than the Gr-myeloid cells from healthy donors (average MFI ~15000 vs. ~5000). In summary our results show that the major immunosuppressive cell population in mesothelioma patients are Gr-MDSCs and they suppress T cell proliferation and activation through release of reactive oxygen species.

#3248

Over-expression of 1-ACBP, B-ACBP and PBR genes in mast cells infiltrating oesophageal squamous cell tumors.

Zodwa Dlamini. _Mangosuthu University of Technology, Durban, South Africa_.

Oesophageal cancer ranks as the sixth most common malignancy in the world, and recent evidence has shown that its incidence is increasing. ACBPs (Acyl-coA binding proteins) act as intracellular carrier-proteins for medium to long chain acyl-coA, mediating fatty acid transport to the mitochondrion for ß-oxidation. ACBPs are also believed to be putative ligands of PBR (peripheral benzodiazepine receptor), and bound to this receptor facilitate mitochondrial membrane permeabilization giving the notion that favours apoptosis. The main aim of the study was to establish the expression patterns of 1-ACBP, B-ACBP, and PBR in oesophageal cancer, and to link their roles with the disease. Probes specific to the genes encoding 1-ACBP, B-ACBP, and PBR were PCR amplified and cloned into pGEM-T easy vectors using gene specific primers. The cloned products were sequenced and screened against the GENBANK database (NCBI) to verify the identity and orientation of cloned products within the vector. Antisense and sense probes were then generated using T7 or SP6 RNA polymerase after digestion with either Pst1 or Apa1, respectively. In situ hybridization and quantitative real-time PCR methods were performed to determine localization and the expression levels of the three genes in oesophageal cancer. All three genes illustrated substantial up-regulation within the malignant tissue sections as compared to normal oesophageal sections, all three transcripts localized specifically to plasma cells and lymphocytes in diseased and normal tissue section. In the diseased tissue B-ACBP and 1-ACBP mRNA localized to endothelial cells of blood vessels in the submucosa. B-ACBP also localized to the nucleus of squamous epithelial cells. PBR localization was indicated in tumour islands of invasive tissue sections. Quantitative RT-PCR also indicated that the expression levels of PBR were higher as compared to the ACBP genes expression in tumours. These results show that 1-ACBP, B-ACBP and PBR play a role in the pathogenesis of oesophageal tumours and also suggest the interaction between these genes with the immune system and that was also confirmed using bioinformatics analysis.

#3249

Regulation of interferon regulatory factor-8 (IRF8) during myelopoiesis is a critical checkpoint for the formation of defective myeloid cells in cancer.

Colleen S. Netherby, Michelle N. Messmer, Scott I. Abrams. _Roswell Park Cancer Institute, Buffalo, NY_.

Aberrant myelopoiesis is commonly observed in patients with solid cancers. This results in the production of myeloid-derived suppressive cells (MDSCs) which promote tumor growth and metastasis. However, the molecular mechanisms underlying MDSC development have remained poorly understood. The transcription factor IRF8 is integral for overseeing myelopoietic fate; high IRF8 expression during normal myelopoiesis favors monocytic differentiation and directly inhibits granulocytic differentiation. We recently showed that tumor-derived STAT3/5-activating cytokines downregulate the expression of IRF8 in myeloid cells leading to the accumulation of MDSCs in the periphery, which are primarily granulocytic. However it is unclear if MDSC production is a consequence of IRF8 downregulation in the periphery or upstream in the bone marrow during myelopoiesis. To that end, we utilized a novel mouse model expressing an IRF8-EGFP fusion protein which allowed us to investigate changes in IRF8 expression during both orthotopic and spontaneous mammary tumor growth and progression. Our results showed that: 1) the total granulocyte-monocyte progenitor (GMP) fraction, as with the peripheral MDSC population, was responsive to tumor burden and greatly expanded with increasing tumor size, and 2) IRF8 expression within the total GMPs significantly decreased during tumor growth, reflecting the expansion of a newly defined primarily IRF8lo granulocyte progenitor (GP) population, and 3) tumor-induced GPs were hyper-responsive not only to G-CSF, but also M-CSF to generate a 'biased' granulocytic MDSC-like phenotype. Expression of IRF8 in other progenitor populations was not affected, suggesting that IRF8lo GMPs/GPs are a key source of the peripheral MDSC response. Lastly, genetically enforced IRF8 expression in myeloid progenitors of the bone marrow constrained aberrant myelopoiesis during tumor growth, resulting in delayed autochthonous tumor growth and reduced spontaneous lung metastasis. Altogether, these data reinforce the notion that modulation of IRF8 in myeloid progenitors is an early consequence of the neoplastic process and a potential target for therapeutic intervention.

NIH R01CA140622

NIH T32CA085183

#3250

Caspase-1 from MDSC promote MyD88 dependent carcinogenesis that is T-cell independent.

Qi Zeng, Jesse R. Qualliotine, Lee Blosser, Drew Pardoll, Young J. Kim. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Myeloid-derived-suppression cells (MDSC) are functionally defined to suppress T-cells in their primary means of immune evasion mechanism in cancer. However, we have discovered that MDSC can have direct, T-cell independent pro-carcinogenic effect on tumor cells. Sorted monocytic CD14+/CD11b+/HLA-DRlow MDSCs from head and neck squamous cell carcinoma (HNSCC) patients were found to increase the proliferation index of HNSCC cells. This induction of tumor proliferation was not dependent on MDSC-tumor cell contact. Exome analysis showed that these human MDSC from cancer patients expressed high levels of inflammasome complex, and supernatant from the MDSCs were found to secrete IL-1b and IL-18. When we probed the functional significance of these inflammasome complex, we found that MDSC's promotion of tumor proliferative index of the tumor was found to be Caspase-1 dependent. To test this in vivo, T-cell depleted caspase-1 null mice showed significant decrease in tumor growth rate. To confirm the importance of myeloid inflammasome signaling in carcinogenesis, we suppressed MyD88 gene in tumor cell line Cal27 and found that the ability of MDSC to promoting tumor proliferation is diminished. Taken together, our findings demonstrate that tumor infiltrating MDSCs may play a prominent role in chronic inflammation associated carcinogenesis. 

## TUMOR BIOLOGY:

### Antiangiogenic Therapy: Inhibitors and Resistance

#3251

A Senescence-like phenotype associates with rapid metastasis promotion following antiangiogenic drug resistance and therapy withdrawal.

Michalis Mastri,1 Amanda Tracz,1 Biao Liu,1 Christina R. Lee,2 John ML Ebos1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Few studies have investigated mechanisms of antiangiogenic drug resistance in mouse models that faithfully recapitulate clinically relevant spontaneous metastatic disease or evaluate the impact of therapy cessation on tumor and stromal cell growth. Generation of such models may be critical to study reported instances of antiangiogenic therapy-induced metastasis in animals that have often proved challenging to confirm in patients. Here we describe the derivation of several human and mouse tumor cell lines obtained from spontaneous metastatic lesions present after surgical removal of primary tumors and following long-term in vivo treatment with sunitinib or axitinib. Selected drug-resistant metastatic (kidney, breast, and melanoma) and non-malignant stromal cell variants (endothelial and fibroblast) were continuously exposed to drug in vitro and then evaluated following both short- and long-term treatment removal (48 hours and 6 months, respectively). Our results show that metastatic drug-resistant cells receiving sustained treatment were re-sensitized to therapy upon orthotopic re-implantation into treatment-naïve animals, suggesting a predominant host-mediated role in therapy failure. However, significant increases in tumor growth and metastatic potential were observed in all models when cells were re-implanted and therapy stopped, suggesting a tumor-dependent pro-metastatic mechanism activated by therapy withdrawal. Whole genome expression analysis for resistant cells on and off treatment revealed reversible and irreversible gene changes implicating a senescence-like phenotype capable of influencing metastatic potential. Senescent-like characteristics included increased cell size, decreased proliferation, cell cycle check-point protein alteration, and therapy-induced SA-β-galactosidase expression, depending on the cell line. Critically, antiangiogenic therapy could induce a senescence associated secretory phenotype (SASP) in both tumor and stroma cells which reversed or persisted following therapy cessation in certain instances. Importantly, we found that interleukin-6 (IL-6) - a major component of the SASP - was upregulated in resistant tumor and stroma cells, but only remained upregulated in stromal cells following therapy withdrawal. These results suggest that antiangiogenic treatment-induced senescence-like changes may contribute to treatment failure and contribute to pro-metastatic growth depending on cell origin (i.e., tumor or stroma) and whether treatment is sustained or stopped.

#3252

Eriocalyxin B (EriB), a natural diterpenoid, inhibits VEGF-induced angiogenesis by suppressing VEGFR2 signaling.

Xu-Nian Zhou,1 Grace Gar-Lee Yue,2 Stephen Kwok-Wing Tsui,1 Han-Dong Sun,3 Kwok-Pui Fung,4 Jian-Xin Pu,3 Clara Bik-San Lau2. 1 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong;_ 2 _Institute of Chinese Medicine, The Chinese University of Hong Kong; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong;_ 3 _State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China;_ 4 _School of Biomedical Sciences, The Chinese University of Hong Kong; Institute of Chinese Medicine; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong_.

Introduction: Angiogenesis, the formation of new capillaries from existing blood vessels, is an essential requirement for tumor growth and metastasis. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, is crucial for the promotion of new vascular sprout formation in vessel stabilization. Previous studies showed that the inhibition of angiogenesis via targeting on VEGF is a promising strategy for the suppression of tumor growth as well as metastasis. Eriocalyxin B (EriB), a natural diterpenoid isolated from Isodon eriocalyx Dunn, exerts anti-tumor activities1,2. Here, the aim of the present study was to determine the effect of EriB on angiogenesis both in vitro and in vivo.

Methods: Human umbilical vein endothelial cells (HUVECs) were used to demonstrate the anti-angiogenic activities of EriB and its underlying mechanisms. The transgenic zebrafish Tg(fli1:EGFP)y1, in which the endothelial cells express enhanced Green Fluorescent Proteins (eGFP), was also applied to evaluate the effect of EriB on angiogenesis in vivo.

Results: Our results in HUVECs showed that EriB exerted no inhibitory effect on cell viability and cell proliferation. However, it could significantly inhibit VEGF-induced cell viability and cell proliferation. In addition, the decreased tube formation, cell migration and cell invasion were also observed. Flow cytometry analysis demonstrated that EriB could induce cell cycle arrest at G1 phase by interference with cyclinD1/CDK4/pRb pathway. Investigation of the signal transduction revealed that EriB was involved in the suppression of VEGFR2 signaling. Results from the zebrafish model demonstrated that EriB treatment could inhibit the formation of sub-intestinal vessels. Besides, genome wide mRNA expression profiling of zebrafish after EriB treatment revealed the alteration of various angiogenic genes. The expressions of several genes such as kdr, pcdh15b, cdkn1a, loxl1, etc. were evaluated by quantitative real-time PCR.

Conclusions: The present study reported the activities of EriB as a novel anti-angiogenic agent. Thus, EriB has the potential of modulating tumor angiogenesis, which may be useful in the treatment of human cancers.

References:

1. Li, L., et al. (2014). Eriocalyxin B-Induced Apoptosis in Pancreatic Adenocarcinoma Cells Through Thiol-Containing Antioxidant Systems and Downstream Signalling Pathways. Curr Mol Med 14(5): 673-689.

2. Zhang, Y. W., et al. (2010). Eriocalyxin B induces apoptosis in lymphoma cells through multiple cellular signaling pathways. Exp Hematol 38(3): 191-201.

#3253

Mast cell-derived granzyme b contributes to resistance against anti-angiogenic therapy.

Mark A. Wroblewski,1 Raimund Bauer,1 Miguel Cubas Córdova,1 Florian Udonta,1 Isabel Ben Batalla,1 Victoria Gensch,1 Stefanie Sawall,1 Jonas S. Waizenegger,1 Julian Pardo Jimeno,2 Klaus Pantel,3 Carsten Bokemeyer,4 Sonja Loges1. 1 _University Medical Center Hamburg-Eppendorf; II. Medical Clinic and Institute of Tumor Biology, Hamburg, Germany;_ 2 _Centro de Investigación Biomédica de Aragón, Zaragoza, Spain;_ 3 _University Medical Center Hamburg-Eppendorf; Institute of Tumor Biology, Hamburg, Germany;_ 4 _University Medical Center Hamburg-Eppendorf; II. Medical Clinic, Hamburg, Germany_.

Significance: Targeted therapies have revolutionized the treatment of cancer. However, efficacy of anti-angiogenic therapies is limited due to significant resistance.

Recent studies showed that the tumor microenvironment is involved in resistance towards targeted anti-angiogenic treatment. Based on the correlation of mast cell (MC) density with tumor growth and angiogenesis we put forward the hypothesis that MC might be implicated in anti-angiogenic therapy resistance.

Methods: C57BL/6J, NSG or MC-deficient KitW-sh (Wsh) mice were subcutaneously injected with 5x105 (Panc02 and EL4) or 1x106 (TD2) cells +/- bone marrow derived MC. Tumors were treated with 20 mg/kg anti-VEGFR2 antibody (DC101) or 25 mg/kg cromoglicic acid (Cromo). BrdU was injected 12 h before sacrifice.

Results: We show that MC alter the proliferative and organizational state of endothelial cells (EC). MC dose-dependently induced EC-proliferation (158 ± 12 %; *p<0.05) and tube formation (290 ± 12 %; *p<0.05). Furthermore, MC increased HUVEC migration by 1.4-fold (*p<0.05) and protected them from AAT in vitro. In MC-deficient mice, tumor growth was reduced by 36 % (*p<0.05) and the efficacy of AAT was increased (WT + DC101: 1420 ± 134 mg; Wsh + DC101: 599 ± 107 mg; *p<0.05). Histomorphometric analyses unraveled that MC-deficiency decreased the numbers of mature pericyte-covered vessels by 80 % (*p<0.05) rendering them more prone for therapy. Indeed, an additive anti-angiogenic effect of MC-deficiency and AAT was observed resulting in reduced microvessel density (MVD) and tumor cell proliferation. This "angiosensitizing" effect could be abrogated by adoptive transfer of bone marrow-derived MC into MC-deficient mice.

In WT mice, AAT only initially reduced the proliferation of tumor vessels by 60 % (*p<0.05), a process that got reverted after long-term treatment as a result of therapy resistance. Intriguingly, this pro-angiogenic rescue phenotype did not occur in MC-deficient mice. By blocking MC degranulation with Cromo we could increase the efficacy of AAT (DC101: 703 ± 48 mg; Cromo + DC101: 386 ± 92 mg; *p<0.05), leading to reduced vessel proliferation, MVD and tumor cell proliferation.

Microarray analysis of tumor-resident MC unraveled increased expression levels of ECM-degrading granzyme b (gzmb) in response to therapy. MC-specific knock down of gzmb rendered tumors more susceptible for AAT and lowered the levels of alternative, pro-angiogenic mediators beside the VEGF-VEGFR2-axis in the tumor microenvironment.

Conclusions: Our results indicate that tumor-resident MC interfere with AAT. We provide evidence that MC-derived gzmb liberates ECM-bound pro-angiogenic factors besides the targeted VEGF-VEGFR2 axis, thereby fine-tuning vessel maturation and proliferation, which ultimately decreases therapeutic efficacy. Importantly, knock down of gzmb and pharmacological inhibition of MC degranulation improved the therapeutic response towards AAT.

#3254

Discovery of histone demethylase KDM5C inactivation as a novel mechanism for tumors resistant to VEGF RTKi via genome-wide in-vivo RNAi.

Azadeh Fahim Golestaneh,1 Maoyun Sun,2 Christian Schwager,1 Zili Tang,1 Stephan Macher-Goeppinger,3 Lila Ma,2 Philip Hahnfeldt,2 Mahmoud Moustafa,1 Wilko Weichert,3 Sascha Pahernik,4 Carsten Grüllich,5 Wilfried Roth,3 Jürgen Debus,1 Lynn Hlatky,2 Amir Abdollahi1. 1 _German Cancer Consortium (DKTK), Molecular- and Translational Radiation Oncology, Heidelberg Ion-beam Therapy Center (HIT), Heidelberg Institute of Radiation Oncology (HIRO), National Center for Tumor Diseases (NCT), Heidelberg University Medical School a, Heidelberg, Germany;_ 2 _Center of Cancer Systems Biology, Tufts University School of Medicine, Boston, MA;_ 3 _Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany;_ 4 _Department of Urology, University of Heidelberg, Heidelberg, Germany;_ 5 _Medical Oncology, Heidelberg University Hospital, Heidelberg, Germany_.

Acquired tumor resistance to vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitors (RTKi) constitutes a major obstacle for sustained efficacy of antiangiogenic cancer therapy. We sought to decipher the molecular landscape of tumor resistance to continuous VEGF-RTKi sunitinib (SU11248, SU) treatment via serial transplantation of a syngeneic LLC/C57bl6 model and genome-wide mouse lentiviral shRNA library (150K complexity). The abundance of each single knock down (kd) was longitudinally traced via molecular barcodes. After three in-vivo panning rounds (p3-SU) tumor cells containing the kd of H3K4 histone demethylase (KDM5C) were 212-fold enriched compared to the in-vivo passaged library without the SU selection pressure (p3-ctrl). Interestingly, KDM5C is among the top 5 frequently mutated genes in clear cell renal cell carcinoma (RCC). SU constitutes a cornerstone in the therapy of metastatic RCC. We hypothesized that KDM5C mutation status might be prognostic for therapy refractoriness of RCC. Indeed, whole exome analysis of the cancer genome atlas (TCGA) RCC collective revealed a trend towards poor overall survival (OS) for patients with mutated KDM5C tumors. In LLC and RCC xenograft models, loss of KDM5C (shKDM5C) was attributed to SU resistance with significantly enhanced hypoxia tolerance, increased proliferation (Ki67, 13-fold, p=0.04) and microvascular density (CD31, 23-fold increase, p<0.02) compared to SU treated shCtrl. We aimed to identify a consensus set of genes differentially expressed in tumors with KDM5C kd/mutation. Applying this KDM5C gene signature to RNAseq data of 533 RCC patients separated the cohort into a poor-prognosis KDM5Cmut-like (median OS 4.5 years) vs. good prognosis KDM5Cwt-like group (median OS not reached, p=0.0001). Genes regulating cancer cell survival, redox-balance and angiogenesis were enriched in this signature. Together, these data indicate a critical role for the epigenetic modulator KDM5C in development of tumor resistance to VEGF-RTKi. The here-identified KDM5C dependent gene-signature might be instructive to prognosticate patients at risk for therapy refractoriness to RTKi.

#3255

Inhibition of intracrine VEGF signaling prevents colorectal cancer cell migration and invasion.

Rajat Bhattacharya, Ling Xia, Fan Fan, Rui Wang, Delphine Boulbes, Xiang-Cang Ye, Lee Ellis. _MD ANDERSON CANCER CENTER, HOUSTON, TX_.

Introduction: Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and vascular function. The mechanisms of VEGF signaling in angiogenesis have been extensively studied. However, its effects on tumor cell function remain to be elucidated. Our studies on depletion of VEGF by somatic knockout of the VEGF gene or by siRNA in human colorectal cancer (CRC) cell lines have demonstrated that loss of VEGF expression led to significantly decreased cell growth, increased apoptosis, and enhanced chemo-sensitivity of CRC cells through an intracrine signaling mechanism. These intracrine effects were mediated through VEGF by regulation of the activity of multiple receptor tyrosine kinases (e.g. EGFR, cMET) and downstream AKT signaling. Similar effects were observed by depletion of VEGF receptor 1 (VEGFR1), supporting the role of a novel intracellular VEGF-VEGFR1 complex in CRC cell survival. Since VEGF signaling has been shown to effect endothelial and tumor cell migration, we attempted to determine if inhibition of intracrine VEGF signaling affected CRC cell motility.

Methods: Migration and invasion of CRC cells, with and without VEGF depletion, were assayed using Boyden chambers and a serum gradient. Changes in cell morphology and epithelial to mesenchymal transition (EMT) markers and markers of cell motility were assessed by microscopy, immunostaining, and western blot analyses.

Results: Depletion of VEGF by siRNAs in multiple CRC cell lines led to a very strong inhibition of migration and invasion of CRC cells. Such inhibition was not observed when CRC cells were treated with bevacizumab to inhibit paracrine or autocrine signaling. Further examination revealed no significant changes in cell morphology. Other than Twist, there were no significant differences between specific markers of EMT indicating that differences in migration and invasion were not due to classic alterations in EMT. However, significant changes in activation of mediators of cell motility were observed. VEGF depleted CRC cells demonstrated significantly lower levels of phosphorylated focal adhesion kinase (p-FAK) compared to cells with normal VEGF expression. Analyses of upstream regulators indicated strong reduction in either p-cMET or p-SRC depending on cell type.

Conclusions: Inhibition of intracrine VEGF signaling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

#3256

Highly potent monoclonal and bispecific anti-angiogenic antibodies to VEGF and angiopoietin-2.

Hangil Park, April Zhang, Yi Ding, Lihong Wang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. _Galaxy Biotech, LLC, Sunnyvale, CA_.

The angiogenic factor Vascular Endothelial Growth Factor (VEGF) is a well-established target for cancer therapy. The humanized monoclonal antibody (mAb) bevacizumab (Avastin®), which binds and neutralizes VEGF, is widely prescribed for the treatment of colon, lung and certain other tumors, but typically provides a relatively modest survival benefit of several months. It is unknown whether the limited benefit is due to redundancy in angiogenic pathways or to the inability of bevacizumab at the doses used to fully neutralize VEGF in the tumor microenvironment. However, the exploration of higher doses of bevacizumab in the clinical setting has been very limited due to lack of incentive to perform dose-ranging studies for an already approved drug, the expense of the drug, and concern about toxicity. To determine whether more effective neutralization of VEGF would be possible and useful, we have therefore used an intensive immunization protocol to generate a new murine mAb designated VE1 that binds VEGF with very high affinity. A humanized form of this mAb, HuVE1, retains the full affinity of VE1 and binds VEGF about 5-fold better than bevacizumab. In side-by-side comparisons, HuVE1 also blocked the binding of VEGF to its receptor with an IC50 that is 5 to 10-fold lower than for bevacizumab, and it correspondingly inhibited VEGF-induced proliferation of HUVEC about 5-fold better than bevacizumab. HuVE1 almost completely blocked growth of primary human hepatocellular carcinoma (HCC) xenografts in one model, and showed a trend toward better efficacy than bevacizumab in a second HCC xenograft model. For potentially even greater anti-angiogenic efficacy, we developed a humanized mAb HuA2T that potently neutralizes angiopoietin-2 (Ang-2), a cytokine that acts through the Tie-2 receptor to promote angiogenesis, especially in combination with VEGF. The synergy between VEGF and Ang-2 provides a strong rationale to develop a bispecific mAb that neutralizes both these factors. We therefore developed such a mAb using the IgG-like bispecific antibody format Bs(scFv)4-IgG consisting of a homodimer of two monomers, each monomer having a single chain Fv from HuVE1 linked to a light chain constant region, and a single chain Fv from HuA2T linked to a heavy chain constant region. While, as is common in construction of bispecific antibodies, this bispecific HuA2T/HuVE1 mAb did lose some binding affinity for VEGF and Ang-2 relative to the original mAbs, its VEGF binding activity was still greater than that of bevacizumab, illustrating the importance of starting with an extremely high activity mAb. Based on its potency in vitro and in animal models, we believe that HuVE1, in either natural or bispecific form, has the potential for greater clinical efficacy than bevacizumab and thus merits further investigation.

#3257

The impact of dual targeting VEGFR2 and Tie-2 on angiogenesis using in vitro model.

Khalid Alhazzani,1 Thanigaivelan Kanagasabai,2 Saad Alobid,1 Appu Rathinavelu1. 1 _College of Pharmacy, Health Professions Division, Nova Southeastern University, Fort Lauderdale, FL;_ 2 _Rumbaugh Goodwin Institute for Cancer Research, Nova Southeastern University, Plantation, FL_.

Angiogenesis is an essential key process in tumor growth, invasion, and metastasis. Several angiogenesis inhibitors have been clinically proven to treat a wide variety of cancer types. These agents target either the vascular endothelial growth factor (VEGF) or the intracellular tyrosine kinase domain of VEGF receptors. Despite the encouraging clinical benefits, there are multiple challenges such as significant toxic effects and rapid development of drug resistance which limit the long-term benefits of VEGF-targeted therapy. Emerging evidence suggests that tumors can bypass VEGF/VEGFR inhibition by up-regulating other pro-angiogenic pathways to compensate for VEGF inhibition. For these reasons, additional strategies to inhibit tumor vasculature are being sought. In this regard, F16, a new compound developed by Rumbaugh-Goodwin Institute for Cancer Research, harbors the potential for suppressing VEGF-driven angiogenesis by selectively blocking the extracellular domain of VEGFR2, which plays a crucial role in endothelial cell proliferation, survival, and migration. F16 has shown a remarkable efficacy in inhibiting VEGFR-associated tyrosine kinase activities. On the other hand, Tie-2 receptor is predominantly expressed in the endothelium and has been shown to facilitate VEGF-mediated angiogenesis. Therefore, we hypothesize that dual targeting of VEGFR2 and Tie-2 using F16 and T2i respectively will result in augmented angiogenesis inhibition. To test our hypothesis, human umbilical vein endothelial cells (HUVECs) were used as a model for our initial validation studies. Our preliminary data indicated that combining F16 and T2i significantly inhibited HUVECs proliferation compared to monotherapies (P<0.001) and 90% inhibition of HUVECs proliferation was observed at 1µM of the drug combination. Moreover, the ability of the indicated drugs to inhibit angiogenesis was further evaluated using a Matrigel-based tube formation assay. Using the scoring system developed by our laboratory, combining F16 and T2i at 1µM scored five pluses, indicating that individual cells were well-separated as compared to monotherapies (three pluses and two pluses respectively). These findings clearly indicated that concurrent blockade of VEGFR2 and Tie-2 represented a new strategy to potentiate angiogenesis inhibition. However, further studies are required to test whether there would be imposing changes in the growth rates of human cancers implanted in a mouse model, and whether these changes are attributed to changes in tumor vasculatures. These studies will ultimately lead us to understand the pharmacodynamics of F16 and Tie-2 inhibitor for better clinical applications in the future. (This research was supported by Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida.)

#3258

Gene expression and mutation analysis in human osteosarcoma xenograft models associated with combination activity of lenvatinib with etoposide and ifosfamide.

Zoltan Dezso,1 Sergei I. Agoulnik,1 Crystal MacKenzie,1 Kaoru Mitsuhashi,2 Kiyoshi Okamoto,2 Junji Matsui,2 Yasuhiro Funahashi2. 1 _Eisai Inc., Andover, MA;_ 2 _Eisai Co., Ltd., Tsukuba, Japan_.

Objectives: Lenvatinib mesilate (lenvatinib) is a selective inhibitor of VEGFR1-3, and other proangiogenic and oncogenic pathway-related receptor tyrosine kinases (RTKs) including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its inhibition of angiogenesis. The triple combination of lenvatinib, ifosfamide, and etoposide was effective against human pediatric osteosarcoma xenografts in nude mice. The objective of the study was to identify genes and pathways associated with tumor response to combination of Lenvatinib with etoposide (ETP) and ifosfamide (IFM).

Methods: Five human osteosarcoma cell lines (143B, HuO9, G292 clone A141B1, Saos-2, HOS) were grown in vitro and were used for DNA isolation and subsequent whole exome sequencing to determine their mutational profiles. Thirty tissue samples from tumor xenografts in nude mice were used for RNA isolation and subsequent transcriptional profiling using RNA sequencing. Each tumor xenograft model was represented by 6 samples isolated from 6 different mice.

Results: We performed mutation and gene expression profiling of 5 human pediatric osteosarcoma xenograft models using whole exome and RNA sequencing. Three of the models (G292, 143B and HOS) showed enhanced antitumor activity when lenvatinib was combined with IFM and ETP. We identified genes and signaling pathways associated with tumor response to the combination treatment. Top three pathways identified were hepatic fibrosis (p=1.7*10-7), molecular mechanism of cancer (p=1.3*10-4) and role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis (p=3.3*10-4). Genes from the pathways were related to cell differentiation and RTK. Hierarchical clustering of the models based on genes from the FGF pathways showed different expression patterns between models. A large fraction of genes from the FGF pathway showed statistically significant differences between the models. For example FGFR1 showed highest expression in G292, FGFR2 in Saos2, FGFR4 in 143B and HOS and FGFR3 in Hu09 and Saos2. The sequencing analysis identified 27 genes with previously reported mutations in TCGA with unique mutation or wild type status in the models with enhanced anti-tumor activity for combination of lenvatinib with IFM and ETP combination.

Conclusions: Gene expression analysis by RNAseq and whole exome sequencing showed variation between the xenograft models. Based on the differences we were able to identify genes and signaling pathways associated with anti-tumor activity of combination treatment of lenvatinib with IFM and ETP compared to combination of IFM and ETP.

#3259

LY3127804, a novel anti-Angiopoietin-2 antibody in combination with an anti-VEGFR2 antibody potently inhibits angiogenesis, tumor growth and metastasis.

Sudhakar R. Chintharlapalli, Johnny E. Croy, Donmienne Leung, Damien Gerald, Jirong Lu, Philip W. Iversen, Linda N. Lee, Lysiane Huber, Jonathan Tetreault, Rowena Almonte-Baldonado, Jianghuai Xu, Bharathi Ramamurthy, Jennifer A. Pereira, Chi-Kin Chow, Axel-Rainer Hanauske, Volker Wacheck, Laura Benjamin, Ling Liu. _Eli Lilly and Company, Indianapolis, IN_.

Angiopoeitin-2 (Ang2) is released from endothelial cells only in response to stimulus (e.g. wound healing, tumor growth) and facilitates blood vessel sprouting and inhibits pericyte-endothelial cell interaction via Tie2 signaling. Combination of an anti-Ang2 antibody and aflibercept, a VEGF trap, has been shown to inhibit tumor growth and decrease tumor vascularity in mouse xenograft tumor models (Daly et al., Cancer Res (2013) 73(1):108). Multiple investigational anti-Ang2 antibody therapies are currently in clinical trials. LY3127804 is a humanized and engineered IgG4 isotype antibody that selectively binds to Ang2 with high affinity and neutralizes Ang2 induced phospho-Tie2. LY3127804 inhibits sprouting angiogenesis and increases pericyte coverage in a mouse developmental retinal angiogenesis model and in mice bearing PC3 xenograft tumors. Combination of LY3127804 and DC101, a potent anti-VEGFR2 antibody, exhibits enhanced efficacy when compared to monotherapy in multiple patient derived xenograft models including NSCLC and ovarian cancers. Anti-Ang2 antibody monotherapy alone resulted in marginal reduction of tumor growth and improved overall survival, while DC101monotherapy had greater reduction in tumor volume with no survival benefit in MDA-MB-231 breast orthotopic model. Combination of anti-Ang2 antibody with anti-VEGFR2 antibody shows reduction in tumor volume and improved overall survival. This robust pre-clinical evidence supports testing the combination of anti-Ang2 and anti-VEGFR2 antibodies in the clinic. LY3127804 is currently in Phase 1 clinical trials (NCT02597036)

#3260

Differential effects of antiangiogenesis treatments on betel-nuts-related head and neck squamous cell carcinoma in Taiwan.

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

Background:

The treatment of head and neck squamous cell carcinoma(HNSCC) in Taiwan is very challenging and might be related to betel-nuts use. Betel-nuts chewing might contribute to (1)strong inflammation, invasion, and angiogenesis; (2)easy recurrence or metastasis; (3) poor response to chemotherapy, radiation, and epidermal growth factor receptor(EGFR) inhibitors.

Purpose:

We try to prove different kinds of anti-angiogenesis treatments will lead to different response on betel-nuts related HNSCC in Taiwan.

Methods:

Different anti-angiogenesis treatments, such as axitinib(VEGFR2 inhibitor), nintedanib(VEGFR2/FGFR inhibitor), and regorafenib(VEGFR2/FGFR/ more other signals inhibitor) were first used to treat (1)HUVEC; (2)HNSCC cell lines(SCC4, SCC9, SCC15, SCC25, FaDu, SAS, KB, Cal27, and TW2.6, betel-nuts related) to evaluate (a) invasion capacity by wound healing; (b) drug sensitivity by MTT assay; (c)synergistic effect with chemotherapy, EGFR inhibitor, and polo-like kinase inhibitor. Western blotting was also used to test signal change by treatments. TW2.6 had already been proved to possess defective p53, p16 loss, and increased Bcl2.

Results:

In our previous study, TW2.6 was resistant to chemotherapy, radiation, EGFR inhibitors, and ani-angiogenesis treamtents, such as axitinib and sunitinib. Axitinib, pure VEGFR2 inhibitor, was found to suppress HUVEC more prominently than nintedanib or regorafenib did. Invasion capacity of all HNSCC cell lines were all blocked by the three drugs but regorafenib did mostly well. However, axitinib had no effect on TW2.6; but nintedanb and regorafenib had moderate response on TW2.6. Besides, nintedanib and regorafenib both would resensitize TW2.6 to respond to chemotherapy, EGFR inhibitor, and polo-like kinase inhibitor again. Western blotting showed mesenchaml differentiation markers(slug, Twist, snail, Axl, c-MET, Vimentin) decreased after nintedanib and regorafenib use, too.

Conclusion:

TW2.6 might reflect treatment refractoriness of betel-nuts related HNSCC in Taiwan. In addition to PI3K/mTOR dual inhibition and polo-like kinase inhibitor with radiation(our studies shown in AACR and ESMO2015), VEGFR2/FGFR dual blockage might be effective on TW2,6 and resensitize TW2.6 to EGFR inhibitor, polo-like kinase inhibitor, & chemotherapy, maybe through the inhibition of mesenchymal transformation. VEGFR2/FGFR dual blockage will be one future combination backbone of betel-nuts related HNSCC.

#3261

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth.

Karla Parra,1 Paloma Valenzuela,1 Alejandra E. Gallegos,1 Natzidielly Lerma,1 Luis Reza,1 Georgialina Rodriguez,1 Urban Emmenegger,2 Marian Manciu,1 Teresa Di Desidero,3 Guido Bocci,3 Mitchell S. Felder,4 Robert A. Kirken,1 Giulio Francia1. 1 _UT El Paso, El Paso, TX;_ 2 _University of Toronto, Toronto, Canada;_ 3 _Univ. of Pisa, Pisa, Italy;_ 4 _William Beaumont, El Paso, TX_.

Although we previously reported that metronomic cyclophosphamide can augment the anti-tumor impact of anti-CTLA-4 immunotherapy, the effectiveness of this strategy to treat drug resistant tumors remains to be evaluated. To that end, the parental, drug sensitive, EMT-6/P murine breast cancer, or its cisplatin (DDP) or cyclophosphamide (CTX) resistant variants, were implanted into Balb/c mice (n=5-8 mice per group), either subcutaneously or orthotopically. Established tumors where monitored for relative growth following treatment with anti-CTLA-4 antibody (mouse anti-CD152, clone 9H10, given at 100ug on day 1 and 35ug on day 6, i.p.), alone or in combination with; a) metronomic CTX (ldCTX; 20mg/kg/day), b) Bolus (150mg/kg) plus ldCTX, or c) sequential treatment with gemcitabine (160mg/kg every 3 days).

EMT-6/P tumors responded to anti-CTLA-4 therapy, but this response was less effective when combined with Bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with a regimen of sequential gemcitabine. The EMT-6/P tumor showed consistent results in subcutaneous tumors and in those that were orthotopically implanted into the mammary fat pad. Tumor responses were less pronounced in drug resistant EMT-6/CTX or EMT-6/DDP tumor models than in the EMT-6/P tumor, but nonetheless even in the drug resistant models CTLA-4 targeting could be effectively combined with metronomic CTX or sequential gemcitabine (p<0.05). A number mice implanted with the parent or with the drug resistant tumors developed spontaneous metastases under continuous therapy, including mice bearing subcutaneous tumors, and about 20% of the mice on the combination therapies showed complete tumor responses. Of these mice, over 90% rejected subsequent EMT/6 tumor grafts, indicating that the anti-CTLA-4 plus metronomic chemotherapy regimens did not impair immunological memory. Furthermore, the efficacy of a therapy of anti-CTLA-4 with sequential continuous gemcitabine was confirmed in an independent syngeneic CT-26 colon cancer model.

We conclude that metronomic chemotherapy can combined with anti-CTLA-4 therapy, both in syngeneic breast and colon cancer models, and in breast tumors selected in vivo for resistance to alkylating agents. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in both chemotherapy naïve tumors and in tumors selected in vivo for resistance to CTX or to DDP, although tumor relapses can occur, in some cases accompanied by the development spontaneous metastases. Our results suggest a strategy for improving the anti-tumor efficacy of anti-CTLA-4 based therapies.

#3262

Effects of lenvatinib mesilate in combination with everolimus on VEGF and FGF-driven angiogenesis and tumor growth.

Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Yusuke Adachi, Kiyoshi Okamoto, Junji Matsui, Yasuhiro Funahashi. _Eisai Co., Ltd., Tsukuba, Japan_.

Lenvatinib mesilate (lenvatinib) is an orally available inhibitor for multiple receptor tyrosine kinases (RTKs) that selectively inhibits the kinase activities of VEGFR1, 2, and 3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1, 2, 3, and 4; PDGFRα; KIT; and RET. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in a Phase 2 study. In this study, we evaluated the effect of lenvatinib in combination with everolimus on VEGF and bFGF-driven angiogenesis to elucidate the mechanism of combination action in preclinical models. Preclinical studies provide a plausible biologic rationale for the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus.

Effects of lenvatinib, everolimus, and its combination on VEGF or bFGF activated intracellular signaling were analyzed in HUVEC by western blotting. Combination effects of lenvatinib and everolimus on VEGF and bFGF-induced proliferation or tube formation of HUVEC were examined using combination indexes (CI). Antitumor activities were tested in the KP-1/VEGF or KP-1/FGF models, where VEGF or FGF-induced tumor angiogenesis and tumor growth were enhanced in nude mice due to overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells.

Lenvatinib inhibited the VEGF or bFGF-driven phosphorylation of Erk1/2 (Thr202/Tyr204), S6K (Thr389), and S6K (Thr421/Ser424), and S6 (Ser235/Ser236), indicating the inhibition of both the MAPK pathway and the mTOR-S6K-S6 pathway. Everolimus inhibited the phosphorylation of S6K (Thr389), S6K (Thr421/Ser424), and S6 (Ser235/Ser236), but not Erk1/2. The combination showed greater inhibition for the phosphorylation of S6K (Thr421/Ser424) and S6 (Ser235/Ser236) than each single agent. Inhibitory activity of the combination at several molar ratios was mostly additive for VEGF-driven proliferation (CI: 0.799-1.167) and mostly synergistic for bFGF-driven tube formation (CI: 0.469-0.741). In the KP-1/VEGF or KP-1/FGF xenogtaft models, lenvatinib, everolimus, and the combination (p.o., qd x 14) significantly inhibited tumor growth compared to vehicle. In addition the combination of lenvatinib (7.5 mg/kg) and everolimus (15 mg/kg) showed significantly greater antitumor activity than higher dose of either lenvatinib (10 mg/kg) or everolimus (30 mg/kg) monotherapy.

These results demonstrated enhancement of the inhibitory activity against VEGF and FGF-induced angiogenesis by the combination of lenvatinib with everolimus, and the synergistic enhancement against bFGF-induced angiogenesis unlike other VEGFR2 TKIs. The vertical inhibition of angiogenic signaling pathways with lenvatinib (RTK) and everolimus (mTOR) may contribute to enhanced antiangiogenic activity by dual targeting of the mTOR-S6K-S6 pathway.

#3263

Dietary supplement 3, 3'-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer.

Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. _New York Medical College, Valhalla, NY_.

Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC's) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC's, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3'-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 µM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 µM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect.

#3264

Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models.

Yusuke Adachi, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Kiyochi Okamoto, Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Yasuhiro Funahashi, Junji Matsui. _Eisai Co., Ltd., Tsukuba, Japan_.

Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; the platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis, and is currently marketed for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in Phase 2 study. The aim of this study is to elucidate the activity of the combination of lenvatinib and everolimus in preclinical human RCC xenograft models.

We examined antitumor activity in two human RCC (A-498 and Caki-1) xenograft models orally treated with lenvatinib (10 mg/kg), everolimus (30 mg/kg), and the combination of lenvatinib and everolimus for 1 or 2 weeks. The antitumor proliferation and antiangiogenic effects were evaluated by immunohistochemistry (IHC) using anti Ki67 antibody and anti CD31 antibody, respectively. The induction of apoptosis was detected by TUNEL assay. To analyze the gene expression profile of tumor samples, microarray analysis were also conducted.

The antitumor activity of the combination of lenvatinib and everolimus was greater than that of either agent administered alone in A-498 and Caki-1 xenograft models. The combination caused tumor regression and had no remarkable body weight loss. IHC analysis revealed decrease of microvessel density in lenvatinib and combination groups, and also decrease in the proportion of proliferative cells in everolimus treated and combination-treated group in A-498 model. In TUNEL assay, significant induction of apoptosis was observed only in the combination-treatment group. The analysis of gene expression profile in A-498 xenograft tumors also supported these results: lenvatinib alone upregulated hypoxia-related genes and everolimus decreased proliferation-related genes. The combination of these 2 drugs induced blends of the gene expression changes caused by each single treatment.

Our results indicate that treatment of lenvatinib in combination with everolimus caused significant antitumor effect by combining the potent antiangiogenic activity of lenvatinib as well as direct antitumor activity of everolimus in A-498 model. These preclinical results provide one of the mechanisms to support the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus.

#3265

Homeobox B9 (HOXB9) sustains anti-VEGF treatment resistance in gastrointestinal tumors.

Carmine Carbone,1 Geny Piro,1 Francesca Simionato,1 Fotios Loupakis,2 Chiara Cremolini,2 Gabriella Fontanini,2 Federica Di Nicolantonio,3 Elisa Moratti,1 Francesca Ligorio,1 Marco Zanotto,1 Raffaela Santoro,1 Maria Mihaela Mina,1 Aldo Scarpa,1 Alberto Bardelli,3 Giampaolo Tortora,1 Davide Melisi1. 1 _University of Verona, Verona, Italy;_ 2 _University of Pisa, Pisa, Italy;_ 3 _University of Torino, Torino, Italy_.

Resistance to anti-angiogenic therapies poses one of the greatest challenges in gastrointestinal tumors research. We recently identified several proinflammatory secreted factors, including interleukin-1 (IL1), CXC ligand (CXCL) 1 and 8, Transforming Growth Factor β (TGFβ)1, and Angiopoietin-like Protein 2 (ANGPTL2), that were overexpressed in murine models of tumors resistant to the anti-VEGF antibody bevacizumab (BEV). These factors induced epithelial-to-mesenchymal transition (EMT) and increased aggressiveness. HOXB9 has been identified as a key transcription factor in common for these factors. Here, we hypothesized that HOXB9 might be responsible for BEV resistance in gastrointestinal tumors.

HOXB9 expression and activation were measured in BEV-sensitive COLO357FG and in their BEV-resistant counterpart FGBR pancreatic cancer cell lines, and in LOVO, MDST8, LIM2099, CCK81, GP5D, and SNUC4 colon cancer cell lines by EMSA and DAPA. Serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 in nude mice bearing tumors were measured by multiplex xMAP technology. Immunohistochemical analyses were performed in pre- vs. post-progression biopsies from colorectal cancer patients receiving BEV.

HOXB9 protein was more expressed and activated in FGBR than did in COLO357FG cells. shRNA to knock down HOXB9 significantly (P<0.01) reduced proinflammatory factors and EMT markers expression, and migration in FGBR cells. Mice bearing HOXB9-silenced tumors had significantly (P<0.01) lower serum levels of IL1, CXCL1 and 8, TGFβ1 and ANGPTL2 than did their respective controls. BEV significantly reduced tumor burden (P≤0.0001) and prolonged survival duration (P=0.0006) in mice bearing HOXB9-silenced FGBR tumors, whereas it was ineffective on FGBR scramble tumors. HOXB9-positive LOVO, MDST8, and LIM2099 cells had significantly higher expression levels of proinflammatory factors and EMT markers, and migration rates than did HOXB9-negative CCK81, GP5D, and SNUC4 colon cancer cell lines. In vivo, BEV significantly reduced tumor burden and prolonged survival duration in mice bearing CCK81 or GP5D tumors (P<0.05), whereas it was ineffective on MDST8 and LIM2099 tumors. HOXB9 shRNA significantly reduced proinflammatory factors and EMT markers expression, and migration in MDST8 cells. BEV significantly prolonged survival duration in mice bearing HOXB9-silenced MDST8 tumors (P=0.0025), but did not on MDST8 scramble tumors. Validation of the differential expression of HOXB9 in biopsies from colorectal patients progressing under BEV therapy will be available at the time of the meeting.

In conclusion, we identified HOXB9 as crucial transcription factor to sustain BEV resistance in gastrointestinal tumors. Silencing of HOXB9 is a promising approach to modulate this resistance. Our results candidate HOXB9 as potential biomarker for selecting patients with colorectal and pancreatic cancer for antiangiogenic therapy.

#3266

Antitumor activity of a combination of lenvatinib mesilate, ifosfamide, and etoposide against human pediatric osteosarcoma cell lines.

Masahiro Matsuki,1 Kiyoshi Okamoto,1 Zoltan Dezso,2 Sergei I. Agoulnik,2 Yasuhiro Funahashi,1 Junji Matsui1. 1 _Eisai Co., Ltd., Tsukuba, Japan;_ 2 _Eisai Inc., Andover, MA_.

Lenvatinib mesilate (lenvatinib) is an orally administered molecular targeted agent that selectively inhibits several receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptors (VEGFR1-3), fibroblast growth factor receptors (FGFR1-4), platelet-derived growth factor receptor (PDGFR) α, RET, and KIT. Lenvatinib has been marketed for the treatment of advanced or differentiated thyroid cancer refractory to radioactive iodine. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis. In this study we evaluated the antitumor activities of lenvatinib in combination with ifosfamide and etoposide against human pediatric osteosarcoma cell lines.

Five human pediatric osteosarcoma cell lines (143B, G-292 clone A141B1, HOS, HuO9, and Saos-2) were used. Antiproliferative activity of lenvatinb and etoposide was evaluated in WST-8-based colorimetric proliferation assay or in soft agar colony formation assay. To evaluate the in vivo antitumor activity, lenvatinib alone, the combination of etoposide and ifosfamide, and the combination of these 3 drugs were administered to mice bearing the human osteosarcoma xenografts.

Etoposide inhibited cell proliferation with IC50 values of a range within 0.31 - 4.1 μmol/L. G-292 clone A141B1 (G-292) had the lowest sensitivity to etoposide. Four human osteosarcoma cell lines out of five were not sensitive to lenvatinib in proliferation assay (IC50 values >10 μmol/L), and only G-292 was susceptible to lenvatinib with IC50 values of 1.3 and 0.059 μmol/L in proliferation assay or soft agar colony formation assay, respectively. G-292 has been reported to have FGFR1 amplification and shows high transcript expression of FGFR1, suggesting that the antiproliferative activity of lenvatinib could be mediated by the FGFR1 blockade. In all xenograft models, tumor volumes with the triple combination treatment were significantly lower than that with vehicle treatment on Day 8. Antitumor effect of the triple combination was significantly more potent than that of lenvatinib alone and the ifosfamide-etoposide combination in 143B, G-292, and HOS, and more potent than that of lenvatinib alone in HuO9 and Saos-2 xenograft models.

The triple combination of lenvatinib, ifosfamide, and etoposide was active against all examined human pediatric osteosarcoma xenografts in mice, and lenvatinib enhanced antitumor activity of the ifosfamide-etoposide combination in 3 out of 5 models. These results support further investigation of the combination of lenvatinib with ifosfamide and etoposide in osteosarcoma. In addition, direct antitumor activity of lenvatinib against the osteosarcoma cells harboring FGFR1 amplification warrants further investigation.

#3267

Eltd1 as an anti-angiogenic therapy against glioma tumors.

Jadith Ziegler,1 Nataliya Smith,2 Debra Saunders,2 Blake Evans,2 James D. Battiste,3 Michael Sughrue,4 Paul Tompkins,4 Jake Sutton,2 Megan Lerner,1 Patricia Coutinho de Souza,5 Kar-Ming Fung,6 Jonathan Wren,2 Rheal Towner2. 1 _The University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _Oklahoma Medical Research Foundation, Oklahoma City, OK;_ 3 _Department of Neurology, OU College of Medicine, Oklahoma City, OK;_ 4 _Department of Neurosurgery, OU College of Medicine, Oklahoma City, OK;_ 5 _Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA;_ 6 _Peggy and Charles Stephenson Cancer Center, Oklahoma City, OK_.

Gliomas consist of up to 80% of malignant brain tumors that are invasive and typically resistant to radiotherapy and chemotherapy. Finding biomarkers to high-grade gliomas can enable better diagnosis and therapeutic intervention for this disease. We have identified ELTD1 as a biomarker for high-grade human gliomas. Here, we report our findings in vivo using anti-ELTD1 (at two different concentrations), Bevacizumab, and IgG antibodies on human G55 xenograft glioma models. Using MRI, we investigate tumor growth, perfusion, tumor blood flow, and microvessel density changes in mice. In addition, survival rates were measured.

Mice were implanted with human G55 xenograft glioma cells and were left untreated, administered anti-ELTD1, (1 mg/kg or 2 mg/kg every 2-3 days) Bevacizumab (2 mg/kg every 2-3 days) or IgG (1 mg/kg every 2-3 days). MRI experiments were performed to assess tumor growth and calculate tumor volumes. In order to assess angiogenesis, representative histology slides were obtained and stained for blood vessels using CD34 antibody and microvessel density (MVD) was then calculated. Finally, cerebral blood flow rates from MR perfusion imaging was obtained as well as MR angiography to determine tumor blood volume from mice in each group.

Our results show a significant decrease in tumor volumes and increase in percent survival for mice treated with 1 mg/kg and 2 mg/kg of ELTD1 antibody compared to untreated mice and IgG treated mice. Mice also had an increase in perfusion, decrease in tumor blood volume and decrease in MVD. Anti-ELTD1 antibody therapy reduced tumor volumes, prolonged life, and overall decreased angiogenesis in our mouse model. Anti-ELTD1 therapy may be an ideal anti-angiogenic treatment for high-grade gliomas.

#3268

Proteomic response in breast cancer treated with neoadjuvant chemotherapy with and without bevacizumab: Reverse phase protein array (RPPA) results from NeoAva - a randomized phase II study.

Mads H. Haugen,1 Ole Christian Lingjaerde,2 Marit Krohn,1 Evita M. Lindholm,1 Laxmi Silwal-Pandit,1 Elin Borgen,3 Øystein Garred,3 Anne Fangberget,3 Marit M. Holmen,3 Ellen Schlichting,3 Helle Skjerven,4 Steinar Lundgren,5 Erik Wist,2 Bjoern Naume,2 Gunhild M. Maelandsmo,1 Yiling Lu,6 Anne-Lise Boerresen-Dale,2 Gordon B. Mills,6 Olav Engebraaten2. 1 _Oslo University Hospital - Institute for Cancer Research, Oslo, Norway;_ 2 _Oslo University Hospital & University of Oslo, Oslo, Norway; _3 _Oslo University Hospital, Oslo, Norway;_ 4 _Vestre Viken Hospital Trust, Drammen, Norway;_ 5 _St. Olavs Hospital & NTNU, Trondheim, Norway; _6 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

BACKGROUND: In this phase II clinical trial, patients with HER2 negative primary tumors of ≥25 mm were treated with neoadjuvant chemotherapy (4 x FEC100 + 12 weeks of taxane-based therapy) and randomized (1:1) to receive bevacizumab or no bevacizumab. Mammography, ultrasound and MR imaging were used for response evaluation, in addition to final pathology assessment.

HYPOTHESIS: RPPA proteomic analyses support identification of molecular mechanisms associated with clinical response to bevacizumab treatment.

METHODS: Tumor responses were evaluable in 132 patients; of which 66 received bevacizumab. Ratio of the tumor size at final pathology assessment, and at inclusion was calculated to obtain a continuous scale of response reflecting the percentage of tumor shrinkage in response to therapy. Tumor material was obtained at screening, 12 weeks into treatment and at surgical removal of tumors at 25 weeks. Lysates from each sample was analyzed on reverse phase protein arrays (RPPA) for expression levels of 210 proteins of which 54 were phospho-specific. Data from protein analyses was compared to previously generated mRNA expression data.

RESULTS: Several proteins were found for which expression prior to treatment (week 0) reflected a better response on tumor shrinkage in the combination treatment arm (chemotherapy+bevacizumab): E.g. good responders had lower PDGFR-beta expression, and this was also observed at the mRNA level, while this result was not identified in the mono treatment arm (chemotherapy alone) on either level. The proteomic response from week 0 to 12 in both treatment arms had an overall similar profile regarding up- and down-regulated proteins; however, the combination treatment (FEC100 + bevacizumab) induced substantially more effect on regulation of each protein. This might reflect the capability of bevacizumab treatment to potentiate the effects of the anthracyclin based chemotherapy from week 0 to 12. Conversely, from week 12-25 (taxane-based therapy + bevacizumab) this effect was lost or even reversed, and reveals a possible need for further studies investigating changes in protein expression and correlation to response of a given treatment. Of particular interest were proteins that switched direction of regulation between the FEC and taxane-based regimes, however, these effects were not confined to the combination treatment and thus probably not due to the added bevacizumab. We are in the process of analyzing the impact of phosphorylation and thus protein activation states on treatment response. The above mentioned results have potentially important clinical relevance and will be further investigated with respect to subtypes and the biological pathways affected by antiangiogenic therapy.

#3269

Recapitulating the BETH adjuvant breast cancer trial (NCT00625898) using clinically accurate orthotopic surgical resection models.

Liam S. Shiels,1 Ian S. Miller,1 Clare Morgan,2 Mattia Cremona,2 Robert Kerbel,3 Bryan Hennessey,2 Norma O'Donovan,4 John Crown,4 Annette T. Byrne1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _RCSI Education & Research Centre, Beaumont Hospital, Dublin, Ireland; _3 _Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada;_ 4 _National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland_.

Background: An overwhelming majority of phase III clinical trials in the oncology setting fail as promising results observed in the 'pre-clinic' are not successfully translated to patients. As such, improved models which more accurately predict outcomes are now required. Herein, we have established orthotopic surgical resection models of Her2+ breast cancer, which replicate the phenotype of clinical post-resection metastasis [1,2]. Using these models we have recapitulated the BETH adjuvant breast cancer trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (TRAST) failed to provide additional benefit in the adjuvant setting. Methods: SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231or HCC1954 cells and palpable tumors resected approximately 5 weeks later. 3 weeks after resection, mice were treated with 10 mg/kg TRAST + 5mg/kg paclitaxel IP once weekly for 6 cycles with or without weekly BVZ (5mg/kg IP). Metastasis was monitored by weekly bioluminescence imaging. Results: Tumor growth and imaging data confirmed that the addition of BVZ to adjuvant TRAST + chemotherapy provided no additional benefit compared with TRAST + chemotherapy alone. Previous pre-clinical studies using inappropriate non-resection models failed to predict this response. Furthermore, Reverse Phase Protein Array analysis of treated tumors implicated HER2, VEGF, mTOR and the intrinsic mitochondrial cell death apoptotic pathways in treatment resistance. Conclusion: In summary, our data provides evidence for pre-clinical models which better predict clinical outcome in the breast cancer adjuvant setting, and which represent an important resource for interrogating resistance pathways and identifying novel biomarker signatures. [1]. Breast. 2013 Aug;22 Suppl 2:S57-65; [2]. Cancer Res. 2013 May 1;73(9):2743-8.This work was performed under Irish HPRA Authorization AE18982 and was supported by the Clinical Cancer Research Trust. ATB receives funding from the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant (CCRC13GAL).

#3270

Myriocin, an inhibitor of serine palmitoyltransferase activity, has the anti-angiogenic effect by attenuating VEGF-induced angiogenesis signaling pathway.

Sun Hee Lee, Yuk Dong Jung, Hye-Eun Lee, You Mie Lee. _Kyungpook National University, Daegu, Republic of Korea_.

Myriocin is a powerful serine palmitoyl transferase inhibitor, by inhibiting of sphingosine biosynthesis. Myriocin is also known to have immunosuppressive activity. Here, we demonstrate the anti-angiogenic effect of myriocin. Myriocin decreased vascular endothelial cell growth factor (VEGF)-induced angiogenic process, that include proliferation, migration, invasion, tube formation and cell adhesion in human umbilical vein endothelial cells (HUVECs). Myriocin down-regulated VEGF-induced VEGFR2, focal adhesion kinase, AKT kinase, rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and matrix metalloproteinase-2 (MMP2) expression. Myriocin greatly reduced new vessel formation in chick chorioallantoic membrane and vessel sprouting in mouse aortic ring assay. Furthermore, VEGF-induced phalloidin expression was inhibited by miyriocin in HUVECs. Our result indicated that myriocin inhibited VEGF-induced proliferation, migration, invasion, tube formation and cell adhesion in vitro by inhibiting VEGFR2-mediated activation of FAK, AKT, ROCK1 and MMP2. Taken together, we suggest that myriocin may act as significant angiogenic inhibitor by inhibiting VEGF-induced signaling pathway.

#3271

Gene expression changes underlying glioblastoma resistance to anti-angiogenic therapy.

William C. Chen,1 Arman Jahangiri,1 Garima Yagnik,1 Maxim Sidorov,1 Jonathan Rick,1 Ruby Kuang,1 Michael DeLay,2 Manish K. Aghi1. 1 _UCSF, San Francisco, CA;_ 2 _Columbia University, New York, NY_.

Introduction: Despite positive pre-clinical and clinical trials, treatment of glioblastoma with bevacizumab has been limited by acquired resistance and transient response. To study gene expression changes underlying tumor resistance and progression during bevacizumab therapy, we performed microarray gene expression analysis on a novel multigenerational xenograft model of acquired bevacizumab resistance.

Methods: Using a two-component normal mixture model, we identified a set of genes exhibiting significant inter-generational variance. Protein-protein interaction scores among these genes were extracted from the String 10 database and used as undirected edge weights in a network representation of the significant gene set. Gene set over-representation (GSO) analysis via ConsensusPathDB of gene clusters identified biologically meaningful clusters and subnetworks mediating distinct functions and molecular pathways.

Results: Gene set enrichment analysis revealed significant overexpression across generations of previously identified gene signatures of the mesenchymal subtype, as well as a tumor mesenchymal metabolic signature. Key mesenchymal markers, including putative tumor-stemness marker CD44, NT5E, SNAI2, and ZEB2, were found to be upregulated across generations. These results suggest tumor progression under bevacizumab challenge to be accompanied by a shift in gene expression towards the mesenchymal subtype, a subtype of GBM associated with enhanced invasiveness, resistance, and worse outcome.

Our analysis also revealed expression changes in pathways related to angiogenesis. Pro-angiogenic genes FGF2, MMP1, HIF1A, UGCG, LPAR1, and ITGB3 were found to be upregulated. Furthermore, GSO analysis identified angiogenesis as a significantly enriched ontology within the inflammatory response and ECM remodeling subnetworks identified by spectral clustering. Angiogenesis related genes identified via GSO included highly upregulated inflammatory mediators such as COX2, IL6, IL1A, upregulated pro-angiogenic factors TGFA, WNT5A, FGF2, and a downregulated anti-angiogenic gene, SPARC.

Conclusions: These results suggest a mesenchymal and pro-angiogenic response to bevacizumab treatment supported by multiple converging pathways involving inflammation, hypoxia, and ECM remodeling. Strategies preventing the evolution of these responses should be developed and tested in the context of our novel xenograft model in order to improve the durability of response to these therapies and allow them to fulfill their therapeutic promise.

#3272

LKB1 loss is associated with resistance to VEGF inhibitors in non-small cell lung cancer (NSCLC).

Irene Guijarro, Alissa Poteete, Chao Yang, Emily Roarty, Monique Nilsson, Huiying Sun, Pan Tong, Edward Chang, Jaime Rodriguez-Canales, Barbara Mino, Edwin Parra, Ignacio Wistuba, Jing Wang, Timothy Heffernan, John V. Heymach. _MD Anderson, Houston, TX_.

Background

LKB1 is a protein kinase that is mutated and down-regulated in 20-30% of NSCLC. LKB1 mutations co-occur with KRAS alterations in 7%-10% of NSCLC, resulting in an aggressive phenotype with short survival, and frequent metastases. Because LKB1 activates AMPK, many of the best known functions of LKB1 are attributed to its ability to control metabolic alterations in the cells. However LKB1 also plays an important role in regulating tumor progression, metastasis and angiogenesis, likely as a compensatory strategy to overcome energetic depletion of tumor microenvironment. Bevacizumab, the anti-VEGF antibody improves PFS in NSCLC patients combined with chemotherapy but the benefit is modest and transient and often patients develop resistance. Our laboratory has identified alterations in cell metabolism and in vasculature of LKB1 deficient tumors when compared to LKB1 wild type. These findings may indicate that loss of LKB1 could alter the tumor vasculature in NSCLC.

Methods

mRNA expression of angiogenesis related genes were analyzed in wild type and LKB1 deficient NSCLC tumors (TCGA). In vitro validation was performed by qPCR and western blot. CD31 IHC was performed to analyze microvasculature density (MVD) in Krasmut and Krasmut LKB1f/f mice tumors. HUVEC tube formation and migration assays were performed with conditioned medium of LKB1 expressing and deficient NSCLC isogeneic cell lines. Xenograft NSCLC models were established via s.c. injection of H460 (LKB1 deficient) and H460 LKB1 (LKB1 expressing) cells in nude mice. Treatment consisting of human and/or mouse bevacizumab and nintedanib were administrated. Tumor volumes were measured and vasculature analysis was performed.

Results

In vitro HUVEC cells exhibited an increase migration and differences in endothelial network formation when incubated with conditioned medium from LKB1 deficient cells compared to LKB1 expressing cells medium (p<0.05). LKB1 deficient cells upregulated hypoxia and energetic stress related genes (HIF1AN, EGLN1, HIF3A, EPAS1 and CA12) and increased the secretion of angiogenesis related factors (VEGF, IL8, endoglin). IHC analysis of baseline CD31 expression of tumors from Krasmut and Krasmut LKB1f/f mutant mice showed no significant differences in MVD. However, anti-angiogenic therapy significantly inhibited tumor progression in LKB1 expressing xenografts but did not show any therapeutic effect in LKB1 deficient tumors. LKB1 expressing xenografts treated with human or mouse bevacizumab, or the combination of both and nintedanib, resulted in a significant decrease in tumor volume (p<0.05). The blockade of human and mouse VEGF showed an optimal therapeutic effect with approximately 70% reduction of tumor volume (p<0.001).

Conclusions

LKB1 deficiency may promote resistance to anti-angiogenic therapy by regulating compensatory angiogenic pathways along metabolic adaptations to energetic stress in NSCLC.

#3273

The efficiency of anti-tumor effect of metformin for gemcitabine-resistant pancreatic adenocarcinoma.

Keiichi Suzuki, Osamu Takeuchi, Yoshiyuki Ishii, Masayoshi Osaku. _Kitasato Institute Hospital, Tokyo, Japan_.

Metformin (MET) is the first-line treatment for type 2 diabetes mellitus. Several epidemiological studies have revealed the anti-cancer effects of metformin (MET), including against pancreatic ductal carcinoma (PDC). Gemcitabine (GEM) has become the standard chemotherapy for PDC but tolerance to GEM becomes an important issue.

We evaluated the anti-tumor effects of MET for GEM-resistant PDC in a xenograft mouse model. For this in vivo study, wild-type BxPC-3 was implanted into both flanks of female BALB/c nude mice. Mice were divided into four groups: (i) control (without any treatment); (ii) GEM-treated group (100 mg/kg); (iii) MET-treated group (600 mg/kg); and (iv) combined treatment group (G+M). Mice were fed for 4 weeks. Estimated tumor volumes and body weights were measured each week. Treatments were initiated 2 weeks after implantation. MET was administrated orally every day. GEM was given by intraperitoneal injection every week.

In the final tumor volumes, the two groups treated with GEM had significantly reduced tumors compared with control, but there were no differences between control and MET groups. The anti-tumor effect of MET for BxG30 (the cell line for GEM-resistant PDC) was evaluated using the method described above. Tumor volumes were decreased significantly in GEM+MET groups compared with control. The treated control ratio (T/C%) was calculated: GEM, 80.2%; MET, 54.0%; GEM+MET, 47.2%. The anti-tumor effect of GEM for BxG30 was limited. The MET group showed satisfactory anti-tumor effects, but T/C% was <50% only in the GEM+MET group. This result revealed that combination therapy had excellent anti-tumor effects even for GEM-resistant PDC.

Western blot analysis was performed to confirm mammalian target of rapamycin (mTOR) expression level was surely inhibited. The results revealed that the expression level of mTOR treated with MET was significantly decreased, but no in GEM group. The expression level of hypoxia-inducible factor 1 (HIF-1) was evaluated by western blot analysis also. The results showed significant inhibition of HIF-1 expression by MET treatment, but not by GEM, again. Then the expression of VEGF mRNA, downstream of HIF-1 signaling pathway, was evaluated by qRT-PCR. The result showed inhibition of VEGF mRNA expression by MET treatment.

Our results showed that MET has a partial anti-tumor effect for PDC. Combination therapy has an excellent effect not only for wild-type PDC but also for GEM-resistant PDC. The mechanism of anti-tumor effect of MET seems to be, at least partly, the inhibition of HIF-1/mTOR signaling pathway. These data suggest that MET could be used to treat PDC.

#3274

VEGF─receptor antagonist, Sugen 5416, sensitizes pulmonary endothelial stem-like cells to estrogens: A microvascular model for the progression of lung cancer.

Mayur Doke, Quentin H. Felty. _Florida International University, Miami, FL_.

Proliferative vascular lesions are an aggressive angiogenic phenotype associated with a poor prognosis in non-small cell lung cancer (NSCLC). Recent studies suggest that CD133+ endothelial stem-like cells are recruited to the angiogenic vascular system of NSCLC; and are associated with tumor stage and progression. Epidemiological studies have reported that women who received hormone replacement therapy (HRT) show an overall lower survival to lung cancer especially NSCLC when compared to women with no HRT. Proliferative vascular lesions have been studied extensively using the VEGF─receptor antagonist, Sugen 5416 (SU5416), plus chronic hypoxia (SuHx) rodent model. Therefore, the aim of this study was to determine whether SU5416 exposure of human pulmonary vascular endothelial cells (ECs) become more sensitive to estrogen-induced cell growth. We exposed human pulmonary endothelial cell line HPMEC-ST1.6R to SU5416 to select for a sub-population of cells which were then treated with 17β-estradiol and/or estrogenic polychlorinated biphenyl 153 (PCB153). We observed a significant increase of 2-3 fold change in cell growth and proliferation as determined by MTT, SRB, BrdU, and FACS analysis when SU5416 treated cells were exposed to estrogenic chemicals. Sugen 5416 treatment of ECs showed an increase in the inhibitor of DNA binding and differentiation protein 3 (ID3) along with stem cell markers CD133, Oct-4, and Sox-2. Immunohistochemistry of lung tissue samples from SuHx rodent model showed increased expression of ID3 in pulmonary vascular lesions that correlated with an increase in stem cell markers Oct-4 and Nanog; and increased VEGFR3+ cells surrounding vessel obliterating lesions from SuHx lung tissue. Our findings demonstrate that SU5416 sensitized cells to estrogens resulting in higher cell growth compared to control. Since VEGFR3 expression levels are reported to correlate with metastasis in NSCLC patients, our findings may be used as a model to study how VEGFR3 signaling can promote the aggressive growth and spread of lung cancer when exposed to estrogenic chemicals. Furthermore, we showed that stem cell markers Oct-4, Sox-2, and Nanog are inducible molecular targets of Sugen 5416 treatment. This finding is significant in that it points toward a potential mechanism of chemical-induced pluripotent stem-like cells. A better understanding of how vascular lesions depend on SU5416 may open new avenues for the prevention and treatment of lung cancer. The presence of stem cell markers does not imply that these cells actually function as stem cells. Instead, we propose that the combination of stem cell marker expression and chemical exposure to Sugen 5416 re-program terminally differentiated ECs to a pluripotent cell that gives rise to a clonal pulmonary vascular lesion that supports the aggressive growth of NSCLC.

#3275

Chemokine CXCL12 promotes vascular compensation in antiangiogenic resistance through CXCR7 in glioblastoma.

Aleksandra B. Gruslova, David Cavasoz, Andrew Brenner. _UTHSCSA, San Antonio, TX_.

Introduction: While antiangiogenic therapy has become an integral part of therapy for malignancy, it has failed to result in a proven survival benefit for glioblastoma (GBM). Recent studies have suggested a role for the chemokine CXCL12 in both de novo and acquired antiangiogenic resistance. To assess the impact of CXCL12 signaling on the vasculature and its ability to recruit progenitor cells, we combined a fluorescent chimeric mouse model and in vivo imaging to capture the chronological interactions among tumor cells (RFP), circulating BMDCs (GFP) and the cerebral vessels (FITC dextran) during tumor growth.

Methods: For chimeric mice, bone marrow from Tie2_GFP mice were transplanted to athymic nude mice after 6Gy irradiation. After 6 weeks U251 glioma cells were stereotactically injected into the brain cortex, followed by installation of a cranial window. In vivo imaging was performed weekly (before, during and after treatment) on a confocal microscope. 3D projected images were processed for the determination of tumor and vasculature characteristics. To validate the role of two CXCL12 receptors we used sunitinib (VEGFR inhibitor), AMD3100 (CXCR4 inhibitor), CCX662 (CXCR7 inhibitor) alone or in combination.

Results: Inhibition of VEGFR by sunitinib leads not only to a significant decrease in the number of small vessels as previously published, but also a statistically significant increase in size of the large vessels. Addition of CXCR7 inhibition to VEGFR inhibition prevented this adaptive vasculomegaly, but did not impact the loss of smaller vessels. In turn, CXCR4 inhibition had no effect on the vasculature. Further, CXCR7 inhibition decreases the number of BMDCs within vasculature, while CXCR4 inhibition decreased the number of cells recruited outside the vessels and into the tumor itself. These tumoral BMDCs are Iba1 positive and suggest a role of CXCR4 in recruitment of microglia. 3D analysis of cranial window images showed that while VEGFR inhibition slows tumor growth, and inhibition of CXCR4 or CXCR7 was not additive to this effect. In fact, inhibition of CXCR4 resulted in significant increase in tumor size perhaps through and impact on infiltration.

Conclusions: Our results suggests a compensatory response of vasculomegaly to trimming of small vessels by sunitinib may be mediated by CXCL12 in a CXCR7 dependent process. CXCR7's effect on the number of BMDCs within the vessels suggests that the impact on vasculomegaly may be due to recruitment of endothelial progenitor cells into vessels or their survival following recruitment. In turn, VEGFR inhibition affects recruitment of progenitor cells outside the vessels in a CXCR4 dependent fashion. We propose that CXCL12 receptors, CXCR4 and CXCR7, have unique roles in response to loss of VEGF signaling and subsequent hypoxia. Further studies into the interplay between VEGFR, CXCL12, compensatory vasculomegaly, and vascular cooption are needed.

#3276

The tissue and cellular destination of therapeutic IgGs in glioblastoma.

Gaelle Muller-Greven,1 Cathleen Carlin,2 Steven Toms,3 Manmeet Ahluwalia,1 Markus Bredel,4 Justin Lathia,1 Jeremy Rich,1 Petra Hamerlik,5 Candece L. Gladson1. 1 _Cleveland Clinic, Cleveland, OH;_ 2 _Case Western Reserve University, Cleveland, OH;_ 3 _Geisinger Medical Center, Danville, PA;_ 4 _University of Alabama, Birmingham, AL;_ 5 _Danish Cancer Society Research Center, Copenhagen, Denmark_.

Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is not known. It has been assumed that bevacizumab solely targets circulating VEGF-A in blood. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized either bevacizumab or human IgG into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab or human IgG via macropinocytosis. Furthermore, bevacizumab or human IgG was largely detected in the Rab4+ "fast" recycling compartment at 5 min, and both were largely detected in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. CSCs (CD133+) from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Administration of bevacizumab to an orthotopic xenograft mouse model of established GBM showed that bevacizumab was partially co-localized with Rab4+ or with LAMP1+ in perivascular tumor cells, consistent with our in vitro findings. Taken together, our data show that in GBM, humanized IgG, including bevacizumab, gains access to the perivascular tumor space and is then macropinocytosed by CSCs and trafficked to a recycling compartment or to the late endosome/lysosome. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients' response to GBM therapy.

#3277

Prion-like protein "Doppel" is a selective therapeutic target for tumoral angiogenesis.

Taslim Al-Hilal,1 Yoosoo Yang,2 In-San Kim,2 Youngro Byun,3 Fakhrul Ahsan1. 1 _Texas Tech University Health Science Center, Amarillo, TX;_ 2 _Korea Institute of Science and Technology, Seoul, Republic of Korea;_ 3 _Seoul National University, Seoul, Republic of Korea_.

Regulating growth factor signaling in a controlled and site-specific manner is the main hurdle that current antiangiogenic treatment modalities face. Here we show that the prion-like protein, doppel, is uniquely found in tumoral endothelial cells (TEC) but not in the normal ECs. Tissue sections containing primary human tumor (lung and colon) and normal tissues were used to study doppel protein expression. Strong expression and clear co-localization of doppel protein with CD34, a classical endothelial marker, was observed in both lung and colon cancer tissues compared to normal tissues. To evaluate doppel expression at the molecular level, we isolated TECs from different tumors derived from mouse xenografts. Extensive analysis showed that doppel was highly and ubiquitously expressed in the vasculature of squamous, breast, lung, and colon cancers. To elucidate the role of doppel in TEC during tumor angiogenesis, we generated a gain-of-function EC model to express doppel in ECs. Inducible overexpression of doppel in ECs correlates with high vessel formation in a spheroid-based EC transplantation technique. To integrate doppel expression with the canonical angiogenic circuits, we studied the mechanism by which doppel regulates angiogenesis. We evidently observed that doppel co-localized and formed complexes with vascular endothelial growth factor receptor 2 (VEGFR2). Also, doppel prolonged the surface residency of VEGFR2 and amplified the responsiveness of VEGF to VEGFR2. Genetic deletion of doppel in TEC not only depleted membrane residency but also induced internalization and degradation of VEGFR2. This indicates that doppel blocking can lead to effective control of VEGF-signaling in TECs and selective inhibition of tumor angiogenesis. Based on the functional role of doppel in angiogenesis and tumor growth inhibition, we generated doppel knockout mice on immunogenic C57BL6 mice. Tumor growth was inhibited in both hetero (Dpl+/-) and homo (Dpl-/-)-type mice compared to their wild-type littermate controls when syngeneic cancer cell lines including melanoma (B16f10) and lymphoma (EL4) or allogeneic colon cancer CT26 cell line were inoculated. Moreover, we developed doppel targeting monoclonal antibodies. Anti-doppel antibodies, 5C7 and 4D6, were effective against both murine CT26 and human HCT116 colon cancer cell lines. Our studies thus demonstrate the success of doppel targeting in inhibiting pathological tumor growth and establish doppel-VEGFR2 interactions as a specific molecular and therapeutic target in tumoral angiogenesis.

#3278

Dopamine inhibits growth of multiple myeloma.

Chandrani Sarkar, Debanjan Chakroborty, Sujit Basu. _Ohio State University, Columbus, OH_.

Introduction: Multiple myeloma (MM), progressive plasma cell tumor leading to overproduction of monoclonal immunoglobulins, osteolytic bone lesions, renal disease and immunodeficiency, is the second most prevalent hematologic malignancy in the United States. Vascular endothelial growth factor-A (VEGF-A) is a critical cytokine responsible for the induction of angiogenesis in several solid and hematological malignancies like MM. It is likely that VEGF-A induced bone marrow (BM) angiogenesis promotes the survival, growth and proliferation of MM .Therefore targeting BM angiogenesis in MM may be effective for the treatment of MM patients

Objective/Hypothesis: Bone marrow stromal cells (BMSC) are believed to play a central role in progression of MM pathogenesis. Along with other factors, these cells overproduce IL-6 that in turn stimulates growth, survival and progression of MM cells. Since it has been shown that VEGF-A can stimulate IL-6 secretion by these cells and because we have demonstrated that neurotransmitter dopamine (DA) can inhibit VEGF-A functions by acting through its D2 receptors (D2R) and BMSC express DAD2R, we therefore hypothesize that DA or D2R agonists have a role in inhibiting VEGF-A mediated IL-6 secretion by BMSC and thereby, MM progression.

Results: Our results indicate that VEGF-A can significantly increase the secretion of IL6 by isolated BMSC cells in vitro. Addition of DA or D2R agonist, quinpirole, to VEGF-A treated BM cells results in suppression of IL-6 production. However treatment with D2R antagonist, eticlopride, prior to treatment with DA results in abrogation of the effect of DA. Similar effects of DA on VEGF-A induced IL-6 secretion was also observed in HS-5 human BM stromal cells. Our results further indicate that VEGF-A induced VEGFR1 phosphorylation is suppressed in HS-5 cells on treatment with D2R agonist. Furthermore, DA also inhibits the proliferation of MM1.S human MM cells in vitro.

Conclusion: DA may be a new and an effective approach to retard the progression of MM. Since anti-angiogenic monotherapy cannot eradicate the disease completely, strategies using DA with more traditional modalities to eradicate the disease at the time of initial treatment might be beneficial. Finally, DA or its D2R agonists are being used in the clinics for several years for the treatment of other disorders, therefore these inexpensive drugs (in comparison to presently available drugs for the treatment of MM) with known and manageable side effects can be used in the clinics in near future for the treatment of both early and late stage MM.

#3279

**The effect of Erlotinib treatment on human angiogenesis** in vitro **.**

Tanja Milosavljevic, Elise Juge, Russ Guidry, Eugene Woltering. _LSU Health New Orleans, Department of Surgery, School of Medicine, New Orleans, LA_.

Background: Angiogenesis is a process by which new capillaries develop from previously formed venules. Epidermal growth factor receptor (EGFR) is a member of ErbB family of receptor tyrosine kinases (TKs). The EGFR signaling pathway is activated in proliferation, angiogenesis, tumor growth and progression. Overexpression of EGFR in many solid human tumors associates with poor prognosis. One of the effective strategies to target Erb receptors is use of small molecule inhibitors of the TK domain, such as Erlotinib. We hypothesized that inhibiting EGFR signaling by Erlotinib treatment will decrease physiologic and pathologic angiogenesis in an in vitro model system.

Methods: Tissue was embedded in a three-dimensional fibrin-thrombin clot, and evaluated for neovessel growth as per our in vitro Human Angiogenesis Model (HAM). Physiologic angiogenesis was studied in two venous tissues, human placental vein (HPV) and inferior vena cava (IVC). Human liver neuroendocrine tumor (NET) tissue modeled pathologic angiogenesis. All tissues were treated with Erlotinib in a dose response manner (1 µM, 10 µM, 100 µM) and evaluated for three angiogenesis parameters: percent initiation (%I), angiogenic growth (AG), and overall angiogenic response (OAR). Concentrations of angiogenesis-relevant ligand-receptor pairs in supernatant were determined by Human Angiogenesis/Growth panel (Milliplex, EMD Millipore). Effect of Erlotinib treatment on EGF pathway genes expression in IVCs and liver NETs was analyzed by TaqMan EGF pathway array. Angiogenesis data was analyzed for significance using Z-test (Primer) and paired t-test (MedCalc).

Results: Selected dose of Erlotinib [10 µM] consistently achieved statistically significant inhibition of both AG (p<0.0001) and OAR (p<0.0001) in all three tissue types compared to the control. Percent inhibition of %I was below 30% for HPV, IVC and liver NETs. Angiogenic growth was inhibited by 45.65% in HPV (p=0.035), 52.14% in IVC (p<0.0001) and 47.59% in liver NETs (p=0.0107) and OAR by 60% in HPV (p=0.105*), 53.61% in IVC (p<0.0001) and 52.27% in liver NET (p=0.0168). Preliminary ligand-receptor pair concentrations in IVCs and liver NETs show opposing response to Erlotinib treatment. Out of 92 EGF pathway-related genes, only twelve exhibited significant expression level changes [≥2-fold] in both IVC and liver NET Erlotinib-treated samples. Among them, only five genes demonstrated the same direction of change in both tissue types. The CAV2, GAB1, RHOD genes were down-regulated; MUC1 and PIK3c2b genes were upregulated.

Conclusions: Erlotinib is an effective antiangiogenic kinase inhibitor in HPV, IVC and liver NETs in-vitro. It impedes physiologic and pathologic angiogenesis via regulation of neovessel growth rather than initiation of sprouts. Five EGF pathway related genes commonly affected in both physiologic and pathologic models by Erlotinib treatment may be essential modulators of human angiogenesis.

#3280

**Imprime PGG synergizes with anti-angiogenic antibodies to repolarize the immune microenvironment, suppressing xenograft tumor growth** in vivo **.**

Kathryn Fraser, Nadine Ottoson, Xiahong Qiu, Anissa Chan, Adria Jonas, Takashi Kangas, Jeremy Graff, Nandita Bose. _Biothera, Eagan, MN_.

Anti-angiogenic antibodies (Ab) such as bevacizumab (αVEGF) and ramucirumab (αVEGFR2) suppress tumor growth by disrupting the leaky, tortuous vasculature characteristic of growing tumors. Recent work now indicates that these Ab may also promote a shift from an immunosuppressive tumor microenvironment to one more permissive for immune recognition and tumor eradication. These data suggest that combining anti-angiogenic Abs with immunotherapies, particularly those that may also drive repolarization of the immunosuppressive tumor microenvironment, may enhance therapeutic efficacy.

Imprime PGG (Imprime) is a β glucan PAMP (Pathogen Associated Molecular Pattern) that has demonstrated promising efficacy in phase 2 randomized clinical trials with the bevacizumab (bev)-based therapy. Preclinical mechanistic work has shown that Imprime can promote repolarization of the suppressive M2 macrophages and MDSCs that typically reside within the tumor microenvironment. We now show that, when combined with DC101 (murine anti-VEGFR2 Ab), Imprime significantly enhances the inhibition of H441 human NSCLC xenograft tumor growth in athymic nude mice. Moreover, we also show that the combination of Imprime plus DC101 promotes a more pronounced and significant shift in myeloid function than either agent alone. Specifically, mice treated with Imprime plus DC101 had reduced numbers of immunosuppressive, splenic MDSCs and an increase in the number of CD68+F4/80+ cells expressing the critical co-stimulatory marker CD86, indicating an increase in activated splenic macrophages. Tumor associated macrophages from these mice also showed significantly increased expression of CD86. qRT-PCR analyses of these tumor tissues likewise revealed that the combination specifically elicited a profound shift in the polarization state of the microenvironment, increasing M1 markers (TNFα, iNOS, IL-6) and decreasing M2 markers (CD206, IL-10, TGFβ and CCL22). Similarly, in H1299 NSCLC xenograft-bearing mice, the addition of Imprime to bev also elicited a profound shift in the polarization state of myeloid cells. Macrophages and neutrophils from spleen and tumor tissue of mice treated with the combination showed significant upregulation of CD86. Moreover, when compared to mice treated only with bev, splenic MDSCs from Imprime plus bev treated mice showed increased iNOS expression and reduced Arg-1 expression- a shift typifying the M1 polarization state. These data reveal that the addition of Imprime to anti-angiogenic Ab therapy prompts a substantial shift in the tumor immune microenvironment in situ and enhances the efficacy of anti-angiogenic therapy.

### Hematological Microenvironment

#3282

Host-derived MMP-13 mediates multiple myeloma-induced osteolysis.

Chen Hao Lo, Gemma Shay, Conor C. Lynch. _H. Lee. Moffitt Cancer Center, Tampa, FL_.

Myeloma cells promote osteolysis and suppress osteogenesis in the bone microenvironment by regulating osteoclasts (OCL) and osteoblasts (OBL), respectively. Patients often suffer painful, osteoporotic bone status as diseases progresses. Bone consists of 90% type-I collagen, which is degraded by OCL derived cathepsin K during regular bone remodeling. While bisphosphonates delay pathological fracture, they have little impact on overall survival; therefore, development of new therapies is crucial. Matrix metalloproteinase-13 (MMP-13) is an enzyme involved in type-I collagen degradation and resorption. Immunohistochemical staining of a panel of human myeloma biopsies (n=15) show high MMP-13 expression predominantly in bone stroma and to a lesser extent in the myeloma compartment. Consistent with previous reports, MMP-13 expression localizes to mesenchymal stromal cells (MSC) and OBLs rather than OCLs. Further supporting these observations, analysis of publically available data sets revealed upregulation of MMP-13 expression in MSCs co-cultured with myeloma cells (1.81 LogFC, p<0.05). Based on this rationale, we examined whether stromal MMP-13 plays a role in myeloma progression.

To test our hypothesis, we generated immune-compromised MMP-13-null mice on a C57BL/6 background for ex vivo and in vivo studies. Mouse myeloma cell line, 5TGM1, homes to the skeleton and progresses upon tail vein injection in this model. Our in vivo studies showed that 5TGM1-bearing MMP-13-null mice had significantly improved overall survival compared to wild-type controls (Mean 39 vs. 43 days; p<0.05). Interestingly, we detected no difference in tumor burden between wild-type and MMP-13-null groups, by bioluminescence or IgG2b quantification. We observed significantly less bone degradation in the MMP-13 null mice compared to controls as determined by μCT and histomorphometry. Surprisingly, however, tartrate-resistant acid phosphatase (TRAP) staining in bone sections showed no difference in OCL numbers and size in vivo, but ex vivo OCL differentiation cultures demonstrated fewer (Mean 89 vs. 16; p<0.05) and smaller (Mean 29 vs. 20 μm; p<0.05) OCLs in the MMP-13-null group. Furthermore, we note that OCLs derived from MMP-13-null bone marrow have a reduced capacity to resorb bone mimetic (24% reduction; p<0.05). Time course analyses indicate OCL formation is delayed in MMP-13-null cultures, and may partially explain observed differences between groups in vivo. Mechanistically, we are assessing MMP specific cross-linked carboxy-terminal telopeptide of type-I collage (ICTP) fragments in sera of tumor-bearing MMP-13-null and wild-type animals. Taken together, the data suggests that host stroma-derived MMP-13 contributes to myeloma progression and bone turnover, by regulating the formation and functionality of OCLs. The outcomes of our study provide rationale for the use of MMP-13 specific inhibitors in the treatment of multiple myeloma.

#3283

APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment.

Yu-Tzu Tai,1 Chirag Acharya,1 Gang An,1 Michele Moschetta,1 Mike Y. Zhong,1 Xiaoyan Feng,1 Michele Cea,1 Antonia Cagnetta,1 Kenneth Wen,1 Hans van Eenennaam,2 Andrea van Elsas,2 Nikhil Munshi,1 Kenneth Anderson1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _BioNovion, Oss, Netherlands_.

Elevated B cell maturation antigen (BCMA) and its ligand a proliferation-inducing ligand (APRIL) may directly advance human multiple myeloma (MM) malignancy in vivo. Here, we first show that BCMA downregulation potently diminishes viability and colony formation of MM cells while BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades and molecules essential for proliferation and anti-apoptosis. Importantly, BCMA itself forces accelerated tumor growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly enhanced CD31/microvessel density and VEGF than paired control tumors. Concurrently, these tumors express increased factors crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as immune inhibition including PD-L1, TGFβ and IL-10 which further triggers 38 IL-10 signaling molecules. In parallel, these identified target genes are induced by paracrine APRIL binding to BCMA in MM cells and they are potently blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells protected by abnormal bone marrow (BM) myeloid cells, i.e., osteoclasts, macrophages, and plasmacytoid dendritic cells. It further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. It prevents in vivo MM cell growth within implanted human bone chips in SCID mice. 01A-inhibited MM cell viability induced by osteoclasts is further enhanced by lenalidomide and bortezomib. Taken together, these results further characterize new molecular mechanisms of APRIL/BCMA-induced in vivo MM progression and immunosuppressive BM microenvironment, strongly supporting targeting this prominent pathway in MM.

#3284

Targeting E-selectin/CXCR4 with GMI-1359 effectively mobilizes bone marrow leukemia cells and enhances FLT3 inhibitor-induced anti-leukemia efficacy in a murine acute myeloid leukemia model.

Weiguo Zhang,1 Hong Mu,1 Qi Zhang,1 Nalini B. Patel,1 William E. Fogler,2 John L. Magnani,2 Michael Andreeff1. 1 _UT M.D. Anderson Cancer Center, Houston, TX;_ 2 _GlycoMimetics, Inc, Rockville, MD_.

FLT3 internal tandem duplication (ITD) mutations are the most common alterations in acute myeloid leukemia (AML) and are associated with poor disease prognosis. Targeted therapy using FLT3-ITD inhibitors showed limited effect in reducing leukemia blasts in bone marrow (BM) than that in peripheral blood. The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents. Therefore, blocking CXCR4 and E-selectin concomitantly with FLT3 inhibition could theoretically eliminate the protection in FLT3-mutant AML patients. We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 showed significant prolongation of survival of mice engrafted with FLT3-ITD mutant MV4-11 leukemia cells. In the present study, we further investigated anti-leukemia effects of dual CXCR4/E-selectin inhibition. Results indicate efficient mobilization of leukemia cells into the circulation by GMI-1359 in a MOLM14-engrafted murine model 2h after drug treatment, which was 3.3-fold (± 0.3) higher compared with the CXCR4 antagonist plerixafor, and 7.4-fold (± 2.7) higher compared with controls. In addition, GMI-1359 also mobilized normal murine leukocytes from the BM, suggesting that GMI-1359 may block interactions of leukemia cells with various BM components. Combination therapy of GMI-1359 and sorafenib significantly reduced leukemia burden (1.9e7 vs. 2.3e9, 1.0e9 and 8.5e7 photons/sec in the combination group versus control, GMI-1359 and sorafenib groups, respectively, at day 20 as determined by Xenogen IVIS bioluminescence Imaging; p < 0.001). Moreover, the combination treatment profoundly reduced leukemia burden in the BM of leukemic mice after a 15-day treatment (1.7e7 vs. 1.6e9, 6.3e8 and 7.0e7 photons/sec, p < 0.01; and 1.76% vs. 70.96 %, 72.8% and 33.8%.leukemia cell engraftment measured as GFP positive leukemic cells in BM by flow cytometry, in the combination group vs. vehicle, GMI-1359 and Sorafenib groups, respectively, p < 0.01). Of note, the combination therapy profoundly extended the median survival of the leukemia-bearing mice from 27 days in the control group to 46.5 days in the combination treatment groups (p < 0.001). Mechanistically, we demonstrated that direct blockade of the interaction of E-selectin-CXCR4/SDF-1α with GMI-1359 was critical for leukemic cell mobilization via the disruption of adhesion to stromal cells. Furthermore, the presence of GMI-1359 enhanced sorafenib-induced apoptosis in FLT3-ITD mutated leukemic cells co-cultured with stromal cells. This effect occurred even under hypoxia, which is characteristic for the leukemic BM microenvironment. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3-mutant AML.

#3285

HTLV-1 viral oncogene Hbz induces leukemia with osteolytic bone involvement in mice.

Alison Esser,1 Dan Rauch,1 Jingyu Xiang,1 John Harding,1 Nicole Kohart,2 Patrick Green,2 Stefan Niewiesk,2 Thomas Rosol,2 Lee Ratner,1 Katherine Weilbaecher1. 1 _Washington University in St. Louis, St Louis, MO;_ 2 _Ohio State University, Columbus, OH_.

Adult T-cell leukemia/lymphoma (ATL) develops in a subset of patients infected with the HTLV-1 virus. Most ATL patients become refractory to chemotherapy and have a median survival time of 6 months. Although uncommon in hematologic malignancies, 80% of ATL patients develop osteolytic lesions and hypercalcemia of malignancy. Bone resident and metastatic tumors release paracrine factors that modulate the bone microenvironment to facilitate disease progression and decrease survival. HTLV-1 encodes 2 viral oncogenes, Tax and Hbz. Tax is critical to ATL development and regulates tumor growth and proliferation in part through trans-activation of NFκB and CREB. We have previously shown Tax expression driven by the Granzymbe B promoter is sufficient for the development of leukemia/lymphoma with osteolytic lesions and hypercalcemia. We and others have shown that Tax alters the expression of paracrine factors that modulate the bone microenvironment through effects on bone forming osteoblasts (OB) and bone resorping osteoclasts (OC). Tax is expressed in early lymphocyte transformation with low expression in advanced ATL. HBZ is expressed early in lymphocyte transformation and throughout ATL progression. We hypothesize that in ATL cells, HTLV-1 viral oncogenes Tax and Hbz cooperate to modulate bone metabolism in a paracrine manner to enhance ATL tumor growth and progression. Mice with Granzyme B driven Hbz expression (T and NK cells) develop leukemia/lymphoproliferative disease in lymph nodes correlating with increased spleen weight. We found that lymphoproliferative disease is also present in the bone marrow. Hbz mice have decreased trabecular bone at 18 months by microCT and radiographic analysis. These data suggest Hbz can alter bone metabolism. Future studies will define the effects of Hbz on bone formation, OB and OC specific effects and tumor progression. Understanding HTLV-1 oncogene modulation of the bone microenvironment will uncover critical pathways in tumor/bone cross talk enabling the development of novel targeted therapies for ATL patients.

#3286

Targeting mir101/ EZH2/NFkb axis by FGFR1 inhibitor in mantle cell lymphoma.

Lalit Sehgal, Neeraj Jain, Tamer Khashab, xin wang, Sattva Neelapu, Felipe Samaniego. _UNIVERSITY OF TEXAS MD ANDERSON CANCER CENTER, HOUSTON, TX_.

Mantle cell lymphoma is classified as distinct non-Hodgkin's lymphoma subtype characterized by the t(11;14)(q13;q32) translocation, which results in overexpression of Cyclin D1. The clinical presentation often includes extra-nodal involvement, of the bone marrow and gut. The prognosis of patients with mantle cell lymphoma (median overall survival, 3-5 years) is poorest among B-cell lymphoma patients. However, MCL pathogenesis has been delayed until the recent development of a tissue culture system, using stromal cells and adipocytes, suitable for propagating primary MCL cells. We hypothesized that tumor microenvironment might activate survival-signaling pathways essential for survival and maintenance of MCL, and targeting the identified signaling pathways might be an ideal for designing of curative treatment strategies. We developed an in-vitro culture system, mimicking bone marrow microenvironment that can be exploited to understand the biology of MCL resistance to death pathways. Primary MCL cells grown in co-culture with bone marrow stromal cells grew faster than cells cultured alone. We identified the soluble factors responsible for survival of MCL cancer stem cells (MCL-IC) that can be targeted to prevent relapse. We found the IL6 secretion triggered FGF-2 autocrine loop in MCL cells contributing to cell survival and growth by regulating mir101/EZH2/NFkb axis. The death signaling pathways inactive in MCL are restored upon therapeutic intervention by specific pathways inhibitors. In conclusion, we delineated three different signaling arm regulated in MCL cells to promote cell survival and resistance. We defined the factors supporting MCL and MCL-ICs survival essential for forming new targeting treatment strategies. To overcome MCL resistance to death pathways we have outlined the hMSCs-mediated support of MCL and identified drug targets to sensitize MCL to apoptosis.

#3287

Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas.

Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. _Division of Hematology, Mayo Clinic, Rochester, MN_.

BACKGROUND

Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function.

METHODS

Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry.

RESULTS

Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue.

CONCLUSIONS

We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies.

#3288

The chemokine receptor CXCR4 and the cannabinoid receptor CB2R form heterodimers in non-Hodgkin lymphoma (NHL) and solid tumors leading to functional crosstalk.

Martina Guerrero,1 Rosa Griera,2 Maria Perez,2 Gaël Roué,1 Joan Bosch,2 Peter J. McCormick,3 Jordi Camps,1 Patricia Perez-Galan1. 1 _IDIBAPS, Barcelona, Spain;_ 2 _University of Barcelona, Barcelona, Spain;_ 3 _University of East Anglia, Norwich, United Kingdom_.

Background: A major hallmark of cancer cells, including both solid tumors and hematological neoplasias, is the ability to disseminate and generate distant/distal metastasis.Two families of proteins that play a role in dissemination and metastasis are the cannabinoid and the chemokine receptors. Moreover, cannabinoids also impair uncontrolled cancer cell growth and induce apoptosis. Both receptors are members of the G-protein coupled receptor (GPCR) super family, and may combine among themselves to generate new and unique biochemical and functional characteristics. We have focused our study in the chemokine receptor 4 (CXCR4), overexpressed in several human cancer types and associated with an aggressive phenotype, and in the cannabinoid receptor 2 (CB2R). Recently, it has been reported that CB2R forms heterodimers with different receptors such as CB1R and HER2. In the case of CB2 with HER2, the co-expression and the formation of these heterodimers correlate with poor prognosis in breast cancer.

Aim: To characterize CXCR4 and CB2 expression and heterodimer formation in non-Hodgkin lymphoma (NHL), colorectal cancer (CRC) and pancreatic cancer, together with their role in tumor cell dissemination and proliferation.

Results: We have characterized the expression of CXCR4 and CB2 in several NHL models (Chronic Lymphocytic Leukemia (CLL), Follicular Lymphoma (FL), Mantle Cell Lymphoma (MCL) and Diffuse Large B-Cell Lymphoma (DLBCL)), colorectal cancer (CRC) and in pancreatic cancer. Flow cytometry analysis showed high CXCR4 expression in most of the cell lines, while CB2R expression was moderate. Noteworthy, hypoxia increased the expression of both CXCR4 and CB2. Preliminary immunohistochemistry results using primary tumors and their matched metastasis indicate that CXCR4 and CB2R expression is heterogenous. By means of Proximity Ligation Assay (PLA), we have uncovered that the formation of CXCR4-CB2R heterodimers in NHL, CRC and pancreatic cancer models. Moreover, we have confirmed that these heterodimers modulate pathway activation and function of both receptors. In this sense, using the CRC cell line SW480 and p42/p44 ERK phosphorylation a as read-out, we have proved that CXCR4 pathway activation by its agonist CXCL12 is decreased by co-treatment with the CB2R antagonist JTE907. Conversely, CB2R activation by its agonist JWH133 is hampered by the CXCR4 antagonist AMD3100. Furthermore, CB2R blockade by JTE907 interferes with CXCL12-induced cell migration assessed by wound healing assay.

Conclusions: We have described for the first time the formation of CXCR4-CB2 heterodimers in NHL, CRC and pancreatic cancer models together with its implications in downstream receptor crosstalk and cellular function. These results establish the proof-of-concept to target CXCR4-CB2 heterodimers as a new therapeutic intervention in these neoplasias.

#3289

Microenvironment modulation and enhancement of cytotoxic therapy by the heparanase inhibitor Roneparstat against human B-non Hodgkin lymphomas.

Massimo Di Nicola,1 Franco Zunino,1 Anna Rossini,1 Giusi Ruggiero,1 Micheleandrea De Cesare,1 Denis Cominetti,1 Monica Tortoreto,1 Cinzia Lanzi,1 Giuliana Cassinelli,1 Roberta Zappasodi,1 Claudio Tripodo,2 Nadia Zaffaroni1. 1 _Istituto Nazionale Tumori, Milan, Italy;_ 2 _University of Palermo, Palermo, Italy_.

Background: The standard chemotherapy treatment for Non-Hodgkin lymphoma (NHL) consists in the combination of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). Because of the relevant treatment-related toxicity and the minor therapeutic effectiveness of CHOP therapy variants, the development of novel therapeutic approaches represents an urgent clinical need. Despite treatment with the anti-CD20 monoclonal antibody Rituximab and CHOP has led to favourable results for CD20+ lymphoma, many patients experienced drug resistance. Based on studies indicating that the malignant behaviour of tumors depends on the interactions between tumor and its microenvironment, we tested the effect of the novel heparanase inhibitor Roneparstat (sigma-tau Research Switzerland) in combination with various clinically relevant agents against B-cell lymphoma models, poorly responsive to conventional agents.

Methods: We assessed the CD20 cell surface expression by flow cytometry, the heparanase (HPA-1) expression levels by immunoblotting, the VEGF spontaneous release in cell supernatant by proteome array in B-NHL models. The therapeutic activity was evaluated in SCID mice xenografted with CD20+ and VEGF+ B-NHL models. The efficacy of the drug treatment was estimated as tumor volume inhibition percentage (TVI %), log10 cell kill (LCK) and complete regression (CR). To gain insight the mechanisms underlying the anti-tumor activity of Roneparstat plus rituximab histopathological/immunoistochemical analyses were performed on treated tumors versus controls.

Results: In a model of aggressive diffuse large B-cell NHL, except for doxorubicin, the combinations cyclophosphamide, dexamethasone and rituximab exhibited significantly superior efficacy over single-agent therapy. The most impressive enhancement of anti-tumor activity was observed with Roneparstat plus rituximab, with a high rate of complete tumor regressions and no evidence of disease at the end of the experiment in 50% of treated animals. Histological analysis revealed inflammatory cell infiltration, stromal destructuration, most pronounced apoptotic changes in Roneparstat plus rituximab-treated tumors versus controls; stromal architecture analysis showed signs of an altered, incomplete reticulin network suggestive of impaired stromal scaffolding that might have promoted the recruitment of complement components (C1q, C5) within tumors increasing the cytotoxic effect of rituximab. This interpretation was also supported by the favourable interaction of Roneparstat with bevacizumab in a Burkitt's lymphoma model characterized by high levels of VEGF..

Conclusion: Our results provide evidence that targeting the tumor microenvironment with Roneparstat may have therapeutic potential in combination with various agents against aggressive lymphoma subtypes.

#3291

The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions.

Gabriela Pavlasova,1 Marek Borsky,2 Vaclav Seda,1 Katerina Cerna,1 Jitka Osickova,2 Michael Doubek,2 Yvona Brychtova,2 Jiri Mayer,2 Sarka Pospisilova,1 Matthew S. Davids,3 Jennifer R. Brown,3 Marek Mraz1. 1 _CEITEC MU, Brno, Czech Republic;_ 2 _Department of Internal Medicine - Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic;_ 3 _Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA_.

The introduction of anti-CD20 antibodies has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin lymphomas. The aim of this study was to analyze molecular pathways that influence expression of CD20 since this is largely unknown. This is of a great clinical interest, as combinatorial therapy of novel BCR-signaling inhibitors ibrutinib and idelalisib currently focuses mainly on the use with anti-CD20 antibodies.

Firstly, we analyzed samples obtained from CLL patients treated with ibrutinib and showed that administration of ibrutinib in vivo leads to CD20 down-modulation (P<0.0001). That implies that CD20 expression might be regulated by a yet unknown mechanism in the context of immune niches. Therefore, we focused on determining the effect of microenvironmental interactions on CD20 expression on malignant B cells. We co-cultured primary CLL cells with the bone marrow stromal cell line HS-5. This revealed that CLL cells co-cultured with HS-5 had higher surface CD20 expression compared to control cells cultured on plastic (P<0.01). Moreover, CLL cells that have been recently released from the lymph node microenvironment to the peripheral blood (CXCR4dimCD5bright) had ~2-fold higher surface CD20 expression when compared to CLL cells circulating in blood of the same patient for a longer time (CXCR4brightCD5dim cells; P<0.0001). Further, sorted CXCR4dimCD5bright CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002) suggesting that changes in CD20 expression were rather due to changes in its mRNA levels than surface modulation. As CD20 expression was decreasing with the transition from CXCR4dim to CXCR4bright CLL cells, we hypothesized that the CXCR4/SDF-1 axis is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 produced by stromal cells, significantly up-regulated surface CD20 (P<0.05). On the contrary, the treatment with plerixafor, CXCR4 antagonist, inhibited this SDF-1α mediated up-regulation of CD20 which proves that SDF-1α binding to CXCR4 is directly involved in CD20 regulation.

Altogether, the CD20 levels are up-regulated within the context of microenvironmental interactions through the CXCR4/SDF-1 axis, and the impairment of microenvironmental interactions mediated by ibrutinib down-regulates CD20 expression. This study reveals a novel regulation of CD20 expression in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination.

Supported by: SoMoPro II-no.4SGA8684; NGS-PTL(306242); EHA Fellowship award; Ministry of Health of CR (16-29622A); Academy of Sciences of CR (16-13334Y); Ministry of Education, Youth and Sports, grant LD15144 (COST CZ); MUNI/A/1028/2015; CZ.1.05/1.1.00/02.0068; G.P. is supported by Ostrava city.

#3292

Manipulating osteoblast differentiation in order to inhibit protection of AML cells within the bone marrow.

Rosalie Sterner, Kimberly Kremer, Amel Dudakovic, Scott Kaufmann, Jennifer Westendorf, Andre van Wijnen, Karen Hedin. _Mayo Clinic, Rochester, MN_.

While standard chemotherapeutics can initially induce remission in patients with acute myelogenous leukemia (AML), patients often relapse in part due to the protective bone marrow microenvironment, which promotes the survival of AML cells. Using an in vitro cell culture model of the bone marrow, our lab recently showed that differentiating osteoblasts potently protect AML cells from apoptosis. In addition, we found that differentiating osteoblasts treated with histone deacetylase inhibitors (HDACi) fail to protect AML cells in this model. Since HDACi decreased the expression of genes required for osteoblast differentiation, we hypothesize that blocking specific stage(s) of osteoblast differentiation may make AML cells within the bone marrow more susceptible to chemotherapy. We are currently disrupting specific osteoblast differentiation stages via molecular or therapeutic drug manipulation and exploring the impact on AML cell survival both in cell culture models of the bone marrow microenvironment and in a mouse model of AML. The results of our studies will characterize novel bone marrow molecular and cellular interactions that promote the survival of AML cells. Targeting these interactions in combination therapies may more completely eradicate the AML cell reservoir and better prevent relapse.

#3293

The TLR-2 signal pathway is enhanced in myeloproliferative neoplasm - related to the increased inflammatory cytokine and pathogenesis.

Guanfang Shi, Maryna Yarotska, Hui chen, Cherif Abdelmalek, Ching Wong, Madhumati Kalaovar, VLadimir Gotlieb, Jen Chin Wang. _Brookdale University Hospital, Brooklyn, NY_.

Introduction

Although MPNs have been regarded as stem cell disease. Recent evidence suggest that chronic inflammation induces a state of chronic oxidative stress with elevated reactive oxygen species (ROS) and then induce alternation in epigenetic changes or DNA mutations.. Also, MPNs are associated with constitutional symptoms and high levels of circulating inflammatory cytokines. The etiology of this increase in inflammatory cytokines remains elusive, we investigated the role of TLRs (Toll-like receptors) and related cytokines in MPN as they are known to play a role in innate and adaptive immunity in inflammation.

Materials and Methods:

TLR assay: TLR-2, 4, 7, 9 quantification was performed by TLR staining of 5×105 mononuclear cells (MNC) which were incubated with 5 μg/ml of conjugated TLR-2, 4, 7, 9 antibody and assayed by flow cytometry.

Monocyte-derived dendritic cells (mdDC) and Inflammatory Cytokines.

mdDC were generated from isolated monocytes (anti-CD14-coated magnetic beads) then by incubation with IL-4 and GM-CSF for 4 days. Immature mdDC (1×104/well) were either left untreated or stimulated with Pam3CSK4, in 96 well plates. After 48h incubation, cells were stained with fluorescence-labeled mAb specific to CD80, CD83 (BD Biosciences). Cytokine levels were determined in supernatants harvested after 48h using the MSD (Meso Scale Discovery) System.

Results. 1) Levels of TLR-2 were significantly elevated in patients with MPNs especially in those with polycythemia vera and essential thrombocythemia, but only marginally elevated in myelofibrosis patients. In all patient subsets, TLR-4, 7, 9 levels did not differ significantly from controls. 2) Maturation of mdDC were done by stimulation of TLR-2 ligand, Pam3CSK4. Higher numbers of CD83+ cells were generated more from patients with elevated TLR levels, confirming these MPN patients have elevated TLR-2 levels which portend the ability to generate more DC by TLR-2 ligand. 3) In patients with elevated TLR-2 level, Pam3CSK4 stimulated more cytokines including IFNγ, IL12p70, TNFα, IL6, IL8, IL10, and IL13 than those patients with low levels of TLR-2. 4). Moreover, patients with elevated TLR-2 levels have more of IL8, TNFα, IL13, IL6, in the plasma, consistent with inflammatory cytokines which is a likely consequence of elevated TLR-2.

Conclusion. TLR-2 and not TLR-4, 7, 9 were found to be significantly elevated in MPNs. Stimulation of the TLR-2 ligand promotes the generation of more mature DCs , greater inflammatory cytokines production and Higher plasma inflammatory cytokines are associated with patients with higher TLR-2 levels. We conclude that TLR-2 is important in the production of inflammatory cytokines in MPNs and this evidence suggests that MPNs are in a TLR induced chronic inflammatory state and may play a significant role in the pathogenesis of MPNs. 

### Microbiome in Cancer

#3294

Treponema denticola, a periodontal pathogen, promotes stemness and migration in oral squamous cell carcinoma.

Pachiyappan Kamarajan, Islam Ateia, Jae M. Shin, J Christopher Fenno, Yvonne L. Kapila. _Univ. of Michigan, Ann Arbor, MI_.

Periodontitis is a chronic infectious disease characterized by inflammation and destruction of periodontal tissues including the periodontal ligament fibers and alveolar bone. Recent epidemiological studies have revealed a significant association between periodontitis and oral cancer. While the precise mechanisms that mediate these associations are not well understood, periodontal pathogens, including Treponema denticola (T. denticola), which are believed to initiate the destructive inflammatory responses and dysbiosis or dysregulation of tissue homeostasis that characterize periodontal disease may contribute to oral cancer. However, knowledge about T. denticola's contribution to oral cancer is limited. Previously, we showed that nisin ZP, a bacteriocin and commonly used food preservative, reduced tumorigenesis in vivo and long term treatment with nisin ZP extended survival. In a separate study, we further showed that nisin ZP exhibits antimicrobial and antibiofilm effects, limiting T. denticola viability. The antimicrobial doses of nisin ZP are two orders of magnitude lower than the antitumor doses. The present study investigated the impact of T. denticola on the stemness, migration, and tumorigenesis of oral squamous cell carcinoma (OSCC), and nisin's potential modulatory effects on these processes. To investigate the role of T. denticola on OSCC tumorigenesis, OSCC cells were treated with T. denticola and with or without nisin ZP then assayed for stemness (orasphere) and migration. Treatment with T. denticola enhanced OSCC orasphere formation and migration without affecting cell viability or inducing apoptosis. Addition of nisin ZP inhibited these T. denticola-mediated processes. These data indicate that the periodontal pathogen T. denticola promotes stemness and migration of OSCC cells, and thereby may contribute to oral cancer tumorigenesis.

#3295

Microbiome profiling and immunological characterization on gastrointestinal tract tumors.

Chao Zhang,1 Kyle Cleveland,1 Felice Schnoll-Sussman,1 Bridget McClure,2 Michelle Bigg,2 Nikolaus Schultz,3 Manish A. Shah,1 Doron Betel1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _New York-Presbyterian Hospital/Weill Cornell Medical Center, New York, NY;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Motivation: Conservative estimates are that around 20% of all malignancies may be attributed to microbiota. Especially in the gastrointestinal (GI) tract, infection can cause chronic inflammation and further influence immune response to create a proinflammatory microenvironment for cancer development. This can account in part for the reason that the immunotherapy in colon cancer is less efficacious than in non-GI tract cancers. We proposed a systematic computational study to profile the microbiome from next generation sequencing of gastric biopsy samples, and evaluate the relationships among microbiome composition, host immune response and genomic characterization.

Method: Patients undergoing upper endoscopy without chronic inflammatory disease or chronic NSAID use were eligible for participation, including cancer/non-cancer, Helicobacter pylori positive and negative patients. In total, 62 endoscopic biopsies from 3 locations in the gastric antrum/body were freshly frozen for whole genome sequencing and transcriptome analysis. In addition, TCGA whole genome sequencing (WGS) data from 37 gastric cancer, 102 colon cancer, and 46 rectal cancer samples were analyzed for microbiome composition.

Results: H. pylori was detected in all positive infection gastric biopsy samples by our pipeline, and the results were successfully validated by qPCR. Comparing to other traditional bacterial identification methods, our pipeline shows a strong capacity for profiling microbiome from low bacterial content biopsy samples. We observed a high positive correlation between the H. pylori content and total non-H. pylori bacteria content. And we also identified significantly differentially expressed immune-related genes between active H. pylori infection and negative samples. Finally, we analyzed collected TCGA WGS data, and identified the dominant species from the different cancer studies, such as Bacteroides fragilis in rectum adenocarcinoma, and Fusobacterium nucleatum in colon adenocarcinoma. The correlation among microbiome composition, the expression of immune-related genes and cancer genomic characterization were also demonstrated in this study.

#3296

Breast mammographic density and the fecal microbiota.

Gieira S. Jones,1 Heather S. Feigelson,2 Jacques Ravel,3 James J. Goedert1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Kaiser Permanante Colorado, Denver, CO;_ 3 _University of Maryland School of Medicine, Baltimore, MD_.

The microbial population (microbiota) of the distal human gut has multiple functions that modulate cancer risk including digestion of nutrients, deconjugation and enterohepatic circulation of hormones and other bile acids, and regulation of immunity and inflammation. We have previously reported that postmenopausal breast cancer cases, compared to controls, have lower fecal microbiota alpha diversity, independent of age, body mass index, and estrogen levels, and that the cases' microbiota is compositionally altered. For deeper understanding of these associations with breast cancer, we compared microbiota metrics to a strong cancer risk factor, breast density, among 48 cancer-free, postmenopausal controls in the Breast and Colon Health study (86% non-Hispanic White, mean age 62 years). In routine screening mammograms, they had the following Breast Imaging Reporting and Data System (BI-RADS, 5th ed.) composition: a) almost entirely fatty (N=4); b) scattered areas of fibroglandular density (N=14); c) heterogeneously dense (N=26); and d) extremely dense (N=4).

As shown in the Table, simple linear regression across these four ordered BI-RADS categories revealed that there was no significant association with fecal microbiota alpha diversity (PD_tree β -0.08, P= 0.56). Breast density in these women also was not associated with microbiota beta diversity (composition) by MiRKAT analysis of un-weighted and weighted UniFrac metrics (P= 0.25 and 0.08, respectively).

We conclude that breast density is unlikely to mediate the association of the gut microbiota with breast cancer in postmenopausal women.

Fecal alpha diversity by mammographic density category

---

BI-RADS Density | No. | Mean (SD) PD_Whole_Tree

Almost Entirely Fatty | 4 | 36.06 (4.22)

Scattered Fibroglandular | 14 | 38.18 (8.25)

Heterogeneously Dense | 26 | 37.19 (4.88)

Extremely Dense | 4 | 38.33 (7.99)

#3297

Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition.

Ishfaq Ahmed,1 Badal Roy,1 Rita-marie McFadden,2 Shrikant Anant,1 Seth Septer,3 Shahid Umar1. 1 _Kansas University Medical Center, Kansas City, KS;_ 2 _University of Kansas, Kansas City, KS;_ 3 _Children's Mercy Hospital, Kansas City, MO_.

Background. Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. Employing Citrobacter rodentium (CR) as a model system to mimic the pathogenic mechanisms of Enteropathogenic and Enterohaemorrhagic E. coli, we showed previously that a functional cross-talk between the Notch and Wnt/β-catenin pathways regulates hyperplasia and/or colitis following CR infection. Aim: To test the hypothesis that CR infection combined with chronic Notch inhibition will alter the composition of the colonic mucus to promote dysbiosis, tight junction disruption, loss of multipotent intestinal stem cells (ISCs) and colitis. Material/methods: Immune-competent NIH:Swiss outbred mice were infected with CR (108 CFUs) and treated with Notch blocker Dibenzazepine [DBZ, i.p. @ 10µmol/Kg body weight]. Mucus composition, fecal 16S rDNA analysis and components of TJ integrity were measured and correlated with changes in components of the Notch pathway. 6% Pectin diet served as a SCFA-delivery system to the colon. Results: In the colons of CR-infected/DBZ-treated mice, mucus analysis revealed dramatic alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected alone that preceded alterations in 16S rDNA microbial community structure. Indeed, mucin degrading and colitogenic bacterium, Akkermansia muciniphila exhibited dramatic increases in the feces of CR-infected and DBZ-treated mice. At the same time, colons from CR-infected/DBZ-treated mice had decreased expression of antimicrobial peptides Angiogenin-4, Retnlβ, Intelectin-1 and Intelectin-2, respectively. Expression levels of both TJ and adherens junction proteins (e.g., Claudin-5, ZO-1, ZO-2, E-Cadherin, β-catenin) also decreased significantly in the colonic crypts of CR-infected/DBZ-treated mice that paralleled loss of Notch signaling. Both C3H/HeN mice that exhibit exaggerated response to CR infection and T- and B-cell deficient Rag-1 mice following CR-infection and DBZ-treatment, exhibited dramatic increases in paracellular permeability, goblet cell metaplasia and exacerbation of colitis. Chronic blockade of the Notch pathway depleted colonic epithelium of ISC markers Dclk1 and Lgr5 concomitant with exacerbation of colitis. 6% Pectin diet ameliorated colitis by restoring barrier integrity via recruitment of Bacteroides vulgatus, Streptococcus gordonii and Lactococcus lactis, bacteria with potential anti-inflammatory functions. Conclusions: 1. These studies help us delineate the mechanistic basis of colitis development in the aftermath of chronic Notch inhibition. 2. Our findings also caution against the use of Notch inhibitors for patients suffering from colitis-associated colon cancer.

#3298

Distinct microbiome populations within breast cancer microenvironments.

Kevin J. Thompson, Jason Sinnwell, Xiaojia Tang, James N. Ingle, Matthew P. Goetz, Krishna R. Kalari. _Mayo Clinic, Rochester, MN_.

INTRODUCTION:

The tumor microenvironment consists of a complicated interplay between a heterogeneous mixture of tumor cells and the surrounding tissue and there is a burgeoning interest in the role of the tissue microbiota within this microenvironment. Our group has investigated the breast tumor microbiome using the unaligned (non-human) reads from 796 RNASequencing data obtained from The Cancer Genome Atlas (TCGA) repository of Breast Cancer.

METHODS:

Unmapped sequences for 720 TCGA tumor samples and 76 paired normal samples were analyzed with Kraken v0.10.5 using the RefSeq 16S and standard genomes. To minimize false discoveries Kraken results were filtered to species that have bacterial genomic and 16S read support. Cluster analysis of the T-distributed Stochastic Neighbor Embedding (tsne) was performed and consensus voting of 26 maximum scoring metrics identified 3 distinct clusters. Differential abundance analysis was performed with the edgeR package. Covariate association analysis was performed for tumor clinical characteristics, as well as sample heterogeneity characteristics.

RESULTS:

There were three microbiome clusters we identified using TCGA breast samples. The average silhouette width of the third cluster was deemed statistically insignificant (0.071), suggesting further stratification of this cluster is necessary. The remaining two TCGA clusters (467) shared an average silhouette width of 0.39, using Bray's dissimilarity index. Differential abundance analysis of the two clusters after Bonferroni correction identified 450 significant species. The biomes were represented by 63 triple negative, 93 HER2+, 258 Luminal, and 50 tumor adjacent normals. Concordant cluster assignment was observed in 93.7 +/- 7.3% of the normal-tumor pairings, averaged across clinical subtypes. We observed clear delineation of biome populations at the order taxonomic level, which was not associated with the clinical subtype or the tumor/tumor adjacent pairings. We found enrichment of Burkholderiales in the first biome and enrichment of Bacillales and Lactobacillales within the second biome. The second biome appears to be deplete of representatives from the Asian population (p value 0.005) and survival associations were observed to be significantly reduced for HER2 patients associated with Biome 1 (p value 0.025). Geneset enrichment analysis of the expression signatures indicated drug metabolism and chemical carcinogenesis pathways to be associated with biome 2.

CONCLUSIONS:

In our study, we observed two significantly different microbiomes among TCGA breast cancer samples that were not associated with the clinical subtypes. Current efforts include validating these biomes, using 16S sequencing on matching fresh frozen paraffin embedded samples. Additionally, we are evaluating the influence of the biomes in independent adjuvant and neo-adjuvant studies with next generation sequencing data.

#3299

Bacterial DNA integrations in the genomes of gastric tumor and adjacent samples.

Kelly Robinson,1 Nikhil Kumar,1 Javier Torres,2 Julie C. Dunning Hotopp1. 1 _Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD;_ 2 _Unidad de Investigación en Enfermedades Infecciosas, UMAE Pediatría, IMSS, México City, Mexico_.

There are 10 times more bacterial cells in the human body than human cells, and various bacteria are known to influence carcinogenesis. While viral DNA integrations in the human genome have been shown to promote carcinogenesis, bacterial DNA integrations (BDI) into the human genome are rarely investigated. Our previous analysis of publicly available sequencing data from the Cancer Genome Atlas showed BDIs from Pseudomonas spp. rRNA into the 5'-UTR of four proto-oncogenes as well as in Ig (the immunoglobulin gene) of gastric cancer samples. However, we were left with many unanswered questions when we were unable to obtain the materials required to validate our findings. Therefore, we sought to sequence a different cohort of gastric cancer patients in order to identify BDIs. Whole exome sequencing was completed on seven tumor samples and six adjacent gastric samples, as well as one intestinal metaplasia sample and one non-atrophic gastritis sample. Four of those samples were also investigated with transcriptomics and whole genome sequencing. Using our previously published BWA-based pipeline, we identified putative BDIs from Helicobacter pylori rRNA into numerous genes in one tumor sample as well as in its adjacent matched sample, including BDIs in the Ig locus. Helicobacter pylori is considered a carcinogen by the World Health Organization due to its ability to promote carcinogenesis in gastric tissue, causing us to speculate that these integrations could be carcinogenic. Similarly to our analysis of the Cancer Genome Atlas data, we also identified a few samples with BDIs from Pseudomonas spp. and about half of the samples possessed BDIs from other bacteria. We identified at least one BDI in each sample regardless of the sequencing data type. Validation of these results is ongoing and will be presented. Given these results, more consideration should be given to BDIs into the human genome when bacterial associations with diseases are suspected.

#3300

Gal-GalNAc overexpressed in colorectal carcinoma mediates attachment and colonization of Fusobacterium nucleatum utilizing the Fap2 lectin.

Jawad H. Abed,1 Johanna Emgård,1 Stella Chaushu,1 Wendy Garrett,2 Gilad Bachrach1. 1 _The Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Recent reports revealed overabundance of oral Fusobacterium nucleatum (Fn) in colorectal carcinoma (CRC) compared to adjacent healthy tissue. This study aims to investigate the molecular mechanisms that mediate CRC colonization by Fn.

Using the orthotopic Colon-26 colorectal cancer model, we found that intravascular injected Fn colonizes CRC more than adjacent healthy tissue. Fusobacterial localization in the colon is tumor-dependent, as injected fusobacteria were undetected in the colons of tumor-free mice. CRC colonization is species specific as the gram-negative, anaerobe and periodontitis-related pathogen Prophyromonas gingivalis, which was shown to be abundant in oral tumors failed to colonize CRC.

Higher levels of D-galactose-β(1-3)-N-acetyl-D-galactosamine (Gal-GalNAc) are known to be detected in CRC. We recently identified the fusobacterial Fap2 outer-surface protein as a galactose binding lectin, suggesting that it might promote binding of fusobacteria to CRC. A fluorescent-labeled Peanut Agglutinin lectin (PNA) detected more Gal-GalNAc in human and mouse CRC compared to adjacent healthy tissues. Binding of FITC-labeled Fn corresponded with the levels of Gal-GalNAc and colocalized with Gal-GalNAc in the tumor. Flow cytometry revealed that Fap2-deficient Fn mutants are impaired in binding to CRC cell lines compared with their WT parent. Soluble GalNAc inhibited the binding of PNA and WT Fn to CRC cells in a dose-dependent manner, but did not affect the residual CRC binding of fap2 mutated Fn. Furthermore, in the colorectal cancer murine model, tumor colonization by Fap2-deficient Fn mutants was significantly lower than that of the Fap2 sufficient parental strain.

Our results demonstrate that the fusobacterial Fap2 lectin binds to Gal-GalNAc on CRC and mediates the tumor-specific attachment of Fn. Understanding the interaction of Fn with CRC, may enable future development of novel diagnostic and therapeutic agents for this disease.

#3301

Breast cancer subtypes have distinct microbial and immune cytolytic transcriptomic signatures.

Chandra Sekhar Pedamallu,1 Matthew Meyerson,1 Akinyemi Ojesina2. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Background: The role of genetic mutations in breast cancer is well documented and has formed the basis for novel therapies. However, the impact of viral and bacterial infection in the pathogenesis of breast cancer is not well understood.

Methods: We analyzed RNA sequencing (RNASeq) data generated by the Cancer Genome Atlas (TCGA) from over 900 pairs of breast tumor and adjacent normal tissues, using our pathogen discovery tool, PathSeq. We developed a novel metric for quantifying and normalizing microbial sequence abundance from RNASeq data by correcting for both the sequencing coverage and relative genome sizes of bacteria in the dataset. We also performed linear discriminant analyses (LDA) to test the hypothesis that the differences between the canonical breast cancer subtypes can be modeled on microbial abundance data. In addition, we interrogated the human gene expression data derived from the RNASeq dataset to determine the relationships between the breast cancer subtypes and immune cytolytic activity (CYT index; defined as the geometric mean of the expression levels of perforin and granzyme).

Results: Viral sequences were rare but we identified bacterial sequences to varying degrees of abundance. Intriguingly, we demonstrated that microbial abundance data can be used to recapitulate the well-established PAM50 gene expression-based subsets. Comparison of tumors and adjacent normal tissues also revealed subtype-associated microbial profiles. In addition, we observed that the basal and Her2 subtypes of breast cancer had a higher frequency of tumors with high CYT index compared with luminal A and B subtypes.

Conclusion: This study has revealed that the various breast cancer subtypes have unique microbial transcriptomic signatures as well as differential immune cytolytic activity profiles. These data suggest the potential for differential responses of breast cancer subsets to immunotherapy.

#3302

High incidence of MAC387 positive cells in the carcinoma tissues of inflammatory breast cancer patients correlate with the detection of multiple human Cytomegalovirus genotypes and invasive properties of the disease.

Hossam Taha Mohamed,1 Ramy Gadalla,1 Sherif Abdel Aziz Ibrahim,1 M. Akram Nouh,2 Mohamed El-Shinawi,3 Robert J. Schneider,4 Mona Mostafa Mohamed1. 1 _Zoology Department, Faculty of Science, Cairo Univiersity, Giza, Egypt;_ 2 _Department of Pathology, National Cancer Institute, Cairo Univiersity, Giza, Egypt;_ 3 _Department of General Surgery, Faculty of Medicine, Ain Shams University, Cairo, Egypt;_ 4 _Department of Microbiology, School of Medicine, New York University, New York, NY_.

Introduction: Previously we showed that the incidence of multiple human cytomegalovirus (HCMV) genotypes in the carcinoma tissues of inflammatory breast cancer (IBC) patients plays essential role in the disease progression. Primary HCMV infection to monocytes induces differentiation and biological turnover of monocytes to macrophages. In addition infected macrophages serves as "mobile vectors" for virus spreading and dissemination to different organs mainly by transendothelial migration. In addition we screened for the infiltration of CD14+ and CD68+ monocytes/macrophages markers in the carcinoma tissues of IBC versus non-IBC patients we showed that of CD14+ cells highly infiltrate tumor microenvironment (TME) of IBC patients compared to non-IBC.

Aims: In the present study we aim to 1) Assess the level of expression of MAC387 protein by monocytes/macrophages infiltrating TME of IBC versus non-IBC patients; 2) Test the correlation between the density of infiltrated MAC387+ cells and the incidence of different HCMV genotypes in carcinoma tissues of IBC versus non-IBC tissues. Since MAC387 found to be more common in cancers characterized by high metastatic properties we will also 3) determine whether the expression of MCA387 correlate with lymph-node metastasis and lymphovascular invasion in IBC versus non-IBC breast cancer patients.

Materials and Methods: A total of 135 breast cancer patients (91 non-IBC and 44 IBC) were enrolled to the present study during the period of January 2012 to September 2015 from Ain Shams university Hospitals. Detection of MAC387 marker was assessed by immunohistochemistry and HCMV genotypes were detected using multiplex PCR methodology.

Results: MAC387+ positive cells were more prevalent in IBC tissues than non-IBC tissues (p= 0.4). Incidence of higher number of MAC387+ cells were positively correlate with higher number of metastatic lymph nodes in both IBC and non-IBC patients r= 0.807 and 0.779 respectively. Moreover, Incidence of higher number of MAC387+ cells found to be positively correlate with lymphovascular invasion in IBC patients r=0622. Detection of multiple HCMV genotypes was statistically higher (p= 0.04) in IBC tissues in comparison with non-IBC tissues. Moreover, triple negative non-IBC and IBC tissues showed higher incidence of multiple HCMV genotypes in comparison with hormonal positive non-IBC and IBC tissues. of the monocytes/macrophages MAC387+ positive cells were more prevalent in IBC tissues showed multiple HCMV genotypes in comparison with IBC tissues showed single HCMV genotype (p= 0.46).

Conclusion: MAC387+ positive cells were more prevalent in IBC tissues and correlate with presence of multiple HCMV genotypes and high invasive properties of the disease.

### Stemness Properties of Breast and Ovarian Cancer

#3303

Identifying a functional correlation between cancer stem cells and 3D clonogenic growth: testing rational combinations of PI3K pathway inhibitors in subtype-specific BC.

Jennifer H. Carlson,1 Nandini Dey,1 Pradip De,1 Lori Friedman,2 Casey Williams,1 Brian Leyland-Jones1. 1 _Avera Research Institute, Sioux Falls, SD;_ 2 _Genentech, San Francisco, CA_.

Introduction: Studying clonogenic behavior of tumor cells in the context of physiologically relevant ECM offers the potential to explore signaling processes which more closely approximate the in vivo microenvironment. This approach is valuable for identification and dissection of the key molecular signaling of cancer cells to test targeted drugs. 3D cell cultures have been recognized as the method of choice for the physiologically relevant modeling of many aspects of malignant cell behavior ex vivo. Kenny et al., 2007 identified that morphologies of breast cancer cell lines in 3D assays correlate with their gene expression profiles and protein expression patterns reflecting the underlying distinct morphologies which were associated with tumor cell invasiveness and metastases-associated phenotypes. Study of the molecular characteristics and clinical significance of CSC in the context of prognostic relevance as well as the understanding the scope of therapeutically targeting CSC within distinct tumor cell populations have revealed that CD44+ cell-specific genes represents SC markers and correlated with decreased patient survival (Fillmore and Kuperwasser, 2008).

Hypothesis: Since 3D clonogenic growth of epithelial tumor cells represents the most appropriate model in vitro, we hypothesized that there is a functional association between phenotypic 3D clonogenic growth and cancer stem cells (CSC) proportion.

Methods: To study the role of CSCs in the 3D clonogenic growth of tumor cells we have used BC and ovarian cell lines.The cells were challenged to form 3D colonies in matrigel and cells were recovered from the 3D colonies after de-gelling using PBS-EDTA buffer. Relative distribution of CD24+, CD44+, CD24- and CD44- expression in cells were determined by flow cytometry (Accuri C6) following recovery of the cells from the colony and compared to cells grown in 2D. We have tested the effect of a rational combination of pan-PI3K pathway inhibitor, GDC-0941 or isoform-specific inhibitors along with AI, trastuzumab, or HRD inhibitors (PARP) in ER+/PIK3CA mutated, HER2+/PIK3CA mutated or PTEN-null TNBC cells.

Results: CD44/CD24 expression level changes between 2D and 3D growth was characterized in individual cell lines. Overall 3D growth led to a reduction in CD44 levels. Rational drug combinations blocked proliferative signals and enhanced apoptosis (cleaved caspase3) as demonstrated by WB, AnnexinV staining, proliferation (Incucyte). The mechanistic details of the effect of this combination on the CD44H/CD24L stem cell populations are being worked out, the results of which will be presented in the meeting.

Significance: Our study reports a methodological breakthrough to determine CD24L/CD44H CSC fraction from live cells following 3D clonogenic transformation.

#3304

Targeting Cripto in breast cancer plasticity.

Peter C. Gray,1 Evan Booker,1 Berhane Hagos,2 Masami Furphy,1 Benjamin T. Spike2. 1 _Salk Institute, La Jolla, CA;_ 2 _Huntsman Cancer Insititute, Salt Lake City, UT_.

The ability of cancer cells to adapt to various extrinsic challenges such as nutrient deprivation, hypoxia and chemotherapy may underlie their tumorigenicity as well as their ability to disseminate to distant organs. Therefore, identifying molecular mechanisms that govern such cellular plasticity may be critical to the development of anti-cancer therapies that target situationally defined tumor stem cells in metastasis, resistance, dormancy and recurrence. We considered the GPI-anchored/secreted Cripto protein to be an excellent target in this regard, as it regulates cellular plasticity during normal development including promotion of EMT, the embryonic stem cell state, and reacquisition of the stem cell state in cellular reprogramming. Cripto also regulates tissue specific stem cells including those of the mammary gland as we recently demonstrated. Due to the rarity of tissue stem cells under homeostasis, Cripto protein is generally low or undetectable in normal adult tissues but it is highly expressed in a wide variety of human tumors including breast cancers where its levels correlate with tumor aggressiveness and poor patient outcomes. Furthermore, inhibition of Cripto expression at the transcript level was recently reported to block metastasis in a mouse mammary tumor model. We have used xenograft assays to demonstrate that the requirement for Cripto function during metastasis, and to a lesser extent in primary tumor growth is conserved in human triple negative breast cancer cells. Critically, we demonstrate this through the use of a biologic/recombinant therapeutic protein specifically engineered to target Cripto. This recombinant protein, ALK4L75A-Fc, is comprised of an Fc-domain fused to a mutant extracellular domain of the natural Cripto binding partner and activin/Nodal type I signaling receptor, ALK4. The relatively modest effect of Cripto blockade on bulk primary tumors relative to metastasis and our inability to identify significant inhibitory effects of ALK4L75A-Fc on these cells in standard in vitro assays suggests that Cripto may only be critical for a subset of cancer cells and/or in specific microenvironmental contexts in the course of tumor progression. Consistent with this, we previously reported that Cripto signaling depends on its cell surface interaction with the stress inducible HSP70 family member and ER chaperone, GRP78 (glucose regulated protein, 78 kDa). Stresses including nutrient deprivation and chemotherapy strongly induce GRP78 expression and increase its levels at the cell surface and we find that nutrient deprivation in vitro elicits sensitivity of the MDA-MB-231 breast cancer cell line to inhibition of migration by ALK4L75A-Fc. Together, these results suggest that Cripto/GRP78 signaling promotes cancer cell plasticity and adaptation to microenvironmental challenge and that ALK4L75A-Fc may have therapeutic potential as an inhibitor of tumor cell plasticity.

#3305

Hippo kinases LATS1/2 control human breast cell fate.

Adrian Britschgi,1 Stephan Duss,1 Sungeun Kim,2 Heike Brinkhaus,1 Joana Pinto Couto,1 Duvini De Silva,1 Loren Miraglia,2 Michael B. Stadler,1 Anthony P. Orth,2 Ghislain M.C. Bonamy,2 Venkateshwar A. Reddy,2 Mohamed Bentires-Alj1. 1 _Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland;_ 2 _Genomics Institute of the Novartis Research Foundation, San Diego, CA_.

Cellular differentiation and lineage commitment in adult tissues is a tightly regulated, yet highly dynamic process. It is of crucial importance to understand how different cell fates are maintained and how cellular plasticity is regulated: In the context of normal development but also in the light of the fact that perturbations acting as cell fate switches are the underlining cause for many human diseases. Still, the regulation of cell fate in many tissues, including the breast, remains largely elusive. We performed a high-content confocal image-based loss-of-function screen for regulators of primary breast cell fate. We infected primary human breast cells with a shRNA tumor suppressor library in serum-free suspension culture in an arrayed format and assessed a variety of phenotypic parameters using automated confocal imaging. We found that removal of tumor-suppressor genes increased the number of progenitor cells and promoted self-renewal potential. Unexpectedly, these effects were particularly linked to an increase in the number of cells of the luminal lineage. Redundant shRNA activity (RSA) and pathway analysis revealed that activation of the Hippo pathway was significantly associated with this increase in self-renewal and the luminal phenotype. We focused on the Hippo core kinases LATS1/2 (LATS), which were among the highest scored hits in the initial screen. Depletion of LATS in normal breast cells derived from several independent donors promoted self-renewal, luminal fate and increased the numbers of luminal progenitors. Luminal progenitors have been shown to include the cell-of-origin of most human breast cancers and supporting this hypothesis, we found that LATS protein expression was low or absent in most human breast cancers. Furthermore, we generated a gene signature of LATS depleted primary breast cells, which revealed a high enrichment in genes belonging to luminal A and B subtypes of breast cancer. Surprisingly, endogenous activation of the Hippo pathway via removal of its core kinases LATS only resulted in modest upregulation of its canonical targets YAP/TAZ, but rather triggered a pronounced increase in the expression of luminal and luminal progenitor genes. These findings challenge the current observation that Hippo effectors YAP/TAZ are associated with mammary basal cell fate and uncover a novel YAP/TAZ-independent role of LATS in the regulation on breast cell fate.

#3306

Wnt pathway dynamics in breast cancer progression.

Kevin Roarty,1 Adam D. Pfefferle,2 Chad J. Creighton,3 Charles M. Perou,2 Jeffrey M. Rosen1. 1 _Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 3 _Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX_.

Background: The canonical Wnt/β-catenin signaling pathway directs self-renewal cues for stem cells in multiple tissues. Through alternative receptors, noncanonical Wnt/β-catenin-independent pathways exist, mediating cellular processes that include planar cell polarity, convergent extension, actin/cytoskeletal rearrangements, and in some contexts, antagonism of canonical Wnt signaling. How different Wnt pathways cooperate within heterogeneous cellular landscapes in breast cancers remains an outstanding question. Within the current study, we investigated the role of the alternative Wnt receptor, Ror2, in mediating Wnt/β-catenin-independent functions during tumor progression.

Experimental design and methods: A TP53 null transplantable GEM model was used in the current study, which exhibits subtype-specific molecular signatures characteristic of human breast cancers. A lentiviral-based shRNA strategy was developed for gene silencing of noncanonical Wnt pathway components in vivo, with emphasis on the alternative Wnt receptor, Ror2. This strategy was combined with pathway-specific Wnt reporters to survey the interplay of canonical and noncanonical Wnt pathways in vivo.

Results: TP53 null mammary tumors differentially expressed the noncanonical Wnt receptor Ror2, with the highest levels of expression evident in basal-like tumors relative to luminal and claudin-low subtypes analyzed. Basal-like tumors also displayed Wnt/β-catenin active populations, which were inversely correlated with Ror2 expression. Lentiviral shRNA silencing of Ror2 within 3 independent basal-like models (T1, T2, 2225L) resulted in elevated Wnt/β-catenin activity along with various phenotypic outcomes, including squamous differentiation. Ror2, therefore, functions to restrain Wnt/β-catenin-dependent signaling in vivo. Despite the similarity in Wnt/β-catenin-dependent signaling outcome upon Ror2 depletion, gene expression profiling of Ror2-intact (shLUC) vs. Ror2-depleted (shRor2) tumors unveiled diverse gene expression changes within basal-like tumor models. Intriguingly, Ror2 silencing within 2225L basal-like tumors resulted in a change in the composition of tumor cell subpopulations. Additionally, gene expression analysis of shLUC vs. shRor2 tumors revealed that genes upregulated in shRor2 tumors were associated with claudin-low features, while genes downregulated were associated with basal-like features, reflecting changes in the cellular landscape upon Ror2 loss. Gene ontology analysis also revealed alterations in actin/cytoskeletal- and cell adhesion-associated genes upon Ror2 depletion.

Conclusion: The current study highlights the differential effects of Ror2 loss within TP53 null basal-like models. In particular, Ror2 can regulate the plasticity between basal-like and claudin-low states, impacting cytoskeletal and cell adhesion dynamics. Current efforts are focused on how these changes impact tumor progression and metastasis.

#3307

Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients.

Eric Lin,1 Lianette Rivera,1 Shamileh Fouladdel,1 Hyeun Joong Yoon,2 Stephanie Guthrie,3 Jacob Weiner,1 Yadwinder S. Deol,1 Evan Keller,1 Vaibhav Sahai,1 Diane M. Simeone,1 Monika L. Burness,1 Ebrahim Azizi,1 Max S. Wicha,1 Sunitha Nagrath1. 1 _University of Michigan, Ann Arbor, MI;_ 2 _South Dakota State University, Brookings, SD;_ 3 _University of Virginia, Charlottesville, VA_.

Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (~1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis.

Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques.

Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples.

Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics.

#3308

Bisphenol A treatment induces hyperplasia in primary and stem cell-generated mammary glands from pregnant mice.

Hakim Bouamar,1 Fuchuang Zhang,2 Xiang Gu,1 Qiaoxiang Dong,2 Lu-Zhe Sun1. 1 _University of Texas Health Science Center, San Antonio, TX;_ 2 _School of Environmental Sciences and Public Health, Wenzhou Medical College, Wenzhou, China, China_.

Breast cancer is a commonly diagnosed cancer during pregnancy. However, how endogenous and exogenous endocrine factors may contribute to the development of pregnancy-associated mammary tumorigenesis is not clear. There is growing evidence that mammary stem cells (MaSCs) may initiate neoplastic transformation when dysregulated in mouse models. We investigated the effect of the environmental endocrine disruptor bisphenol A (BPA) on mouse mammary gland morphology, epithelial cell composition, pre-neoplastic lesions, and the regenerative function of MaSCs. Pregnant FVB mice with GFP transgene on Day E8.5 were implanted with osmotic pumps that constantly release BPA at 0, 25 or 250 ng/kg/day for 28 days and the mice were euthanized one month after weaning. In agreement with the literature, we observed an abnormality of the morphology of the mammary gland after BPA treatment characterized by higher duct density and abnormal secondary and tertiary branching. Quantification of percent hyperplastic mammary ducts in H&E-stained tissue slides revealed a significant increase of ducts with hyperplastic lesions after BPA treatment, particularly with the low dose. To investigate the effects of BPA treatment on MaSCs, we used enzyme digestion to isolate the CD24hi/CD49f+ luminal epithelial cells (also termed as colony forming cell or CFC) and the CD24+/CD49fhi basal epithelial cells (also termed as mammary repopulating unit or MRU) from mammary gland tissues by FACS and found no significant difference in percent of luminal or basal cell population after BPA treatment. Because the basal cells are enriched with MaSCs that can form mammospheres in suspension culture and subsequently form solid 3D organoids when cultured in Matrigel, we transplanted the solid 3D organoids into cleared mammary fat pads of syngeneic FVB mice and immune-compromised nude mice to examine how BPA treatment might alter MaSC function. Significantly, similar to the results from the primary mammary glands, the regenerated mammary glands by MaSCs from mice treated with the low dose of BPA showed increased duct density, secondary and tertiary branching, and a significantly greater number of hyperplastic lesions. Taken together, our study demonstrated that BPA exposure at very low dose could induce pre-neoplastic lesions in the mammary gland of pregnant mice, apparently by directly targeting MaSCs, and implicates BPA as an exogenous endocrine factor that may promote pregnancy-associated mammary tumorigenesis.

#3309

Novel small molecule ONC201 induces cell death and targets chemotherapy-resistant cancer stem-like cells in triple negative breast cancer.

Marie D. Baumeister,1 Jessica Wagner,2 Varun V. Prabhu,3 Christina LB Kline,4 Bora Lim,5 Josh E. Allen,6 David T. Dicker,4 Wafik S. El-Deiry4. 1 _Temple University School of Medicine, Philadelphia, PA;_ 2 _Temple University, Philadelphia, PA;_ 3 _Penn State Hershey Cancer Institute, Hershey, PA;_ 4 _Fox Chase Cancer Center, Philadelphia, PA;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _Oncoceutics, Inc, Philadelphia, PA_.

Breast cancer is the most commonly diagnosed cancer in US women today. Triple negative breast cancer (TNBC) accounts for 15% of all breast cancers that are diagnosed and has a poor prognosis. TNBC has a high recurrence rate following treatment with chemotherapy and radiation compared to other breast cancer subtypes. Many tumors, including those of the breast, contain a population of stem-like cells known as cancer stem cells (CSCs). The CSC population is capable of self-renewal and has been shown to be resistant to chemotherapy and radiation. These observations suggest that the CSCs may be responsible for tumor relapse after initial response to therapy. We previously identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10. This compound functions through the ATF4/CHOP pathway to upregulate DR5 (Kline et. al., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Science Translational Medicine, 2013). ONC201 recently completed its first-in-human clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-NCI-EORTC meeting). ONC201 is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We have shown that ONC201 is able to decrease formation of colonospheres as well as deplete colorectal CSC markers in vitro. We have also showed that treatment with ONC201 prevents colorectal CSCs from initiating xenograft tumor growth in mice. (Prabhu et. al Cancer Research., 2015). The goal of this work was to investigate the efficacy of ONC201 in triple negative breast cancer cells, as well as to determine if ONC201 was able to target breast CSCs. We used cell viability assays to determine that the IC50 values for 6 TNBC cell types are in the low micromolar range, doses which are achievable based on human pharmacokinetic data. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in TNBC cells. Levels of TRAIL receptor DR5 are increased on the cell surface in TNBC cells following ONC201 treatment. In addition we show that ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We have observed that ONC201 depletes Aldefluor+ TNBC cells in vitro. ONC201 inhibits formation of mammospheres from TNBC CSC-like cells while paclitaxel, a chemotherapeutic drug used to treat TNBC, does not. Our findings suggest that ONC201 exerts cytotoxic effects against TNBC cell types and that ONC201 may target paclitaxel resistant CSCs. Our work contributes to the development of a preclinical rationale for the use of ONC201 as a novel therapy for triple negative breast cancers.

#3310

Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure.

Shamileh Fouladdel, Tahra Luther, Kaitlin Harvey, Rachel Martin-Trevino, Jill Granger, Ebrahim Azizi, Max S. Wicha. _University of Michigan, Ann Arbor, MI_.

Most tumors are hierarchically organized and driven by a population of cells that display stem cell properties. These cancer stem cells (CSCs) have been characterized by in vitro assays such as sphere formation and in vivo by tumor initiation. In addition cell surface markers have been utilized to enrich for CSC populations. More recently a subtype of CSCs have been reported to display autofluorescence (AF), a property mediated by cellular uptake and concentration of riboflavin. In order to characterize the molecular heterogeneity of CSCs, we studied AF positive sorted single cells in different subtypes of breast cancer cell lines and also patient derived xenograft (PDX) breast tumor cells. Exposure of cell lines and PDXs to riboflavin significantly increased the percentage of AF+ cells 3-5 fold. These sorted AF+ cells were enriched for tumorsphere forming capacity in vitro. Flow cytometry analysis revealed partial overlap of AF+ cells with the previously characterized EMT-CSC phenotype CD44+/CD24- cells. We also utilized the microfluidic C1 and BioMark HD instruments to determine the expression patterns of 96 target genes using TaqMan assays in a multiplex RT-qPCR setting at single cell resolution. Single cell gene expression data showed a distinct signature in AF+ cells with high expression levels of MKI67, PCNA, ABCG2, BRCA1, Jag2, EZH2, and MMP9 genes. Since AF is mediated by specific cellular uptake and concentration of riboflavin, and the report that riboflavin is capable of generating cytotoxic free radicals upon light exposure, we determined whether this property could be exploited to selectively target CSCs. The AF+ cells were cultured in the presence and absence of riboflavin, exposed to visible light and analyzed by MTT cytotoxicity assay. Data revealed significant cytotoxicity of riboflavin on AF+ cells that were exposed to visible light. In conclusion, AF+ cells show distinct cancer stem cell features and are sensitive to light-activated riboflavin. These findings will help better understanding of tumor biology to identify new target therapies for treatment of cancer patients.

#3311

Autocrine and paracrine IL-4 maintains breast cancer stem cells traits via RAS/MAPK/DUSP pathway.

Alice Turdo, Miriam Gaggianesi, Tiziana Apuzzo, Antonina Benfante, Aurora Chinnici, Alessandro Giammona, Simone Di Franco, Giorgio Stassi, Matilde Todaro. _University of Palermo, Palermo, Italy_.

Background:

Despite the advent of successful treatment of localized malignancies, metastatic cancer still lacks efficacious therapeutic approaches, including breast cancer. It is now well established that within malignant tumors Cancer Stem Cells (CSCs) constitute a unique cell subset that fuel and succeed at tumor growth and metastases formation. Tumor microenvironment sustains CSCs characteristics, making the molecular mechanisms driving tumor progression and recurrence more complex and difficult to elucidate. Furthermore, the interaction that occurs between CSCs and the nearby stroma has proven to enhance the aggressive behavior of several carcinomas through the secretion of microenvironmental cytokines. In this context, IL-4 has already been described to promote survival of cancer cells through the up-regulation of several anti-apoptotic factors. However, still little is known about its role in promoting breast cancer progression.

Results:

Here we show for the first time that autocrine and paracrine production of IL-4 regulates breast CSCs (BCSCs) features, including cell proliferation, motility and cytoskeletal organization via RAS/MAPK pathway. Interestingly, relief from IL-4 impaired BCSCs proliferation, colony forming efficiency and in vivo tumor formation, while it fostered the expression of the dual specificity phosphatase-4 (DUSP4) in triple-negative basal-like BCSCs, leading to the decrease of CD44+/CD24- population. DUSP4 is commonly deficient in the most aggressive breast cancers, such as the basal-like subtype. Likewise, we observed that the enforced expression of DUSP4 increases the CD24+ compartment in basal-like BCSCs, determining also a dramatic decrease of their proliferation, colony forming efficiency, invasiveness and metastases formation. Contrarily, in luminal-like BCSCs, DUSP4 suppression favors BCSCs cell traits, including their tumorigenic and metastagenic properties.

Methods:

Patient-derived BCSCs were obtained by digestion of breast cancer tissues and plated in serum-free media with bFGF and EGF. DUSP4 were inserted into the p-Lenti expression vector. Stable DUSP4 knockdown was produced by lentiviral transduction of the pGFP-C vector. IL-4 function was impaired by using a high affinity IL-4Rα antagonist. To assess tumorigenicity and metastases formation, BCSCs were suspended in matrigel and injected either orthotopically or intra caudal in NOD/SCID mice.

Conclusions:

These findings will shed light on the molecular basis of cancer progression and on the complex crosstalk occurring between tumor and its microenvironment. The identification of tumor-related molecular events, such as the IL-4 activated signaling, might be clinically exploited as therapeutic targets in the adjuvant setting and synergize the effect of conventional chemotherapy in patients affected by breast cancers with limited therapeutic options, such as triple-negative breast cancers.

#3312

Transcriptional regulation of chemokine receptor 4 (CXCR4) by nuclear respiratory factor 1 (NRF1) controls estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells.

Jayanta Kumar Das, Deodutta Roy. _Florida International University, Miami, FL_.

Chemokine receptor 4 (CXCR4) is involved in the maintenance normal stem cells and metastasis of cancer stem cells. NRF1 is one of the major transcription factor that controls transcription of CXCR4. The functional regulation of transcription of NRF1 target genes has not been explored in breast cancer. In this study, we have investigated whether transcriptional regulation of CXCR4 by NRF1 regulates estrogen-induced malignant transformation of breast epithelial cells to breast cancer stem cells (CSCs). We have previously shown that NRF1 may be involved in 17 β-estradiol (E2) induced malignant transformation of breast epithelial cells, however, the mechanism of transcriptional regulation of the NRF1 target genes remains unknown. In this study we showed that NRF1 and E2 jointly contributed in reprogramming of breast epithelial cells to CSCs. Levels of breast cancer stem cell markers (CD44+CD24+ALDH1+CD133+) were significantly increased by E2 treatment in NRF1 overexpressing cells compared to cells transfected with vector receiving E2. E2-induced increases of spheroid formation, cell survival and growth of cancer stem cells were modulated by functional gain or loss of NRF1. For morphological details we used a holographic microscope (HoloMonitor M4), we observed that E2 generated breast cancer stem cells with CD44+CD24+ALDH1+CD133+ markers produced large live spheroids in NRF1 overexpressing cells compared to cells transfected with vector receiving E2. Overexpression of NRF1 promoted the transition of E2-treated breast epithelial MCF-10A cells to mesenchymal stem cell-like phenotype. The ChIP qPCR, RT-qPCR, Western blotting and immunofluorescence microscopic assays showed that NRF1 mediated transcriptional changes of its target genes CXCR4, BNIP3, and DJ-1 correlated with malignant phenotypic changes. The suppression of NRF-1 activity induced growth arrest and apoptosis, and reduced stemness, growth and tumorigenic property of cancer stem cells. In summary, our findings for the first time showed that transcriptional regulation of CXCR4 by NRF1 may contribute in the induction of a pre-malignant phenotype by estrogen presumably by promoting generation of breast cancer stem cells. These data suggest NRF1 as an emerging potential target for therapeutic intervention against breast cancer.

#3313

Pharmacologic targeting of Aurora-A kinase restores ERα expression and endocrine sensitivity through impairment of breast cancer stemness.

Antonino B. D'Assoro, Tufia Haddad, Matthew Goetz, Mary Kuffel, John Hawse, Vera Suman, Jeffrey Salisbury, James Ingle, Evanthia Galanis. _Mayo Clinic College of Medicine, Rochester, MN_.

Background: Intrinsic resistance has been linked to ER down-regulation and poor clinical outcome in breast tumors. Aurora-A kinase (AURKA) promotes distant metastases through activation of epithelial to mesenchymal transition (EMT) programming and expansion of breast tumor initiating cells (BTICs) harboring a CD44high/CD24low phenotype. Moreover, AURKA induces down-regulation of ERα expression and resistance to endocrine therapy that is mediated by SMAD5 transcription factor (D'Assoro et al., Oncogene 2014: 33:599-610). The aim of this study was to evaluate the role of AURKA in endocrine resistance induced by breast cancer stemness and ER down-regulation.

Materials and Methods: We employed parental MCF-7 and variant MCF-7 cells ex-vivo (vMCF- 7/LCC9, vMCF-7/FR) that have high endogenous levels of AURKA, are ERlow, and exhibit in vivo resistance to fulvestrant. Real-Time Cell Proliferation and Apoptosis assays were performed to assess response to endocrine and/or AURKA-targeted therapy using the innovative IncuCyte ZOOM System. Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to generate mammospheres. Expression of ER, CD44 and CD24 markers was characterized by FACS analysis and immunofluorescence.

Results: Pharmacologic inhibition of AURKA activity with the small molecule, alisertib, significantly restored sensitivity to fulvestrant in vMCF-7/LCC9 and vMCF-7/FR cells. To establish whether fulvestrant resistance was linked to enrichment of BTICs, breast cancer cells were cultured under non-adherent conditions to generate mammospheres. vMCF-7/FR cells generated mammospheres at higher efficiency compared to parental cells suggesting that vMCF-7/FR cells exhibited high self-renewal activity. vMCF-7/FR-derived mammospheres displayed a stem cell-like CD44high/CD24low phenotype that was further characterized by low levels of ER expression and resistance to fulvestrant. When vMCF-7 FR-derived mammospheres were treated with alisertib alone or combined with fulvestrant, the combination was more effective in mediating restoration of endocrine sensitivity. The phenotype of the alisertib-treated mammospheres was functionally linked to loss of CD44 expression, restoration of CD24 and ER expression, and sensitivity to fulvestrant.

Conclusions: These findings provide further evidence that AURKA activity plays a central role in breast cancer progression through the genesis and clonal expansion of BTICs that are characterized by ER down-regulation and resistance to endocrine therapy. These results are important for the treatment of endocrine resistant breast tumors because they provide the rationale for the development of an innovative therapeutic approach to combine endocrine therapy with a targeted inhibitor of AURKA activity.

#3314

Targeting Hsp90 affects stem cell signaling in triple negative breast cancer.

Prabhu Ramamoorthy, Parthasarathy Rangarajan, Roy Jensen, Shrikant Anant. _University of Kansas Medical Center, Kansas City, KS_.

Background: Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. Many studies have also suggested that breast cancer's ability to proliferate, progress, and spread is due to the presence of a rare subpopulation of cancer stem cells. The aim of this study is to investigate the efficacy of HSP90 inhibitors celastrol and triptolide (from the Chinese herb "Thunder God of Vine" (Tripterygium wilfordii) on stem cells as a novel therapy for TNBC.

Methods: We performed in vitro studies using the TNBC cell lines BT20 and MDA-MB-231. We performed mammosphere assays to determine self-renewal capacity. For in vivo, we injected BT20 cells into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively.

Results: Both celastrol and triptolide significantly suppressed mammosphere size and number. Furthermore, expression of breast cancer stem cell markers DCLK1, ALDH1 and CD133 were significantly reduced in the two cell lines following treatment. Flow cytometry also suggested a significant reduction in DCLK1+, ALDH+ and CD133+ cells. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. In cells treated with either celastrol or triptolide, there was a significant reduction in activated Notch intracellullar domain (NICD), and its downstream target Hes-1. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control, vector transfected cells in reducing 2D cell proliferation or 3D mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. Furthermore, inhibition of Notch signaling pathway by using Υ-secretase inhibitor DAPT shows further reduction in mammosphere formation and Notch and its downstream target gene. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size and weight of tumors in mice treated with celastrol or triptolide. Western blot data showed that there is a decrease in activated Notch-1 protein and stem cell marker, DCLK1+ and ALDH+ in celastrol and triptolide treated xenograft tissue.

Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling.

#3315

Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target.

Mousumi Majumder,1 Xiping Xin,1 Ling Liu,1 Elena Tutunea-Fatan,1 Mauricio Rodriguez-Torres,1 Krista Vincent,1 Andrew Deweyert,1 Lynne-Marie Postovit,2 David Hess,1 Peeyush K. Lala1. 1 _Univ. of Western Ontario, London, Ontario, Canada;_ 2 _University of Alberta, Edmonton, Alberta, Canada_.

Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH and WNT up-regulation. EP4 antagonist (EP4A) treatment ablated SLCs both in vitro and in vivo. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 human breast cancer cell lines (named MCF-7-COX-2 and SKBR-3-COX-2) resulted in aggressive phenotypes: increased migration/invasion/proliferation, EMT, elevated SLCs, evidenced by spheroid formation for successive generations, increased ALDH activity and co-expression of COX-2/SLC markers. These changes were reversed with COX-2 inhibitor or EP4A, indicating dependence on COX-2/EP4 activities. COX-2 overexpression or EP4 agonist treatment of COX-2 low cells caused up-regulation of NOTCH/WNT pathway genes, blocked with PI3K/AKT inhibitors. Supporting above findings, micro-array analysis

showed up-regulation of numerous SLC-regulatory and EMT-associated genes in MCF-7-COX-2 cells. MCF-7-COX-2 cells showed increased orthotopic tumorigenicity and spontaneous multi-organ metastases in NOD/SCID/IL-2Rγ-deficient mice for successive generations with limiting cell inocula. Orthotopic tumors showed significant up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4 knockdown or EP4A treatment. COX-2, EP4 and ALDH1A expression in situ in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. High COX-2/EP4 expression was linked with poor survival. Thus EP4 represents a novel SLC-ablative target in human breast cancer. (Supported by a grant of the OICR to PKL and a TBCRU fellowship to MM)

#3316

The effects of dopamine receptor 2 on breast cancer tumor initiating cells.

Matthew Tegowski, Charlene Santos, Albert Baldwin. _The University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Despite advances in early detection and treatment of breast cancers, metastatic disease remains a significant problem and a large cause of mortality in the US. Although tumors are characterized as a mass of uncontrolled cell division, it is thought that only small populations of cells, known as tumor initiating cells (TICs), are primarily responsible for metastasis. TICs are less differentiated than most tumor cells and many TICs show an epithelial-to-mesenchymal transition (EMT). Recently, the FDA-approved drug thioridazine, a dopamine receptor 2 (DRD2) antagonist, has been shown to inhibit pluripotency gene expression only in transformed stem cells. To test whether DRD2 can regulate TICs in breast cancer cells, two main assays were performed. The tumorsphere assay is used to measure TIC activity within a population of cells in vitro. In this assay, single cells are cultured in suspension, in the absence of serum. Cells must escape anoikis and grow without cell-cell contacts or the assortment of factors contained in serum. However, the gold standard in vivo assay to measure tumor initiation is the transplantation assay at limiting dilutions. Cells are injected into immunocompromised mice in decreasing numbers and the number of tumors each population of cells is capable of forming is measured. In this study the tumorsphere assay and transplantation assay both show that DRD2 supports TICs in breast cancers. Interestingly, our data suggest that DRD2 regulates TICs and cellular differentiation through the activity of the STAT3 transcription factor. Activation of STAT3 has been shown to induce gene expression changes that support TICs and upregulate the pluripotency factor OCT4. STAT3 activity can also induce EMT and resistance to some therapeutics. By inducing the expression of its own activator, IL-6, STAT3 can initiate a feed forward loop to maintain continual expression to promote TICs, EMT, and drug resistance. Halting the continual activation of STAT3 in these cells should be an important objective. Our data supports the concept that DRD2 may represent a new, and druggable target for therapies to decrease metastasis and/or recurrence by interfering with STAT3 and inhibiting TIC-like activities.

#3317

Tumor microenvironment of metastasis (TMEM) in breast cancer patients represents a stem cell niche; likely mediated by the Wnt pathway.

Gargi Bandyopadhyaya,1 Eli Grunblatt,1 Sweta Roy,1 Nathan Agi,1 Esther Adler,2 Joan Jones,3 John S. Condeelis,3 Maja H. Oktay,3 Sumanta Goswami3. 1 _Yeshiva University, New York, NY;_ 2 _Montefiore Medical Center, Bronx, NY;_ 3 _Albert Einstein College of Medicine, Bronx, NY_.

Cell surface biomarkers CD44, CD24, CD133 and intracellular biomarker ALDH1 have been used to identify breast cancer stem cells (BCSC). BCSC are clinically significant because they are resistant to chemotherapy and in very small numbers can initiate tumor growth and metastasis. Recent studies suggest that juxtacrine signaling from monocytes and macrophages support the BCSC niche. Macrophages represent one of the components of the micro-anatomical sites of hematogenous dissemination of breast cancer cells called Tumor MicroEnvironment of Metastasis (TMEM) which are predictors of metastatic outcome in patients. We hypothesize that TMEM rich tumor microenvironments support BCSCs which are a prerequisite for systemic metastatic outgrowths.

We used flow cytometry to quantify CD44high/CD24low cells, as well as mRNA fluorescent in situ hybridization (FISH) and qRT-PCR to quantify CD133 and ALDH1 expression in breast cancer cells from 50 invasive ductal carcinomas obtained from patients' cancer excisions by fine needle aspiration (FNA) before formalin fixation. Formalin-fixed paraffin embedded tissue from each sample was also analyzed for TMEM score using triple immunohistochemistry and the stem cell marker expression was compared to TMEM scores for each corresponding cancer excision.

We observed very strong correlation between the percentage of CD44high/CD24low cells and TMEM scores (r=0.91), as well as the percentage of CD133 and ALDH1 expressing cells with TMEM scores (r=0.88 and 0.86 respectively). FISH results were validated using qRT-PCR and showed very strong correlation with TMEM scores (r=0.76 and 0.73 for CD133 and ALDH1 respectively).

Wnt/β-catenin signaling pathway is involved in stem cell generation and pathogenesis of various types of cancer. Aberrant activation of Wnt-signaling pathway in normal stem cells can promote their transformation into cancer stem cells. We hypothesize that interactions between cancer cells and macrophages, and/ or cancer cells and endothelial cells (i.e. both interactions among the cellular components of TMEM) induce cancer stemness via Wnt/-β catenin pathway. Indeed, co-culture of mouse macrophages (BAC) with MCF-7 cells, as well as human endothelial cells (HUVEC) with MCF-7, significantly increased the percentage of cells expressing stem cells markers, while salinomycin, selective inhibitor of breast cancer stem cell growth and progression, significantly reduced BAC or HUVEC induced increase in the percentage of cancer stem cells.

Our findings indicate that TMEM-rich microenvironments support breast cancer stem cells likely via Wnt signaling involving juxtacrine cancer cell-macrophage and cancer cell-endothelial cell interactions.

#3318

Exploring the utility of natural and synthetic resveratrol derivatives for bone regrowth following loss due to breast cancer therapies.

Evan Robert, Lyndsay V. Rhodes. _Florida Gulf Coast University, Fort Myers, FL_.

Many of the most widely used therapies for the treatment of breast cancer have been associated with enhanced rate of bone loss, including chemotherapies and hormone therapies, due to alteration of normal hormone signaling. Bone loss is due to a shift in the balance between osteoblast and osteoclast cells that are derived from a common precursor, mesenchymal stem cells (MSC), found in the bone marrow. Estrogen is an important mediator of both hormone-responsive breast cancers and normal bone development and regeneration, favoring MSC differentiation down the osteoblast lineage. Stilbenes, defense compounds produced by plants including red grapes, peanuts, and blueberries, have been popularized in recent years based on observed health benefits. Resveratrol, the most widely studied stilbene, has been credited with many health benefits including cardiovascular health, anti-cancer and antioxidant effects, and inhibition of obesity and diabetes. Many stilbenes, including resveratrol can be classified as phytoestrogens due to their ability to bind and alter the activity of the estrogen receptor. A series of 28 stilbene compounds, including resveratrol and pterostilbene, were obtained by the U.S. Department of Agriculture and screened for effects on MSC differentiation. MSC were treated with stilbene compounds (both E and Z isoforms) and cultured in either osteogenic or adipogenic differentiation media for 7-21 days. Following differentiation, cells were stained with (1) Alizarin Red S to identify calcium deposits or (2) Oil Red O to identify lipid droplets. Several compounds were found to stimulate osteogenesis while inhibiting adipogenesis and were selected for further study and development. Realtime PCR for known genes associated with osteogenesis (ALPL, RUNX2, OCN, OPN) and adipogenesis (ADIPOQ, GLUT4, LEP, PPARG) were used to confirm differentiation and gain insight into the mechanism of altered differentiation. Unlike estrogen, stilbenes do not appear to negatively impact breast and reproductive organs. Stilbenes thus represent a novel option for inducing a preferential shift of MSCs toward the osteogenic lineage while potentially avoiding the negative health effects of estrogen. Targeting the differentiation of MSC to favor bone formation represents a viable target for the development of novel therapeutics for bone loss following cancer treatment. Further, identifying compounds that stimulate osteogenesis while repressing adipogenesis could benefit other metabolic disorders like obesity associated with increased breast cancer risk.

#3319

Characterization of the CD44+/CD49f+/CD24- subpopulation in basal-like DCIS cells.

Nadire Duru, Ramkishore Gernapudi, Pang-Kuo Lo, Yuan Yao, Benjamin Wolfson, Yongshu Zhang, Qun Zhou. _University of Maryland, Baltimore, MD_.

The molecular mechanisms responsible for Ductal Carcinoma In Situ (DCIS)-Invasive Ductal Carcinoma (IDC) transition have yet to be elucidated. Due to the lack of molecularly targeted therapies, basal-like DCIS has a high risk of recurrence and progression to invasive and metastatic cancers. In this study, using a human DCIS cell line model and a novel single-cell clonogenic approach, we have identified and characterized the aggressive clones derived from single cells with the CD49f+/CD44+/CD24- surface markers. We found that aggressive clones had enhanced self-renewal, migratory and invasive capacities. The aggressive clones had elevated ALDH activity, lower global DNA methylation and increased expression of stem cell related genes including OCT4 and SOX2. Further mechanistic studies showed that linc-ROR and miR-10b were overexpressed in aggressive clones and are key regulators of their self-renewal and invasive abilities. Finally, we confirmed our in vitro results in vivo, demonstrating that aggressive clones were capable of forming tumors in nude mice, whereas non-aggressive clones were not. We believe that characterization of aggressive clones within the heterogeneous CD49f+/CD44+/CD24- DCIS population and having linc-ROR and miR10b as additional markers to distinguish them from non-aggressive clones would provide the basis to develop new chemoprevention agents.

#3320

Human bone marrow- and adipose-mesenchymal stem cells secrete inflammatory cytokines and growth factors in the presence of breast tumor cells.

Brandi Talkington, Leah Figurski, Yuya Kudo, Kelsey Sadlek, Linda Vona-Davis. _West Virginia University Randolph Cancer Center, Morgantown, WV_.

Introduction: Stem cell and adipocyte-epithelial interactions are mediated by tumor-enhancing factors such as cytokines, chemokines, and growth factors. The objective of our study was to identify novel secreted cytokines and growth factors common to mesenchymal stem cells and adipocytes and breast tumor cells using protein array analysis. Methods: Bone marrow (BM-MSC) or adipose (ASC) mesenchymal stem cells or their preadipocytes were co-cultured indirectly with MDA-MB-231 invasive breast cancer cells. Conditioned media was collected to compare protein expression levels using a cytokine antibody array. Signals were visualized, scanned, and quantitated by densitometry. Ingenuity Pathway Analysis mapped direct and indirect relationships between highly expressed proteins involved in obesity and the inflammation. Results: Within a week, stem cells derived from either bone marrow or adipose tissue differentiated into preadipocytes, as evident by staining with oil red O solution. Greater upregulation of cytokine and growth factor expression was observed when mesenchymal stem cells or preadipocytes were cultured in the presence of breast cancer cells. In BM-MSCs, IL-6, IL-6R, FGF9, ADIPOQ, and CXCL1 were the top five upregulated proteins, and IL13, IL2, CCL8, CXCL9, and TNF were the top five downregulated proteins when cultured with breast cancer cells. IL13, IL2, TNF, IL6, IL6R, and ADIPOQ are obesity-related while CXCL1, IL6, IL6R, ADIPOQ, CCL8, CXCL9, TNF, IL2, and IL13 are involved in inflammation. In ASCs, CXCL5, CCL5, IFNG, FGF7, and PDGF BB were the top five upregulated proteins, and CXCL11, ICAM3, IL6R, CCL16, and EGF were the top five downregulated proteins when cultured with breast cancer cells. CXCL11, IL6R, and CCL5 are obesity-related while IFNG, EGF, CCL16, IL6R, and CXCL11 are involved in the inflammatory response. IL7, CXCL3, ANG, CXCL11, and IGFBP1 were the top five upregulated proteins, and TNF, CCL18, IGFBP2, CCL15, and CCL20 were the top five downregulated proteins when BM-MSCs were differentiated into preadipocytes. CCL16, IL17, ICAM3, AGRP, and TNFSF18 were the top five upregulated proteins, and CCL3, IL6, FGF9, NAP1L4, and CSF1 were the top five downregulated proteins when ASCs were differentiated into preadipocytes. Conclusions: We identified a number of metabolic and inflammatory cytokines and growth factors common to bone and adipose tissue that could be involved in reciprocal communication between stem cells, their preadipocytes, and invasive breast cancer cells. A number of unique cytokines and growth factors were differentially expressed and under investigation for their roles in breast cancer.

#3321

PUMA expression targets αvβ3/Slug+ "stem-like" tumor cells to prevent breast cancer progression independent of subtype.

Qi Sun, Jacqueline Lesperance, Jay S. Desgrosellier. _UCSD Moores Cancer Center, La Jolla, CA_.

Breast cancer cells bearing similarities to normal mammary stem cells represent some of the most aggressive tumors cells. Here, we show that αvβ3/Slug+ "stem-like" tumor cells are highly vulnerable to cell death induced by PUMA (p53-upregulated modulator of apoptosis). In human breast cancers, we were surprised to find that αvβ3/Slug+ cells represent a unique subset of tumor cells associated with progression that are present in all major histological subtypes. Analysis of "stem-like" cell lines showed that αvβ3 directs an EMT-independent role for Slug in suppressing PUMA expression and promoting tumor cell survival and stemness. In fact, αvβ3 is both necessary and sufficient for PUMA suppression and PUMA knockdown specifically rescued defective colony formation and tumor initiation caused by decreased β3 expression. Inducing PUMA expression with specific therapies selectively ablated αvβ3/Slug+ cells, preventing the formation of new colonies or tumors, with no effect on established disease. These new findings define an unexpected role for αvβ3/Slug-mediated PUMA suppression in driving stemness and suggest that inducing PUMA with specific therapies can selectively target "stem-like" breast cancer cells to prevent aggressive disease.

#3322

Estrogen and nuclear respiratory factor 1 act as joint mediators of redox modulation and stem cell aging that contribute in the pathogenesis of breast cancer.

Juan Vazquez, Deodutta Roy, Jayant Das. _Florida International University, Miami, FL_.

Deterioration of adult stem cell function with increasing age is a risk factor of breast cancer. Metastasis of breast cancer seems to be also influenced by aging or senescent cells overexpressing mitophagy/autophagy genes. Recently, estrogen, ROS, mitochondria and NRF1 activity have been implicated with stem cell aging, health and longevity. The exact underlying mechanisms of how aging of stem cells may increase the susceptibility to estrogen-induced carcinogenicity remain unknown. The main objective of this study was to examine whether estrogen, through activation of NRF1-mediated redox modulation and autophagic genes involved in senescence of stem cells, contributes in the development of breast cancer. To test this hypothesis, first we investigated the role of NRF1 to induce stemness by re-programming of breast epithelial cells (MCF-10A) and breast cancer cells (MCF-7 and MDA-MB231). We found that NRF1 promoted pluripotency markers: Oct4, Sox 2, and Nanog compare to MCF10A, MCF-7 or MDAMB 231 control cells. Loss of E-cadherin expression and gain of N-cadherin and vimentin expression in NRF1 overexpressing cells suggested that NRF1 promotes epithelial- mesenchymal transition. NRF1 promoted the ability to differentiate in vitro into a variety of cells (e.g chondrocytes, neurons, and smooth muscle cells). This suggested that NRF1 promotes multilineage differentiation potentials. Our data also showed that higher expression of NRF1 increased sizes of spheroids and ROS levels compared to controls. Mitophagy/Autophagy that regulates clearance of damaged mitochondria/apoptotic cells has important functions in regulating stem cell aging and tumorigenesis. Therefore, we examined the effect of estrogen on aging-associated diseases-related genes in NRF1 overexpressing as well as NRF1 silenced cells. Autophagy/Mitophagy/Apoptosis-related genes - BNIP3, PINK1, DJ-1 and LKB1 were altered in response to exposure of a carcinogenic concentration of estrogen. This effect of estrogen on these genes was further amplified by NRF1 overexpression and was inhibited by loss of NRF1. In summary, estrogen and NRF1 act as joint mediators of stem cell fate and reprogramming through a panel of critical stem cell proteins involved in modulating senescence and cell death. NRF1 seems to function as a rheostat to coordinate various cellular processes, such as resistance to apoptosis, autophagy and mitophagy of stem cells required for the stem cell aging as well as for the pathogenesis of breast cancer.

#3323

Breast cancer stem cell culture and enrichment using poly(Ɛ-caprolactone) 3D scaffolds.

Marc Rabionet,1 Sonia Palomeras,1 Ines Ferrer,2 Ariadna Sarrats,1 Ariadna Giro-Perafita,1 Maria Luisa Garcia-Romeu,2 Joaquim Ciurana,2 Teresa Puig1. 1 _TargetsLab (Oncology Unit); Medical Sciences Dept.; Faculty of Medicine, University of Girona, Girona, Spain;_ 2 _Department of Mechanical Engineering and Industrial Construction, University of Girona, Girona, Spain_.

Introduction: Cancer stem cell (CSC) population displays self-renewal capabilities, resistance to conventional therapies and a tendency for post-treatment recurrence. Increasing knowledge about CSCs phenotype and functions is needed to investigate new therapeutic strategies against CSC population. Poly(Ɛ-CaproLactone) (PCL) as biocompatible and free toxic dye polymer, have been used to manufacture scaffolds, solid structures suitable for 3D cancer cell culture. It has been described that scaffold cell culture enhances the CSCs population.

Experimental procedures: A RepRap BCN3D+ printer and 3.5 mm PCL wire were used to fabricate circular scaffolds. PCL design and fabrication parameters were first determined and then optimized considering several product features of the resulting scaffolds. MCF7 breast carcinoma cell line was used to assess scaffolds adequacy for 3D cell culture. To evaluate CSC enrichment the Mammosphere Forming Index (MFI) was performed in 2D and 3D MCF7 cultures.

Summary of the new, unpublished data: Results shown that the 60º scaffolds were more suitable for 3D culture than the 45º and 90º ones. Moreover, 3D culture experiments both, in adherent and non-adherent conditions, showed a significant increase on MFI compared to 2D cultures (control). Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and to enrich the CSCs population. Basically cells population growth with a 3D culture better and higher in value as it is demonstrated within the work.

Conclusions: Thus, 3D cell culture with PCL scaffolds could be useful to improve cancer cell culture and to enrich the CSCs population for further pre-clinical studies on CSC molecular characterization and treatment.

#3324

Salinomycin possesses anti-tumor activity and inhibits breast cancer stem-like cells via inhibition of STAT3 activation in triple-negative breast cancer cells.

Ji Young Kim, Hyunsook An, Eunhye Oh, Youngkwan Cho, Nahyun Lee, Jae Hong Seo. _Korea University, Seoul, Republic of Korea_.

Cancer stem cells (CSCs) play important roles in the formation, growth and recurrence of tumors, particularly following therapeutic intervention. Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44+/CD24- stem-like population. Salinomycin has received recent attention for its ability to target breast cancer stem cells (BCSCs), but the mechanisms of action involved are not fully understood. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in TNBC. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44+/CD24- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. TUNEL analysis of MDA-MB-231-derived xenografts revealed that salinomycin administration elicited a significant reduction in tumor growth with a marked downregulation of CD44, ALDH1 and phopspho-STAT3 levels, but seemingly without the induction of apoptosis. Our findings shed further light on the mechanisms responsible for salinomycin's effects on triple-negative breast cancer stem cells.

#3325

Combination of DNMT and HDAC inhibitors reprogram cancer stem cell signaling to overcome drug resistance.

Rajneesh Pathania, Ravindra B. Kolhe, Sabarish Ramachandran, Gurusamy Mariappan, Priyanka Thakur, Puttur D. Prasad, Vadivel Ganapathy, Muthusamy Thangaraju. _Georgia Regents University, Augusta, GA_.

In recent years, impressive technical advancements have been made in the isolation and validation of the mammary stem cells (MaSCs) and cancer stem cells (CSCs). However, the signaling pathways that regulate stem cell self-renewal are largely unknown. Further, CSCs are believed to contribute to resistance to chemotherapy and radiation therapy. However, an effective therapeutic strategy to overcome this resistance is yet to be identified. We have recently discovered that the DNA methyltransferases, especially DNMT1, play critical role in MaSCs and CSCs self-renewal and targeted deletion of this gene impaired mammary tumor formation by inhibiting CSCs formation. However, the molecular mechanism(s) by which DNMTs control CSCs and the therapeutic relevance of DNMTs inhibitors in regulation of CSCs and overcome drug resistance are also largely unknown. In this study, using MMTV-Neu-Tg mouse mammary tumor model, we found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which leads to activation of un-activated Neu-Tg into transformed tumor forming phenotype. Combination of 5-Azacytidine, a DNMT inhibitor, and butyrate, a HDAC inhibitor, markedly reduces CSCs and consequently increases the overall survival of the animal. RNA-seq analysis of the CSCs treated with 5-AzaC+butyrate provides evidence that combined inhibition of DNMTs and HDACs reduces CSCs pool in the mammary gland by blocking growth-promoting signaling molecules like RAD51AP1 and SPC25. RAD51AP1 and SPC25, which are known to play a key role in DNA damage repair and kinetochore assembly, are significantly overexpressed in breast tumor tissues and associated with decreased overall patients' survival. Further, these two genes are overexpressed in Tamoxifen and Taxol resistant human breast cancer cell lines. Functional inactivation of these genes in breast caner cells facilitates chemotherapy-induced apoptosis and reduces tumor growth. Overall, our studies provide strong evidence that breast CSCs (both basal stem cells and luminal progenitor cells) are susceptible for genetic and epigenetic modifications and associated with resistance to chemo- and radiotherapy. Thus, combination of DNMT and HDAC inhibitors can serve as an effective therapeutic strategy to block mammary tumor growth and to overcome drug resistance by inhibiting CSCs.

#3327

The tumor associated sialyltransferase ST6Gal-I promotes a cancer stem cell phenotype and upregulates stem-related transcription factors.

Matthew J. Schultz,1 Andrew T. Holdbrooks,1 Asmi Chakraborty,1 William E. Grizzle,1 Charles N. Landen,2 Donald J. Buchsbaum,1 Michael G. Conner,1 Rebecca C. Arend,1 Karina J. Yoon,1 Chris A. Klug,1 Daniel C. Bullard,1 Robert A. Kesterson,1 Patsy G. Oliver,1 Amber K. O'Connor,3 Bradley K. Yoder,1 Susan L. Bellis1. 1 _The University of Alabama at Birmingham, Birmingham, AL;_ 2 _University of Virgina, Charlottesville, VA;_ 3 _Children's National, Washington, DC_.

Altered glycosylation is a key hallmark of tumor cells; still, the role of individual glycosyltransferases remains unclear. ST6Gal-I is a tumor-associated sialyltransferase which catalyzes the addition of a sialic acid sugar to substrate glycoproteins. Addition of the negatively-charged sialic acid by ST6Gal-I has been shown to alter receptor conformation, clustering, and surface retention, leading to changes in downstream signaling. In this study we assayed ST6Gal-I by immunohistochemistry and report the great majority of patient ovarian and pancreatic tumors express this enzyme. In contrast, the normal epithelium expresses minimal ST6Gal-I. Enzyme expression in ovarian cancers is enriched during metastasis and correlates with worse progression-free and overall survival. Recent evidence points to ST6Gal-I activity in stem/progenitor cells. In light of this, we investigated whether ST6Gal-I functionally promotes a cancer stem cell (CSC) phenotype, i.e. resistance to chemotherapy, survival as tumorspheroids, and ability to initiate tumors. We previously reported that ST6Gal-I activity confers resistance to cisplatin; we now show its activity additionally confers resistance to gemcitabine in pancreatic tumor cells. ST6Gal-I expressing cells are enriched in patient derived xenografts (PDX) treated with gemcitabine suggesting that these cells preferentially survive chemotherapy in vivo. In addition to chemoresistance, ST6Gal-I promotes the growth of pancreatic and ovarian cell lines in tumorspheroid culture. Moreover, ST6Gal-I expressing primary tumor cells isolated from ovarian cancer ascites or PDX tumors survive in tumorspheroid culture, whereas ST6Gal-I negative cells do not. Conversely, forced expression of ST6Gal-I protects tumor cells exposed to the ascites fluid milieu in vitro, while non-ST6Gal-I expressing cells succumb to this inflammatory environment. In a limiting dilution tumor initiating assay, ST6Gal-I expressing cells have a higher tumor incidence and form larger tumors compared to cells with ST6Gal-I knockdown. We next created a conditional mouse model with forced ST6Gal-I expression in the intestinal tract and used AOM-DSS chemically-induced carcinogenesis model to evaluate tumor formation. Compared with wildtype mice, ST6Gal-I knock-in mice have a greater tumor burden, evidenced by increased tumor number and area. As a novel mechanistic link beteween ST6Gal-I and the CSC phenotype, direct modulation of ST6Gal-I levels in tumor cells regulates the expression of stem-related transcription factors, Sox9 and Slug, implicated in tumor progression. The finding that a distinct glycosyltransferase governs the expression of key transcription factors highlights the tumor glycome as a driving factor in CSC behavior.

#3328

MYC and MCL1 cooperatively promote chemotherapy-resistant cancer stem cells through regulation of mitochondrial biogenesis and oxidative phosphorylation.

Kyung-min Lee, Jennifer Giltnane, Justin Balko, Carlos Arteaga. _Vanderbilt University, Nashville, TN_.

Triple negative breast cancer is the most virulent subtype of this malignancy. Although initially responsive to cytotoxic chemotherapy, most patients with TNBC eventually develop drug-resistant disease. We previously reported co-amplification of MYC and MCL1 in a cohort of TNBCs after neoadjuvant chemotherapy (Balko et al. Cancer Discov. 2014). In addition, MYC and MCL1 are overexpressed in paclitaxel-resistant compared to paclitaxel-sensitive TNBC cells. We hypothesized that MYC and MCL1 play a role in chemotherapy resistance. TNBC cell lines with claudin-low gene expression exhibited the highest levels of MYC and MCL1 mRNAs. This molecular subtype of breast cancer is enriched for cancer stem cells (CSCs) and exhibits resistance to neoadjuvant chemotherapy. Knockdown of both MYC and MCL1 by siRNA in SUM159PT and MDA-MB-436 TNBC cells significantly reduced mammosphere formation and CSC markers such as ALDH and CD44high/CD24low. Conversely, overexpression of ectopic MYC and MCL1 in MDA-MB-468 cells increased mammosphere formation and CSC markers. It has been reported that mitochondrial oxidative phosphorylation (OXPHOS) is enhanced in CSCs. Thus, we investigated a role for MYC and MCL1 in mitochondrial OXPHOS using Seahorse XFe extracellular flux analyzer. CSCs with high expression of MYC and MCL1 sorted by flow cytometry exhibited increased mitochondrial OXPHOS compared to non-CSCs. Transfection of MYC and MCL1 expression vectors into MDA-MB-468 cells enhanced mitochondrial OXPHOS activity. Conversely, knockdown of MYC and MCL1 in SUM159PT and MDA-MB-436 cells reduced mitochondrial OXPHOS activity. MYC mediated these effects by promoting mitochondrial biogenesis whereas MCL1 potentiated mitochondrial OXPHOS via localization into the mitochondrial matrix, independent of its ability to associate with BH3-domain containing anti-apoptotic proteins (BAD, BCL2, BCL-XL). Deletion of the mitochondrial target sequence (MTS) of MCL1 impaired its ability to increase mitochondrial OXPHOS and mammosphere formation. Reactive oxygen species (ROS), which are produced in CSCs as a by-product of activated mitochondrial OXPHOS, were abrogated by MYC and MCL1 siRNA. To determine the effect of ROS on CSCs, we treated SUM159PT and MDA-MB-436 cells with hydrogen peroxide (H2O2). Treatment with H2O2 enriched CSCs, which showed up-regulation of several cancer stem cell genes such as Nanog and IL-8. Enrichment of CSCs by H2O2 was diminished by n-acetylcysteine (NAC), an antioxidant. These data suggest that MYC and MCL1 co-amplification confers drug resistance to TNBC via mitochondrial OXPHOS-mediated enrichment of CSCs. Moreover, MYC and MCL1 stimulate H2O2 production, which potentiates CSCs. Thus, targeting mitochondrial OXPHOS and H2O2 may be an effect strategy to delay or reverse resistance to chemotherapy in MYC and MCL1 amplified TNBC.

#3329

Increasing the efficacy of cancer stem cell-based vaccine in the PD-L1/PD-1 blockade.

Yangyang Hu,1 Yang Xia,1 Xin Chen,1 Li Zhou,1 Yangyi Bao,2 Shiang Huang,3 Xiubao Ren,4 Elaine Hurt,5 Robert E. Hollingsworth,5 Alfred E. Chang,1 Max S. Wicha,1 Qiao Li1. 1 _University of Michigan Comprehensive Cancer Center, Ann Arbor, MI;_ 2 _The Third Affiliated Hospital of Anhui Medical University, China;_ 3 _Center for Stem Cell Research and Application, Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, China;_ 4 _Department of Biotherapy, Tianjin University Cancer Institute and Hospital, China;_ 5 _MedImmune Inc., Gaithersburg, MD_.

We have previously reported the development of a strategy to target the cancer stem cell (CSC) populations in melanoma and squamous cell carcinoma using CSC lysate-pulsed dendritic cells (DCs). Using mouse models we demonstrated that DCs pulsed with CSCs enriched by virtue of their expression of the CSC marker ALDH (termed CSC-DC) significantly inhibited tumor growth. However, CSC-DC vaccine therapy alone may not be sufficient to overcome the immunosuppressive components of the tumor microenvironment. CSC-mediated suppression of T cells has been reported in a process involving the PD-L1/PD-1 axis. Immunologically targeting CSCs while simultaneously blocking PD-L1/PD-1-mendiated immune suppression may significantly enhance the outcome of current immunotherapies of cancer. To test this hypothesis, we investigated the effect of using anti-PD-L1 to block the immunosuppression during the CSC-DC vaccination to augment the therapeutic efficacy of using each regimen alone. We used 4T1 cell line, a mammary carcinoma syngeneic to BALB/c mice, and found that 4T1 tumors contain approx. 6-10% ALDEFLUOR+/ALDHhigh cells, and that these cells are enriched for tumor initiating capacity compared to bulk or ALDEFLUOR-/ALDHlow cells. Inoculating 4T1 cells into the mammary fat pad induces the development of spontaneous pulmonary metastases. ALDHhigh 4T1 CSCs expressed PD-L1. 4T1 ALDHhigh CSC-DC vaccine + anti-PD-L1 administration significantly reduced pulmonary metastases and prolonged survival compared with 4T1 ALDHhigh CSC-DC vaccine or anti-PD-L1 administration alone. Anti-PD-L1 administration increased systematic anti-4T1 CSC immunity induced by CSC-DC vaccine. This is evident by the increased production of total IgG in the serum samples collected from the 4T1-bearing animals subjected to 4T1 CSC-DC vaccination with anti-PD-L1 administration. The CSC-reactivity/specificity of the immune sera was demonstrated by their specific binding to the ALDHhigh vs. ALDHlow 4T1 cells in flow cytometry. Importantly, the immunological consequence of such binding was Ab-mediated ALDHhigh 4T1 CSC lysis via complement dependent cytotoxicity. In addition, CTLs generated from the splenocytes harvested from the hosts subjected to 4T1 CSC-DC vaccination with anti-PD-L1 administration killed ALDHhigh 4T1 CSCs significantly more than ALDHlow 4T1 cells. These results are supportive of the conclusion that administration of anti-PD-L1 as an adjuvant could significantly boost the therapeutic efficacy of the CSC-DC vaccine, and that this effect was due to both antibody and T cell-mediated anti-CSC immunity.

#3330

Tumor angiogenesis and cancer stem cells in serous adenocarcinoma of the ovary.

Syama Krishna Priya, Rohit P. Nagare, V S. Sneha, C Sidhanth, S Bindhya, P Manasa, Trivadi S. Ganesan. _Cancer Institute (WIA), Chennai, India_.

Angiogenesis is required for tumor growth and metastasis. The conventional view of tumour angiogenesis is that tumours get their blood supply from the neighbouring normal stroma. However, recently the origin of tumour endothelial cells or pericytes in part has been shown to be derived from cancer stem cells (CSC) in glioma. The spread of ovarian cancer (SOC) is different and the origin of endothelial cells in the tumor is not known. Using spheroids as an in vitro model (which is enriched for CSC), we have evaluated the role of CSC in primary malignant cells (PMCs) from patients with serous adenocarcinoma of ovary (n=30) cultured under endothelial conditions. The expression of endothelial markers (CD31, CD105, and CLEC14a) was evaluated by flow cytometry. Further, the ability of CSC to express endothelial markers under appropriate growth conditions was also evaluated with Bevacizumab (Avastin) or Cediranib which antagonize VEGF or its receptor (VEGFR2) respectively. In addition, functional assays such as uptake of Dil labelled acetylated low density lipoprotein (Dil-ac-LDL) and expression of endothelial nitric oxide synthase (e-NOS) was performed to assess the endothelial phenotype. The localization of tumour blood vessels and CSC in primary ovarian tumors was examined by double immunohistochemistry of CD31/CD105/CLEC14a and ALDH1a1. Primary malignant cells (n=30) grown in endothelial growth medium (EGM) showed significantly higher expression of CD105 (mean 20.8%, p = 0.001) and CLEC14a (mean 5.3%, p = 0.012) and a co-expression of CD105/CLEC14a (mean 1.6%, p= 0.012) than that of control (mean, 12.5%, 2.5% and 0.95% respectively). The co-expression of ALDH1a1 with endothelial markers, CD31, (mean, 1.58%), CD105, (mean 0.7%), CLEC14a (mean 0.44%) in primary malignant cells (n=5), denovo, suggest that there is a small proportion of cells which are in transit to form endothelial cells. When the primary malignant cells were grown as spheroids in endothelial conditions in the presence or absence of Avastin (1 µg/µl), there was reduction in the expression of CD105 (mean, 12.5% (EGM) and 7.9% (Avastin), P = 0.056) and CLEC14a (mean. 5.5% (EGM) and 1.5% (Avastin), P = 0.04). However, when the spheroids were cultured in presence of cediranib (10 nM), the expression of CD105 was not reduced (Mean, 2.53% (EGM) and 4% (cediranib). The cells grown in endothelial conditions showed uptake of Dil-ac-LDL (n=3) and expressed e-NOS (n=3), confirming their endothelial phenotype. The double immunostaining with ALDH1a1 and CD31/CD105 demonstrated that the blood vessels were in proximity to ALDH1A1+ cells in primary serous adenocarcinoma of the ovary (n=5). These results suggest that a proportion of endothelial cells (probably 20%) could be derived from CSC in serous ovarian carcinoma and the VEGF pathway has an important role. This property of CSC to contribute to tumour angiogenesis can be inhibited.

#3331

RP6530, a dual PI3K δ/γ inhibitor attenuates cancer stem cell proliferation in serous adenocarcinoma of ovary.

Sneha S,1 Nivetha R,1 Srikant Viswanadha,2 Swaroop Vakkalanka,3 Ganesan TS1. 1 _Cancer Institute, Chennai, India;_ 2 _Incozen Therapeutics Pvt. Ltd., Hyderabad, India;_ 3 _Rhizen Pharmaceuticals SA, Chennai, Switzerland_.

The overall survival of patients with advanced serous adenocarcinoma of the ovary (EOC) has not significantly improved for the past 3 decades despite advances in treatment. In part, this is due to resistance of malignant cells and it is currently hypothesized that this could be due to persistence of cancer stem cells (CSC). Elimination of these cells may improve the survival of patients. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor that demonstrated high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. A Phase-1 dose-escalation study to evaluate the safety and efficacy of RP6530 in patients with hematological malignancies in currently underway at several sites in Europe. The objective of this study was to test the efficacy of RP6530 against the CSC of EOC. A spheroid assay enriched for CSC was employed for this study. Initially, we have analyzed 3 cell lines (PEO4, OVCAR-5 and CAOV-2) with and without addition of 5 µM RP6530. In addition, MTT assay for cell viability was performed for the molecule to evaluate for non-selective cytotoxicity. To confirm that the target was CSC, we performed analysis of Side population (SP) based on H33342 dye exclusion. In the screening assay (triplicate and repeated thrice), RP6530 significantly inhibited spheroid formation in PEO4 and OVCAR-5 (p<0.05) and CAOV-2 (p<0.0005). In the cell viability assay, RP6530 had no cytotoxic effect in any of the cell lines. To evaluate the effect of the compound on CSC, we performed analysis of SP cells with Hoechst 33342 at 355 nm by flow cytometry. RP6530 either caused a reduction in the fraction of SP cells (PEO4 and CAOV-2) or was neutral (OVCAR-5) indicating that the compound preferentially targets CSC in ovarian cancer cell lines. Further in vitro and in vivo experiments are ongoing to conclusively prove this effect.

#3332

Targeting cancer stem cells in ovarian cancer.

Sham S. Kakar, Mariusz Z. Ratajczak. _University of Louisville, Louisville, KY_.

Ovarian cancer is a disease commonly complicated by the presence of ascites in the abdominal cavity which represents a major clinical problem. Currently, the treatment for ovarian cancer entails cytoreductive surgery followed by chemotherapy, mainly cisplatin or carboplatin combined with paclitaxel. Although this regimen is initially effective in a high percentage of cases, unfortunately within few months of initial treatment, tumor relapse occurs because of platinum-resistance. This is attributed mainly due to the presence of cancer stem cells (CSCs) present in ascites which are chemo-resistant and responsible for recurrence of cancer. In our preliminary studies, we show for the first time that withaferin A (WFA), a bioactive compound isolated from the plant Withania somnifera targets putative CSCs. Treatment of nude mice bearing orthotopic ovarian tumors with WFA resulted in a 70% to 80% reduction in tumor growth, complete inhibition of metastasis, and a significant elimination of the cells expressing CSCs markers - CD44, CD24, CD34, CD117, ALDH1 and Oct4 and down regulation of Notch1 and its downstream signaling genes (Hes1 and Hey1) reported to play crucial role for self-renewal and maintenance of CSCs. In addition, WFA also resulted in a significant inhibition of securin expression an "oncogene" important in ovarian tumorigenesis. In contrast, treatment of mice with CIS alone had the opposite effects, causing an increase in cells expressing CSC markers and Notch1 signaling pathway. However, combining of WFA with CIS showed enhanced effects on suppression of securin as well as elimination of CSCs. This may explain the development of platinum-resistance and recurrence of cancer in patients treated with first line chemotherapy. Based on this information, we hypothesize that a combination of WFA with CIS should target ovarian cancer cells as well as cancer stem cells by reducing securin and CSCs thereby reducing CIS resistance and recurrence of ovarian cancer. In our study, we used cells from ascites from patients with recurrent ovarian cancer and determine the effect of WFA alone and in combination with CIS on tumorigenic functions of ascites cells and CSCs from ascites in vitro and in vivo, and defined the molecular mechanisms associated with self-renewal, maintenance and recurrence of cancer in relation to expression of securin. Because the two agents act synergistically, this combination may minimize side effects as well as induction of cisplatin resistance and recurrence of cancer. In addition, this study will justify the initiation of phase I/phase II clinical trials to assess potential toxicity and efficacy of WFA/CIS combination in advanced/recurrent ovarian cancer patients.

### Stemness Properties of Leukemias and Carcinomas

#3334

KLF4 promotes self-renewal by repressing DYRK2-mediated degradation of c-Myc in leukemic stem cells: development of targeted therapy.

Chun Shik Park,1 Ye Shen,1 Koramit Suppipat,1 Julie Tomolonis,1 Monica Puppi,1 Toni-Ann Mistretta,1 Leyuan Ma,2 Michael Green,3 Daniel Lacorazza1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _University of Massachusetts, Massachusetts, MA;_ 3 _University of Massachusetts, Worcester, MA_.

Chronic myeloid leukemia (CML) is the first blood cancer known to originate from a single hematopoietic stem cell (HSC) by expression of BCR-ABL, a product of the chromosomal translocation t(9;22), that slowly progress to a lethal fast-growing leukemia caused by malignant reprogramming of progenitor cells (blast crisis). Although CML can be successfully managed with targeted therapy by suppressing BCR-ABL kinase activity with tyrosine kinase inhibitors (TKI), patients remain in remission as long as they adhere to a lifelong treatment. The leukemic stem cell (LSC) population emerges as a key 'CML reservoir' that escapes TKI therapy by developing BCR-ABL-independent mechanisms of self-renewal and survival. LSC still remains an elusive target because of our poor understanding of specific self-renewal mechanisms and inability to selectively eliminate LSC without damaging normal hematopoiesis. Thus, there is a need for alternative drugs for relapse patients to prevent reactivation of BCR-ABL-positive LSC after stopping chemotherapy or emergence of chemoresistance and as frontline therapy to achieve treatment-free remission. We found that somatic deletion of the transcriptional factor Krüppel-like factor 4 (KLF4) in BCR-ABL(p210)-induced CML severely impaired disease maintenance. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSCs in bone marrow and the spleen and impaired ability of LSCs to recapitulate leukemia in secondary recipients. This data suggest that KLF4 promotes self-renewal of LSCs whereas serial transplantation indicates that KLF4 restricts stress self-renewal of normal HSCs and thus inhibition of KLF4 function would impair LSC self-renewal without altering blood production. Analyses of global gene expression in purified LSCs and genome-wide binding of KLF4 in a murine CML cell line revealed that KLF4 represses the gene encoding for the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2). Immunoblots revealed elevated levels of DYRK2 protein in KLF4-deficient LSCs purified from the bone marrow of CML mice. Because phosphorylation of c-Myc and p53 by DYRK2 induces proteosomal degradation and cell death, respectively, we found that DYRK2 upregulation in KLF4-deficient LSCs was associated with a reduction of c-Myc protein and increased cleavage of PARP. As a proof-of-principle of the therapeutic potential of this finding, we explored the efficacy of vitamin K3 to eradicate LSCs by inhibiting the ubiquitin E3 ligase SIAH2 in charge of DYRK2 degradation. Vitamin K3 efficiently reduced cell viability in a panel of human-derived CML cell lines by inducing Dyrk2 expression and apoptosis. The identification of Dyrk2 as a critical mediator of LSC downfall is an innovative paradigm poised to support the development of LSC-specific therapy to induce treatment-free remission in CML patients.

#3335

The high cMyc expression characterizes a small cell subpopulation in ALK-positive anaplastic large cell lymphoma that carry cancer stem-like properties.

Chengsheng Wu, Haifeng Zhang, Nidhi Gupta, Alshareef Abdulraheem, Qian Wang, Yunghsin Huang, Raymond Lai. _University of Alberta, Edmonton, Alberta, Canada_.

We have previously identified two phenotypically distinct cell subpopulations in ALK+ anaplastic large cell lymphoma (ALK+ALCL) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and chemoresistant than reporter unresponsive (RU) cells. The regulation of the RU/RR dichotomy remains to be elusive. Our bioinformatics analysis of the SRR2 sequence suggests that cMyc may be an important factor. In support of this concept, we found a substantially higher expression of cMyc and more cMyc and Sox2-SRR2 probe bindings in RR cells than in RU cells. Inhibition of cMyc in RR cells significantly decreased SRR2 luciferase activity, and overexpression of cMyc in RU cells significantly increased SRR2 luciferase activity. Correlating with this, cMyc inhibition in RR cells significantly attenuated the clonogenicity and sensitized cells to doxorubicin; while overexpression of cMyc in RU cells conferred to the potentiated clonogenicity and chemoresistance. Importantly, we found cMyc not only maintains Sox2 protein expression, but also promotes Sox2 SRR2 probe binding and its transcriptional activity. Moreover, we found the highly active Wnt canonical pathway (WCP) contributes to the high expression of cMyc in RR cells. Intriguingly, we observed a positive regulatory loop in RR cells, in which Sox2 up-regulates Wnt2B, which can activate the WCP and cMyc. Furthermore, the significance of cMyc in this context was further highlighted by the fact that RU cells with stably transfected c-MYC are phenotypically similar with RR cells in terms of the elevated activation of WCP and increased tumorigenicity in vivo. Lastly, we found cMyc is heterogeneously expressed in ALK+ALCL tumor cells (n=5) by immunohistochemcial staining, and it co-localizes with active β-catenin, a marker of active WCP, in tumor cells by immunofluorescence assay. In conclusion, cMyc is a key regulatory factor in the determination of RU/RR dichotomy, which is linked to tumorigenic potential and chemo-resistance in ALK+ALCL.

#3336

ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling.

Kaitlyn A. Dorsett, Susan L. Bellis. _University of Alabama at Birmingham, Birmingham, AL_.

Acute myelogenous leukemia (AML) is characterized by an increased number of myeloid cells in the bone marrow. These cells are arrested in their maturation process, frequently resulting in hematopoietic insufficiency. Recent advancements in molecular genetics have identified new markers that may be useful in diagnosing AML, including mutant c-kit (CD117). c-kit is a receptor tyrosine kinase and activating mutations in this receptor have been identified as a functional driver of AML and other myeloproliferative diseases. c-kit also plays a major role in maintaining normal stem cell pools. Prior studies suggest that ST6Gal-I, a unique glycosyltransferase upregulated in many cancers, promotes a stem-like cell phenotype in epithelial tumor cells. ST6Gal-I is a sialyltransferase that adds a negatively charged sialic acid in an α2-6 linkage to membrane glycoproteins. In this study, we show c-kit to be a substrate of ST6Gal-I, with increased sialylation correlating to increased c-kit activation. In order to study the effects of sialylation on signaling, we forced constitutive expression of ST6Gal-I in the U937 monocytic leukemia cell line. c-kit signaling was then induced by treating U937 cells with stem cell factor (SCF), the c-kit ligand. Here we show enhanced SCF-mediated c-kit activation, indicated by increased levels of phospho-c-kit in ST6Gal-I overexpressing cells. This, in turn, is associated with increased activation of downstream effecters of c-kit signaling, phospho-Erk and phospho-Akt. We further hypothesize that ST6Gal-I might play a role in the cell differentiation process. To test the effects of sialylation on differentiation, we used a well-established model to induce monocyte cell differentiation, treatment with the phorbol ester, PMA. PMA is known to induce macrophage differentiation in U937 cells, evidenced by exit from the cell cycle and upregulation of differentiation markers, including CD11b. Macrophage differentiation induces downregulation of endogenous ST6Gal-I, thereby reducing surface sialylation of specific receptors. However, forced overexpression of ST6Gal-I inhibits PMA-induced exit from the cell cycle, suggesting that ST6Gal-I acts to maintain cells in an undifferentiated state. To further understand the effects of receptor sialylation on PMA-induced differentiation, we used flow cytometric analysis to evaluate surface markers on U937 cells with or without ST6Gal-I overexpression. We show that forced expression of ST6Gal-I inhibits PMA-induced upregulation of CD11b, whereas c-kit expression is elevated in ST6Gal-I overexpressing cells relative to parental cells. These results suggest a novel mechanism by which ST6Gal-I contributes to an undifferentiated state in part through the regulation of the c-kit receptor.

#3337

Targeting acute myeloid leukemia via anti-IL1RAP antibodies.

Ping Jiang,1 Bob Y. Liu,1 Jen Huang,1 Jennifer Lu,1 Sharmili Roy,1 Madhavi Mishra,1 Xiaoxian Zhao,2 Jeffrey Lin,2 Eric D. Hsi,2 Jagath R. Junutula1. 1 _Cellerant Therapeutics, San Carlos, CA;_ 2 _2Robert J. Tomsich Pathology & Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH_.

The current standard of care for Acute Myeloid Leukemia (AML) is largely ineffective in terms of achieving long term remission. This is mostly due to inability to remove all of the leukemic cells, and there is accumulating evidence to support the remaining leukemic cells are Leukemic Stem Cells (LSC) which are resistant to standard chemotherapy. We and others (Barreyro L. et. al. 2012) have discovered that IL-1 receptor accessory protein (IL1RAP) is overexpressed on LSC and their progenitors, but not on normal hematopoietic stem cells (HSC). Moreover, knockdown/knockout of IL1RAP expression greatly decreases growth/survival of AML cells. Thus, we have screened anti-IL1RAP therapeutic antibodies by applying FACS based analysis and Complement-Dependent Cytotoxicity (CDC) assays on both AML cell lines and primary patient samples. In a parallel effort, we also applied a functional IL-1β-induced NF-κB signaling assay to identify function-blocking anti-IL1RAP antibodies. Colony Formation Cell (CFC) assay results demonstrated that lead IL1RAP antibodies can significantly impede growth of both AML blast cells and leukemic stem cell-like cells. In contrast, normal HSCs are unaffected by these antibodies. Lead anti-IL1RAP antibodies also showed tumor growth inhibition in an established EOL-1 orthotopic tumor model. One major challenge of targeting IL1RAP by an antibody is that a large amount of IL1RAP protein is secreted into human serum, serving as a potential "sink" that inhibits IL1RAP antibody efficacy. We addressed this by developing a novel IL1RAP antibody that binds preferentially to the membrane-bound IL1RAP protein as compared to the secreted form. We will discuss different therapeutic development strategies to further advance the utilization of these lead anti-IL1RAP therapeutic antibodies for treating AML and other myeloid leukemic indications.

#3338

Pacritinib reduces human myeloid leukemia stem cell maintance in a defined niche.

Larisa Balaian, Anna Kulijian, Edward D. Ball, Catriona H.M Jamieson. _UCSD, La Jolla, CA_.

Introduction

Pacritinib, a potent clinical small molecule inhibitor of JAK2, it also suppresses signaling through wild-type and mutant FLT3, IRAK1, and CSF1R. Pacritinib does not cause marrow suppression and has demonstrated single agent activity preclinically in myelofibrosis and other myeloid neoplasms including AML and CMML. Stromal protection was not observed. However, the capacity of pacritinib to eradicate therapy resistant leukemia stem cells (LSC), residing in the bone marrow niche, had not been examined. Thus, we investigated the impact of pacritinib alone or in combination with standard of care therapy on primary blast crisis chronic myeloid leukemia (BC CML), myelofibrosis (MF) and AML LSC survival and self-renewal in a stem cell supportive niche.

Methods

Genetically engineered mouse bone marrow fibroblasts producing human SCF, IL3 and G-CSF were used as stromal monolayers to support LSC survival and self-renewal. Human primary CD34+ cells were selected from BC CML (n=5), MF (n=5) and relapsed AML (n=4) before and after clinical treatment with azacitidine. As a control, CD34+ cells from age matched normal bone marrow (a-NBM, n=4) were used for the co-culture. Survival and self-renewal of the cells were investigated by colony forming and replating assays. Pacritinib was used at concentrations ranging from 10 to 50 nM alone and in combination with 1 nM dasatinib .

Results

Pacritinib alone induced dose-dependent inhibition of self-renewal in a-NBM, AML, MF and CML-BC, with the optimal concentration of 20nM leading to IC50 diversity in the response between normal and leukemia progenitors. AML and MF responded uniformly and inhibition reached 50% at 10nM concentration. BC CML cells were more divergent: 40% demonstrated >50% inhibition, in another 40% it was 20-50% and in 20% inhibition was <20%. Combined treatment with the low dose of dasatinib and pacritinib doses of 10 or 20 nM resulted in a significant (p<0.001, Anova) difference in self-renewal of all BC CML cells, raising a possibility of an additive/synergistic mechanisms. AML cells collected before and after clinical treatment with azacitidine uniformly showed a significant decrease in self-renewal starting with 10 nM pacritinib alone and combined treatment with dasatinib did not enhance the inhibition, suggesting a prospect of using pacritinib as a single agent in the treatment of relapsed AML.

Conclusions

Together these data indicate that possibly through inhibition of CSF1 and IRAK1 signaling in addition to suppression of JAK2, even in the presence of a LSC supportive niche, readily clinically achievable low nM concentrations of pacritinib alone are effective in reducing self-renewal of MF and relapsed AML. However, a combination of dasatinib and pacritinib is required to eliminate self-renewing LSC in BC CML with minimal toxicity toward normal progenitors. Targeting niche-dependent signaling could represent a robust avenue for treatment of refractory myeloid leukemia.

#3339

Single-cell analysis reveals molecular mechanisms of NRAS-mediated leukemia stem cell self-renewal in a murine model of AML.

Rebecca S. LaRue, Klara E. Noble-Orcutt, Conner Hansen, Ngoc Ha, David A. Largaespada, Zohar Sachs. _University of Minnesota, Minneapolis, MN_.

Acute myeloid leukemia (AML) is frequently fatal because patients who initially respond to chemotherapy eventually relapse. Most anticancer therapies are designed to inhibit proliferation. Yet, in hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al., Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to eradicate this disease. NRASG12V is required for self-renewal in a murine AML model (Sachs et al., Blood 2014). To study NRAS-mediated leukemia self-renewal, we use a transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, NRASG12V oncogene (Kim et al., Blood 2009). Doxycycline abolishes NRASG12V expression leading to leukemia remission. We hypothesize that NRAS-activated pathways required for self-renewal are limited to a subpopulation of LSCs. To identify the NRASG12V-mediated self-renewing subpopulation, we performed single-cell RNA sequencing on the LSC-enriched cells from our AML model. Hierarchical clustering of LSC single-cell data identified three discrete profiles. Comparing the profiles of NRASG12V-expressing LSCs ("RAS-on") to doxycycline-treated LSCs ("RAS-Off") revealed that two of the three LSC-expression profiles are lost when NRASG12V is withdrawn. These data suggest that these two profiles are NRASG12V-dependent consistent with an earlier report that activated NRAS exerts bimodal effects on HSCs (Li et al., Nature 2013). One of these LSC subpopulations preferentially expresses genes associated with leukemia self-renewal. On the basis of this single-cell gene expression data, we identfied cell surface markers (CD36 and CD69) that delineate the two NRASG12V-responsive LSC-subpopulations. We sorted LSCs based on CD36 and CD69 status and found that CD36-CD69+ LSCs (the group that expresses self-renewal gene expression profiles) harbor nearly all of the colony-forming capacity of the LSCs, forming an average of 13 colonies versus 0.33 colonies for CD69- LSCs and versus 0.11 colonies for non-LSCs (per 10,000 cells plated, p < 0.00001 for each comparison). Accordingly, in vivo mouse reconstituting experiments show that the CD36-CD69+ LSC is the only LSC subgroup that can reconstitute the leukemia in mice (p < 0.005). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature. Using mass cytometry to query the activation status of signaling pathways simulteneously with multiple immunophenotypic markers, we show that Ki67Low LSCs (the putative self-renewing LSCs) preferentially express increased levels of β-catenin and Myc. These data implicating AML self-renewal pathways can provide precise molecular targets for treating this deadly disease.

#3340

Doublecortin-like kinase 1 marks cancer stem-like cells and modulates drug-resistance, self-renewal, and tumorigenesis in renal cancer.

Yang Ge,1 Nathaniel Weygant,2 Dongfeng Qu,2 Randal May,2 William L. Berry,2 Parthasarathy Chandrakesan,2 James J. Tomasek,2 Guangyu An,1 Courtney W. Houchen2. 1 _Beijing Chao-Yang hospital, Capital Medical University, Beijing, China;_ 2 _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Introduction

Doublecortin-like kinase 1 (DCLK1), is a tumor stem cell-specific marker, expressed in many cancers and highly correlated to cancer initiation, EMT and progression. We recently reported DCLK1's epigenetic dysregulation and specific overexpression in clear cell renal carcinoma (RCC). In this study, we assessed DCLK1's value as a therapeutic target in RCC progression.

Materials and Methods

RCC cell line Caki-2 was infected with Lentivirus to overexpress DCLK1. Moreover, DCLK1 gene expression was knocked down in ACHN and Caki-2 cell lines using small interfering RNA. Gene and protein expression levels were measured by real time RT-PCR and Western blotting respectively. Proliferation and drug resistance was determined by MTT assay. Cell cycle analysis and sorting of DCLK1+/DCLK1- cells were completed by FACS. Clonogenicity was evaluated using colony formation assay in matrigel.

Results

Overexpression of DCLK1 increases Caki-2 cell proliferation, but despite DCLK1's microtubule localization, there was no evidence of altered cell cycle status. Aldehyde dehydrogenase (ALDH), one of only a few known stem cell markers in RCC, was highly upregulated (>5-fold) by DCLK1 overexpression and downregulated (2-fold) by knockdown of DCLK1 in RCC cells. FACS results also indicated the existence of small ALDH and DCLK1 double-positive populations in both Caki-2 (1.51%) and ACHN (0.54%) RCC cells. Furthermore, there was approximately 7% more ALDH+ cells in DCLK1 overexpressing Caki-2 cells compared to control. In further support of molecular hallmarks, we observed increased expression of HIF-1α and Vimentin mRNA and protein as well as enhanced clonogenic capacity of ACHN and Caki-2 RCC cell lines in matrigel at baseline and after 72 h treatment with 0.5 μM Sunitinib. Immunocytochemistry of the spheroids suggested that DCLK1 enhances colony formation ability by triggering expression of pluripotency factors NANOG and POU5F1/OCT4. Since DCLK1 is known to have an extracellular domain we performed FACS sorting to determine if we could isolate a viable DCLK1+ population from the ACHN cell line. ACHN-DCLK1+ cells had >1.5 fold higher relative colony number and larger sphere size in three-dimensional clonogenic assay compared to ACHN-DCLK1- cells. Finally, we performed MTT assays demonstrating that overexpression of DCLK1 caused Caki-2 cells to become resistant to Sunitinib, Everolimus, and Temsirolimus compared to controls after 48 h treatment. Converesely, knockdown of DCLK1 sensitized both ACHN and Caki-2 cells to Sunitinib and Sorafenib after 48 hours treatment.

Conclusion

DCLK1 modulates proliferation, stemness, tumorigenesis and resistance to FDA-approved drugs, and marks a population of stem-like cells in RCC. These findings suggest that targeting DCLK1 may be a novel primary or adjuvant therapeutic strategy in RCC.

#3341

Microarray analysis of gastrointestinal stromal tumor-originated spheroids.

Hirotoshi Kikuchi, Shinichiro Miyazaki, Tomohiro Murakami, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Manabu Ohta, Takanori Sakaguchi, Hiroyuki Konno. _Hamamatsu Univ. School of Medicine, Hamamatsu, Japan_.

Background: Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit are thought to occur during tumor progression. Although sequential therapy with tyrosine kinase inhibitors (TKIs), imatinib mesylate, sunitinib malate and regorafenib, is an effective and standard treatment strategy for recurrent or metastatic GISTs, it is difficult to cure recurrent GISTs with TKIs alone. It has been reported that viable cells are often seen in tumors resected in response to imatinib therapy and they may cause refractory tumors after interruption of imatinib in responding patients with advanced GISTs. Therefore targeting these persistent clones, which may have stem cell characteristics, can be a novel treatment strategy to cure recurrent or metastatic GISTs. In this study, we generated spheroids from resected GIST and performed microarray analysis to identify gene sets typically expressed in GIST-originated spheroid (GISTOS).

Methods: According to the previous report of cancer tissue originated spheroid (CTOS) (Kondo J et al. PNAS 108:6235-40, 2011), GISTOS was prepared from fresh samples of 6 primary gastric GISTs. Total RNAs were extracted from 6 GISTOS and their original tumors and utilized for cDNA microarray analysis. None of the patients had received imatinib therapy before surgery. Gene expression levels were compared between GISTOSs and their original tumors.

Results: Hierarchical clustering revealed two distinct clusters, and distinguished GISTOS from original tumors. Gene set enrichment analysis (GSEA) revealed that the gene sets CISPLATIN_RESISTSNCE, METASTASIS_STROMAs and TUMOR_INVASIVENESS_2D were upregulated and gene sets CELL_CYCLE_G1_S and MIR21_TARGETS were downregulated in GISTOSs compared with original tumors. These gene sets appear to reflect distinctive features of GISTOS that consists of enriched stem cell populations.

Conclusions: We generated GIST-originated spheroids. Microarray analysis revealed gene sets up-regulated and down-regulated in GISTOS. These gene sets appear to reflect distinctive features of GISTOS and may serve as novel molecular markers of stem cells in GIST.

#3342

Galiellalactone derivative targets stem cell population in ENZ-resistant prostate cancer through inhibition of STAT3.

Daksh Thaper, Sepideh Vahid, Kate Gibson, Micaela Janse Van Rensburg, Alistair Davies, Krisi Ketola, Martin Johansson, Jennifer L. Bishop, Amina Zoubeidi. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Background: Androgen ablation remains the most effective therapy for patients with advanced disease. Unfortunately, most patients progress to castrate resistant prostate cancer (CRPC) characterized with hyper-activation of the androgen receptor (AR). Enzalutamide, a potent AR inhibitor showed efficacy by prolonging survival in CRPC patients. However, ENZ resistance (ENZR) rapidly occurs in patients and in our pre-clinical model targeting AR in ENZ resistant tumors with a 3rd generation AR inhibitor is short lived. The role of signal transducer and activator of transcription (STAT) 3 in the progression of prostate cancer is well established and has been repeatedly linked with maintenance of a stem cell phenotype across cancers. Additionally, drug resistance has been hypothesized to occur via enrichment of cancer stem-like cells (CSC), a phenotype associated with poor survival in patients. Therefore, we propose a shift of focus to target CSC phenotype using small molecule inhibitor of STAT3 called GPA500, instead of the AR axis to deal with ENZ resistance.

Methods and Results: We developed a unique model of ENZ-resistance and found that cell lines derived from serially passaged ENZR tumors displayed broad genetic diversity and differential AR activity. Notably, the cell lines 42DENZR and 42FENZR are PSAlow, harbor an expanded CSC population and have STAT3 hyper-activation compared to CRPC controls measured by STAT3-luc reporter. Accordingly, Crystal Violet and MTT proliferation assays showed that 42DENZR and 42FENZR cell lines were more sensitive to the STAT3 inhibitor GPA500 compared to CRPC control. Targeting the ENZ-R cells with GPA500 reduced mRNA levels of CD133, CD44, SOX2, OCT4 and Nanog. The reduction of these CSC markers was accompanied by reduced self-renewal capacity measured by spheroid assay. Cytometry analysis revealed that treatment with GPA500 reduced α2β1+, CD44+ and CD133+ (CSC) population in the 42D and 42F cells.

Conclusion: In this study, we provide pre-clinical proof that targeting the STAT3 pathway using GPA500 in ENZ resistance as well as CRPC reduces cell proliferation as well as expression of stem cell markers.

Impact: Exploring mechanisms of ENZR resistance serves a critical unmet need in PCa oncology, as the number of men with ENZ resistant CRPC continues to rise. Targeting STAT3 with the small molecule inhibitor GPA500 may provide an effective method to treat ENZR patients or delay the emergence of ENZ resistance in CRPC by reducing the emergence of stem cells.

#3343

Targeting the Lin28A/B axis reverts stem cell-like phenotype and tumor-initiating properties in prostate cancer.

Domenico Albino,1 Gianluca Civenni,1 Cecilia Dallavalle,1 Martina Roos,2 Hartmut Jahns,2 Jonathan Hall,2 Giuseppina M. Carbone,1 Carlo V. Catapano1. 1 _Institute of Oncology Research (IOR), IOSI, Bellinzona, Switzerland;_ 2 _Institute of Pharmaceutical Sciences, ETHZ, Zurich, Switzerland_.

Cancer stem cells (CSCs) represent the most tumorigenic, metastatic and therapy resistant cell subpopulation within human tumors. Current therapies target bulk tumor cells while spare the most aggressive CSC subpopulation. Understanding the mechanisms responsible for the acquisition and maintenance of the CSC phenotype will help to identify new strategies to target CSCs. In this study, we describe a link between deregulated expression of the ETS transcription factor ESE3/EHF and upregulation of Lin28A and Lin28B in prostate CSC-enriched subpopulations. Furthermore, using various cell line models and in vitro/in vivo experimental systems we demonstrate the efficacy of targeting the Lin28A/B axis for selective elimination of cancer cells with tumor-initiating and stem-like properties. Mechanistically, we found that ESE3/EHF represses transcription of Lin28A and Lin28B in normal prostate epithelial cells. Downregulation of ESE3/EHF in prostate tumors led to upregulation of Lin28A and Lin28B and consequent reduction of microRNAs (miRNAs) of the let-7 family, which exert tumor suppressor functions. These events promoted cell transformation and expansion of the prostate CSC subpopulation. Conversely, targeting Lin28A/Lin28B with small interfering RNAs (siRNAs) in transformed prostate epithelial cells and prostate cancer cell lines restored the levels of let-7 miRNAs, decreased expression of several CSC marker genes, and restrained tumor-sphere formation and self-renewal properties in vitro and tumor-initiating capability in vivo. Notably, systemic treatment with a siRNA targeting Lin28B reduced growth of prostate tumor xenografts in mice. This was associated with a significant contraction of the CSC subpopulation in tumor xenografts, as demonstrated by the reduced content of ex vivo tumor-sphere forming cells, reduced expression of CSC marker genes, and upregulation of let-7 miRNAs. Furthermore, tumor cells derived from siLin28B-treated xenografts exhibited reduced in vivo tumor-initiating and self-renewal capability, in line with a persistent loss of CSC properties. Collectively, these data establish the Lin28/let-7 axis as a critical element in malignant transformation and acquisition of tumor-initiating and stem-like properties and identify a valid therapeutic strategy to antagonize CSCs in human prostate cancer.

#3344

Targeting the treatment-induced developmental reprogramming process that facilitates progression of prostate cancer.

Mannan Nouri,1 Amy Anne Lubik,1 Josselin Caradec,1 Na Li,1 Sarah Truong,1 Brett G. Hollier,2 Ralph Buttyan1. 1 _The Vancouver Prostate Centre, Vancouver, British Columbia, Canada;_ 2 _Australian Prostate Cancer Research Centre of Queensland, Brisbane, Australia_.

Metastatic prostate cancer (PCa) develops resistance to hormone therapies through androgen receptor (AR) dependent and independent mechanisms. Previously we described a remarkable and reversible adaptive cellular plasticity that permits adherent AR+ PCa cells (LNCaP, VCaP, LAPC4 and CWR22rv1) to efficiently reprogram to non-adherent AR-/lo cells by exposure to an androgen-free, custom-formulated stem cell medium (STM). STM-reprogrammed cells grew as rosettes/spheroids on conventional tissue culture treated dishes, were resistant to enzalutamide compared to parental cells and had high tumor forming efficiency when xenografted into castrated and intact male mice. Reprogrammed cells overexpressed genes associated with the stem cell state, expressed cell surface antigens characteristic of neural crest stem cells (NRCAM, HNK-1, CD271 and CD29) and showed increased in vitro invasive behaviour through a matrix-coated membrane and increased invasion in vivo into zebrafish following yolk-sac xenografting. When re-exposed to serum-containing mediums, reprogrammed cells could be re-differentiated back to AR+ prostate-like cancer cells or to other differentiated cell lineages derived from neural/neural crest stem cells (neural-, oligodendrocyte-, glia- or osteoblast-like). Gene expression profiling data from reprogrammed cells was analyzed to identify activation of key signalling pathways associated with this transition and outcomes were confirmed by qPCR (for mRNAs) and Western blotting (for protein levels). Inhibitors of Bmi1 (PTC-209), PI-3-kinase (LY294002), Akt (MK2206) or NF-κB (Copper-DDC) were employed to determine whether they interfered with acquired non-adherent (spheroidal) colony formation in the LNCaP model system exposed to STM. Results indicated that key molecules in the non-classical NF-κB pathway (RelB and p100) were upregulated in the transition to AR-/lo stem-like cells and that Bmi1 and phospho-Akt proteins were also consistently overexpressed in all cell models in response to reprogramming. Inhibitors of Bmi1, PI-3-kinase, and Akt significantly suppressed the ability of LNCaP cells to form non-adherent spheroids in the STM, while an inhibitor of NF-κB was able to reduce the number and size of spheroids. The results support the idea that this adaptive and reversible tumor cell plasticity that generates resistance to androgen deprivation is targetable by agents directed at key genes associated with the developmental reprogramming process described previously.

#3345

Targeting the YAP1/COX2/SOX2 signaling axis eliminates cancer stem cells in urothelial carcinoma.

Akira Oki, Mohammad Hoque. _Johns Hopkins University, Baltimore, MD_.

Urothelial cancer stem cells (CSCs) contribute to tumor maintenance and resistance to therapy and accumulated evidence suggest that chronic carcinogen exposure induce "stemness". Therapeutic targeting of CSCs could improve treatment response and prolong patient survival. Here we used our recently published in vitro chronic arsenic (As) exposed models to characterize the property of urothelial CSCs due to arsenic exposure. We hypothesized that urothelial stem cells have a survival selection advantage during carcinogen exposure such as As, and it facilitates their malignant transformation and in acquiring selective phenotypes similar to CSC.

Arsenic-exposed cells displayed more aggressive phenotype than arsenic unexposed cells in a time dependent manner as determined by MTT, invasion, and wound healing assay. In gene set enrichment analysis of expression array of chronic arsenic exposed and unexposed cells; EGFR, COX2 and YAP1 were top-ranked oncogenic signature based on enrichment score in As-exposed cells. Further analysis indicated that several known basal cell markers such as KRT5, KRT6A, and KRT6C etc were overexpressed in As exposed cells in comparison with As unexposed cells. Because the presence of urothelial CSCs in basal-type provides a biological explanation for their aggressive behaviors, we assessed the influence of As on generating CSC phenotypes. As exposure was associated with overabundance of potential CSCs characterized by sphere formation, self-renewal capacity, redifferentiation, and chemotherapy resistance. To explore global association of As exposure and CSC generation, we used the Human Stem Cell RT² Profiler™ PCR Array and found that SOX2 has been gradually overexpressed in line with acquired spheroid formation and self-renewal capacities. SOX2 is frequently overexpressed in numerous urothelial carcinoma (UC) cell lines as well as in arsenic-exposed cells, especially in the spheroid cells. Stable silencing of SOX2 reduces in vitro CSCs properties with reduce expression of CSCs associated molecules such as NANOG,OCT4 etc and also in vivo tumorigenicity. COX2 and YAP1 are also frequently overexpressed in UC cell lines, and inhibition of COX2 or YAP1 reduced SOX2 expression. Interestingly, the inhibition of COX2 and YAP1 expression inversely induced YAP1 and COX2 expression, respectively. Combination treatment with COX2 and YAP1 inhibitors reduced SOX2 expression and suppressed spheroid formation significantly than either inhibitor alone. In xenograft model, the combination treatment suppressed the tumor growth rate, but not either inhibitor alone. In conclusion, chronic As exposure induces CSCs with SOX2 overexpression, one of the most important CSC factor for UC. COX2 and YAP1 coordinately regulate SOX2 expression, and mutually compensate for the reduction of expression of SOX2 to maintain CSCs. Thus targeting the COX2/YAP/SOX2 signaling axis eliminates urothelial CSCs.

#3346

Testing the cancer stem cell hypothesis using the hybrid spheroid assay and Koch's postulates.

Christopher S. Lange, Talal Syed, Mike Zhang, Christian Sabalvaro, Hana I. Lim, Ghadir Salame, Bhargava Chitti, Ifeanyi Ilonzo, Arsalaan Ansari, Michelle Chan, Catherine Li, Jing Xu, Xiaotong Chen. _SUNY Downstate Medical Center, Brooklyn, NY_.

We tested the Cancer Stem Cell (CSC) hypothesis using the patented Hybrid Spheroid (HS) Assay (HSA) and by applying Koch's postulates to test its validity. The HSA is an in vitro assay that enables one to take a viable sample of an individual patient's tumor, make a single cell suspension, mix it in known proportions with human fibroblasts (AG1522) and dispense a known number of cells of the mixture into each well of Ultra Low Attachment (ULA) 96-well plates to agglomerate into 1 HS/well, each containing an average of <1 CSC. The HS provides an analog of the CSC niche, enabling the CSC to proliferate (with some daughters differentiating into Amplifying Transit Cells (ATCs)) and undergo 10-15 symmetric divisions before exhausting the nutrients. This satisfies the McCulloch and Till (spleen colony assay) requirements for a stem cell.

Applying Koch's postulates: (1) Does the patient's tumor contain cells with CSC properties? Yes: (a) The HSs grow to a size consistent with containing a CSC, (b) those that grew contain cells expressing ≥ 1 OKSM factor(s), and (c) such HSs contain differentiated progeny of the initial CSC. (2) Can we isolate and propagate these cells? In progress: testing HS serial growth, dissociation and reculture. (3) Can these cells induce the tumor in vivo? In progress: testing by xenografting individual CSC-containing HSs in NSG mice. (4) Do these cells contain and express specific gene products that give them CSC properties? Yes: Demonstrated for Oct4 and Nanog. (5) If we disrupt these genes, do the cells lose their CSC properties? In progress: (shRNA). (6) If we eliminate the CSCs do we eliminate the cancer? Yes: tested by (a) mathematical modeling of single CSC growth, differentiation of some progeny into ATCs capable of a limited number of divisions, that then become terminal non-proliferative tumor cells in the HSA; if a single CSC survives/remains, the HS will grow, i.e., the cancer will recur (63% for an average of 1 CSC in a HS, with a Poisson distribution, assuming that niche sites remain available), and (b) by measuring surviving fractions after exposure to radiation and/or chemotherapy agents (where sterilization of the CSC prevents the HS from growing ≥ 10 divisions.

The HSA was applied to tumor samples taken from individual endometrial adenocarcinoma patients and correctly predicted, based on CSC radioresistance, which patients would fail their standard-of-care treatments.

Conclusion: The CSC hypothesis is validated in the HSA.

#3347

Establishment of four novel HPV-16 positive cell lines of invasive cervical carcinoma of Indian origin and characterization of CD133 positive cancer stem cells.

Shifa Javed, Bal Krishan Sharma, Sanjeev Kumar Sharma, Swati Sood, Rashmi Bagga, Shalmoli Bhattacharyya, Radhika Srinivasan. _Post Graduate Institute of Medical Education and Research, Chandigarh, India_.

Background and Aim: Cancer of the cervix is the 2nd most common cancer in Indian women. Cell lines are essential in vitro models in cancer research. Till date, available cell lines in cervical cancer were highly passaged, developed from Caucasian, Black or Asian origin; hence, establishment of a low-passage cervical cancer derived cell line was the first aim of this study. Cancer stem cells (CSCs) are proposed to be responsible for metastasis and therapy resistance. Previously, we reported CD133 to be a putative marker for CSC, and they were further characterized.

Materials and Methods: 25 women were recruited after informed consent and approval from the Institute Ethics committee. A small portion of the biopsy was collected in DMEM/F12 media and subjected to primary culture. Successful propagation was possible in 4 cases. Their morphology, epithelial nature and HPV status were evaluated. Variable Number Tandem Repeat (VNTR) assay, cell cycle and kinetic studies were performed. Tissue expression of CD133 and CD49f stem cell markers was evaluated by direct immunofluorescence on frozen tumor samples. Adherent cells were sorted into CD133+ vs CD133- tumor bulk from all 4 cell lines by FACS. These two populations were evaluated for differences in tumorosphere formation, chemosensitivity to cisplatinum, expression of stemness (SOX2, OCT4, NANOG) and EMT (SNAIL, SLUG, TWIST, VIMENTIN and E-CADHERIN) markers at the transcript level.

Observations: Four cell lines designated RSBS-9, RSBS-14, RSBS-23 derived from non-keratinizing squamous cell carcinoma and RSBS-43 derived from adenocarcinoma cervix were established and passaged up to 50 times. The epithelial nature of cell lines was confirmed by cytokeratin and epithelial membrane antigen positivity. VNTR assay confirmed derivation of cell lines from respective parental tissue sample. All 4 cell lines were HPV-16positive. The cell doubling time was approximately 48 hours. Immunofluorescence on tissue samples revealed diffuse positivity for CD49f indicating that it may not represent a CSC marker as previously reported whereas CD133 staining showed scattered positive cells. No particular location of CD133+ CSC was observed. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorosphere formation; however, no significant difference in the chemosensitivity to cisplatinum was seen. Real time quantitation of transcripts of stemness and EMT markers revealed CD133+ cells showing upregulation of EMT markers VIMENTIN, SNAIL, SLUG in the RSBS-23 cell line and of VIMENTIN and TWIST in RSBS-9 cell line whereas there was no significant difference in the RSBS-14 and RSBS-43 cell lines for any of the markers studied.

Conclusion: Low passage cell lines are useful model systems for understanding the biology of cervical cancer. CD133+ Cancer Stem cells exhibit some EMT markers in 2 of the 4 cell lines established.

#3348

Expression of putative cancer stem cell markers in fallopian tube epithelium from women at increased risk of developing high-grade serous ovarian cancer.

Anna Ebbesson,1 Susanne Malander,2 Päivi Kannisto,2 Anita Ringberg,3 Anna Måsbäck,2 Ingrid A. Hedenfalk1. 1 _Lund University, Lund, Sweden;_ 2 _Skåne University Hospital, Lund, Sweden;_ 3 _Skåne University Hospital, Malmö, Sweden_.

Pre-neoplastic p53 signature containing lesions, termed serous tubal intraepithelial carcinomas (STICs), have been identified in the fimbriae of women predisposed to ovarian cancer and in patients with sporadic high grade serous ovarian cancer (HGSOC). These findings instigated the theory that HGSOC originates from fallopian tube epithelium (FTE) and not from ovarian surface epithelium (OSE), and has now become an accepted paradigm for HGSOC genesis. However, only few studies have investigated the stem cell niche in the human fallopian tube. Instead, most research on ovarian cancer stem cells has focused on cells isolated from primary ovarian tumors or cell lines.

In this study we performed immunohistochemistry to explore the presence of putative ovarian cancer stem cells in fallopian tubes from women who had undergone a risk reducing salpingo-oophorectomy (RRSO) due to a germline BRCA1/2 mutation and/or strong family history. Sections of frozen tissue from 14 RRSO patients (10 BRCA1/2 mutation carriers, 4 tested negative for BRCA1/2 mutations) were used in immunohistochemistry for p53 and the proposed cancer stem cell markers ALDH1, CD44, CD117, CD133, LGR5 and SOX2.

Presence of a p53 signature (>12 consecutive cell nuclei in normal appearing FTE) was observed in 1 of 10 BRCA1/2 mutation carriers and none of the non-carriers. ALDH1 and CD44 positive staining of varying intensity was found in both FTE cells and stromal cells within the fallopian tube mucosa in all cases. On the other hand, no CD117 positive epithelial cells were observed in any of the samples, though strongly stained CD117+ mast cells were present in all sections. Most FTE cells were weakly positive for CD133 in all sections, while only one case showed positive LGR5 staining of FTE cells. Finally, SOX2 positive FTE cells were found in all samples. In most cases, the fimbriated end of the fallopian tube harbored more SOX2 positive cells than the more proximal end.

This is an initial attempt at visualizing proposed ovarian cancer stem cells in the fallopian tubes of women predisposed to developing HGSOC. Further research is needed to validate the significance of these findings, including investigating additional markers, testing different antibodies and performing multiple labeling experiments. Based on the currently prevailing theory that HGSOC originates from STICs in the fimbriated end of the fallopian tube, we find SOX2 to be the most promising candidate for further investigations, since SOX2 was the only marker found to be differentially expressed throughout the fallopian tube. Elucidating the cell of origin and etiology of HGSOC has important implications for early detection and preventive therapeutic approaches.

#3349

Differential expression of CD44 and CD24 markers discriminates the epithelioid from fibroblastoid subset in a sarcomatoid renal cell carcinoma cell line: evidence of the existence of cancer stem cells in both subsets.

Chin-Hsuan Hsieh,1 Cheng-Keng Chuang,1 See-Tung Pang,2 Samuel Chien,3 Paul Lin,3 Shuen-Kuei Liao4. 1 _Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Taipei, Taiwan;_ 3 _Kai Fu Biotech Co. Ltd., Guanzhou, China;_ 4 _Taipei Medical University, Taipei, Taiwan_.

The co-existence of epithelioid and fibroblastoid subsets in the human sarcomatoid renal carcinoma cell line, RCC52, has been demonstrated previously based on clonal studies, in which only the epithelioid subset was found to be tumorigenic in NOD/SCID mice. Since only few clonal isolates were used to analyze in that study, we argued that the tumorigenicity results might not be truly reflective of this tumor line. We therefore in this study determined to use cytofluorometrically sorted cells to re-evaluate certain biological parameters including xenotransplantation. Using two monoclonal antibodies to CD44 and CD24 markers, we succeeded in identification and isolation of the two populations, and showed the CD44bright/CD24dim and CD44bright/CD24bright phenotypes corresponding to the epithelioid and fibroblastoid subsets, respectively. At variance with the results of clonal studies, both cytofluorometrically sorted subsets displayed different but significant degrees of tumorigenicities, indicating that each subset harbored its own cancer stem cells (CSCs). In consistence with the tumorigenicity findings, both sorted subsets showed in vitro migratory/invasive potential with greater ability exhibited by the CD44bright/CD24bright subset which was associated with higher expression of MMP-7, -8 and TIMP-1 transcripts. On the other hand, the CD44bright/CD24dim subset was found to be associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Furthermore, both sorted subsets expressed the RCC stem cell marker CD105 in a small proportion of cells (4-5%), and were able to covert to PAX2+ clear cells in areas in the xenografts to a greater extent by the CD44bright/CD24dim subset, highlighting high plasticity of each subset in different ways. Collectively, our findings confirm the co-existence of two morphologically and phenotypically distinct subsets, and document the presence of CSCs in both subsets for the first time in sarcomatoid RCC. The CSCs/mCSCs in the two subsets should therefore be considered as emerging therapeutic targets in the design of future treatment strategies for this subtype of renal cancer.

#3350

Verification of mechanism that CSC markers are implicated in poor prognosis for pancreatic ductal adenocarcinoma.

Kota Arima, Takatsugu Ishimoto, Hirohisa Okabe, Yuki Kitano, Risa Inoue, Kensuke Yamamura, Takayoshi Kaida, Takaaki Higashi, Katsunobu Taki, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

Background: Cancer stem cells (CSCs) refer to a subset of tumor cells that have self-renewal ability and generate plenty of non-CSC cells that comprise a tumor. In addition, CSCs play crucial roles in various processes during tumor progression and metastasis as well. A number of CSC markers candidates have been explored to date, and Aldehyde dehydrogenase 1 (ALDH1), c-Met, and CD44 have been identified as CSC markers in pancreatic ductal adenocarcinoma (PDAC). On the other hand, prostaglandin E2 (PGE2) is one of metabolites in arachidonate cascade and is implicated in the expansion of hematopoietic and tissue stem cell fraction. The aim of this study is to single out the most important CSC marker and elucidate the functional role of PGE2 for CSC expansion in PDAC.

Methods: Three CSC markers (ALDH1, CD44, and c-Met) expression was examined by immunohistochemistry in 121 primary surgical specimens of PDAC and analyzed a relationship with clinicopathological factors and clinical outcomes. The clonogenic growth potential of CSC marker-positive PDAC cells was assessed in vitro by growth assays and sphere formation assays. We next investigated the expression of CSC markers and self-renewal related genes in PDAC cell lines with PGE2 or 15-PGDH inhibitor treatment. We further conducted functional experiments using siRNA to identify the critical molecule in PDAC progression.

Results: A high level of ALDH1 expression was detected in 63 of the 121 cases, and was significantly associated with large tumor size and poor prognosis in PDAC patiants. On the other hand, CD44 and c-Met expression were not associated with the prognosis. Among CSC markers, the expression of ALDH1 was significantly increased by PGE2 treatment in PDAC cells. By suppressing ALDH1 expression by siRNA, growth and sphere formation potential were inhibited in ALDH1 high-expressing PDAC cells. In contrast, the expression of ALDH1 was remarkably increased by PGE2 or 15-PGDH inhibitor treatment in ALDH1 low-expressing PDAC cells. Finally, we found that Nanog and Oct-4 were down-stream molecules of PGE2-ALDH1 signaling and played crucial roles for PDAC cell expansion.

Conclusion: Our results demonstrated that PGE2 positively regulated ALDH1 expression, and the growth and sphere formation potential were promoted by increasing ALDH1 expression, resulting in poor prognosis of PDAC patients. Inhibiting PGE2-ALDH1 signaling could lead to the suppression of tumor growth in PDAC patients.

#3351

Differential glycosylation of extracellular matrix specifically modulates lung cancer initiating cells subsets.

Giulia Bertolini,1 Cecilia Gardelli,1 Francesca Andriani,1 Massimo Moro,1 Laura Russo,2 Laura Cipolla,2 Gabriella Sozzi,1 Luca Roz1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _University of Milan-Bicocca, Milan, Italy_.

In lung cancer CD133+ cells have properties of cancer initiating cells (CICs) as stemness features, high tumorigenic potential and chemoresistance. Within the CD133+ CICs, we have recently identified a specific subset, defined as CD133+CXCR4+EpCAM-, endowed with high dissemination and metastatic potential (metastasis initiating cells, MICs). In normal tissues stem cells reside in specialized niches composed of both stromal cells and extracellular matrix proteins (ECM) while factors responsible for CICs maintenance and modulation are relatively unknown. Collagen, the most abundant protein of ECM, plays a pivotal role in mediating regulation of adhesion, survival and proliferation of tumor cells. The relevance of collagen glycosylation, a fundamental post-translational modification controlling several biological processes, remains however largely unexplored.

To investigate the effects of interactions between tumor cells and differentially glycosylated ECM epitopes, we cultured primary (LT73) and established lung cancer cell lines (A549, H460) on type I collagen films neo-glycosylated with glucose, galactose or sialic acid residues. Our results showed a general increase of CICs subsets, evaluated by flow cytometry, in tumor cells cultured on glycosylated collagen compared to pristine collagen or tissue culture plates with a concomitant increased expression of stemness-related genes. Particularly, we observed that collagen functionalized with glucose had the highest efficiency in enriching for MICs (3-fold change). Analysis of proliferation and viability of tumor cells cultured on collagen films showed that glucose residues were particularly proficient in causing G1 phase growth arrest and cell death compared to pristine collagen resulting in an overall decrease in the bulk population and CICs enrichment. In PKH label-retention assays, glucose-glycosylated collagen also selected and increased the fraction of PHK-labeled quiescent tumor cells, enriched for MICs component, confirming the preferential selection/survival of MICs subset. Using LT73 cell line depleted for CD133+ cells, we then proved that CICs increase was also due to a de novo generation of CD133+ cells and in particular of CD133+CXCR4+EpCAM- metastatic subset through non-CICs to CICs conversion. The immunophenotypic increase of CD133+ cells was functionally validated in vivo by a limiting dilution tumorigenic assay that estimated a 4.6 higher frequency of CICs in LT73 cells cultured on glucose-glycosylated collagen compared to control group. Moreover tumors derived from cells exposed to glucose residues retained a higher contents of MICs associated with an increased dissemination potential compared to controls.

Our results suggest that differential collagen glycosylation could play an essential role in the creation of a niche favorable for the generation and selection/survival of lung metastasis initiating cells.

#3352

Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) regulates the tumor initiating cell phenotype through SOX2 expression and modulates treatment response to anti-cancer drugs in lung adenocarcinoma.

Maria P. Wong, Jing Liu, Zhi-Jie Xiao, Si-Qi Wang, Vicky Pc Tin. _The University of Hong Kong, Hong Kong, Hong Kong_.

Lung cancer is the most lethal of all cancers world-wide and the majority of patients require chemotherapy or targeted therapy at presentation. Survival is compromised by drug resistance through multiple mechanisms such as by-pass mutations, micro-environment activation of survival programs and induction of tumor initiating cells (TIC). Induction of TIC phenotypes could be accompanied by upregulation of embryonic or developmental pathways but molecular mechanisms are incompletely understood. Calcium/calmodulin-dependent protein kinase II alpha (CaMK2A) is a multifunctional serine/threonine kinase involved in growth and stress signals integration. A tumor-supportive role has been reported in leukaemia and thyroid cancer but reports on its involvement in TIC regulation are limited. We investigated the role and molecular mechanism of CaMK2A in lung cancer TIC regulation using in vitro and in vivo models. The results showed CaMK2A was overexpressed in lung cancer cell lines and human lung adenocarcinomas compared to normal lung. In cancer cells with stable CaMK2A-knockdown, in vitro reduction in tumor spheres formation, anchorage independent growth and increased reactive oxygen species, as well as in vivo reduction of xenograft tumorigenicity were observed. The opposite effects were observed in cancer cells with stable CaMK2A over-expression. Further, specific pharmacological inhibition of CaMK2A by KN93 led to significantly reduced IC50 for gefitinib in lung cancer cells harboring activating EGFR mutation (HCC827) and in HCC827 exogenously induced for gefitinib resistance. In contrast, IC50 of cisplatin treatment was increased in cancer cells with genetically-induced CaMK2A over-expression. Moreover, CaMK2A knockdown led to reduced mRNA expressions of pluripotency genes (SOX2, NANOG, OCT4) and TIC markers (ALDH, CD166). These results suggested CaMK2A is involved in lung tumorigenicity through TIC regulation. To further investigate the molecular mechanism, we showed tumorigenicity was restored in xenografts with CaMK2A knockdown rescued with SOX2 over-expression. In CaMK2A knockdown cells, reduced SOX2 expression was associated with increased H3K27me3 repressive histone mark, which has not been reported in the literature. Taken together, we have shown CaMK2A plays a role in lung adenocarcinoma and TIC maintenance through histone modification and regulation of SOX2 expression. Targeting TIC through CaMK2A modulation might be useful for overcoming drug resistance and improving long term lung cancer survival.

#3353

Cancer stem cell markers (ALDH1 and CD133) expression could be associated with a poor prognosis in the patients with lung adenocarcinoma.

Takeaki Miyata,1 Takashi Yoshimatsu,2 Atsushi Sekimura,3 Tetsuya So,4 Naohiro Nose,5 Tsunehiro Oyama,6 Hisao Nagaya,1 Akinobu Gotoh,1 Takeaki Miyata,2 Tsunehiro Oyama1. 1 _Hyogo College of Medicine, Nishinomiya-City, Japan;_ 2 _Fukuoka-Wajiro Hospital, Fukuoka-City, Japan;_ 3 _Shin-Takeo Hospital, Takeo-City, Japan;_ 4 _Shin-Komonji Hospital, Kitakyusyu-City, Japan;_ 5 _Shin-Yukuhashi Hospital, Yukuhashi-City, Japan;_ 6 _Imamitsu Home Care Clinic, Kitakyusyu-City, Japan_.

[Introduction and Purpose] Cancer stem cells (CSCs) may have abilities of self-renewal and multi-potent differentiation and be responsible for tumor initiation, progression, metastasis and highly resistant to radiation or chemotherapy. Aldehyde dehydrogenase 1 (ALDH1) enzymes are a family of intracellular enzymes that participate in cellular detoxification, differentiation and drug resistance through the oxidation of cellular aldehydes. The biochemical function of CD133 currently remains unclear, but its expression on the cell surface has been demonstrated to be a specific marker for CSCs in many malignant tumors. ALDH1 and CD133 have been identified as putative CSCs marker in patients with lung adenocarcinoma (ad patients) (Miyata et al, 2015). In this study, we investigated the relationship CSCs markers (ALDH1 and CD133 expression) and various clinical parameters in ad patients. We also showed that the expression of CSCs markers (ALDH1 and CD133 expression) related with prognostic potential in ad patients. [Materials and Methods] We examined 92 of 154 (59.7%) in Japanese ad patients, who underwent surgical resection in Fukuoka-Wajiro Hosp. Those 92 ad sections were performed immunohistochemical (IHC) staining for ALDH1 and CD133 using a standard immunoperoxidase technique. The staining intensity of cytoplasmic staining of ALDH1 was scored as 0, 1, 2, or 3, corresponding to negative, weak, intermediate, or strong immunoreactivity, respectively. Percentage of cells with positive ALDH1 was graded as 0 to 100%. The ALDH1-score was assigned to each case by multiplying the intensity score by the each percentage of cells staining. The ALDH1-score was calculated as follows: H = (% of cells that stained at intensity category 1 × 1) + (% of cells that stained at intensity category 2 × 2) + (% of cells that stained at intensity category 3 × 3). We defined as positive cases when more than 100 of the ALDH1-score were calculated. We also defined as CD133 positive cases when more than 10% of tumor was stained (negative cases; > 10% positivity, positive cases; < 10% positivity). [Results] The mean of the ALDH1-score from 92 cases was 52.1. The ALDH1-score of stage II-IV disease (59.1) seemed to be higher than that of stage I disease (48.7). ALDH1-score positive cases seemed to be associated with a poorer survival according to a survival curve (p=0.06). CD133 positive cases were significantly associated with a poorer survival according to a survival curve (p<0.01), although there were no relationship between CD133 expression and ALDH1-score. [Conclusion] ALDH1 and CD133 can act as a prognostic marker for stem cells in human lung adenocarcinoma. To determine the ALDH1 and CD133 expression in human lung adenocarcinoma is helpful for targeted therapies against stem cells.

#3354

Characterization of lung tumorspheres by gene expression and flow cytometry: differential expression in CSC-related markers and signaling pathways.

Ester Munera-Maravilla,1 Alejandro Herreros-Pomares,1 Alicia Martínez-Romero,2 Sandra Tejedor,2 Silvia Calabuig-Fariñas,3 Eloísa Jantus-Lewintre,4 Rut Lucas,5 Eva Escorihuela,1 Rosa Farràs,2 Carlos Camps6. 1 _Fundación Investigación Hospital General Universitario, Valencia, Spain;_ 2 _Centro de Investigación Príncipe Felipe, Valencia, Spain;_ 3 _Fundación Investigación Hospital General Universitario; Department of Pathology, Universitat de València, Valencia, Spain;_ 4 _Fundación Investigación Hospital General Universitario; Department of Biotechnology, Universidad Politécnica de Valencia, Valencia, Spain;_ 5 _Department of Science History and Documentation. Universitat de València, Valencia, Spain;_ 6 _Fundación Investigación Hospital General Universitario; Department of Medicine, Universitat de València; Department of Medical Oncology, Consorcio Hospital General Universitario de Valencia, Valencia, Spain_.

Chemoresistance, progression and metastasis have made of lung cancer the first cause of cancer mortality. These features were linked to a subpopulation of cells, named cancer stem cells (CSCs), which remain largely unknown. The aim of this study was to isolate and characterize CSCs from lung cancer cell-lines and tumor-tissue from resectable non-small cell lung cancer (NSCLC).

Methods: Tumor cells from resected NSCLC and cell lines (H1650, H1993, A549, and PC9) were grown in monolayer and as spheroids. RTqPCR was performed to analyze the mRNA expression of CSCs-related genes: CSC-markers (EPCAM1, ALDH1A1, CD166, ABCG2, CD44, CD133); pluripotency genes (KLF4, OCT4, NANOG, SOX2, MYC, CCND1); Notch pathway (NOTCH1, NOTCH3, HEY1); Wnt pathway (WNT1, WNT5A, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1). ACTB and CDKN1B were used as endogenous controls for relative expression calculation. The expression of lung stem cell markers EpCAM, CD166, E-cadherin, CD90, CD44, CD34, CD133 and ABCG2 was assessed by flow cytometry. The tumor-initiating cell capacity of selected lung-spheres was tested in vivo to confirm tumorigenicity.

Results: Lung-tumorspheres had increased expression of EPCAM, CD44 and ALDH1A1 (p= 0.028, p= 0.021 and p= 0.043, respectively) when compared to cells grown in adherence. Likewise, NANOG, KLF4 and OCT4 tended to be more expressed in tumorspheres. Relative gene expression of NOTCH1 was also higher in spheroids than in monolayer cells (p= 0.028), in concordance with the same tendency observed in NOTCH3. Similarly, RTqPCR analysis revealed a possible activation of the canonical Wnt pathway in tumorspheres, with high expression levels of the downstream effector gene CCND1 (p = 0.05), along with a repression of DKK1 inhibitor. Regarding to the non-canonical Wnt pathway, its activator WNT5A showed lower expression levels in spheroids compared to monolayer-culture cells. Concerning the expression levels of Hedgehog pathway`s genes, we found that SMO and PTCH1 were underexpressed in lung-tumorspheres compared with their paired adherent-cultured cells (p = 0.028 and

p=0.069). Flow cytometry revealed that EpCAM and CD44 were highly expressed in lungspheres obtained from cell lines and primary tumors. The expression of CD166 differed among the cell lines. Furthermore, EpCAM+/CD90- subpopulation were the ones able to induce tumor in xenotrasplant mouse model demostrating tumor-initiating capacity in vivo.

Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of EpCAM and others CSC markers. Genes related to Notch and Wnt signaling pathways were more expressed in spheroids compared to the cells grown in adherence, suggesting both pathways as interesting lung-CSC targets.

Supported by grants RD12/0036/0025 from RTICC, PI12-02838 and PI12-0956 from ISCIII and SEOM/2012.[E.M.-M., A.H.-P. and A.M.-R. contributed equally to this work.]

#3355

Opening up heterochromatin by histone deacetylase inhibition improves response to DNA damage in lung cancer tumor initiating cells.

Lovisa Lundholm,1 Petra Hååg,2 Mina Eriksson,1 Beata Brzozowska,1 Rolf Lewensohn,2 Andrzej Wojcik,1 Kristina Viktorsson2. 1 _Stockholm University, Stockholm, Sweden;_ 2 _Karolinska Institutet, Stockholm, Sweden_.

Tumors are suggested to be highly responsive to epigenetic alterations such as histone modifications and DNA methylation. We have previously reported a resistant phenotype of sphere-forming non-small cell lung cancer (NSCLC) tumor initiating cells (TICs) with an impaired activation of DNA-damage response proteins. Here we set out to analyze chromatin compactness since a heterochromatic structure has been postulated to constitute a barrier to DNA damage and repair. We show that certain NSCLC and small cell lung cancer (SCLC) TICs have elevated levels of the heterochromatin markers heterochromatin protein 1γ (HP1γ) and trimethylated lysine 9 of histone 3 (H3K9me3). Chromatin modulators were tested and histone deacetylase inhibitors (HDACi) vorinostat, panobinostat and trichostatin A reduced the cell viability of NSCLC TICs compared to bulk cells after 72 h. We could also demonstrate that TICs are more responsive to HDAC inhibitors since the euchromatin marker acetylated lysine 8 of histone H4 (H4K8ac) was increased after vorinostat and trichostatin A in NSCLC TICs but not in bulk cells. Pretreatment with HDACi inhibitors sensitized NSCLC TICs to cisplatin and ionizing irradiation. Accordingly, pretreatment with vorinostat increased the phosphorylation of the DNA-damage response proteins ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) upon irradiation in NSCLC TICs, whereas bulk cells already had a higher level with radiation only. In conclusion, we demonstrate that lung cancer TICs display a heterochromatic structure compared to bulk cells and that NSCLC TICs have an enhanced sensitization upon DNA damage inflicted by cisplatin or radiation after HDACi pretreatment.

#3356

Characterization and enrichment of head and neck cancer stem cells suggests differences between HPV+ and HPV- subtypes of HNSCC cells.

Anirban Chatterjee, Kwangok Nickel, Randall J. Kimple. _University of Wisconsin-Madison, Madison, WI_.

Head and neck squamous cell carcinomas (HNSCC) are the sixth most prevalent type of malignancy worldwide. Despite recent advances in multidisciplinary treatments, the overall survival and quality of life of patients with advanced HNSCC have not improved considerably over the past decade. Recent reports have demonstrated that tumors contain a small subpopulation of cells called cancer stem-like cells (CSCs), which are responsible for tumor maintenance and metastasis. CSCs have the ability to self-renew and are responsible for tumorigenesis, progression, metastasis, and relapse after treatment. Recent studies demonstrate that even established HNSCC cell lines contain a definite sub-population of CSC providing us with the opportunity to discerning their roles in progression, treatment, and relapse of the disease.

In vitro Sphere Forming Assays (using ultra-low attachment plate and serum free media) were employed as the key method of CSC enrichment. qPCR analyses, aldehyde dehydrogenase (ALDH) activity, Side Population Phenotype assay (SPP) were used to confirm the results of the primary analyses. To confirm an in vivo CSC phenotype, xenograft tumor formation, through subcutaneous injection in immunocompromised mice was done.

In vitro sphere forming assay followed by subsequent passaging of the sphere enriched cells (SECs) have successfully demonstrated the existence and enrichment of a CSC sub-population in HNSCC cell lines. Findings suggest a differential existence of CSCs related to the HPV status of the cell. ALDH assay and SPP assay confirmed the results of sphere formation. qPCR analyses of the SECs have confirmed enrichment in several stemness marker genes (Oct-4, Sox2, Nanog, Bmi-1, cMyc, KLF4, Beta catenin etc.), also following the identical differential pattern. Various EMT markers genes (Vimentin, Fibronectin, E-cadherin, N-cadherin, Snail, Slug, Twist, ZO-1 etc.) were also assessed, portraying a significant level of expression in CSC populations. SECs subcutaneously injected in immunocompromised mice formed tumors with as few as 50 cells.

We have demonstrated that a definite subpopulation of CSCs exists in HNSCC cell lines. Significant differences were seen between the HPV+ and HPV- subtypes. Analyses of EMT marker genes and other molecular pathways in CSC confirm the differences between HPV+ and HPV- cells too following the similar pattern. In vivo tumor xenograft formation establishes the heightened tumorigenicity of CSCs/SECs.

#3357

Role of miRNA dynamics in governing cancer stem cell properties via CD44v6/Nanog/ PTEN axis in head and neck cancer: Modulating the master regulators.

Shanaya Patel, Dr. Rakesh Rawal. _The Gujarat Cancer & Research Institute, Ahmedabad, India_.

Background:

Head and Neck Squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality worldwide. Late diagnosis, metastasis and therapeutic resistance are major confounders for the poor 5 year survival rate and are attributed to the existence of Cancer Stem Cells (CSCs). Several evidences have demonstrated the key role of nicotine in Tobacco related Cancers; however, the plausibility of nicotine modulating CSC markers still needs to be elucidated. Propelling evidences strongly suggest that HNSCC is a genetic disease, regulated by epigenetic mechanisms. Thus, understanding the role of miRNAs in regulation of CSCs and its contributions to improvise the prognostic and therapeutic implications is of great importance.

Methodology:

Orosphere was generated from primary habituated and non-habituated HNSCC tumors using CD44+ immunomagnetic cell separation and ex-vivo expansion. Characterization of these in-vitro models to evaluate spheroid formation ability and expression profile of CSC markers (CD44v3, CD44v6, BMI1 and Nanog) was carried out using quantitative Real Time PCR. Further, miRNA profiling of the upstream regulators (miR5423p, miR34a and miR21) was conducted in order to understand the miRNA mediated pathway that regulate these CSC features in tumor development, metastasis and response to chemotherapy. Correlation analysis of these miRNA profile along with the expression levels of its CSC target gene, were analyzed in specimens from patients with HNSCC.

Result:

Orosphere assay facilitated the propagation of CD44+ cells that retained CSC phenotype and self-renewal property. In-vitro model of habituated tumor showed an increased spheroid forming efficiency as compared to non-habituated models. In addition, these models showed higher expressions of CSC markers (CD44v3, CD44v6, Nanog & BMI1) and a significantly reduced expression of PTEN and ATM as compared to the healthy individual; however the habituated and non-habituated were found to have a trivial difference in their behavioral patterns. Furthermore, a significantly increased expression of upstream miRNA was observed which eventually leads to the modulation CD44 interaction with Nanog/PTEN, thus forming an unexplored axis which can have prognostic and therapeutic implications. Clinically, altered miRNA expression not only positively correlated with CSC expression patterns but also associated with increased progression of the disease.

Conclusion:

Collectively, this study indicates that miR5423p and miR34a targets the HA-induced CD44v6 -Nanog-PTEN axis playing a vital role in regulating the CSC properties comprising of self renewal, clonal proliferation and resistance to chemotherapy in Head and Neck Cancers. Furthermore, our results depict an undisputable role of these miRNA mediated pathways in the development of tobacco related carcinogenesis

#3358

Expression of ALDH1, Bmi-1 and CD24 in human oral squamous cell carcinoma and its relationship with clinical factors.

Tetsuya Tamatani, Natsumi Takamaru, Kenji Fujisawa, Hirokazu Nagai, Youji Miyamoto. _Institute of Biomedical Sciences, Tokushima Univ. Graduate School, Tokushima, Japan_.

[Background] A cancer stem cell (cancer initiating cell, CSC) is considered capable of self -replication, self-differentiation, drug resistance, and immune evasion. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including aldehyde dehydrogenase (ALDH) 1, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and CD24, and their expression has been reported to be the potential prognostic values. However, the prognostic values of ALDH1, Bmi1, and CD24 expression in patients with oral cancer are less understood.

[Purpose] The aims of present study were to evaluate the expression of ALDH1, Bmi1, and CD24 in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, the classification of invasion mode, cerebral lymph node metastasis and disease-free survival rate.

[Materials and Methods] Tissue specimens were obtains from 103 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess ALDH1, Bmi1, and CD24 protein using at least 10% staining-positive cells as the definition of positive staining.

[Results] Immunohistochemical analysis of 103 cases showed that 31 cases (30%) expressed ALDH1. There was no relationship between ALDH1 expression and clinical stages or metastasis. However, there was significant association between ALDH1 expression and histological differentiation, or classification of invasion mode or disease-free survival rate. Otherwise, 39 cases (38%) cases of 103 OSCC patients expressed Bmi-1. There was significant association between Bmi-1 expression and invasion mode or disease-free survival rate. Sixty four cases (62%) expressed CD24. There was significant association between CD24 expression and histological differentiation.

[Conclusions] These findings suggested that the expression of ALDH1 and Bmi-1 as CSC markers in OSCC may be good marker indicating survival in patients with OSCC.

#3359

Expression of stem cell markers Oct3/4 and Nanog in the head and neck squamous carcinoma cells and its clinical implications for delayed neck metastasis in stage I/II tongue squamous cell carcinoma.

Noboru Habu,1 Yorihisa Imanishi,1 Kaori Kameyama,1 Masayuki Shimoda,1 Yutaka Tokumaru,1 Koji Sakamoto,1 Ryoichi Fujii,1 Seiji Shigetomi,1 Kuninori Otsuka,1 Yoichiro Sato,1 Yoshihiro Watanabe,1 Hiroyuki Ozawa,1 Toshiki Tomita,1 Masato Fujii,2 Kaoru Ogawa1. 1 _Keio University, School of Medicine, Tokyo, Japan;_ 2 _National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan_.

Background: The side population (SP) of cancer cells is reportedly enriched with cancer stem cells (CSCs), however, the functional role and clinical relevance of CSC marker molecules upregulated in the SP of head and neck squamous carcinoma (HNSCC) cells are yet to be elucidated. Patients with clinical stage I/II (T1-2N0M0) tongue squamous cell carcinoma (TSCC) typically undergo partial glossectomy alone; however, development of delayed neck metastasis (DNM) tends to reduce their survival. In the present study, we aimed to determine the CSC markers in the SP of HNSCC cells along with their functions in cellular behaviors, and to clarify the association of these markers with DNM.

Methods: Flow cytometry was applied to isolate SP from main population (MP) in HNSCC cells (SCC4, SAS, and HSC4). The expression of the CSC markers was examined by semi-quantitative RT-PCR and immunocytochemistry. In vitro proliferation, migration, and invasion assays were performed to assess cellular behaviors. Clinicopathological factors and immunohistochemical expressions of Oct3/4 and Nanog were evaluated using surgical specimens from 50 patients with stage I/II TSCC who underwent partial glossectomy alone.

Results: SPs were isolated in all three cell lines examined. Expression levels of Oct3/4 and Nanog were higher in SP cells than MP cells. Additionally, cell migration and invasion abilities were higher in SP cells than MP cells, whereas there was no difference in proliferation. Univariate analysis showed that expression of Oct3/4 and Nanog, vascular invasion, muscular invasion, and mode of invasion were significantly correlated with DNM. Multivariate logistic regression revealed that Oct3/4 expression (risk ratio = 14.78, p = 0.002) and vascular invasion (risk ratio = 12.93, p = 0.017) were independently predictive of DNM. Regarding the diagnostic performance, Oct3/4 showed the highest accuracy, sensitivity, and negative predictive value of 82.0%, 61.5%, and 86.8%, respectively, while vascular invasion showed the highest specificity and positive predictive value of 94.6% and 71.4%, respectively.

Conclusion: These results suggest that Oct3/4 and Nanog represent probable CSC markers in HNSCC, which contribute to the development of DNM in part by enhancing cell motility and invasiveness. Moreover, along with vascular invasion, expression of Oct3/4 in the primary cancer cells can be considered a potential predictor for selecting patients at high risk of developing DNM.

#3360

EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngealcarcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

Chang-Fu Yang,1 Guang-Da Yang,2 Tie-Jun Huang,3 Jia-Ling Huang,4 Chao-Nan Qian,5 Bi-Jun Huang5. 1 _Department of Cancer Chemotherapy, Gaozhou People's Hospital, Gaozhou, China;_ 2 _Department of Cancer Chemotherapy, Zengcheng People's Hospital, Guangzhou, China;_ 3 _Department of Nuclear Medicine, the Second People's Hospital of Shenzhen, Shenzhen, Shenzhen, China;_ 4 _Department of Pathology, Saint Barnabas Medical Center, New Jersey, NJ;_ 5 _State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China_.

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44+/High stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition (EMT)-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered PI3K/AKT pathway, which subsequently stimulated expression of CSC markers, development of side population (SP) and tumor sphere formation. This suggests that PI3K/AKT pathway plays an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44+/High cells. In addition, LMP1 greatly increased expression of miR-21 and down-regulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by a miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and up-regulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and play a key role in LMP1-induced CSCs in NPC.

#3361

Targeting tumor/cancer stem cell marker DCLK1 for the treatment of hepatocellular carcinoma and erlotinib-resistant lung adenocarcinoma using Z-3,5,4'-Trimethoxystilbene (Z-TMS).

Naushad Ali, Charles B. Nguyen, Sanam Husain, Parthasarathy Chandrakesan, Randal May, Sripathi Sureban, Nathaniel Weygant, Dongfeng Qu, Danny N. Dhanasekaran, Michael Bronze, Courtney Houchen. _University of Oklahoma Health Sciences Center, Oklahoma City, OK_.

Introduction: Tumor/cancer-initiating stem-like cells (CSCs) exhibit self-renewal and unlimited capacity for proliferation and differentiation. These cells also display propensity to reproduce original tumor after metastasis and confer resistance to anti-cancer therapies. Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, has been shown to possess subsets of CSCs generally marked by EpCAM and CD133 expression. We have extensively investigated doublecortin-like kinase (DCLK1) CSC marker in advanced liver diseases including cancers of liver, colon and pancreas. Our published data suggest that DCLK1 promotes inflammatory and oncogenic pathways including epithelial-mesenchymal transition. These observations prompted us to find means for targeting DCLK1-positive cells and to investigate the impacts of interference with DCLK1 on tumor growth.

Methods: Cell cycle analysis of DCLK1-positive cancer cells was carried out using propidium iodide staining followed by flow cytometry. EGFR kinase assay was performed using the ADP-Glo kinase assay. Immunohistochemical staining, confocal microscopy, real-time PCR and Western blot were used for detection of CSC markers in HCC. Specific anti-DCLK1 shRNAs and a resveratrol analogue, Z-TMS, were used to inhibit DCLK1-dependent tumor growth and cell migration.

Results: DCLK1 is extensively expressed in human hepatocytes-derived tumor xenografts and in the liver tissues of patients with HCC whereas normal human liver and isolated hepatocytes lack the protein expression. HCC Patients (n= 369) overexpressing DCLK1 in liver showed approximately 3 times reduction in 5-year survival rate. Z-TMS (1 μM) inhibited tumor-like growth of hepatoma cells in a magnetic levitation-based culture model. The drug induced bundling of DCLK1 with microtubules in hepatoma cells and blocked cell cycle progression at G2/M phase. Similar drug treatment of hepatoma cells triggered downregulation of CDK1, induced p21cip1/waf1 expression, and inhibited Akt-Ser473 phosphorylation. In addition, Z-TMS significantly reduced survival of erlotinib-resistant lung carcinoma (H1975) cells that contain a T790M mutation in EGFR in the 0.1 µM to 1.0 µM range within 48 hr whereas erlotinib failed to reduce cell survival even though it was fully active against the EGFR kinase domain (aa 695-1210).

Conclusions: The Z-TMS's broad-range anti-tumor activities are likely attributed to its ability of targeting DCLK1-microtubule dynamics, cell migration, cell cycle and the Akt signaling pathway. These properties may also be responsible for its inhibitory effects on erlotinib-resistant lung cancer.

#3362

Expression of CD44v9, a cancer stem cell marker, in human hepatocellular carcinoma.

Fumitaka Wada. _Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Fukuoka, Japan_.

Background: CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. This protein has recently been identified as one of the crucial cell surface markers for cancer stem cells (CSCs) in several types of tumor. It has recently been reported that CD44 variant 9 (CD44v9) interacted with the glutamate-cystine transporter xCT, thereby contributing to increased intracellular synthesis of glutathione (GSH), a strong scavenger of reactive oxygen species (ROS). CSCs having such mechanism are highly resistant to ROS (Cancer Cell 2011). Of note, the function of xCT is inhibited by sulfasalazine (Leukemia 2001), a commonly used drug for ulcerative colitis.

Aim: The aims of this study were to assess 1) the expression levels of CD44v9 and xCT in human HCC tissues and HCC cell lines, and 2) possible synergistic anti-tumor effect of cisplatin and sulfasalazine on HCC cells.

Methods: Thirty HCC tissues were subjected to immunohistochemistry for CD44v9 and xCT. The human HCC cell lines HAK-1A and HAK-1B were used. HAK-1B was an aggressive sister cell line derived from HAK-1A, a well-differentiated one (Hepatology 1993). The cells were incubated with cisplatin with or without sulfasalazine at concentrations of 0, 50, 100, and 200 µM under attached and sphere-forming conditions. Western blot analysis was employed to evaluate protein expression levels.

Results: Membranous localization of CD44v9 was found in 2 HCC tissues, that were moderately and poorly differentiated ones. In such cases, the standard isoform CD44s was also strongly expressed. Expression levels of xCT were ubiquitously demonstrated in all HCC cases studied; however, it was robustly upregulated in sphere-formed HAK-1B cells, in concert with the upregulation of CD44v9 in these cells. Sphere formation of HAK-1B was significantly inhibited by co-treatment with cisplatin and sulfasalazine in a dose-dependent manner.

Conclusions: The CD44v9-xCT system was demonstrated in less differentiated HCC tissues. Indeed, poorly differentiated HCC cells (HAK-1B) under sphere formation robustly expressed both CD44v9 and xCT, suggesting that CSC-like cells favored this system. To overcome the ROS resistance of CSCs, delivering sulfasalazine in combination with anti-cancer drugs may open a new avenue to a promising treatment of chemorefractory HCC. 

### Tumor Angiogenesis: Host Interactions and the Tumor Microenvironment

#3363

Resistin promotes tumor angiogenesis and lymphangiogenesis through c-Src/PI3K/AKT signaling pathway in human chondrosarcoma cells.

Meng-Ju Chi, Hsiao-Chi Tsai, Chih-Hsin Tang. _China Medical University, Taichung City, Taiwan_.

Chondrosarcoma is a kind of commonly found bone malignant tumor. The development of distant metastasis often leads to a significant decline in overall survival, and there are no specific therapeutic methods for it until today. For tumors to metastasize, angiogenesis and lymphangiogenesis are both important steps in the early periods. Resistin was discovered as an adipocyte-secreting adipokine, which may play a critical role in modulating cancer pathogenesis. However, the role of resistin in the angiogenesis and lymphangiogenesis of human chondrosarcoma is largely unknown. In our lab, we found that the expression of resistin was higher in chondrosarcoma biopsy tissues than in normal cartilage. We also confirmed that treated cells with resistin increased VEGFA and VEGFC expression, promoting angiogenesis as well as lymphangiogenesis in human chondrosarcoma cells. Moreover, the inhibitors and siRNAs of c-Src, PI3K, and AKT reduced the resistin-increased cell angiogenesis and lymphangiogenesis. Our results indicate that resistin promotes chondrosarcoma angiogenesis and lymphangiogenesis by the increasing VEGFA and VEGFC expression through the activation of the c-Src/PI3K/AKT signaling pathway expression. Therefore, resistin may represent a potential novel molecular therapeutic target for chondrosarcoma therapeutic treatment.

#3364

Adaptive mechanisms upon LRP1 repression: Impact on sphingosine-1-phosphate-mediated signalling and migration of brain endothelial cells.

Amélie Vézina, Cyndia Charfi, Alain Zgheib, Borhane Annabi. _Université du Québec à Montréal, Montréal, Quebec, Canada_.

Switches in the sphingolipid metabolism have recently been associated with oncogenic transformation, and a role for the low density lipoprotein receptor related protein (LRP1) in the sphingosine-1-phosphate (S1P) proangiogenic signalling inferred. Although S1P is a key determinant of blood-brain barrier (BBB) permeability, the interdependent signalling crosstalk with LRP1 in brain endothelial cells (EC) remains unclear. Here, we tested whether any adaptive LRP1/S1P interdependence mechanisms are required in a SV40-immortalized human brain microvascular EC (HBMEC) model which closely mimics the brain tumor endothelium phenotype. Transient LRP1 silencing and stable LRP1-/- HBMEC were generated and increased expression of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor which regulates the transcription of all five S1P receptors members, was observed upon stable but not transient LRP1 repression. Migration of EC isolated from Lrp1(EC)-/- mice and of stable LRP1-/- HBMEC became unresponsive to S1P, in part explained by altered ERK and p38 MAPK pathways, whereas it remained unaltered in transient in vitro LRP1 repression. Adaptive diminished expressions of S1P1, S1P3, and S1P5, and increased S1P2 were observed in stable LRP1-/- HBMEC and in brain EC isolated from Lrp1(EC)-/- mice. Finally, gene silencing strategies highlighted the need for S1P3 for proper S1P-mediated HBMEC migration. Collectively, our study highlights an unexpected adaptive transcriptional crosstalk between LRP1 and specific S1P receptors which accounts for S1P signalling in brain EC. Targeting of the LRP1/S1P signalling axis at the BBB may thus be considered in future therapeutic strategies.

#3365

Provasculogenic effects of alcohol and estrogen: implications for breast cancer development.

Rachana R. Maniyar,1 Robert B. Bednarczyk,1 Ghada M. Ben Rahoma,1 Neha Tuli,1 Abraham Mittelman,1 Raj K. Tiwari,1 Robert Suriano2. 1 _New York Medical College, Valhalla, NY;_ 2 _Mount Saint Vincent, Bronx, NY_.

Over 200,000 cases of breast cancer are diagnosed within the United States, resulting in over 40,000 deaths annually. Of the many lifestyle habits that are associated with cancer development, various epidemiological observations have provided a strong link between increased alcohol consumption and breast cancer progression. Specifically, this observation was made in women on estrogen replacement therapy thus suggesting a possible synergistic link between alcohol and estrogen. Our laboratory has demonstrated that estrogen increases breast cancer neo-vascularization both in vivo and in vitro. Provided that alcohol and estrogen can potentially synergize to promote breast cancer progression, we hypothesize that this progression is attributed to an increase in tumor neo-vascularization. In the current study, Tg-1 murine mammary carcinoma cells were treated with varying concentrations of alcohol +/- estrogen and analyzed for angiogenesis markers, such as VEGF and eNOS, and the pro-proliferation marker, MEK. At 24 hours post treatment, VEGF and eNOS were upregulated in response to both estrogen and alcohol compared to untreated cells and cells treated with either alcohol or estrogen alone. MEK levels remained constant through the conditions with a marginal upregulation in presence of alcohol and estrogen when compared to untreated cells. To further characterize neo-vascularization, we performed a scratch wound assay using the murine endothelial cell line, SVEC4-10, which was cultured in Tg1-1 conditioned media. The scratch wound assay results indicated enhanced migration of endothelial cells in response to conditioned media from Tg1-1 cells cultured in the presence of both alcohol and estrogen. SVEC4-10, treated with Tg-1-1 conditioned media were processed by Western blot analyses for MEK expression. MEK was upregulated in SVEC4-10 cells cultured in the media obtained from cells cultured with both alcohol and estrogen, indicating enhanced proliferation. Our results indicate a pro-neovasculogenic effect elicited by alcohol and estrogen, as determined by enhanced proliferation and migration of endothelial as well as increased expression of the pro-vasculogenic markers, VEGF and eNOS. Future experiments will be aimed to characterize the expression of other essential pro-vasculogenic markers, such as basic fibroblast growth factor (bFGF) and angiopoietin (Ang). The identification of these cellular and metabolic markers will establish a biochemical link between estrogen activity and signal transduction induced cellular effects.

#3366

NCL targeting impairs the progression of pancreatic ductal adenocarcinoma and promotes tumor vessel normalization through Ang-2 inhibition.

Maud-Emmanuelle Gilles,1 Federica Maione,2 Mélissande Cossutta,1 Gilles Carpentier,1 Laure Caruana,1 Silvia Di Maria,1 Damien Destouches,1 Ksenya Shchors,3 Christopher Prochasson,4 Anne Couvelard,4 José Courty,1 Enrico Giraudo,2 Ilaria Cascone1. 1 _University of Paris Est, Créteil, Val de Marne, France;_ 2 _IRCCS, Turin, Italy;_ 3 _Swiss Institute for Experimental Cancer Research (ISREC), Lausanne, Switzerland;_ 4 _Hopital Bichat, Paris, France_.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor, mostly resistant to the standard treatments. NCL is overexpressed in cancers and its inhibition impairs tumor growth. Herein we described, that NCL was overexpressed in human specimens of PDAC, and low NCL staining patients had increased overall survival. Previously, we described a family of multivalent pseudopeptides binding to NCL and inhibiting tumour growth. Here, NCL antagonist N6L, strongly impaired tumor growth, liver metastasis formation and angiogenesis in an orthothopic mouse model of PDAC. N6L inhibited both human and mouse tumor cell proliferation and invasion. Proteome analysis of endothelial cell secreted proteins showed that NCL inhibition decreased Ang-2 levels and switched a pro-angiogenic signature. Importantly, Ang-2 levels were decreased in plasma of N6L-treated PDAC mice. The analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion in parallel to an inhibition of tumor hypoxia. As consequence of N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the anti-tumor properties of gemcitabine in PDAC mice.

In conclusion, NCL inhibition is a new anti-tumor therapeutic strategy that dually blocks tumor progression and normalizes tumor vessels improving the delivery and efficacy of chemotherapeutic drugs in PDAC cancers. Moreover, we identified Ang-2 as a potential target and suitable response biomarker for N6L treatment in PDAC.

#3367

Overexpression of neuropilin-1 exacerbates endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma.

Pratiek N. Matkar,1 Krishna K. Singh,1 Gerald J. Prud'homme,1 David W. Hedley,2 Howard Leong-Poi1. 1 _St. Michael's Hospital, Toronto, Ontario, Canada;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. Recently, endothelial cells have been shown to display an extreme form of plasticity that serves as an important source of fibroblasts in several pathological disorders, including cancer. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation via endothelial-to-mesenchymal transition (EndMT), thereby identifying EndMT as a potential therapeutic target against fibrosis. Previous studies have implicated angiogenic co-receptor, neuropilin-1 (NRP-1) also as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the driving mechanisms linking NRP-1 and EndMT in cancer remain unexplored. We therefore hypothesized that the over-expressed NRP-1 may exacerbate TGFβ1-mediated EndMT as a source of fibrosis in PDAC.

Methods: NRP-1 over-expression studies were performed using lentivirus (Applied Biological Materials Inc.) in human umbilical vein endothelial cells (HUVECs), at baseline and after TGFβ1 stimulation (10 ng/mL). Total RNA was extracted using TRIzol® reagent and protein using RIPA buffer (both Invitrogen). Real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green (Bio-Rad) following manufacturer's protocols. Markers of EndMT, fibrosis and TGFβ1 signaling were evaluated by RT-PCR, western blotting, Masson's trichrome staining and immunocytochemistry. Similarly, expression of NRP-1, EndMT and fibrosis markers was assessed in human PDAC xenografts to establish a correlation between NRP-1 levels with EndMT and fibrosis.

Results: RT-PCR, western blotting and tube formation assay in HUVECs confirmed successful NRP-1 over-expression. TGFβ1-stimulated HUVECs demonstrated a distinct change from "cobblestone-like endothelial cell morphology" to an enlarged spindle-shaped appearance consistent with a "fibroblast-like morphology", accompanied by rearrangement of the cytoskeleton. Moreover, over-expression of NRP-1 in HUVECs exacerbated TGFβ1-induced EndMT as demonstrated by loss of endothelial and gain of mesenchymal markers, and was further accompanied by changes in microscopic cellular characteristics consistent with EndMT. NRP-1 over-expression also up-regulated TGFβ1 signaling and expression of pro-fibrotic genes. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis markers at mRNA and protein levels in human PDAC xenografts.

Conclusions: Overall, these results define a previously undetermined role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis in tumours and advocate NRP-1 as a potential therapeutic target to reduce tumour fibrosis and PDAC progression.

#3368

TIMP-1 and angiogenesis: A role for CDH5 and eNOS.

Sampa Ghoshal-Gupta, Ian A. Coe, Ammar Kutiyanawalla, Byung R. Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. _Georgia Regents University, Augusta, GA_.

Tissue inhibitors of metalloproteinases (TIMP) have emerged as diverse molecules with novel roles in apoptosis, angiogenesis and metastasis. TIMP-1, a well-documented inhibitor of apoptosis, has been shown to inhibit or promote angiogenesis. In recent years, a strong association has also been demonstrated between high levels of TIMP-1 and poor prognosis in many cancers. It has been recognized as a multifunctional protein affecting cell growth and angiogenesis. Conflicting studies have shown it to function either as a negative or a positive regulator of angiogenesis. In earlier studies, we have documented that lung adenocarcinoma cell line H2009, when transfected to overexpress TIMP-1 and injected into nude mice resulted in larger, more aggressive tumors with increased microvessel density, which was supported by in vitro angiogenesis assays whereby enhanced capillary network formation was seen. We have also shown that TIMP-1 expression levels can be correlated with KRAS independency in non-small-cell lung carcinoma (NSCLC) cell lines harboring KRAS mutations. The present study was undertaken to address the role of TIMP-1 in angiogenesis in the context of KRAS in NSCLC cell lines. KRAS dependent cell line H2009 and its TIMP-1 overexpressing clone HB1 and a KRAS independent cell line A549 and its TIMP-1 knockdown (KD) clone SH3 were examined by angiogenesis specific PCR array. Comparison of the angiogenesis associated profiles of these cell lines identified a marked increase in vascular endothelial cadherin CDH5 in TIMP-1 over-expressing clone HB1. KRAS independent A549 cells expressed high level of CDH5, however its TIMP-1 KD clone exhibited a marked decrease in CDH5 level. A similar profile was identified for endothelial nitric oxide synthase (eNOS/NOS3). CDH5 is an indispensable element of normal vascular development and maintenance, and any perturbations of normal levels will impact vessel sprouting and growth. eNOS is primarily active through production of nitric oxide, maintaining vascular tone as well as expressing anti-proliferative and antithrombotic properties. We confirmed our earlier observation that serum free conditioned media (SFCM) from TIMP-1 overexpressing HBI cells caused increased and more complex network formation. SFCM from A549 TIMP-1 KD SH3 clone showed a marked inhibition of endothelial network formation compared to SFCM from A549 cells. To confirm that TIMP-1 was responsible for the changes observed we purified secretory TIMP-1 by combined fast protein liquid chromatography and gel filtration from another high TIMP-1 producing NSCLC cell line (H460) and also observed more capillary network formation in human umbilical vein endothelial cells. Our studies suggest that TIMP-1 modulates angiogenesis by impacting CDH5 and eNOS positively. These results define an important function of TIMP-1 in angiogenesis and provide additional therapeutic targets for managing NSCLC.

#3369

Tumor-microenvironment-dependent imprinting of endothelial cells in human colorectal carcinoma.

Michael Stürzl,1 Andrea Liebl,1 Vera S. Schellerer,1 Manuela Schütz,1 Patrick Kölbel,1 Ute Schaal,1 Lisa Haep,1 Ludger Klein-Hitpass,2 Timan T. Rau,1 Barbara Dietel,1 Valerie Meniel,3 Alan R. Clarke,3 Susanne Merkel,1 Roland S. Croner,1 Werner Hohenberger,1 Elisabeth Naschberger1. 1 _Univ. of Erlangen-Nuremberg, Erlangen, Germany;_ 2 _Institute of Cell Biology, Essen, Germany;_ 3 _European Cancer Stem Cell Research Institute, Cardiff, United Kingdom_.

The tumor microenvironment (TME) and tumor cell heterogeneity contribute significantly to tumor pathogenesis. In contrast, potential stromal cell heterogeneity induced by the TME and its impact on human tumorigenesis has not been conclusively investigated. Therefore, pure tumor endothelial cells (TECs) from human colorectal carcinomas (CRCs) with differential TMEs (favorable Th1-TME and worse control-TME) were isolated and significantly different gene clusters reflecting the tumorigenic and angiogenic impact attributed to the respective TMEs were identified by transcriptome analysis. SPARCL1 - the most strongly upregulated gene in Th1-TME-derived TECs - was analyzed to prove this concept. SPARCL1 was expressed by endothelial cells (EC) in colon tissues, most prominently in mature normal vessels, similarly in CRC with Th1-TME and lost towards more aggressive and angiogenic control tumors. SPARCL1 was induced in confluent, quiescent EC in culture but absent or resolved in angiogenically active EC. SPARCL1 inhibited proliferation, migration and sprouting of cultivated EC in vitro. In human CRC tissues SPARCL1 expressing vessels were highly covered by mural cells and of larger vessel size (perimeter and area), indicting more progressed maturation. Similarly the vessels in the colon of wild type mice as compared to SPARCL1/Sc1 knockout mice exhibited a more mature vessel structure. In conclusion, exploiting cellular memory processes we demonstrated TME-dependent intratumoral TEC heterogeneity in CRC and identified SPARCL1 as novel EC-derived protein regulating vessel maturation and homeostasis.

#3370

Metabolic profiling of the tumor interstitial fluid using 1H MRS: contribution of breast cancer subtypes and VEGF overexpression.

Louis Dore-Savard, Santosh K. Bharti, Aleksander S. Popel, Zaver M. Bhujwalla. _Johns Hopkins University, Baltimore, MD_.

One of the least examined, and yet critically important factors of the tumor microenvironment (TME) is the tumor interstitial fluid (TIF) that contains the tumor secretome. This fluid surrounds cancer and stromal cells and contains various cytokines, and nutritional and molecular factors that shape the outcome of nearly all aspects of tumor angiogenesis, growth, metastasis, and response to treatment. As mining of targets to treat cancer expands into the TME, the TIF represents an important source of identifying new targets in cancer treatment. Angiogenesis, one of the major hallmarks of cancer, is essential for cancers to establish vasculature for growth and hematogenous metastasis. To further understand the TIF and plasma metabolite content in breast cancer, and the role of VEGF in modifying metabolite concentrations in the TME, here we used 1H MRS to characterize metabolites in TIF collected from the triple negative MDA-MB-231 and the ER-positive MCF-7 human breast cancer xenografts with and without VEGF overexpression implanted in SCID mice. We created a collection chamber to collect IF from the tumors. The chamber was implanted with small tumor pieces in the subcutaneous space until the tumor encompassed the chamber (4 to 12 weeks depending on cell types). The tumor was then excised and the TIF and blood plasma were collected from euthanized mice. We also collected normal subcutaneous IF (SCIF) using the same chamber in healthy mice. 1H spectra showed several differences in metabolites in TIF compared to SCIF, between MDA-MB-231 and MCF-7 TIF, and with VEGF overexpression. We observed a consumption of amino acids in the TME with decreases ranging from 17% (glycine) to 77% (methionine) compared to normal SCIF. Lipids, especially polyunsaturated fatty acids, were markedly increased by VEGF overexpression in both MCF-7 and MDA-MB-231 TIF compared to the wild type TIF. Choline metabolism was also modified by VEGF overexpression and we measured increases of 69% and 20% in free choline in MDA-MB-231_VEGF and MCF-7_VEGF respectively, compared to their wild type counterpart. A stable glucose concentration was associated with a decrease in lactate (30%) and pyruvate (44%) in MDA-MB-231 tumors while an opposite pattern was observed in MDA-MB-231_VEGF TIF with decreased glucose (55%) and increased lactate (109%). Ketonic metabolism was also modified in those tumors. Beta-hydroxybutyrate and acetoacetate were higher (81% and 46% increase vs SCIF, respectively) in MDA-MB-231 TIF while acetone was increased in both VEGF-overexpressing TIF compared to the wild type (+48% for MDA-MB-231_VEGF and +44% for MCF-7_VEGF). The TIF represents an importance source of information to understand mechanisms that drive aggressiveness, and identify new targets for diagnosis and therapy of cancer.

#3371

Genetic, phenotypic and functional characterisation of vasculogenic mimicry in small-cell lung cancer.

Francesca Trapani,1 Stuart Williamson,1 Robert L. Metcalf,1 Hui Sun Leong,1 Benjamin Abbott,1 Jenny Antonello,1 Cassandra Hodgkinson,1 Lynsey Franklin,1 Mary J. Hendrix,2 Richard E b Seftor,2 Elisabeth Seftor,2 Dominic Rothwell,1 Ged Brady,1 Crispin Miller,1 Fiona H. Blackhall,3 Kathryn L. Simpson,1 Caroline Dive1. 1 _CRUK Manchester, Manchester, United Kingdom;_ 2 _Stanley Manne Children's Research Institute, Robert H Lurie Comprehensive Cancer Center, Chicago, IL;_ 3 _The Christie NHS Foundation Trust, Manchester, United Kingdom_.

Background: Despite a good initial response to chemotherapy, most small cell lung cancer (SCLC) patients relapse with drug resistant disease. Targeting tumor vasculature in SCLC with anti-angiogenic drugs produced disappointing results, therefore angiogenesis-independent tumor vascularisation pathways warrant further investigation. Vasculogenic mimicry (VM) is the ability of tumor cells to mimic endothelial cells by trans-endothelial differentiation, characterised by increased expression of vascular markers including VE-Cadherin. We previously demonstrated that VM correlates with poor Overall Survival in Limited Stage SCLC patients and sought to phenotypically and genetically characterise VM vessels using SCLC Circulating Tumour Cells (CTCs) and CTC-Derived eXplant (CDX) models1 and to explore the functional significance of VE-Cadherin for VM formation in vitro and in vivo.Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in tumors from 10 CDX models. Laser Capture Microdissection (LCM) and Copy Number Alteration (CNA) analysis was performed on CDX regions with high and low levels of VM. VE-Cadherin expression in SCLC CTCs was evaluated following ISET microfiltration of patients' blood and Immunofluorescence staining for DAPI, CD45, pan Cytokeratin and VE-Cadherin. VE-Cadherin function was evaluated in vitro by Matrigel network assay using H446 SCLC cells and H446 shRNA VE-Cadherin knockdown (KD) cell lines, and in vivo by growth as xenografts, further treated with Cisplatin.

Results: VM was present in CDX models and LCM followed by CNA analysis of VM vessels confirmed their SCLC origin. ISET microfiltration and immunofluorescent staining of CTCs from 37/38 SCLC patients revealed VE-Cadherin as a putative VM biomarker in SCLC CTCs. VE-Cadherin shRNA KD in VM competent H446 SCLC cells abrogated their ability to form VM networks in vitro and in vivo. Cisplatin treatment of mice bearing H446 VE-Cadherin KD tumors resulted in reduced cisplatin binding compared to parental H446 tumors.

Conclusions: VM is present in CDX models and co-localises with VE-Cadherin expression. CNA confirms that VM vessels originate from tumour, and these VM-enriched regions bear a unique chromosomal signature compared to the low-VM regions. VE-Cadherin is required for network formation in vitro and VE-Cadherin levels and VM vessel numbers are positively correlated in vivo. Moreover, levels of VM in tumors had significant impact on both tumour growth kinetics and cisplatin delivery which has implications for drug senstivity. Our future research will interrogate the VM signaling pathway in SCLC and its role in chemosensitivity and establish feasibility for therapeutic targeting.

#3372

Semaphorin 3A normalizes the tumor vasculature and impairs tumor progression in a Nrp-1-independent manner.

Federica Maione,1 Cristina Basilico,1 Elisa Vigna,1 Mauro Giacca,2 Guido Serini,1 Enrico Giraudo3. 1 _Candiolo Cancer Institute, IRCCS, Candiolo, Italy;_ 2 _ICGEB, Trieste, Italy;_ 3 _Candiolo Cancer Institute, IRCCS, and Department of Science and Drug Technology, University of Turin, Candiolo, Italy_.

It is widely described that tumor vessel normalization, occurring in response to certain anti-angiogenic therapies, represents a remarkably advantageous anti-cancer strategy (1). We have demonstrated that Semaphorin 3A (Sema3A), an axon guidance cue part of class 3 semaphorins family, is an endogenous angiogenic inhibitor able to efficiently impair tumor progression, prolong the survival and normalize the tumor vasculature in different mouse models of spontaneous tumorigenesis (2). Moreover, we recently showed that Sema3A, by extending the normalization window and abrogating tumor hypoxia, overcame the resistance to the anti-angiogenic therapy inhibiting metastasis dissemination (3).

Stemming from these findings we sought to investigate the molecular mechanisms of vessel normalization and metastasis inhibition induced by Sema3A. Interestingly, by confocal microscope and western blot analysis, in a co-culture systems of human endothelial cells (ECs) and pericytes grown in contact, we observed that Sema3A dramatically down-modulated its receptor Nrp-1 in both cell types, with the consequent over-expression of PDGF-B and Ang-1, known to promote vessel maturation. Moreover, a wide screening of different genes and pathways modulated in the ECs/pericyte co-cultures revealed that the most modulated was the HGF/Met pathway. In fact, we observed that c-Met phosphorylation was impaired in FACS-sorted ECs co-cultured with human pericytes, compared to ECs grown as single layer. To better investigate the specific role of Sema3A in modulating HGF/Met activation in vessels, we detected a strong inhibition of HGF-induced Met phosphorylation in Nrp-1 silenced ECs induced by Sema3A, suggesting that this semaphorin could directly interfere with Met signaling. Notably, Sema3A impaired HGF-induced Met phosphorylation, not only in ECs, but also in several Nrp-1-silenced gastric, lung and pancreatic tumor cell lines, inducing apoptosis and blocking the invasiveness. Finally, treating an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC) with adeno-associate virus (AAV)-8 expressing Sema3A, we observed a strong inhibition of tumor growth, a dramatic reduction of liver metastasis and a normalized and perfused tumor vessels phenotype. Remarkably, we found that Sema3A strongly and specifically inhibited Met activation in both tumor cells and vessels, in parallel to a down-modulation of Nrp-1.

We conclude that Sema3A normalizes the tumor vasculature and blocks cancer progression in a Nrp-1-independent manner, in part by inhibiting HGF/Met pathway.

References

1. Jain RK, et al. Cancer Cell. 2014; 26:605-22.

2. Maione F., et al. J. Clin. Invest. 2009; 119:3356-72.

3. Maione F., et al. J. Clin Invest. 2012; 122:1832-48.

#3373

TGF-β1 signaling mediated lymphangiogenesis in gastric cancer.

Kyung Ho Pak,1 Jae-Ho Cheong,2 Ki Chung Park2. 1 _Hallym University, Seoul, Republic of Korea;_ 2 _Yonsei University, Seoul, Republic of Korea_.

Background

Recent evidence showed that Transforming growth factor (TGF)-β1 could have important role for gastric cancer progression and metastasis. However, the role of TGF- β1 for lymph node metastasis and lymphangiogenesis, the important steps of gastric cancer progression, is largely unknown. To investigate the role of TGF-β1 on lymphangiogenesis and its molecular mechanisms is the main purpose of this study.

Methods

We selected two gastric cancer cell-line models, MKN45 and KATOIII, which hold appropriate characteristics for the current study. We checked the possibility of the autocrine function of TGF-β1 for lymphangiogenesis in these gastric cancer cell-lines. We investigated the changes of TGF-β1 signaling molecules and VEGF-C expression in responding to TGF-β1 and its inhibitor. To examine the binding of Smad3 to the promoter region of VEGF-C, we performed electrophoretic mobility shift assay (EMSA). Both Smad-dependent and Smad-independent pathways were also examined to elucidate which pathways were involved in lymphangiogenesis. Finally, tube forming assay was performed to validate phenotypical effect of TGF-β1 on lymphangiogenesis.

Results

Two gastric cancer cell-line models, MKN45 and KATOIII, showed autocrine regulation of TGF-β1. TGF-β1 signaling was transduced to VEGF-C, mediated by Smad3. Smad3 was bound to the promoter of VEGF-C which was confirmed with EMSA. Based on EMSA results, KATOIII showed stronger reaction between Smad3 and VEGF-C promoter than MKN45 did. In MKN45, TGF-β1 also transduced its signal to VEGF-C through PI3k-Akt pathway, one of the Smad-independent pathway. Lymphatic endothelial cells cultured in the condition media of MKN45 and KATOIII underwent tubule structure formation. The growth of HLEC was accelerated with TGF-β1 mixed conditioned media, while failed to induce tubular structure in the presence of TGF-β1 inhibitor. The expression level of secreted VEGF-C was as same in responding to TGF-β1 and its inhibitor as the lymphatic tube forming results in gastric cancer cells.

Conclusion

TGF-β1 could promote lymphangiogenesis through VEGF-C expression in gastric

cancer cell line models. TGF-β1 signaling could be transduced through either

Smad-mediated or non- Smad-mediated depending on gastric cancer cell-line

characteristics.

#3374

GM-CSF and MMP9 are key regulators of the effect of adipose progenitor cells over breast cancer onset and metastatic progression in obesity.

Francesca Reggiani, Valentina Labanca, Giovanna Talarico, Stefania Orecchioni, Patrizia Mancuso, Francesco Bertolini. _European Inst. of Oncology, Milan, Italy_.

We recently described a human cell population with progenitor-like phenotype (CD45-CD34+), resident in the white adipose tissue (WAT) and able to promote local and metastatic breast cancer (BC) progression and angiogenesis (Orecchioni et al., 2013). The molecular mechanism involved in this interaction has been so far elusive. An extensive screening of candidate molecules related to angiogenesis, inflammation, motility and invasiveness revealed that two proteins are significantly up-regulated in WAT-derived progenitors following culture with BC cells: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Matrix metallopeptidase 9 (MMP9). In vivo, both proteins were overexpressed in orthotopic models of human BC co-injected with human WAT progenitors. The inhibition of GM-CSF by a monoclonal antibody in diet-induced, obese BC mice led to a reduced intratumor vascularization and a strong impairment of WAT immunosuppressive microenvironment, targeting mainly myeloid cells such as macrophages and myeloid derived suppressor cells (MDSCs) in peritumoral WAT. Circulating levels of monocytes and of CD4+CD25brightCD127low/neg T-regulatory cells (T-regs) were also reduced in treated mice. Similarly, soluble immunosuppressive factors, such as IL-10 and IL-5, and CD274 (PD-L1) were reduced in tumors and WAT collected from immune competent mice neutralized for GM-CSF, confirming the crucial role of the factor in promoting tumor immune escape. This resulted in a significantly reduced local BC growth and lower metastatic progression in vivo. All these findings challenge the clinical use of GM-CSF. In the same syngeneic model, MMP9 inhibition reduced neoplastic angiogenesis and significantly decreased local and metastatic tumor growth, without altering immune cells composition in tumor microenvironment. The combined inhibition of GM-CSF and MMP9 was synergic in impairing angiogenesis, local and metastatic BC growth in diet-induced obese orthotopic BC models, indicating a potential complementary role in tumor spread. As we recently reported that Metformin targets both BC cells and the neoplastic WAT environment (Orecchioni et al., 2015), we investigated Metformin effect over GM-CSF and MMP9 expression. Metformin inhibited GM-CSF and MMP9 up-release from WAT progenitors in vitro. Circulating GM-CSF was significantly impaired in BC xenografts administered with Metformin and MMP9 expression was also affected by the treatment. Collectively these results indicate GM-CSF and MMP9 as key candidates involved in the pro-tumorigenic effect of WAT progenitors on BC in a setting of obesity. The comparison between Metformin and GM-CSF/MMP9 specific inhibition is currently under investigation.

#3375

Activation of the acid sphingomyelinase pathway as a potential mechanism of chemotherapy-induced normal tissue vasculature damage.

Aviram Mizrachi. _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Background: Several classes of chemotherapy confer substantial risk for late-term vascular morbidity. We aimed to investigate the mechanisms of chemotherapy-induced vascular toxicity in normal tissues.

Methods: The effect of Doxorubicin and Cisplatin has been studied in-vitro using bovine aortic endothelial cells (BAEC) as a model of normal tissue vascular endothelium. Specifically, we investigated activation of the acid sphingomyelinase (ASMase) pathway, which may lead to endothelial dysfunction (vasoconstriction) and apoptosis following incubation with different concentrations of Doxorubicin and Cisplatin. ASMase activity was measured using radioenzymatic assay with [N-methyl-14C] sphingomyelin. Cellular Reactive Oxygen Species (ROS) production was assessed using the 2',7'-dichlorofluorescin diacetate (DCFDA) detection assay kit (Abcam®). Endothelial apoptosis was quantified using the 3,5-bis-benzamide assay (Sigma-Aldrich®). For the in-vivo studies, mouse femoral arterial blood flow was measured in real-time during and after Doxorubicin (8 mg/kg intravenously) or Cisplatin (8 mg/kg) administration. Visualization of ovarian and femoral microvasculature was obtained by fluorescence optical imaging system, equipped with a confocal fiber microscope (Cell-viZio).

Results: Incubation of BAEC with Doxorubicin (0.25 µM or 0.50 µM) resulted in increased ASMase activity, production of ROS and induction of apoptosis in a dose-dependent fashion. Cisplatin (25 µM or 50 µM) generated significantly reduced effects in BAEC yet apoptosis and ROS were notable. In-vivo endoscopic fibered confocal microscopy following chemotherapy administration revealed a differential pattern between the two agents. While post Doxorubicin injection, there was a marked reduction in fluorescent signal in small vessels (<15 µM) indicating vessel constriction and disintegration of vessel's wall in larger vessels, Cisplatin treatment resulted in very minor vascular changes.

Conclusions: These findings suggest that chemotherapy-induced vascular damage in normal tissues may represent different modes of toxicity in vivo and in vitro, while is mediated via activation of the ASMase pathway in response to Doxorubicin and to a lesser extent in response to Cisplatin.

#3376

Molecular drivers of endothelial hypoxic adaption during angiogenesis deciphered using the Berg Interrogative Biology® platform.

Tony E. Walshe, Justin Bourdelais, Viatcheslav R. Akmaev, Leonardo O. Rodrigues, Socheata Lao, Stephane Gesta, Michael A. Kiebish, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. _Berg, LLC, Framingham, MA_.

Angiogenesis is a key feature of tumor progression providing oxygen and nutrients required for tumor cell growth, hypoxia being a major driver of this phenomenon. A systems biology approach using the Berg Interrogative Biology® platform was implemented to identify drivers of the endothelial cell (EC) angiogenic response to hypoxia. To determine the proteomic profile of proliferating ECs and non-proliferating confluent ECs exposed to normoxia or hypoxia, a functional proteomic approach employing activity-based probes and phosphorylation analysis was utilized. Kinases and ATPases were labeled with ATP-binding domain enrichment probes and titanium dioxide enrichment of phosphopeptides was employed for capture of protein phosphorylation events. Phenotypic assays including proliferation rates, apoptosis, mitochondrial superoxide and ROS/NO signaling were included as a measure of EC phenotype. Comparative proteomics, kinase activity, phosphoproteomics and assay data were integrated using an AI based Bayesian Network Inference approach to investigate causal signaling networks in order to elucidate the complexity and dynamics of angiogenesis and more specifically, the role of hypoxia in driving intracellular signaling in response to changes in oxygen tension. High confidence causal networks identified novel proteins that modulate the EC hypoxic response, validation of which are in process. Previously characterized proteins that are responsive to hypoxia were also identified in the hypoxic, but not normoxic signaling networks, namely Aldoc, Rac1, mTor, Cav-1 and Bax. Endothelial proliferation rate is closely related to both hydrogen peroxide and nitric oxide signaling, as has previously been reported. Novel regulatory networks that determine these interactions were identified. Using the Berg Interrogative Biology® platform, we are deciphering both the effects of the hypoxic microenvironment, and the unique characteristics of proliferating ECs by applying integrated functional proteomic assays and a systems approach to determine global changes in intracellular signaling in response to hypoxia.

#3377

Bevacizumab-improved distribution of paclitaxel in ovarian cancer xenografts potentiates antitumor efficacy.

Marta Cesca,1 Lavinia Morosi,1 Alexander Berndt,2 Ilaria Fuso Nerini,1 Alessandra Decio,1 Massimo Zucchetti,1 Raffaella Giavazzi1. 1 _IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 2 _Institute of Pathology, University Hospital, Jena, Germany_.

Angiogenesis inhibitors have shown antitumor efficacy when combined with chemotherapy. It has been proposed that this potentiation is related to better drug delivery to the tumor due to improved vessel functionality. This work combined classical pharmacokinetics and imaging analysis to study the delivery and distribution of paclitaxel after bevacizumab in ovarian cancer.

The study was conducted on ovarian cancer models (A2780-1A9 and IGROV-1) xenografted orthotopically under the bursa of the nude mouse ovary. Mice bearing tumors in the ovary, treated with bevacizumab (150μg/mouse) and 24 hours later with paclitaxel (60mg/kg), were sacrificed and tumor excised for analysis, or monitored for anti-tumor activity. Paclitaxel spatial distribution after bevacizumab was analyzed by MALDI-Mass Spectrometry Imaging (MALDI-MSI) on frozen tumor slices, and compared with adjacent histochemical images stained for CD31 (vessel analysis) and Ki-67 (proliferation/necrosis); total paclitaxel in tumor homogenates, liver and plasma was evaluated by high-performance liquid chromatography (HPLC). Tumor growth of A2780-1A9 and IGROV-1, carrying the firefly luciferase gene luc2, was monitored by Bioluminescence imaging (BLI) and survival recorded.

Bevacizumab decreased tumor vessel number/size compared to vehicle treated mice, but potentiated the antitumor activity of paclitaxel in both models. MALDI-MSI showed that after bevacizumab, paclitaxel was more homogenously distributed and mainly in actively proliferating areas of the tumor where vessels were uniformly diffused, despite paclitaxel concentration did not increase after antiangiogenic drug. No changes in paclitaxel systemic exposure was found in normal tissue. These findings suggest that a more uniform distribution of paclitaxel in the tumor of the mouse ovary is responsible for the better antitumor activity of the combination.

A.D. and I.F.N. are the recipients of a fellowship from the Italian Foundation for Cancer Research (FIRC).

#3378

Using multiplex immunoassays for profiling the circulating and tissue angiogenic biomarkers in mouse and human cancer.

Wen-Rong Lie, Jehangir Mistry. _EMD Millipore Corp., St. Charles, MO_.

Monitoring the expression levels of multiple pro- and anti-angiogenic factors in circulation and tumor microenvironment is crucial in examining the role of angiogenesis in cancer pathological processes or in identifying potential biomarkers for oncology drug development. The Luminex xMAP technology allows simultaneous detection of multiple proteins in a single sample and has been used widely in cancer biomarker discovery. Using the Luminex xMAP technology, we developed two Human Angiogenesis Panels (17-plex and 20-plex) and a Mouse Angiogenesis Panel (27-plex) for the simultaneous detection of multiple angiogenic biomarkers in less than 50 μL of human or mouse samples. We report here the protein biomarker profiles of these 37 human angiogenic factors in 1) serum samples, collected from breast cancer and colorectal cancer patients and normal donors, 2) tumor extracts, collected from breast tumor and colorectal tumor extracts and their matched adjacent normal tissue, 3) conditioned media in cultured tumor cells and stromal cells, and in addition, the protein biomarker profiles of 27 mouse angiogenic factors in the conditioned media of various mouse tumor cells and stromal cells. For example, of the 37 human angiogenesis serum protein markers tested, 16 biomarkers [angiopoietin-2 (ANGPT-2), EGF, endoglin, follistatin, G-CSF, HGF, PLGF, sAXL, PDGF-AB/BB, osteopontin (OPN), sHGFR/c-Met, suPAR, sTie-2, sHer2, soluble neuropilin-1 (sNRP-1), and tenascin-C] showed significant difference in expression levels between normal and breast cancer serum samples (p<0.05) and 13 biomarkers [ANGPT-2, EGF, PLGF, sAXL, sHer3, OPN, thrombospondin-2 (TSP-2), sHGFR/c-Met, suPAR, sTie-2, sNRP-1, sIL-6R, and tenascin-C] showed differential expression levels between normal and colorectal cancer serum samples (p<0.05). Altogether, the results of this study demonstrate the utility of these human and mouse angiogenesis multiplex panels as a useful tool in studying circulating and tissue protein biomarker profiles in cancer research.

#3379

Tumor-induced remote extracellular matrix network orientation steers angiogenesis.

Erik H. Danen. _Leiden University, Leiden, Netherlands_.

Tumor angiogenesis promotes tumor growth and metastasis. Here, we use automated sequential microprinting of tumor and endothelial cells in extracellular matrix (ECM) scaffolds to study its mechanical aspects. Quantitative reflection microscopy shows that tumor spheroids induce radial orientation of the surrounding collagen fiber network up to a distance of five times their radius. Across a panel of ~20 different human tumor cell lines, remote collagen orientation is correlated with local tumor cell migration behavior. Tumor induced collagen orientation requires contractility but is remarkably resistant to depletion of collagen-binding integrins. Microvascular endothelial cells undergo directional migration towards tumor spheroids once they are within the tumor-oriented collagen fiber network. Laser ablation experiments indicate that an intact physical connection of the oriented network with the tumor spheroid is required for mechanical sensing by the endothelial cells. Together our findings show that remote physical manipulation of the ECM network by the tumor steers angiogenesis.

#3380

Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells.

Monica Burgett,1 Justin Lathia,1 Patrick Roth,2 Amy Nowacki,1 Elena Pugacheva,3 Ping Huang,1 Amit Vasanji,4 Li Meizhang,1 Tatiana Byzova,1 Tom Mikkelsen,5 Shideng Bao,1 Jeremy Rich,1 Michael Weller,2 Candece Gladson1. 1 _The Cleveland Clinic Lerner Research Institute, Cleveland, OH;_ 2 _University Hospital, Zurich, Switzerland;_ 3 _West Virginia University, Morgantown, WV;_ 4 _Image IQ, Inc., Cleveland, OH;_ 5 _Henry Ford Hospital, Detroit, MI_.

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM), which requires endothelial cell (EC) migration. Here, we show that angiogenesis can also be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and ECs. The direct interaction between CSCs and ECs was mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. Using in vitro assays, we found that this interaction increased network formation and migration-associated signaling events in ECs, including activation of integrin αvβ3, FAK, bone marrow tyrosine kinase on chromosome X (BMX), p130CAS, ERK and JNK. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that upstream and downstream effector activation was not attributed to a secreted factor(s). Activation of αvβ3 and BMX was required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells (consistent with CSCs) in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results show that direct interactions between CSCs and ECs have potent effects on EC migration and reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

#3381

Genetic regulation of melanoma latency and angiogenesis.

Michael S. Rogers,1 Amy E. Birsner,2 Robert J. D'Amato1. 1 _Boston Children's Hospital / Harvard Medical School, Boston, MA;_ 2 _Boston Children's Hospital, Boston, MA_.

Susceptibility to melanoma varies >20-fold among different populations. Polymorphisms in pigment production genes such as ASIP, MC1R, OCA2, SLC45A2, and TYR are prominent among those that affect such susceptibility. In addition to modifying the amount of UV light reaching the nucleus or by affecting the production of DNA-damaging oxygen radicals by melanin and its byproducts, we recently identified a third mechanism by which pigment production genes may affect susceptibility to melanoma. We have mapped genetic polymorphisms in inbred mice that affect the angiogenic response to bFGF and VEGF identified the Tyr and Oca2 genes as regulators of angiogenesis. This finding indicates that pigment production genes may also affect melanoma by impinging on tumor angiogenic support, and is consistent with the observation that pigment production by the melanoma itself affects whether or not the tumor will grow vertically. Candidate gene studies also reveal that host polymorphisms in angiogenesis regulators such as VEGF influence the susceptibility to, and three-dimensional growth of, melanoma.

Given the key role of angiogenesis in melanoma growth, we reasoned that genetic differences in host angiogenic responsiveness would affect some aspects of the natural history of melanoma, such as tumor growth rate. However, contrary to expectation, our data indicate that the major effect of such variations in host angiogenic response is not on growth rate, but rather on the latent period before a tumor initiates exponential growth. Specifically, we initiated subcutaneous B16F10 tumors in mice with differing angiogenic responsiveness: low-angiogenic C57BL/6J or high-angiogenic B6129PF1 (in both strains B16F10 melanoma is seen as "self"). We found that final tumor growth rates are not significantly different, but the latency (i.e. the time lag between tumor implantation and the initiation of macroscopic tumor growth) is significantly longer in the low-angiogenic C57BL/6J mice. This observation was confirmed using an array of BXD recombinant inbred mice of varying angiogenic responsiveness. Again, higher angiogenic responsiveness correlated with a shorter average lifespan; a result that was not caused by an increase in tumor growth rate, but rather a shorter latent period. Ongoing studies using CRISPR-induced alleles of pigment-production genes are designed to assess the effect of additional individual genes on angiogenesis and tumor latency, thereby identifying novel genes that affect both processes.

We and others have shown that induction of angiogenesis is associated with conversion of dormant tumors to exponential growth. These studies show that induction of angiogenesis is more readily accomplished in hosts with high angiogenic responsiveness. This suggests that patients with high angiogenic responsiveness may experience earlier metastatic relapse. Therefore, identification of such patients would allow increased surveillance, timelier therapy, and thereby longer life.

#3382

A physiological model of the tumor microenvironment for screening drug delivery systems.

Yuan Tang,1 Fariborz Soroush,1 Sudhir Deosarkar,1 Bin Wang,2 Balabhaskar Prabhakarpandian,3 Mohammad Kiani1. 1 _Temple University, Philadelphia, PA;_ 2 _Widener University, Chester, PA;_ 3 _CFD Research Corporation, Huntsville, AL_.

Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. Tumor microvasculature has many unique features including unusual transport characteristics, high interstitial pressure, and enhanced permeability and retention (EPR) effect. Current in vitro models of tumor drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. The objective of this study is to develop and characterize a synthetic 3D solid tumor - endothelium model in a novel microfluidic platform that mimics the tumor microenvironment observed in vivo.

The novel synthetic tumor model consists of a vascular network of tumor derived endothelial cells forming a complete lumen in communication with 3D solid tumors. Primary human breast tumor associated endothelial cells (HBTAECs) were co-cultured under physiological fluid flow conditions with 3D tumor cells from metastastic (MDA-MB-231) or non-metastatic (MCF-7) origins to study the effect of metastatic potential on the integrity of the adjacent endothelial cell lining. Extravasation of fluorescently labeled liposomes across the endothelium to the tumors was measured following HBTAECs treatment with normal media, tumor conditioned media (TCM), or TNF-α. Tight junction formation was characterized using ZO-1 immunostaining.

MDA-MB-231 cells quickly invaded into the vascular network from their primary culture location whereas MCF-7 cells rarely grew beyond the boundary of tumor origin. Permeability of liposomes across the endothelium was significantly higher with TCM treatment compared to both control and TNF-α treated cells (P < 0.01, one-way ANOVA). ZO-1 staining indicated strong tight junction formation when HBTAECs were treated with normal media as compared to TCM or TNF-α.

We have successfully established an in vitro 3D tumor - endothelial cell co-culture model in a novel microfluidic platform that closely mimics the tumor microenvironment in vivo. This system reproduces the tumor permeability and retention (EPR) effect on a chip. This realistic in vitro model can have great potential in applications such as cell-cell/cell-drug carrier interaction studies, drug delivery carrier screening, and drug efficacy testing.

#3383

Tumor vasculature normalization by recoupling nitric oxide synthase with a tetrahydrobiopterin precursor.

Christopher Rabender, Ninu Bruno, Ross B. Mikkelsen. _Virginia Commonwealth Univ., Richmond, VA_.

The abnormal, irregular nature of tumor vasculature has been well documented. The aberrant nature of the vasculature results in uneven, heterogeneous blood flow and leaky, hemorrhagic blood vessels. Due to the unusual nature of tumor vessels, areas of hypoxia tend to develop that contribute to radioresistance and inefficiency of therapeutic drug delivery. Our current studies examine the role of nitric oxide synthase (NOS) in tumor vasculature. NOS has been demonstrated to be "uncoupled" in tumor cells due to reduced levels of tetrahydrobiopterin (BH4), a necessary cofactor, resulting in peroxynitrite (ONOO-) formation in lieu of nitric oxide (NO). NO signaling is critical for vascular function and thus uncoupling of eNOS in the endothelial cells may partly explain the poor vasculature structure found within solid tumors. Having previously demonstrated that NOS can be "recoupled" and NO production restored through treatment of tumor cells with Sepiapterin (SP), a BH4 precursor, we examined whether SP could normalize tumor vasculature, promoting radiosensitivity and improving drug uptake. Multispectral optoacoustic tomography analysis of both flank tumor xenografts and a spontaneous breast tumor model (MMTV) demonstrate that SP given orally significantly enhances the percent of oxyhemoglobin in the tumor. Immunohistochemical analysis of tumors treated with SP showed a significant reduction in CD31 staining and a significant increase in smooth muscle actin (SMA), both hallmarks of vascular normalization. Ex vivo analysis of tumors revealed that the enhanced tumor oxygenation resulted in over a two-fold increase in radiation induced cell killing. These preliminary data demonstrate great potential for SP as an adjuvant in the treatment of cancer, especially when we take into consideration that SP has been demonstrated to be cytotoxic to both breast and colon tumors. Future studies will examine drug uptake in tumors as well as the mechanism behind the vascular normalization.

#3384

IGFBP5-derived peptide as a novel angiogenesis inhibitor for treatment of ovarian cancer.

Jeong-Won Lee, Jae Ryoung Hwang, Young-Jae Cho, Yoo-Young Lee, Chel Hun Choi, Duk-Soo Bae, Byoung-Gie Kim. _Samsung Medical Center, Seoul, Republic of Korea_.

Insulin-like growth factor-binding protein 5 (IGFBP5) plays a role in cell growth, differentiation, and apoptosis. We found that IGFBP5 was markedly downregulated in ovarian cancer tissue, and that its overexpression in cancer cells induced cell death. In this study, we undertook to evaluate the functional significance of each region of IGFBP5 as a tumor suppressor of ovarian cancer. We found that the C-terminal region of IGFBP5 inhibited tumor growth in a 2774 cell xenograft mouse model, and that expression of VEGF, IL-6, and TNF-α were inhibited in 2774 cells stably expressing the C-terminus of IGFBP5. In order to evaluate its effects on tumor suppression, a peptide derived from the C-terminus of IGFBP5 (BP5-C) was synthesized. As a control, a peptide mutated in the IGF-binding site of BP5-C (BP5-Cmut), as well as peptides derived from the IGFBP2 C-terminus and heparin-binding site were also synthesized. Of these peptides, BP5-C and BP5-Cmut inhibited VEGF expression and NF-kB activity. Furthermore, BP5-C inhibited angiogenesis in an in vitro and an ex vivo system, consisting of HUVEC tube formation and rat aortic ring blood vessel sprouting, respectively. The in vivo effect of BP5-C on tumor growth was studied using i.p. injection of ovarian cancer 2774 cells into mice, as well as with a patient-derived xenograft mouse model. BP5-C peptide significantly inhibited tumor growth, angiogenesis, and VEGF expression in both xenograft models. These results suggest that the C-terminus of IGFBP5 exerts an anti-cancer activity by inhibiting angiogenesis via downregulation of VEGF in an IGF-independent manner, and may be considered as a novel angiogenesis inhibitor for the treatment of ovarian cancer.

### Tumor Angiogenesis: Mediators and Mechanisms

#3385

Identification of tumor endothelium-related genes in colorectal cancer.

Akira Yorozu,1 Eiichiro Yamamoto,2 Reo Maruyama,1 Masahiro Kai,1 Toshihiko Nishidate,3 Tomohisa Furuhata,3 Tamotsu Sugai,4 Hiromu Suzuki1. 1 _Dept. Mol. Biol., Sapporo Med. Univ., Sapporo, Japan;_ 2 _Dept. Gastroenterol. Rheumatol. Clin. Immunol., Sapporo Med. Univ., Sapporo, Japan;_ 3 _Dept. Surg, Surg. Oncol. & Sci., Sapporo Med. Univ, Sapporo, Japan; _4 _Dept. Diagnostic Mol. Path., Iwate Med. Univ, Sapporo, Japan_.

Background and Aims:

Angiogenesis is a hallmark of cancer development that has been considered an attractive therapeutic target. In this study, we aimed to unravel the molecular mechanism of tumor angiogenesis in colorectal cancer (CRC).

Materials and Methods:

We isolated endothelial and epithelial cells from surgically resected 14 human CRC tissues by using antibodies against endothelial (CD146) and epithelial markers (EpCAM). RNA sequencing was carried out using 3 pairs of normal and tumor endothelial cells. Expression of the genes was validated using quantitative RT-PCR, immunohistochemistry. Functions of a selected gene were carried analyzed by tumor conditioned medium (TCM) experiments, tube formation assay, gene expression microarray and cell cycle analysis.

Results:

Through RNA-seq analysis, we identified 18 genes, which were upregulated in the endothelial cells isolated from CRC tissues. We further validated the results by performing quantitative RT-PCR and immunohistochemical analysis in a larger series of clinical samples, and identified gene A as a novel candidate of the tumor endothelium-related gene. Expression of gene A was also upregulated in human umbilical vein endothelial cells (HUVECs) treated with TCM obtained from CRC cell lines. Knockdown of gene A in HUVECs suppressed in vitro tube formation and induced G1 cell cycle arrest. Microarray analysis revealed that gene A knockdown induced expression changes of approximately 300 genes in HUVECs, and gene ontology analysis showed that genes related to cell cycle or cell division were significantly enriched in the affected genes. To confirm our findings in vivo, we co-transplanted CRC cells with HUVECs into nude mice. We found that gene A knockdown in HUVECs resulted in reduced micro vessel formations in the xenograft tissues.

Conclusion:

We identified elevated expression of gene A in tumor endothelial cells of primary colorectal cancer tissues. Our results suggest that gene A may play an important role in the angiogenesis in colorectal cancer, and that it could be a potential therapeutic target.

#3386

Serum VEGF-A may predict prognosis in patients with uterine cervical cancer.

Mayumi Sawada,1 Tetsuro Oishi,1 Hiroaki Komatsu,1 Hiroaki Itamochi,2 Michiko Nonaka,1 Seitya Sato,2 Jun Chikumi,1 Sinya Sato,1 Sinya Sato,1 Muneaki Shimada,1 Tasuku Harada1. 1 _Tottori University, Yonago, Japan;_ 2 _Iwate Medical University, Morioka, Japan_.

Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including uterine cervical cancer (CC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with CC.

Methods: A total of 115 patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB to IIB CC who were treated at Tottori University Hospital between 2006 and 2015 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. Survival analysis was performed with 86 patients treated between 2006 and 2013. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from CC patients.

Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 313, 8404 and 67.2 pg/ml, respectively. The levels of VEGF-A in patients with lymph node involvement were significantly higher than those in patients without it. On the contrary, the levels of VEGFR1 in patients with lymph node involvement were significantly lower than those in patients without it. Both histological types and FIGO stage were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the maximum tumor diameter. There was a significant negative correlation between VEGFR1 levels and the maximum tumor diameter. The overall survival rate was significantly lower in FIGO stage IIB than stage IB- IIA patients. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (92.9% vs. 72.8%, P = 0.014). Both VEGFR1 and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors.

Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for CC.

#3387

Serum VEGF-A may predict prognosis in patients with epithelial ovarian cancer.

Hiroaki Komatsu,1 Tetsuro Oishi,1 Hiroaki Itamochi,2 Mayumi Sawada,1 Michiko Nonaka,1 Seiya Sato,2 Jun Chikumi,1 Shinya Sato,1 Muneaki Shimada,1 Tasuku Harada1. 1 _Tottori University, Yonago, Japan;_ 2 _Iwate Medical University, Morioka, Japan_.

Objective: Angiogenesis is one of the processes that is critical for the growth, invasion and metastasis of solid tumors, including epithelial ovarian cancer (EOC). The vascular endothelial growth factor (VEGF) family is one of the major pathways involved in tumor angiogenesis. The aim of this study was to determine whether serum levels of these angiogenic factors could be used as biomarkers in patients with EOC.

Methods: A total of 133 patients with EOC who were treated at Tottori University Hospital between 2006 and 2014 were enrolled in this study. The study was approved by the Institutional Review Board of the School of Medicine of Tottori University. All patients gave written informed consent before the collection of specimens according to institutional guidelines. Serum samples were collected before initial surgery and levels of VEGF-A, -C and VEGFR1 were analyzed by enzyme-linked immunosorbent assay (ELISA). We evaluated the association between the levels of VEGF-A, -C, and VEGFR1 and clinicopathologic variables. We also determined the mRNA expression VEGF-A, -C, and VEGFR1 by real-time RT-PCR in fresh frozen tumors and the protein expression by immunohistochemical staining in paraffin-embedded tumors from EOC patients. Additionally, serum samples were collected before and after bevacizumab treatment, and levels of angiogenic factors were examined.

Results: The mRNA and protein expressions of VEGF-A, -C, and VEGFR1 were strongly observed in the cancer cells. Median levels of serum VEGF-A, -C and VEGFR1 were 284, 4861 and 417 pg/ml, respectively. The levels of VEGF-A ans VEGFR1 in patients at stage III-IV were significantly higher than those in patients at stage I-II. Both histologic subtypes and lymph node involvement were not related to levels of these angiogenic factors. We found a significant positive correlation between VEGF-A levels and the ascitic volume. The overall survival rate was significantly lower in FIGO stage III or IV patients with EOC. We set the cutoff value of these factors at the median levels of each angiogenic factors. The 5-year survival rate for patients with high VEGF-A levels was significantly lower than those with low levels (60.7% vs. 45.5%, P = 0.029). Both VEGFR1 and VEGF-C levels were not related to outcome of patients. Multivariate analysis revealed that serum VEGF-A level and FIGO stage were independent prognostic factors. VEGF-A levels dramatically decreased after treatment with bevacizumab.

Conclusion: These results suggest that serum VEGF-A may be a promising prognostic biomarker for EOC. Further study is needed to determine if could be predictive biomarker.

#3388

Genetic prediction of VEGF-A plasma levels in cancer patients.

Federico Innocenti,1 Chen Jiang,2 Alexander Sibley,2 Amy Etheridge,1 Yoichi Furukawa,3 Michiaki Kubo,3 Hedy L. Kindler,4 Alan P. Venook,5 Herber I. Hurwitz,6 Andrew B. Nixon,6 Kouros Owzar6. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Alliance Statistics and Data Center, Duke University Medical Center, Durham, NC;_ 3 _Center for Genomic Medicine, RIKEN, Yokohama, Japan;_ 4 _University of Chicago, Chicago, IL;_ 5 _University of California at San Francisco, San Francisco, CA;_ 6 _Duke Cancer Institute, Duke University Medical Center, Durham, NC_.

ABSTRACT

Background: Angiogenesis is an essential event in tumor growth, progression and metastasis and is strongly regulated by multiple VEGF ligands and receptors. We sought to discover genetic variants that could predict levels of circulating angiogenic proteins in cancer patients prior to receiving therapy.

Methods: EDTA plasma was collected at baseline (before treatment) in 216 treatment naïve pancreatic cancer patients (CALGB 80303, discovery) and 149 treatment naïve colorectal cancer patients (CALGB 80203, validation). Thirty-one angiogenic factors were measured by a multiplexed ELISA assay. Genetic variants associated with levels of each of the 31 proteins were selected from a genome-wide genotyping of 484,523 common variants in CALGB 80303. Using a cut off of p<10-8 for association, variants were selected from CALGB 80303, genotyped in CALGB 80203 and associations tested with the same protein levels. The Jonckheere-Terpstra test was used. Additionally, mRNA was extracted from formalin-fixed, paraffin-embedded primary tumors from patients in CALGB 80203 and tissue levels of VEGF-A analyzed by RT-PCR.

Results: In CALGB 80303, three genetic variants passed the p<10-8 cut off for significance: rs2284284 for MCP1, rs7504372 for VEGFC, and rs7767396 for VEFG-A levels. Of these, only rs7767396 (A>G) for VEFG-A levels was validated in CALGB 80203 (p=1.23x10-5). Patients with the AA genotype exhibited 2.2-fold higher VEGF-A levels than AG patients, who had 1.2-fold higher levels than GG patients. rs7767396 was not associated with VEGF-A mRNA levels from the primary tumors of patients in CALGB 80203 (p>0.05). rs7767396 is a common variant (frequency of 49% in Europeans), is located about 150 Kb 3' to the VEGFA gene, and is predicted by HaploReg to disrupt the binding motifs of two transcription factors, NF-AT1 and ZBRK1.

Conclusions: A common genetic variant predicts the levels of circulating VEGF-A in cancer patients. A similar effect has been also shown in non-cancerous individuals (Debette S et al., Circ Res, 2011). Due to the central role of VEFG-A in the pathophysiology of many conditions, genetic testing could predict patients who have high versus low levels, potentially helping to guide the use of anti-angiogenesis therapies.

#3389

Androgen deprivation therapy potentiates the efficacy of vascular targeted photodynamic therapy of prostate cancer xenografts.

Kwanghee Kim,1 Philip A. Watson,1 Sylvia Jebiwott,1 Alexander J. Somma,1 Stephen P. La Rosa,1 Dipti Mehta,1 Katie S. Murray,1 Hans Lilja,1 David Ulmert,1 Avigdor Scherz,2 Jonathan Coleman1. 1 _MSKCC, New York, NY;_ 2 _The Weizmann Institute of Science, Rehovot, Israel_.

Focal therapies for prostate cancer offer the possibility of less side effects than more aggressive interventions for organ-confined tumors. Vascular targeted photodynamic therapy (VTP) is being investigated in phase II and phase III clinical trials, and relies upon rapid, free radical-mediated destruction of tumor vasculature following photoactivation of a systemic prodrug. In a phase II trial, >80% of patients did not have detectable cancer in prostate biopsies taken 6 months after VTP treatment. Although these early results are encouraging, VTP efficacy could potentially be improved upon by combining with other therapies. Using the LNCaP-AR prostate cancer model system, we employed microarrays and Gene Set Enrichment Analysis (GSEA) to identify potential druggable pathways active in tumors exposed to VTP. Three to six hours post-VTP, androgen responsive gene sets were enriched, suggesting that the androgen receptor (AR) may be a viable target in combination with VTP. We tested this hypothesis in mice bearing LNCaP-AR xenograft tumors by using the AR pathway inhibitors degarelix (to achieve pharmacological castration) or enzalutamide (AR antagonist), alone or in combination with VTP. Degarelix was administered as a single 0.5-1 mg dose 3 days prior to initiation of VTP, while enzalutamide was given daily for two weeks total both before and after VTP. Serum measurements of prostate specific antigen (PSA) were used as a readout of AR activity. Compared to either AR pathway inhibitor or VTP used alone, degarelix or enzalutamide in combination with VTP significantly inhibited tumor growth. A sharp decline in serum PSA confirmed AR inhibition. Combined degarelix plus VTP treated tumors displayed intense TUNEL staining 7 days post-treatment, supporting an increased apoptotic frequency underlying the effect on tumor inhibition. In prostate cancer treated with VTP, compensatory acute upregulation of pro-survival AR signaling may occur. These findings may warrant investigation of combination with androgen deprivation therapy in future clinical trials utilizing VTP for prostate cancer.

#3390

CTHRC1 promotes angiogenesis by recruiting Tie2-expressing monocytes to pancreatic tumors.

Seongyea Jo, Jaemin Lee, jinhoi song. _Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea_.

CTHRC1 (collagen triple helix repeat-containing 1), a protein secreted during the tissue repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 plays an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here, we demonstrate a novel role of CTHRC1 as a potent endothelial activator, promoting angiogenesis by recruiting bone marrow-derived cells into the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, consistent with observed increases in neovascularization in vivo. Moreover, rCTHRC1 induced upregulation of angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody abrogated Ang-2 expression in ECs in vitro. Moreover, CTHRC1-neutralizing antibody treatment in a xenograft mouse model reduced tumor burden and infiltration of TEMs in tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the conclusion that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers.

#3391

SCUBE2 is a co-receptor for VEGFR2 in tumor angiogenesis.

Yuh-Charn Lin, Ruey-Bing Yang. _Academia Sinica, Taipei, Taiwan_.

Signal peptide-complement protein C1r/C1s, Uegf, and Bmp1 (CUB)-epidermal growth factor (EGF) domain-containing protein 2 (SCUBE2) is a peripheral membrane protein expressed in normal tissue and tumor vascular endothelial cells (ECs); however, its role in angiogenesis remains poorly understood. In this study, we show that endothelial SCUBE2 is upregulated by hypoxia and acts as a co-receptor for VEGFR2 to facilitate VEGF binding to VEGFR2 and augment its signals including VEGFR2 phosphorylation and p44/42 MAPKs/Akt activation, thus promotes cell proliferation and tubule formation in ECs. While physiological angiogenesis remained normal in the endothelial ablation of Scube2 in mice, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density. To simulate the angiogenic environment of the tumor, Scube2-deficient ECs were isolated and propagated in vitro with VEGF. Mutant ECs exhibited marked reduction in binding of VEGF, proliferation and sprouting responses to VEGF as well as downstream signal activation. Our results reveal that SCUBE2 might act as a novel co-receptor for VEGFR2, and point that targeting such SCUBE2 function in human ECs may represent a potential anti-tumor strategy by inhibiting tumor angiogenesis.

#3392

Lung cancer cell derived extracellular vesicles carrying DNAs, RNAs and proteins that may trigger oncogenic signals and promote angiogenesis.

Chien-Chung Lin, Wei-Lun Huang, Wu-Chou Su. _Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan_.

Extracellular vesicles (EVs) secreted from tumor cells have been implicated in modulating the tumor microenvironment changes. In this study, we demonstrated that EVs secreted by lung cancer cell are loaded with several cargos, such as DNA, RNA, cytokines, and growth factors. After the EVs had been taken up by recipient cells, the cargos might activate oncogenic pathways. We firstly isolated EVs from cell-free medium of lung cancer cells lines (AS2, PC9) and malignant pleural effusion (MPE) from patients with lung cancer. Isolated EVs were confirmed by the morphology and size in transmission electron microscopy (TEM) and the presentation of specific EV markers (Alix, CD63, and Tsg101). We found that several RNA species with enrichment of small RNAs and mutated EGFR DNA in the EVs of PC9 cells. After labeling with the green fluorescent dye Cellvue, the uptake of EVs by AS2 cells was imaged by the fluorescent microscopy. With these data, we further investigated the role of EVs in regulating the tumor microenvironment. We found the content of IL-6 in EVs, secreted from AS2 and CL1-5 cells, increased gradually after changing culture medium. And after uptake of EVs by recipient cells the cargos induced higher phsophorylation of Stat3 and AKT compared to the treatment with serum-free medium. Previously, we have shown that IL-6/Stat3/tissue factor (TF)/VEGF pathway enhances lung cancer angiogenesis, metastasis and the generation of MPE. In the current study, we demonstrated that EVs from lung cancer cells and in the MPE contain high level of VEGF and TF. And treatment with the EVs, containing high VEGF and TF, induced more prominent extravasation than cell free medium did in Miles assays. In conclusion, we successfully isolated EVs not only from culture supernatants but also from MPE of lung cancer patients. The EVs contain many biologically active molecules that may modulate behaviors of cancer cells and cells in tumor microenvironment.

#3393

miR-34b: A regulator of VEGF-A, Notch1 and Bcl-2 in thyroid carcinoma.

Hamidreza Maroof, Ali Salajegheh, Alfred KY Lam. _Griffith University, Gold Coast, QLD, Australia_.

Background: Thyroid cancer is the most common endocrine malignancy accounting for >91% of endocrine malignancy and 1.8% of all recently distinguished cancer reports, with an incidence continuing to rise globally in recent decades and current treatment strategies are not potent enough for patients. Therefore, novel and more effective treatment are outstandingly required. New strategy for treatment is target therapy, which not only hold cancer-specific expression but also limits side effects. miR-34b as part of p53 tumor suppressor network, plays crucial role in many physiological and pathological processes including cancer initiation, tumor progression and cancer angiogenesis process.

Objectives: In this study, we evaluate the functional roles of miR-34b in 2 thyroid cancer cell lines for its potential in modulating angiogenesis and proliferation in thyroid malignancies.

Methods: Expression levels of miR-34b was determined in metastasizing human papillary thyroid carcinoma (B-CPAP) and human undifferentiated thyroid carcinomas (MB-1) cell lines by qPCR. Exogenous miR-34b was transfected to thyroid cancer cell lines to investigate its effect on predominant genes involved in angiogenesis, cell cycle and apoptosis regulation including VEGF-A and Bcl-2 and Notch1. Confocal laser scanning microscopy (CLSM) and western blot techniques were performed to illustrate the protein expression changes. To demonstrate the perturbation of cell cycle and apoptosis pathways through exogenous miR-34b, fluorescence-activated cell sorting (FACS) was performed.

Results: Significant underexpression of miR-34b was established in B-CPAP and MB-1 cell lines while outstanding overexpression of VEGF-A, Bcl-2 and Notch1 were detected. After transfection with miR-34b, significant drop in concentration of VEGF-A, Bcl-2 and Notch1 were noticed compared with controls in CLSM and western blot analysis (p< 0.05).

Cell cycle analysis demonstrated that 48 hours after ectopic induction of miR-34b, significant impairment in proliferation of B-CPAP and MB-1 was induced through arresting cancer cell proliferation in G0-G1 phase (14.27% ±3.50 for B-CPAP and 13.61% ±0.16 for MB-1) (p<0.05). Apoptosis assay also revealed that miR-34b, induced cell death by increasing early and late apoptosis events, compared with controls (2.20% ±0.16 for B-CPAP and 7.87% ±0.92 for MB-1) (p<0.05) .

Conclusion: These results pave the way that miR-34b may be involved in balancing tumour angiogenesis, cell proliferation and apoptosis in thyroid cancer. These could potentially occur via direct modulation of downstream targets such as VEGF-A, Bcl-2 and Notch1 for their role in those events. The future of this research will investigate the regulatory role of miR-34b in Papillary and undifferentiated thyroid carcinoma through larger scale in vivo and clinical trial studies which shall potentially improve the clinical presentation of aggressive malignancies.

#3394

Bevacizumab enhances the antitumor effect of BRAFV600E inhibition in colorectal and melanoma xenograft models.

Valentina Comunanza, Davide Corà, Emanuele Middonti, Federica Di Nicolantonio, Enzo Medico, Dario Sangiolo, Federico Bussolino. _Dipartimento di Oncologia (Università di Torino) - Istituto di Candiolo IRCCs, Candiolo (TO), Italy_.

BRAF inhibitors (BRAFi) have shown improved response rate and overall survival (OS) compared to standard chemotherapy in metastatic melanoma. Combinations of BRAF and MEK targeted agents further improve progression-free survival (PFS) and OS when compared with single-agent BRAFi, but the development of resistance remains a major obstacle to long-term disease control. Thus, additional strategies must be exploited to increase the efficacy of BRAF-MEK blockade in metastatic melanoma and circumvent or delay the onset of resistance.

We have previously demonstrated in murine xenograft models of BRAF mutant melanomas and colorectal cancers (CRC) that the vemurafenib analog, PLX4720, has cytostatic activity and induces vascular normalization by changing the pro-angiogenic program triggered by the oncogenic mutation (Bottos A et al., 2012;109(6):E353-9). We hypothesized that BRAF-targeted inhibitors could cooperate with anti-angiogenic regimens in the treatment of BRAF-mutant melanoma and colorectal tumors.

Here, we demonstrated that when associated with the VEGFA neutralizing antibody bevacizumab (COMBO), PLX4720 shows enhanced anti-cancer effects and induces a de novo transcriptional signature in BRAF mutant melanoma and colorectal xenografts. Interestingly, in the COMBO, bevacizumab does not alter the improvement in blood perfusion and tumor vessel normalization observed with PLX4720 alone. Although targeted inhibition of either BRAF or VEGFA initially suppresses the tumor growth, only combined inhibition of both pathways results in an evident apoptotic effect. Furthermore, bevacizumab delays the onset of resistance to PLX4720 by recruiting macrophages with a M1-like phenotype and inducing a remodeling of the extracellular matrix. The latter is characterized by a reduction in collagen deposition and of cancer-associated fibroblasts (CAFs), known to sustain cancer progression. The effect of bevacizumab on acquired resistance to PLX4720 is transient and depends on the presence of M1 macrophages. Indeed, 3/6 xenografts treated with COMBO start re-growing after 6 weeks with a dramatic reduction of infiltrating macrophages and an increase of CAFs.

Collectively, our findings offer a new perspective for the management of clinical resistance to BRAF inhibitors and offer a rationale to combine BRAFV600E inhibitors with anti-angiogenic regimens.

#3395

EphrinB2 controls vessel pruning through STAT1-JNK3 signaling.

Ombretta Salvucci,1 Hidetaka Ohnuki,1 Dragan Maric,2 Acker-Palmer Amparo,3 Giovanna Tosato1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD;_ 3 _Institute of Cell Biology and Neuroscience and BMLS, Goethe University Frankfurt, Frankfurt am Main, Germany_.

Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Using both in vitro and in vivo models of vascular development, we found that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. On ocular hyaloid vessels in phospho-EphrinB2-deficient mice (5Y/5Y mice), this regulation depends upon phosphotyrosine-EphrinB2 signaling repressing c-jun N-terminal kinase 3 activity via SHP2/JAK2/STAT1. Functional experiments show that JNK3 activation causes endothelial cell death. Physiological pruning of hyaloid vessel in JNK3 knock-out mice is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. We have thus identified a new pathway where EphrinB2/JNK3 signaling emerges as a key regulator of endothelial cell survival and post-angiogenic vessel remodeling. Furthermore we explored the role of this novel pathway in tumor angiogenesis. In the B16/F10 melanoma mouse tumor model, EphrinB is strongly phosphorylated in the tumor vasculature. We silenced tumor-derived EphB4 and found it to be a critical inducer of this phosphorylation. Surprisingly, tumor growth was increased when EphrinB2 phosphorylation was reduced in the tumor vasculature. All these results together establish a role for phosphotyrosine-dependent EphrinB2 signaling in the vascular system, suggesting that EphrinB2 is a potential therapeutic target for modulation of physiologic and pathologic angiogenesis.

#3396

Purinergic regulation of tumor-mediated angiogenesis: A role for exosomal NM23.

Senny Nordmeier, Suzann Duan, Iain L.O. Buxton. _University of Nevada, Reno, Reno, NV_.

Exogenous nucleoside diphosphate kinase (eNDPK or NM23) has been shown to promote endothelial cell proliferation and migration and tumor-mediated angiogenesis. This is facilitated by its transphosphorylase activity, in which a gamma terminal phosphate group from a triphosphate nucleoside is transferred to a diphosphate nucleoside, resulting in elevated ATP levels. ATP can active purinergic receptors (P2Y1) on adjacent endothelial cells to promote angiogenesis. We previously reported that eNDPK was detected in conditioned media of breast cancer cells and in the serum of breast cancer patients (Yokdang, et al., 2011; Yokdang, et al., 2015). This suggests that eNDPK may have a role in tumorigenesis, but the mechanism by which eNDPK is released into the extracellular environment remains unknown. It is known that all cell types secrete small vesicles called exosomes, 30-100 nm wide vesicles of endocytic origin. We hypothesize that exosomal eNDPK secreted by breast cancer cells targets and promotes endothelial cell tubulogenesis and direct inhibition of eNDPK and P2Y1 activity attenuates angiogenesis. Exosomes were purified from human breast cancer cells, MDA-MB-231 (231) and non-tumorigenic human mammary epithelial cells (HME1) using ExoQuick TC. Transmission electron microscopy, mass spectrometry, and flow cytometry were performed to characterize the exosomes. Purified exosomes labeled with GFP-tagged tetraspanin (CD63) were introduced to human umbilical vein endothelial cells (HUVEC) to detect and measure tubulogenesis formation. We show that 231 secreted exosomes contain eNDPK and HUVEC cells treated with NDPK in the presence of ADP and GTP form tubules. In addition, fluorescently-labeled exosomes are associated with HUVEC tubules. The role of exosomal NDPK released by cancer cells in promoting angiogenesis and tumorigenesis may be used as a potential biomarker and target for treatment in breast cancer management. For future investigations of exosomal eNDPK, this study suggests that breast cancer exosomes promote endothelial tubulogenesis in a P2Y receptor-dependent manner.

#3397

Intrinsic factors from patient's tumor help neovascularization in an ex vivo platform.

Baraneedharan Ulaganathan,1 Allen Thayakumar B,1 Manoj Rajappa,2 Ayyappan Velu,2 Arun Prasath,2 Kamal Ameer,2 Nilesh Brijwani,2 Padhma Radhakrishnan,3 Biswanath Majumder,2 Pradip K. Majumder,3 Saravanan Thiyagarajan2. 1 _Mitra Biotech Inc, Zion, IL;_ 2 _Mitra Biotech Private Limited, Bangalore, India;_ 3 _Mitra Biotech, Boston, MA_.

Neovascularization is considered as a major event during embryonic development, found to be deregulated in several pathological conditions including malignancies. Factors secreted by tumor cells trigger a cascade of events which in turn play a critical role in the initiation and sprouting of tumor-specific-sustained-vasculature subsequently led to dramatic expansion of primary tumors, invasion and metastasis. Increasing evidence suggested that modulation of tumor cell specific intrinsic mechanisms which are accountable for tumor vasculogenesis would have therapeutic benefits to the patients even at later stages of disease. Therefore screening of such potential anti-angiogenic agents necessitates engineering a system where tumor vasculogenic environment is well preserved at micro-architecture level close to that of patient's native settings. Such a platform developed by Mitra Biotech, CANScriptTM demonstrated a high level of correlation to clinical outcome for many chemotherapeutics as well as targeted drugs in multiple solid and hematological cancers. Our CANScriptTM platform was being effectively validated for the retention of vascular phenotypes along with their immune and stromal counterparts, and their associated signaling networks. When patient derived tumor specimens were cultured in a two chambers microplates with human umbilical vein endothelial cells' (HUVECs) in the presence of autologous ligands and customized matrix support, the native mediators released from tumor cells instigated complete formation of mature sprouts within 24 hours compared to HUVEC cells alone. In this platform we also assessed the anti-tumor efficacy of a known anti-angiogenic agents. The response to VEGF inhibitor was in turn measured by functional parameters like tumor morphology, anti-proliferative and apoptotic effects in combination with anti-angiogenic activity for the human tumors enrolled in the study. Treatment with VEGF inhibitor alone resulted in the abrogation of angiogenic crosstalk and transmission of signals from tumor cells to vessels. In addition to capturing anti-tumor efficacy of drugs using this platform technology, we were also able to dissect and better understand intervening strategies targeting tumor angiogenesis and vasculogenesis mechanisms under ex vivo conditions recreating in vivo like milieu.

#3398

MCPIP1 affects growth, angiogenesis and metastasis of clear cell renal cell carcinoma.

Katarzyna Miekus, Paulina Marona, Jolanta Jura. _Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Cracow, Poland_.

Introduction

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinomas. During diagnosis, metastasis is found in over 30% of patients. Despite conducted targeted therapy, survival time of patients with metastatic renal cell carcinoma ranges from three months to two years. Recent reports indicate an important role of inflammatory and angiogenic processes in the ccRCC development. Monocyte Chemotactic Protein-1 Induced Protein (MCPIP-1) is a novel multifunctional modulator involved in the regulation of inflammatory response, apoptosis and angiogenesis. However its role in tumorigenesis is unknown.

Aim

The main objective of our study was to determine the role of anti-inflammatory protein MCPIP1 in growth, angiogenesis and metastasis of clear cell renal cell carcinoma.

Materials and methods

ccRCC cell lines (Caki-1 and Caki-2) were transduced with MCPIP1 shRNA and control shRNA lentiviral vectors and selected with puromycin. The level of genes and protein involved in proliferation, angiogenesis and metastasis were studied by real-time PCR, western blot and flow cytometry. Chemotaxis and invasion assay were performed to check migration and invasion. To study the level of proangiogenic factors ELISA and LUMINEX assays were used. NOD-SCID mice were used in in vivo experiments.

Results

Our study shows that MCPIP1 downregulation increases proliferation rate, survival and migration properties of ccRCC cells. Additionally reduction of MCPIP1 protein level increases the expression of pro-angiogenic factors such as SDF1, VEGF and IL8 what was paralleled by an increased migration of endothelial cells toward conditioned media from MCPIP1 deficient ccRCC cells. We also demonstrate, that silencing of MCPIP1 protein in ccRCC leads to an increase in tumor growth in vivo, after subcutaneous injection of NOD-SCID mice. We observed a significant increase in the number and the area of blood vessels in tumors formed by cells with downregulation of MCPIP1 and greater ability to metastasize to the lungs.

Conclusions

We showed that reduction of MCPIP1 protein level in ccRCC cells is crucial for tumor growth, blood vessel formation in the emerging tumor and metastasis. Moreover, ccRCC cells with downregulation of MCPIP1 secreted factors and proteins essential for the process of angiogenesis. Obtained results may contribute to increased understanding of the biology of clear cell renal cell carcinoma, which in the future may help in identifying new, more effective therapeutic purposes or improving existing ones.

Acknowledgement

This study was supported by research grant from the National Science Centre to KM 2013/09/D/NZ/00249 and grant from the Jagiellonian University BMN 7/2015. Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education

#3399

CTGF promotes tumor angiogenesis process in osteosarcoma through induction of angiopoietin-2.

Yu-che Cheng, Hsiao-Chi Tsai, Tzu-Wei Tan, Chih-Hsin Tang. _China Medical University, Taichung, Taiwan_.

Osteosarcoma is the most common primary malignant bone tumor. Angiogenesis is essential for the tumor growth. Angiopoietin-2 (Angpt2) plays a critical role in angiogenesis and tumor progression. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the angiogenesis of certain human cancer cells. However, the effect of CTGF on Angpt2 expression in human osteosarcoma cells is mostly unknown. In this study, we found that overexpression of CTGF in osteosarcoma promoted angiogenesis in vitro and in vivo by tube formation assay, and Matrigel plug assay. Knockdown of CTGF obviously reduced expression of Angpt2 and angiogenesis. Recently, microRNAs (miRNAs) have been demonstrated that functions as oncogenes or angiogenesis regulators in human cancer. Here, we also found that overexpression of CTGF significantly decreased miR-543 in osteosarcoma cells. Transfection of miR-543 mimic reduced Angpt2 expression and angiogenesis. Taken together, CTGF promotes angiogenesis via up-regulation of Angpt2 by decreasing miR-543 in human osteosarcoma. These findings certify that CTGF functions as a tumor enhancer gene, suggesting that CTGF may be a potential therapeutic target for osteosarcoma patients.

#3400

An angiogenesis gene signature points to active TGF-beta/JAK signaling pathways in a subset of human pancreatic ductal adenocarcinoma cancer patients that are distinct from pathways in pancreatic neuroendocrine tumors.

Kelly E. Craven, Jesse Gore, Julie L. Wilson, Murray Korc. _Indiana University School of Medicine, Indianapolis, IN_.

Pancreatic Ductal Adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival of 7%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density, and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting.

By analyzing the recently expanded TCGA (The Cancer Genome Atlas) dataset, we report here that an angiogenesis gene signature is present in ~35% of PDACs and is mostly distinct from an angiogenesis signature present in PNETs. Additionally, principal component analysis (PCA) of the entire or angiogenic PDAC and PNET transcriptomes from TCGA indicates that there are large differences in gene expression between these two tumor types. For example, PDACs exhibit a transcriptome that reflects active TGF-β signaling, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Functionally, targeting the TGF-β type I receptor (TβRI) kinase with SB505124 and JAK1-2 with ruxolitinib blocks proliferative cross-talk between human pancreatic cancer cells and human endothelial cells. Tumors from the KRC (oncogenic Kras, deleted Rb1) PDAC mouse model show superior enrichment and differential expression of the angiogenic gene signature compared to tumors from the KPC (oncogenic Kras, mutated Trp53) PDAC mouse model. Moreover, treatment of KRC and KPC mice with ruxolitinib suppresses murine PDAC progression in KRC mice but not in KPC mice. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an angiogenesis gene signature should be explored in the clinic and that this could lead to improved responses to anti-angiogenic therapy in PDAC.

#3401

Impact of VEGF and VEGFR polymorphisms on neuroendocrine tumors of the gastro-entero-pancreatic system (GEPNETs) outcome.

Rossana Berardi,1 Mariangela Torniai,1 Silvia Pagliaretta,1 Silvia Rinaldi,1 Francesca Morgese,1 Stefano Partelli,2 Miriam Caramanti,1 Azzurra Onofri,1 Vanessa Polenta,1 Sonia Pasquini,3 Massimo Falconi,2 Stefano Cascinu4. 1 _Università Politecnica delle Marche, Ancona, Italy;_ 2 _Ospedale San Raffaele, Milano, Italy;_ 3 _Università di Bologna, Bologna, Italy;_ 4 _Università di Modena, Modena, Italy_.

Introduction: Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are uncommon neoplasms including a wide range of anatomical, clinical, histological and molecular malignant entities. Improving our understanding of the molecular biology of GEP-NETs represents a key challenge in the treatment of these rare tumors. Therefore we aimed to analyze genotypes of VEGF-A, VEGFR2 and Flt4 in GEP-NETs and their potential correlation with the risk of these tumors and/or with the outcome.

Methods: The genomic DNA of 28 consecutive patients with GEP-NETs treated at our Institution was extracted from paraffinembedded tissue. We selected polymorphisms in the following genes: VEGF-A (rs2010963G>C, rs699947A>C), VEGFR-2 (rs2305948C>T, rs1870377T>A) and VEGFR-3 (rs307826T>C, rs307821C>A). Gene polymorphisms were determined by Real-Time PCR using TaqMan assays.

Results: Median age was 58 (range 33-78) and M/F ratio was 1/1. Twenty-one out of the 28 patients had NETs of the pancreas. The allele frequency of VEGFR2 rs2305948C and of VEGFA rs2010963G showed a trend of lower frequency than in general population (82% vs. 92% and 60.3% vs. 68.8%, respectively, p<0.001). The following factors significantly correlated with a poorer overall survival (p<0.05): VEGFA rs699947C, VEGFA rs2010963A, VEGFR-2 rs1870377T/A, VEGFR-2 rs2305948C/T, VEGFR-3 rs307821C and VEGFR-3 rs307826T.

Conclusion: Our results suggest, for the first time, that inherited abnormalities in VEGF pathway influence the risk and aggressiveness of GEP-NETs. Within March 2016 final data about all the enrolled 64 patients will be also available. These results should be validated prospectively in order to optimize the diagnosis and treatment of these patients.

#3402

Ephrin B2 and Dll-4 mediated co-dependence of tumor and endothelial progenitor cells in human hepatocellular carcinoma.

Rajshekhar A. Kore,1 Meenakshi Upreti,2 Azemat Jamshidi-Parsian,1 Rudd P.M. Dings,1 Issam Makhoul,1 Robert J. Griffin1. 1 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 2 _University of Kentucky, Lexington, KY_.

Hepatocellular carcinoma (HCC) is a highly vascularized tumor, which preferentially recruits new blood vessels from the hepatic artery to fuel its growth, through mechanisms not well understood. Here, we examined the intercellular cross-talk of HepG2 (human HCC) and endothelial progenitor cells (EPC) in transwell co-cultures to mimic initial tumor parenchyma and stroma interactions. We present evidence showing that, in culture, HepG2 cells secrete factors to induce an 'activated' EPC phenotype, namely, an increase in survival, growth, proliferation and migration rates. EPC cells showed a significant increase in growth (~40%) and survival (~80%) when co-cultured with HepG2 cells. Simultaneously, activated EPCs improved the viability of HepG2 cells. A major mediator of intercellular crosstalk are the secreted microvesicles called exosomes. Exosomes constitute a set of distinct nanovesicles (30-100 nm), endosomal in origin, constitutively secreted by many cells. They are involved in intercellular communication by transferring proteins and RNA from one cell to another. Intercellular crosstalk between these HepG2 cells and EPCs cells lead to qualitative and quantitative changes in the exosomal cargo secreted by the respective cells. The exosomal cargo constituting various proteins and genetic material have been demonstrated to be involved in processes which promote remodeling of extracellular matrix (ECM), biological adhesion and pathways modulating developmental processes and progression of inflammation, leading to cancer growth and development. In other work with glioma cell-derived exosomes has shown the presence of Ephrin A and B receptors which suggests a prominent role of secreted exosomes in stimulating and guiding tumor angiogenesis, a hallmark characteristic of glioblastoma multiforme (GBM). Additionally, our results with co-cultures of tumor and endothelial cells show that angiogenesis signaling in EPC appears to be induced by HepG2 cells via elevated endothelial Ephrin B2 and DLL-4 expression. Overall, our results demonstrate the co-dependence of HCC and EPC intercellular crosstalk, likely accomplished via secreted exosomes, in the initial stages of HCC establishment and stimulation of angiogenesis and development, thus a promising target for new clinical strategies.

#3403

Amphiregulin promotes vascular endothelial growth factor-dependent angiogenesis in human chondrosarcoma cells.

Yu-Wen Huang, Hsiao-Chi Tsai, Chih-Hsin Tang. _China Medical University, Taichung, Taiwan_.

Chondrosarcoma is the second most common sarcoma in bone malignancy and is characterized by a high metastatic potential. Angiogenesis has been considered an important role in the cancer metastasis. Therefore, a better understanding of angiogenic and metastatic pathways are needed. Amphiregulin (AR) has been implicated in tumor metastasis and angiogenesis. However, the relationship of amphiregulin with vascular endothelial growth factor A (VEGF-A) expression and angiogenesis in human chondrosarcoma cells is mostly unknown. In this study, we found that the expression of amphiregulin and VEGF-A were correlated with tumor stage and were significantly higher than that in the normal cartilage. VEGF-A expression increased which depend on time and dose dependent of amphiregulin. Exogenous amphiregulin with chondrosarcoma cells promoted VEGF-A expression and subsequently increased migration, proliferation and tube formation in endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs). We also found that amphiregulin increased VEGF-A expression and angiogenesis through focal adhesion kinase (FAK), Src and protein kinase C (PKC) signaling pathway. Knockdown of amphiregulin decreased VEGF-A expression in chondrosarcoma cells. The angiogenesis effects in Matrigel plug nude mice model in vivo was also abolished in amphiregulin-knockdown cells. Taken together, these results indicate that amphiregulin occurs through FAK, Src, and PKC pathway, resulting in the activation of VEGF-A expression and contributing to the angiogenesis of human chondrosarcoma cells.

## EPIDEMIOLOGY:

### Epidemiology of Cancer Prognosis and Survival

#3404

Thrombocytosis and epithelial ovarian cancer survival: relevant time frame of platelet count measurement and diagnostic threshold for thrombocytosis.

Gabriella D. Cozzi, Jacob M. Samuel, Jason T. Fromal, Spencer Keene, Marta A. Crispens, Dineo Khabele, Alicia C. Beeghly-Fadiel. _Vanderbilt University, Nashville, TN_.

INTRODUCTION: Ovarian cancer is rapidly progressive and lethal disease. Thrombocytosis has been associated with poor prognosis; mechanisms underlying this association include paracrine signaling between platelets and cancer cells, resulting in tumor cell proliferation. However, the relevant timing of platelet measurement in relation to diagnostic operation and a threshold to define thrombocytosis as a prognostic indicator have not been evaluated.

METHODS: Tumor Registry confirmed epithelial ovarian and fallopian tube cancer cases with pre-diagnosis platelet counts were identified from the Synthetic Derivative, a de-identified mirror of electronic medical records from the Vanderbilt University Medical Center. Thrombocytosis was defined with two thresholds: platelet count greater than 400 or 450 x 109/L. The relevant time frame of platelet measurement was evaluated within 5 time intervals: on the date of diagnosis, and 1, 2, 4, and 8 weeks before and including the date of diagnosis. Prognostic significance was evaluated using Cox proportional hazards regression to calculate hazard ratios (HR) and confidence intervals (CI); adjustment included age, stage, grade, and histologic subtype of disease.

RESULTS: Measured platelet values were available for 136, 241, 280, 297, and 304 cases within the five time intervals. The prevalence of thrombocytosis was 31.6, 39.4, 38.2, 38.1, and 37.5% for 400 x 109/L, and 19.9, 26.1, 25.7, 24.9, and 25.0% for 450 x 109/L across the five intervals, respectively. Thrombocytosis was significantly associated with higher stage and grade, regardless of threshold used. In both unadjusted and multivariable adjusted analysis, thrombocytosis was significantly associated with worse survival. Associations were similar across thresholds and time frames, but were generally larger on the date of diagnosis (HR(400): 1.96, 95% CI 1.23-3.13; HR(450): 1.90, 95% CI: 1.12-3.23), and smaller as time to diagnosis increased up to 8 weeks (HR(400): 1.54, 95% CI: 1.14-2.09; HR(450): 1.55, 95% CI: 1.11-2.15).

CONCLUSIONS: Depending upon the threshold used and the timing of measurement, thrombocytosis was identified in 20-40% of epithelial ovarian cancer cases. Regardless of threshold or timing, preoperative thrombocytosis was associated with more aggressive disease characteristics and was an independent negative prognostic factor. These preliminary results support use of a lower thrombocytosis threshold and indicate that platelet count measurements collected up to 8 week preoperatively may be used to inform ovarian cancer prognosis.

#3405

The analysis of TRPV6 expression in lymphoid tumours.

Ashley M. DiPasquale,1 Tarek Rahmeh,2 Lauren Andrew,1 Jane Agar,2 Kim Miller,2 Matthew Finniss,1 Alli Murugesan,3 Anthony Reiman1. 1 _Dalhousie University, Saint John, New Brunswick, Canada;_ 2 _Saint John Regional Hospital, Saint John, New Brunswick, Canada;_ 3 _University of New Brunswick, Saint John, New Brunswick, Canada_.

Introduction:

TRPV6 is a calcium channel that is found overexpressed in various malignancies and is correlated with the prognosis of patients with prostate cancer. TRPV6 targeted anticancer therapies are in development. We are interested in the potential of TRPV6 as a potential biomarker and therapeutic target in lymphoma. This retrospective study examines the expression levels of TRPV6 in various lymphoid tumour types, and correlates expression levels with grade, prognostic scores, and survival rates.

Methods:

A clinical-pathological database was constructed using the health records and lymphoid tumor samples of patients diagnosed with diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma (FL), small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL), and Hodgkin's lymphoma (HL). Immunohistochemical studies were performed on lymphoid tumor samples to analyze the level of TRPV6 expression. Tumor samples were graded on a 3-point scale. A score of +3 indicated dense staining with high expression levels, and a score of +1 indicated minimal staining with low expression levels. TRPV6 expression levels were correlated with prognostic scores (IPI, FLIPI) and survival rates using Chi-squared, Mantel-Cox, and Kaplan-Meier survival curves. Descriptive statistics were used to describe patient demographics.

Results:

We found high TRPV6 expression levels in DLBCL and HL tumor samples with > 40% of samples scoring +3. We found low TRPV6 expression levels in FL and SLL/CLL with > 40% of samples scoring +1. We found no significant correlation between the level of TRPV6 expression and prognostic scores or survival rates in any of the lymphoma subtypes studied. In follicular lymphoma tumor samples, it was noted on observation that the large tumor cells generally stained +3 while the small tumor cells generally stained +1.

Conclusion:

This retrospective study showed DLBCL and HL tumors have high levels of TRPV6 expression. It also showed that TRPV6 is found highly expressed in the large transformed cells (centroblasts) in follicular lymphoma. These results demonstrate a need to further assess the role of TRPV6 in the aggressiveness of follicular lymphoma, as well as the prognosis and survival in DLBCL and HL with a larger sample size. Further studies of the potential of TRPV6 as a therapeutic target in lymphoma will focus on DLBCL, high grade/transformed FL, and HL.

#3406

The identification of serum cytokine inflammatory markers as classifiers of lung cancer mortality for stage I lung adenocarcinoma: a retrospective cohort study.

Claire L. Meaney,1 Adriana Zingone,1 Derek Brown,1 Yunkai Yu,2 Liang Cao,2 Brid M. Ryan1. 1 _Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD;_ 2 _Molecular Targets Core, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD_.

Lung cancer is the leading cause of cancer-related mortality worldwide. Low-dose CT (LDCT) imaging is now recommended to screen high-risk lung cancer individuals in the USA. The sensitivity of LDCT has resulted in increased detection of stage I lung cancer and a 20% reduction in lung cancer mortality. The current standard of care for stage 1 lung cancer patients is surgery alone. However, between 20% and 30% of these patients will develop recurrence and therefore are in need of further treatment upon diagnosis.

This study aims to explore and validate biomarkers to identify patients at high-risk of mortality so that additional treatment modalities can be offered at time of diagnosis. Our recent work on a small panel of circulating cytokines identified elevated levels of IL-6, a pro-inflammatory cytokine, as an indicator of poor survival. To further examine the potential of inflammatory biomarkers as prognostic indicators, 125 stage I lung adenocarcinoma cases were selected from the National Cancer Institute-Maryland lung cancer case-control study. This is an on-going prospective study of non-small cell lung cancer based in the greater Baltimore region of the USA.

A panel of 33 inflammatory markers was measured for each case using the Mesoscale V-Plex assay. The magnitude of association between serum inflammatory marker expression levels and lung cancer-specific survival was tested using multivariable Cox proportional hazards regression modeling (Stata 12.0 statistical software). All calculations were adjusted for age, gender, stage and smoking status. Maximum follow up time was 15 years.

Five analytes were significantly associated with shorter survival. In concordance with the previous study, IL-6 was again associated with shorter survival (HR, 2.63; 95% CI, 1.25-5.56). Other cytokines associated with shorter survival included CRP (HR, 2.25; 95% CI, 1.09-4.65), Eotaxin-3 (HR, 2.20; 95% CI, 0.97-4.86), IL-12p40 (HR 1.98; 95% C.I. 1.00 - 3.91), and IL-17 (HR, 2.22; 95% CI, 1.08-4.58). Although IL6 and CRP were positively correlated (Rho=0.451, P<0.0001), the association between IL-6 and survival was independent of CRP (HR, 2.46; 95% CI, 1.19-5.09).

These results support the potential of cytokine markers as a prognostic tool to further classify stage I lung cancer and thus identify patients in need of additional treatment. The associations identified here justify further investigation of a novel, combined cytokine prognostic classifier to serve as a robust predictor of stage I lung adenocarcinoma survival.

#3407

Gene expression subtypes of high grade serous ovarian cancer in African American women.

Jennifer A. Doherty,1 Casey S. Greene,2 James E. Rudd,3 Laura J. Tafe,3 Anthony J. Alberg,4 Elisa V. Bandera,5 Jill Barnholtz-Sloan,6 Melissa Bondy,7 Michele L. Cote,8 Ellen Funkhouser,9 Patricia G. Moorman,10 Edward S. Peters,11 Ann G. Schwartz,8 Paul Terry,12 Rex Bentley,13 Andrew Berchuck,14 Jeffrey R. Marks,15 Joellen M. Schildkraut16. 1 _Department of Epidemiology, The Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 2 _Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA;_ 3 _The Geisel School of Medicine at Dartmouth, Lebanon, NH;_ 4 _Hollings Cancer Center and Department of Public Health Sciences, Medical University of South Carolina, Charleston, SC;_ 5 _Department of Population Science, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 6 _Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH;_ 7 _Cancer Prevention and Population Sciences Program, Baylor College of Medicine, Houston, TX;_ 8 _Department of Oncology and the Karmanos Cancer Institute Population Studies and Disparities Research Program, Detroit, MI;_ 9 _Division of Preventive Medicine, University of Alabama at Birmingham, Birmingham, AL;_ 10 _Duke Cancer Institute, Duke University Medical Center, Durham, NC;_ 11 _Epidemiology Program, Louisiana State University Health Sciences Center School of Public Health, New Orleans, LA;_ 12 _Department of Medicine, University of Tennessee Medical Center-Knoxville, Knoxville, TN;_ 13 _Department of Pathology, Duke University, Durham, NC;_ 14 _Department of Obstetrics and Gynecology, Duke University, Durham, NC;_ 15 _Department of Surgery, Duke University, Durham, NC;_ 16 _Department of Public Health Sciences, University of Virginia, Charlottesville, VA_.

Ovarian cancer accounts for 5% of cancer deaths and is the fifth leading cause of cancer death in women in the United States. While incidence is higher in European American (EA) than African American (AA) women, five-year survival is worse for AA women (36%) than EA women (44%). Access to appropriate surgery and treatment is a major contributor but does not completely explain this disparity. The Cancer Genome Atlas (TCGA) identified four gene expression-based subtypes of the most common and lethal histotype, high grade serous carcinoma (HGSC): mesenchymal, proliferative, differentiated, and immunoreactive. We sought to characterize similarities and differences in gene expression-based subtypes arising in AA and EA women to determine whether there are underlying biologic features that may influence survival. We performed two distinct analyses, first using TCGA data and second using cases from the population-based African American Cancer Epidemiology Study (AACES). For both we summarized differential expression patterns for each subtype with moderated t statistic vectors for >10,000 genes using Significance Analysis of Microarrays. We calculated Pearson's correlations of these vectors to determine concordance of expression patterns between subtypes across EA and AA women. In TCGA, we observed correlations of subtype-specific expression patterns between the 24 AA and 475 EA tumors of 0.52-0.60 for each of the four subtypes. Thus, while analogous subtypes can be identified in AA and EA women, the magnitude of these correlations suggests that there are potential differences in gene expression patterns between AA and EA tumors that are assigned to the same subtype. We generated additional data from 58 AACES HGSC cases using the Affymetrix Human Transcriptome Array 2.0. Instead of assigning these tumors to previously-defined subtypes, we clustered samples to identify four subtypes de novo. We observed concordance with two of the TCGA subtypes; correlations for the mesenchymal-like and proliferative-like subtypes were 0.56-0.65. The mesenchymal-like subtype was more common in these AA women than in the TCGA EA women (33% versus 25%), and the proliferative-like subtype was marginally less common (14% versus 19%). Concordance for the differentiated-like subtype was considerably lower, at 0.21, and this subtype was less common in AA than EA women (19% versus 34%). Another subtype comprising 34% of the AA samples was only weakly correlated (-0.21-0.10) with any of the TCGA subtypes, suggesting that it is a novel subtype. The limited data available on HGSC in AA women suggest that at least two subtypes are comparable to those in EA women but differ in prevalence, and that there may be a novel subtype in AA women that does not strongly correspond to those described in EA women.

#3408

Oncogenic SOX2 signaling in bladder cancer.

Chia-Chang Wu,1 Yu-Fan Chiu,2 Yu-Ting Chou,2 Yuan-Hung Wang3. 1 _Department of Urology, Taipei Medical University-Shuang Ho Hospital, New Taipei City, Taiwan;_ 2 _Department of Life Science and Institute of Biotechnology, National Tsing Hua University, HsinChu City, Taiwan;_ 3 _Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical Uni, New Taipei City, Taiwan_.

The intriguing phenomenon that cancer cells have biological features similar to stem cells suggests that cancer and stem cells may share the same regulatory signaling pathway. We found that SOX2, a master transcriptional factor controlling self-renewal of stem cells, was highly expressed in a subgroup of transitional cell carcinoma of bladder cancer, correlating with the advanced pathological grade. Moreover, SOX2 expression was associated with poor overall survival and recurrence-free survival outcomes in patients with bladder cancer. Knockdown of SOX2 in SOX2-high 5637 bladder cancer cells attenuated cell growth. Consistently, ectopic expression of SOX2 in SOX2-low T24 bladder cancer cells endowed cells with increased cell proliferation. Spheroid assay showed that SOX2 expression encouraged spheroid-forming ability of bladder cancer cells in low serum condition, demonstrating the involvement of SOX2 signaling in the maintenance of stemness in bladder cancer cells. Immunoblotting revealed that the expression of SOX2 induced phosphorylation of AKT in the serum-free condition, supporting that SOX2 regulates bladder cancer cell survival. Gene expression microarray analysis showed that SOX2 expression induced IGF2, which was further confirmed by Q-PCR analysis in bladder cancer cells. Chi-square test assay showed that IGF2 expression correlated with the advanced tumor stages in bladder cancer. Pharmacological inhibition of IGF2 signaling attenuated cell growth in SOX2-postive bladder cancer cells. Our findings support the notion that SOX2-IGF2 signaling axis confers aggressiveness in bladder cancer cells with potentials as biomarkers and therapeutic targets for bladder cancer intervention.

#3409

Nonsynonymous functional variants in DNA repair genes in sporadic colorectal cancer: searching for predictive and prognostic markers.

Katerina Jiraskova,1 Jana Slyskova,2 Fabio Rosa,3 Cornelia Di Gaetano,3 Barbara Pardini,3 Veronika Vymetalkova,2 Pavel Vodicka2. 1 _Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles University, Prague, Czech Republic;_ 2 _Institute of Experimental Medicine AS CR, v. v. i., Prague, Czech Republic;_ 3 _Human Genetics Foundation, Turin, Italy_.

Colorectal cancer (CRC) has one of the highest mortality due to the late diagnosis and a lack of proper predictive and prognostic markers. According to the current knowledge, DNA repair processes are involved both in the onset of CRC and in the treatment efficacy.

In the present study, we analyzed the link between functional genetic polymorphisms (SNPs) in DNA repair genes covering the main DNA repair pathways (relevant for therapy response) in relation with the risk of CRC and clinical outcomes. Our set of candidate polymorphisms was selected according to different functional and genomic databases (FSNP, GAD, Linkage Disequilibrium, Gerp++, SiPhy). FSNP database provides integrated information about the functional effects of SNPs which are predicted and indicated at several levels (protein coding, splicing regulation, transcriptional regulation, post translation). We have focused on those affecting protein coding. We hypothesize that these modified proteins modulate the function/efficiency of DNA repair, and thus may have an effect on CRC.

Sixteen polymorphisms in twelve DNA repair genes (C19orf40, EME1, FANCI, MUS81, NEIL3, POLE, POLN, POLQ, RAD51D, REV1, REV3L, RPA1) were analyzed in DNA samples of 1080 cases and 1442 controls from the Czech Republic. Clinical data at diagnosis and complete information on follow up were provided for all patients.

Genetic variations in several DNA repair genes were associated with clinical outcome. In particular, CRC patients carrying the AG heterozygous genotype for rs5030755 in RPA1 gene displayed a longer survival and decreased recurrence risk (Overall Survival (OS): HR 0.74; 95% CI 0.56-0.98; p=0.04 and Event-Free Survival (EFS): HR 0.72; 95% CI 0.54-0.95; p=0.02). This association with better OS was more pronounced in individuals with colon and sigmoideum cancer (HR 0.58; 95% CI 0.4-0.83; p=0.0035).

Understanding the SNPs effect on the treatment response, resulting ultimately into low-cost and low-invasive markers, is regarded as very important for the possibility to tailor patient specific treatment strategy. Individualized therapy will eventually help to improve therapeutic efficacy and to minimize toxicities. The present results identified plausible candidate DNA repair gene variants potentially affecting clinical outcome in relation to CRC patient's survival.

Supported by grant GA UK 112515, GA CR 15-14789S

#3410

**Downregulation of** let 7a 5p **predicts lymph node metastasis and prognosis in colorectal cancer: implications for chemotherapy.**

Ya-Wen Cheng,1 Tsang-Pai Liu,1 Po-Li Wei,2 Huei Lee1. 1 _Taipei Medical Univ., Taipei, Taiwan;_ 2 _Taipei Medical University Hospital, Taipei, Taiwan_.

Background. Colorectal cancer guidelines recommend adjuvant chemotherapy is based on the number of lymph nodes metastasis. Let-7a-5p is a microRNA which could inhibits migration, invasion and epithelial-mesenchymal transition by targeting HMGA2. The aim of this study was to investigate the role of let-7a-5p in clinical impact of colorectal cancer (CRC).

Methods. One hundred ninety-two CRC patients were enrolled. The expression of let-7a-5p and HMGA2 in serums and tumor tissues were analyzed by real-time PCR and immunohistochemistry. Univariate (Kaplan-Meier) analysis was used to analyze primary outcomes included 5-year overall survival and tumor recurrence.

Results. The expression of let-7a-5p in tumor tissues was significantly negative correlated with tumor size, stage, and lymph node metastasis in CRC patients (p= 0.024 for tumor size, p=<0.0001 for stage, and p<0.0001 for lymph node metastasis). There was a negative correlation between levels of let-7a-5p and the HMGA2 gene (p <0.0001), especially in CRC patients with wildtype APC gene. Patients with let-7a-5p low / HMGA2 high had poorer overall survival (OS) and disease-free survival (DFS) rates than those with let-7a-5p high / HMGA2 high, let-7a-5p high / HMGA2 low, and let-7a-5p low /HMGA2 low groups. In addition, the expression levels of let-7a-5p in PBMCs were positive correlated with in tumor tissues of CRC patients.

Conclusion. We suggest that down-regulation of let-7a-5p in PBMCs and tumor tissues patients could be used to predicts lymph node metastasis and prognosis impact on chemotherapy of colorectal cancer

#3411

Role of mucin gene variations in microRNA binding sites in modulating colorectal cancer susceptibility and clinical outcomes.

Petra Bendova,1 Veronika Vymetalkova,1 Barbara Pardini,2 Fabio Rosa,2 Cornelia di Gaetano,2 Ludmila Vodickova,1 Tomas Buchler,3 Alessio Naccarati,2 Pavel Vodicka1. 1 _The Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic;_ 2 _Human Genetics Foundation, Turin, Italy;_ 3 _Department of Oncology, Thomayer Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic_.

Mucins, high molecular weight glycoproteins predominantly expressed at the epithelial surface of tissues, provide protection for colon surface under normal physiological conditions. Mucinous colorectal carcinoma is generally defined as having greater than 50% of the tumor area with a mucinous differentiation by histologic examination.

MiRNAs have recently emerged as important regulators for altered mucin expression during malignant development. MiRNAs are short non-coding RNAs that regulate gene expression by binding to the 3'untranslated regions (3'UTR) of target mRNA thereby hampering protein translation or inducing mRNA destabilization. Aberrant miRNA expression and/or function are frequently observed in colorectal cancer (CRC). Polymorphisms in miRNA binding sites may affect miRNA binding to target genes, resulting in differential mRNA and protein expression and susceptibility to common diseases. We hypothesize that variations in mucin genes may modulate signaling response affecting cancer susceptibility, cancer survival and efficacy of chemotherapy.

Thirteen polymorphisms in nine mucin genes (MUC6, MUC7, MUC13, MUC14, MUC15, MUC17, MUC20, MUC21 and MUC24) were analyzed in DNA samples of 1111 cases and 1469 controls from the Czech Republic. Investigated variants were also studied in association with clinical outcome in all patients provided with detailed information on follow up.

Genetic variations in mucin genes were associated with clinical outcome. In particular, CRC patients carrying the CC genotype for rs886403 in MUC21 gene displayed a shorter survival and higher recurrence risk (HR 1.69; 95% CI 1.13-2.46; p=0.01 and EFS: HR 1.99; 95% CI 1.38-2.84; p=0.0002, resp.). The observed association was strikingly pronounced in colon cancer patients while individuals with rectal cancer and carrying variant CC genotype of rs4729655 in MUC17 displayed better overall survival (HR 0.27; 95% CI 0.14-0.54; p=0.0002).

Expanding our knowledge on mucin involvement in CRC may help us better understand the etiopathogenesis of this disease and thereby contribute to the development of new treatment strategies. The present results identified plausible candidate SNPs potentially affecting miRNAs that target mucin genes in relation to CRC patients' survival.

Work supported by IGA MZ: NT 15-26535A

#3412

Prognostic significance of plasma ghrelin in patients with gastric cancer.

Saeed Soleyman-Jahi, Kazem Zendehdel, Afshin Abdirad, Amir Afraz Fallah, Sevil Ghasemi, Fatemeh Sadeghi. _Cancer Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran_.

Background: Different prognosis observed for patients of the same clinical stage in gastric cancer, emphasizes the fact that new biological prognostic factors are needed to complement clinical staging. We aimed to investigate the prognostic significance of plasma ghrelin in gastric cancer patients.

Methods: In this prospective study, we included 83 gastric cancer patients from Cancer institute of Tehran, Iran. All the patients were candidates for gastrectomy with or without preoperative neoadjuvant chemotherapy (PNC). The patients were followed for three years. Demographic, clinical and para clinical data were registered. Using an ELISA based assay plasma levels of total and active ghrelin were assessed prior to and one week after the operation. Univariate and multivariate COX analyses were used to investigate the independent predictors of patients overall survival.

Results: The mean (±SD) age of the patients was 60.3±13.6. Sixty-five (78.3%) patients were male. Majority of the patients had gastric adenocarcinoma (95.2%). Thirty-seven (44.6%) patients had TNM stage II or I. Poorly differentiated grade was detected in 29 (34.9%) of the patients. Forty-eight (57.8%) patients received PNC before the operation. Mean (±SD) survival was 529 ± 373 days. Mean level of the plasma total ghrelin was 269.8±268.7 and 108.7±91.2 (pg/ml) before and after the operation, respectively (P<0.001). Mean level of the plasma active ghrelin was 68.0 ± 63.8 and 44.0± 29.6 (pg/ml) before and after the operation, respectively (P<0.001). Patients in the upper half or highest quartile of postoperative total ghrelin had better survival compared to the corresponding subgroups (Log rank test P=0.02 and P<0.001, respectively). Multivariate COX model revealed that late TNM stage (HR = 2.93, 95% CI: 1.37-6.27), no PNC (HR = 2.18, 95% CI: 1.08-4.37), history of significant weight-loss (HR = 2.32, 95% CI: 1.04-5.00) and lower quartile (≤38 pg/ml) of postoperative plasma total ghrelin (HR = 3.74, 95% CI: 1.45-9.63) predicted poor survival of patients. The level of other ghrelins did not show independent prediction of survival.

Conclusion: Our findings showed that low level of plasma total ghrelin after the gastrectomy could independently predict poor survival in gastric cancer patients. This emphasizes both prognostic and therapeutic significance of this biomarker in gastric cancer.

#3413

Supplement use and chemotherapy-induced peripheral neuropathy in breast cancer patients treated on SWOG study S0221.

Gary R. Zirpoli,1 Susan E. McCann,1 Lara E. Sucheston-Campbell,1 Dawn L. Hershman,2 Gregory Ciupak,1 Warren Davis,1 Joseph M. Unger,3 Halle C.F. Moore,4 James A. Stewart,5 Claudine Isaacs,6 Timothy J. Hobday,7 Muhammad Salim,8 Robert B. Livingston,9 Gabriel N. Hortobagyi,10 Julie R. Gralow,11 Daniel F. Hayes,12 G. Thomas Budd,4 Kathy S. Albain,13 Christine B. Ambrosone1. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Columbia University, New York, NY;_ 3 _SWOG Statistical Center, Seattle, WA;_ 4 _Cleveland Clinic, Cleveland, OH;_ 5 _Baystate Medical Center, Springfield, MA;_ 6 _Lombardi Comprehensive Cancer Center, Washington, DC;_ 7 _Mayo Clinic, Rochester, MN;_ 8 _Allan Blair Cancer Centre, Regina, Saskatchewan, Canada;_ 9 _Arizona Cancer Center, Tucson, AZ;_ 10 _MD Anderson Cancer Center, Houston, TX;_ 11 _Seattle Cancer Care Alliance, Seattle, WA;_ 12 _University of Michigan, Ann Arbor, MI;_ 13 _Loyola University Chicago Cardinal Bernardin Cancer Center, Maywood, IL_.

Introduction. Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of paclitaxel treatment. There is conflicting data on the benefits and harms of supplement use for the treatment and prevention of CIPN. We examined dietary supplement use before diagnosis and during treatment in relation to CIPN among breast cancer patients registered to a clinical trial (SWOG 0221; ClinicalTrials.gov Identifier: NCT00070564) before diagnosis and during treatment in relation to CIPN.

Methods. At trial registration (baseline), breast cancer patients (n = 1,225) completed questionnaires to capture use before and at diagnosis of multivitamins and other dietary supplements. Of these patients, 1,068 completed a 6-month follow-up questionnaire to capture use during treatment. We examined the use of multivitamins, vitamins C, D, E, B6, and B12, folic acid, iron, calcium, glucosamine , and combined fish oil, eicosapentaenoic acid (EPA), omega-3, flaxseed and cod liver oil at these timepoints in relation to CIPN. CIPN was assessed via the National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) and the Functional Assessment of Cancer Therapy/Gynecologic Oncology Group Neurotoxicity (FACT/GOG-Ntx) subscale. Odds ratios and 95% confidence intervals were computed using unconditional logistic regression.

Results. Multivitamin use before diagnosis was associated with reduced symptoms of CIPN (CTCAE adjusted OR = 0.60, 95% CI = 0.42-0.87; FACT/GOG-Ntx adjusted OR = 0.78, 95% CI = 0.61-1.00). Although in a similar direction as use prior to diagnosis, multivitamin use during treatment was less strongly associated with CIPN (CTCAE adjusted OR = 0.73, 95% CI = 0.49-1.08; FACT/GOG-Ntx 0.77, 95% CI = 0.60-0.99). Other supplement use, either before diagnosis or during treatment, was not associated with CIPN.

Conclusions. Our results indicate that supplement use during treatment may not appreciably affect experience of CIPN symptoms. Future analyses of the relationships between supplement use and survival outcomes will better inform patient decision making.

Acknowledgments: supported by R01 CA116395 (CBA); S0221 supported, in part, by National Cancer Institute/Division of Cancer Prevention SWOG NCORP Research Base grant 5UG1CA189974-02; National Cancer Institute (NCI), National Clinical Trials Network (NCTN): CA180888, CA180819; and in part by Amgen, Inc.

#3414

Ethnic disparities in breast cancer survival in Sarawak, Malaysia.

Hyuna Sung,1 C.R. Beena Devi,2 Jennifer Guida,1 Tieng Swee Tang,2 William F. Anderson,1 Xiaohong R. Yang1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Sarawak General Hospital, Kuching, Sarawak, Malaysia_.

Introduction: There are three distinct South-East Asian ethnic groups in Sarawak, Malaysia (Chinese, Malay, and natives) whose breast cancer incidence rates and the distribution of tumor subtypes were reported to be significantly different. It remains unclear whether survival varies among breast cancer cases in these ethnic groups. We aimed to evaluate the relationship between survival and ethnicity when taking into account tumor characteristics.

Methods: We analyzed incident breast cancer cases who were enrolled and treated in Sarawak General Hospital, Malaysia, between 1992 and 2014. The association of ethnicity with survival (relapse-free survival and overall survival) was evaluated using multivariate Cox-proportional hazard model. Subgroup analysis was conducted for each tumor subtype defined by hormone receptor (HR+, ER+ or PR+; HR-, ER- and PR-) and HER2 status (HR+/HER2-, HR+/HER2+, HR-/HER2-, and HR-/HER2+).

Results: Among 2,758 eligible cases (median follow-up years, 2.90 years), there were 636 deceased and 423 relapsed cases. While there was no significant difference in disease recurrence, overall survival varied significantly by ethnicity. Compared with Chinese women, Malay women had a higher proportion of mortality (HR = 1.50; 95% CI = 1.14-1.96; P = 0.0037). This disparity remained significant only among cases with HR+ tumors (HR = 1.91; 95% CI = 1.17-3.11; P = 0.0091 among HR+/HER2-; HR =2.74; 95% CI = 1.30-5.77; P=0.0079 among HR+/HER2+). There was no difference in survival between Chinese and native women overall or stratified by subtype.

Conclusions: Our results showed that Malay women experienced worse survival compared to Chinese women particularly among cases with HR+ tumors. Future studies are needed to clarify the underlying factors for this association in order to develop intervention strategies to reduce disparity.

#3415

Identifying lifestyle and genetic factors to prevent recurrence of non-muscle invasive bladder cancer in a prospective cohort study at Kaiser Permanente (The Be-Well Study).

Marilyn L. Kwan,1 Lawrence H. Kushi,1 Virginia P. Quinn,2 Nirupa R. Ghai,2 Janise M. Roh,1 Tracy Becerra,2 Adriana Martinez,1 Kimberly L. Cannavale,2 Alexander S. Carruth,2 Valerie S. Lee,1 Isaac J. Ergas,1 Ronald K. Loo,2 David S. Aaronson,1 Yuesheng Zhang,3 Christine B. Ambrosone,3 Li Tang3. 1 _Kaiser Permanente Northern California, Oakland, CA;_ 2 _Kaiser Permanente Southern California, Pasadena, CA;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Bladder cancer is one of the top 10 incident cancers. Most cases (75%) are diagnosed as non-muscle invasive disease (NMID), yet NMID typically recurs (70%) and a subset (25%) progresses to muscle-invasive disease. The Be-Well Study is an NCI-funded collaborative, multi-center prospective cohort study, with NMID bladder cancer patients enrolled at Kaiser Permanente Northern (KPNC) and Southern California (KPSC) and bioassays performed at Roswell Park Cancer Institute (RPCI). The goal is to examine diet and lifestyle factors and prognosis, with an emphasis on cruciferous vegetable (CV) intake and their unique isothiocyanate (ITC) content, the modifying effect of polymorphisms of ITC-metabolizing genes, and interactions with treatment. Our prior work suggests that dietary ITCs may prevent disease recurrence and progression in NMID patients.

Methods: Newly-diagnosed patients with NMID (Ta, Tis, T1), who are English-speaking, KP members, and ≥21 years of age, are ascertained rapidly from electronic pathology reports and enrolled on average 2.6 months post-diagnosis. Baseline participation consists of a telephone interview including a food frequency questionnaire focused on CV intake, and providing blood and urine samples. Patients will be contacted for follow-up interviews and urine samples at 12 and 24 months. Smoking, medication use, occupational exposures, physical activity, quality of life, and urinary function are also queried. Biospecimens are processed and assayed at RPCI. Strong support for Be-Well by KP urologists will promote dissemination of study results in patient care and recommendations.

Results: Recruitment began in February 2015. To date, 222 patients have completed the baseline interview, representing 78% male and 22% female, and 81% White, 7% Black, 5% Hispanic, 3% Asian, and 4% Other. Urine specimens have been collected from 82% of consented patients. Blood specimens have been collected from 85% of KPNC patients, and collection at KPSC began in November 2015. The 12-month follow-up interview and outcome ascertainment for disease recurrence and progression are scheduled to begin in February 2016.

Conclusions: The Be-Well Study is poised to be the largest and most comprehensive study to answer critical questions related to prognosis, quality of life, and care in patients diagnosed with early-stage bladder cancer.

#3416

Association between pre-diagnostic circulating 25-hydroxyvitamin D and cancer survival.

Stephanie J. Weinstein,1 Alison Mondul,2 Demetrius Albanes1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _University of Michigan, Ann Arbor, MI_.

Background: Vitamin D has many anti-cancer properties such as reducing angiogenesis, inflammation, and cell proliferation, and enhancing apoptosis and cellular differentiation. Epidemiological studies are inconsistent across cancer sites, showing inverse, positive, and null associations with 25-hydroxyvitamin D [25(OH)D] blood concentrations (i.e., the accepted biomarker of vitamin D status). We previously reported that higher 25(OH)D status was related to improved prostate cancer survival, yet few other studies have examined pre-diagnostic vitamin D concentrations and survival after a cancer diagnosis.

Methods: Based on measured prospective circulating 25(OH)D from several case-control sets nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of Finnish men, we examined serum 25(OH)D (DiaSorin LIAISON 25(OH)D TOTAL assay) in relation to overall and site-specific cancer survival among 3,740 men and 1,946 cancer deaths occurring through December, 2013. Multivariate-adjusted proportional hazard regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association between 25(OH)D and survival from malignant melanoma, non-Hodgkin lymphoma, and cancers of the colorectum, lung, prostate, kidney, stomach, oropharynx, larynx, bladder, esophagus, liver, and pancreas.

Results: Baseline serum 25(OH)D concentrations were significantly higher among men who were alive at the end of follow-up compared with men who died from their cancer (medians 35.0 vs. 32.1 nmol/L, respectively; p=0.003). Higher baseline 25(OH)D was associated with lower overall cancer death (HR=0.75, 95% CI 0.65-0.87 for highest vs. lowest quintile, p-trend 0.0001). Excluding the prostate cancer cases did not materially alter this association (HR=0.79, 95% CI 0.67-0.82 for highest vs. lowest quintile, p-trend 0.006). Organ site-specific survival showed higher 25(OH)D to be associated with lower risk of death from the following cancers: prostate (Q5 vs. Q1 HR=0.74, 95% CI 0.53-1.03), renal (Q5 vs. Q1 HR=0.58, 95% CI 0.32-1.04), stomach (Q5 vs. Q1 HR=0.49, 95% CI 0.30-0.82), oropharynx/larynx (Q5 vs. Q1 HR=0.55, 95% CI 0.28-1.06), and melanoma (T3 vs. T1 HR=0.41, 95% CI 0.18-0.94), and higher risk of death from esophageal cancer (higher vs. below median HR=1.80, 95% CI 1.04-3.12).

Conclusion: Higher pre-diagnostic serum 25(OH)D concentrations were associated with improved overall cancer survival, as well as improved survival for prostate, renal, stomach, oropharyngeal and laryngeal cancers and malignant melanoma, but poorer esophageal cancer survival. The apparent paradox of higher vitamin D status being associated with improved survival, but not necessarily reduced incidence, of these particular cancers deserves further study in other prospective populations.

#3417

Peripheral CD8+CD28-suppressive T lymphocytes act as a prognosticator among breast cancer patients with adoptive T-cell immunotherapy.

Qingkun Song,1 Jun Ren,1 Jing Yu,2 Xiaoli Wang,1 Xinna Zhou,1 Herbert Kim Lyerly3. 1 _Beijing Shijitan Hospital, Capital Medical University, Beijing, China;_ 2 _Peking University Cancer Hospital & Institute, Beijing, China; _3 _Duke University Medical Center, Durham, NC_.

Introduction: This study aimed to assess the prognostic value of CD8+CD28- T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy.

Methods: 217 patients participated in the follow-up study. CD8+CD28- proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD8+CD28- proportion more than 24.2% and less than or equal to 24.2% in peripheral T cells.

Results

The median follow-up period was 15 months. Median time to progression was 11.8 months among patients with CD8+CD28- proportion less than or equal to 24.2% and 7.1 months among patients with CD8+CD28- proportion more than 24.2% (p<0.05). With further adjustments, CD8+CD28- proportion of more than 24.2% produced a HR being 2.13 (95%CI 1.45, 3.12) for progression. Patients with CD8+CD28- proportion of less than or equal to 24.2% had median overall survival of 36.2 months, 10.6-month longer than patients with higher CD8+CD28- proportion (p<0.05). With further adjustments, CD8+CD28- proportion of more than 24.2% produced a 86% higher risk for death (HR=1.86, 95%CI 1.07, 3.24).

Conclusion

Peripheral CD8+CD28- proportion had a negative association with progression-free and overall survival among breast cancer patients receiving adoptive T cell immunotherapy. CD8+CD28- suppressive T cell was a potential biomarker for breast cancer patients' prognosis.

#3418

Differential prognostic effect of smoking and multivitamin use on lung cancer survival by sex.

Mi Yang,1 Hyun-Kyung Oh,1 Young Mog Shim,2 Myung-Hee Shin1. 1 _Sungkyunkwan Univ. School of Medicine, Suwon, Republic of Korea;_ 2 _Samsung Medical Center, Sungkyunkwan Univ. School of Medicine, Seoul, Republic of Korea_.

Incidence of lung cancer is decreasing among men and increasing among women in developed countries and also in Korea. However, mortality of lung cancer is still the highest among male cancers. Smoking is one of the group 1 carcinogens for lung cancer development, but its prognostic effect on lung cancer survival is not well established. Beta-carotene supplement increased the risk of lung cancer among smokers but the role of multivitamin supplement on the risk and survival of lung cancer is unclear either. We aimed to evaluate the association between smoking and multivitamin use on lung cancer survival by sex. We interviewed 910 pathologically confirmed lung cancer patients who were diagnosed between 2010 and 2012 in Samsung Medical Center. Questionnaire included current smoking status, age at first smoking, daily smoking amount, alcohol intake, past medical history, and multivitamin use. Pathological type, stage, and treatment information was collected from the electronic medical records. We followed the patients until December 31, 2012. Hazard Ratios (HR) and 95% Confidence Intervals (CI) were estimated using Cox's proportional hazard model (SAS9.4). We found significant difference in age at diagnosis, pathological type and stage between male and female patients; Female patients were younger, smoked less, used more multivitamin, and had more adenocarcinoma and earlier stage cancer than male patients. Smoking increased the risk of lung cancer mortality among female patients only. HR for lung cancer mortality for those who smoked more than 40 pack-years compared to those who never smoked was 5.64 (95%CI=1.43, 22.28) in women and 1.17 (95%CI=0.61, 2.26) in men (p-interaction=0.01) when adjusted for age, stage, and pathologic type. Multivitamin use also increased the risk of lung cancer mortality among female patients only. HR for lung cancer mortality for multivitamin users was 4.14(95%CI=1.77, 9.73) in women and 1.46 (95%CI=0.71, 3.00) in men (p-interaction=0.17). The association between multivitamin use and lung cancer mortality was strongest among female non-smokers (HR=4.10, 95%CI=1.72, 9.77). In conclusion, smoking and multivitamin use worsen the survival of female lung cancer. Prognostic effect of multivitamin use may be stronger among non-smokers.

#3419

Diabetes medication use in association with survival among patients of breast, colorectal, lung, and gastric cancer.

Michelle L. Baglia,1 Yong-Bing Xiang,2 Gong Yang,1 Tao Zheng,3 Honglan Li,2 Mingrong You,1 Yong Cui,1 Yu-Tang Gao,2 Wei Zheng,1 Xiao-Ou Shu1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Shanghai Cancer Institute, Shanghai, China;_ 3 _Changning District Health Information Center, Shanghai, China_.

Background: Diabetes is associated with an increased risk of several cancers and overall mortality and cancer mortality. Previous studies have suggested that metformin use may decrease cancer mortality, though findings have been inconsistent. We examined metformin and other diabetes medication use and survival from breast, colorectal, lung, and gastric cancers with respect to timing of diabetes medication initiation. Methods: Electronic medical record (EMR) data on diabetes medication use was extracted for 2,890 participants from the Shanghai Men's Health Study (SMHS) and Shanghai Women's Health Study (SWHS) with incident breast (n=633), colorectal (n=892), lung (n=822), or gastric (n=543) cancers diagnosed after 2004. Individuals with and without diabetes diagnosis were analyzed for the association between diabetes medication use (metformin, sulfonylureas, and insulin) and cancer survival using Cox proportional hazards models. Results: After adjustment for patient and clinical characteristics, ever use of any diabetes medication was associated with a decrease in all-cause mortality among all four cancer types (HR=0.84, 95% CI: 0.74, 0.94). Compared to non-users of any diabetes drug, lower mortality was observed among all cancer patients who ever took metformin (HR=0.78, 95% CI: 0.65, 0.93) or sulfonylureas (HR=0.80, 95% CI: 0.69, 0.93). When stratified by initiation of diabetes medication use with respect to cancer diagnosis, the association was significant for both metformin and sulfonylureas use, but only among those who initiated use after cancer diagnosis. When cancers were analyzed individually, significant associations were observed for lung and colorectal cancer cases for metformin or sulfonylureas use among those who initiated use after cancer diagnosis. The inverse associations were predominantly observed among those whose diabetes diagnosis could be verified by EMR. Diabetes medication use was not significantly associated with survival from breast or gastric cancer. Conclusions: Use of metformin or sulfonylureas was associated with improved survival among lung and colorectal cancer patients. The association was primarily observed among those who initiated diabetes medication use after cancer diagnosis. While a possible survival time bias can't be excluded, additional investigation on the topic is needed given the potential translational impact if our finding were proved to be true.

#3420

Clinical prognostic factors in adult with astrocytoma: Historic cohort.

Talia Wegman-Ostrosky,1 Nancy Reynoso Noverón,1 Sonia Iliana Mejía-Pérez,2 Thalía Estefania Sánchez-Correa MD,2 Rosa María Álvarez,1 Bernardo Cacho,1 Luis A. Montalvo,1 Teresita Corona2. 1 _Instituto Nacional de Cancerología, Mexico City, Mexico;_ 2 _Instituto Nacional de Neurologia y Neurocirugia, Mexico City, Mexico_.

Introduction: Malignant tumors of the central nervous system contribute extremely to cancer mortality especially in the high risk age groups. In USA are the second and the fifth leading cause of cancer mortality in men and women aged 20 to 39 years respectively. Astrocytomas are the most common and lethal tumors of the CNS, being the most common grade IV (glioblastoma) (4.37 per 100,000 population).

The prognosis of astrocytomas can be predicted by specific clinical factors allowing neurosurgeons and neuro-oncologists to define the best treatment for each patient. Some patients' features like age, gender, performance status and tumor localization have been studied as potential prognostic factors. In Mexico there are few reports on demographic, clinical characteristics and prognostic factors in adults.

Objective: To explore the clinical prognostic factors for adults affected with astrocytoma

Methods: Using a historic cohort, we selected by simple randomization, 155 clinical files from patients with astrocytoma. The main outcome variable was overall survival time (OS). To identify clinical prognosis factors we used: bivariate analysis, Cox regression models, log rank test and Kaplan-Meier survival function.

Results: The mean age at diagnosis was 45.7 years. Compared with other studies, our population was 15 years younger when diagnosed. Analysis by stage in grade II, III and IV also showed a younger age of presentation. The OS was 15 months, 9% (n=14) presented a survival of 2 years and 3% of 3 years. Kaplan-Meier survival estimate showed that the variables grade, Karnofsky Performance Status (KPS)>70, the type of resection, chemotherapy, radiotherapy, alcohol consumption, familiar history of cancer and clinical presentation were significantly associated to survival time. Using proportional Hazard Model the variables: age, grade IV, resection, chemotherapy + radiotherapy and KPS where prognosis factors.

Conclusion: Astrocytoma in our study was present in young adults. The OS was 15 months, 9% (n=14) presented a survival of 2 years and 3% of 3 years. The variables alcoholism, family history of cancer and clinical presentation influenced significantly survival time, and showed a tendency in the mortality analyses.

#3421

Epidemiology and survival in patients with extragastric signet ring carcinoma.

Chul Kim, Susanna Ulahannan, Julius Strauss, Jaydira Del Rivero, Austin Duffy, Tim F. Greten, Oxana V. Makarova-Rusher. _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Background: Signet ring carcinoma (SRC) is a distinct histological phenotype of adenocarcinoma. There are only a few published studies specifying the epidemiology of SRC with extragastric presentation. The purpose of our study was to define the most common primary sites of extragastric SRC, determine the incidence, and to compare survival by primary site and disease stage.

Methods: The Surveillance Epidemiology and End Result (SEER) database was examined from 2000 to 2012 in order to identify SRC histology (8490) and determine its most common primary sites, incidence, and survival by site and stage. The five most common primary extragastric sites were identified by utilizing ICD-0-3/WHO 2008 classification. Age-adjusted incidence rates for extragastric SRC were calculated and compared to gastric SRC. Relative survival (RS) and overall survival (OS) at 1 and 3 years were analyzed by primary site and stage using Kaplan-Meier method. Chi-square test was used for categorical variables.

Results: A total of 24,522 histologically confirmed cases of SRC were identified, and SRC comprised 0.5% of all malignant neoplasms. Among cases with known histological grade, 89.7% had poorly differentiated tumors. Overall, digestive system origin was recorded for 90% of SRC cases. Approximately half (44.2%) of primary SRC tumors were detected outside of the stomach. The most common primary sites for extragastric SRC were colon (40.5 %), esophagus (11.9%), rectum (9.8%), lung/bronchus (7.3%), and pancreas (4.7%). The incidence rates for common extragastric SRC were much lower than for gastric SRC, and were higher for males than females (p<0.001). Incidence profile differed by gender as breast was among top five SRC anatomical sites for females. Clinically, distant metastatic disease was more often diagnosed in patients with pancreatic (66.3%) and lung/bronchus (73.8%) SRC than SRC of the stomach (43.5%) (p<0.01). Survival varied substantially by primary site. Pancreatic SRC had the worst survival and colorectal the most favorable. For example, among patients with stage IV disease, 1-year RS was 43.9% (95% CI=41.1-46.6) for colorectal, 25.5% (95% CI=20.6-30.6) for lung/bronchus, 20.9% (95% CI=16.2-26.1) for esophageal, and only 10.7% (95% CI=6.5-15.9) for pancreatic primary SRC. In concordance with 1-year RS, 3-year RS was 9.9% (95% CI=8.2-11.9) for colorectal vs. 0.0% for pancreatic SRC.

Conclusion: Our study indicated that extragastric SRC most commonly occurs in the colorectum, esophagus and lung/bronchus. We confirmed that the primary site substantially impacts survival. Thus, development of unique molecular or histologic markers may help to identify the organ of origin and thereby determine prognosis in these phenotypically similar neoplasms.

#3422

The influence of genetic and clinical factors on the outcome following a diagnosis of small cell carcinoma of the ovary, hypercalcemic type.

Leora Witkowski,1 Catherine Goudie,1 Pilar Ramos,2 Anthony N. Karnezis,3 Talia Boshari,4 Patricia Pautier,5 Michel Longy,6 James A. Knost,7 Emmanouil Saloustros,8 W Glenn McCluggage,9 Martin Hasselblatt,10 William P. Hendricks,2 David Huntsman,3 Douglas A. Levine,1 Jeffrey Trent,2 William D. Foulkes1. 1 _McGill University, Montreal, Quebec, Canada;_ 2 _Translational Genomics Research Institute, Phoenix, AZ;_ 3 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 4 _Lady Davis Institute, Montreal, Quebec, Canada;_ 5 _Gustave Roussy, Villejuif, France;_ 6 _Institut Bergonié, Bordeaux, France;_ 7 _Illinois Cancer Care, Peoria, IL;_ 8 _University Hospital of Heraklion, Crete, Greece;_ 9 _Belfast Health and Social Care Trust, Belfast, United Kingdom;_ 10 _University Hospital Münster, Münster, Germany_.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is the most common undifferentiated ovarian tumor diagnosed in women below the age of 40. While it is uncommon, with only ~500 cases reported, it is an extremely aggressive tumor, with long term survival rates of <30% in early stage disease. Recently, we discovered that SCCOHT is a monogenic disease, attributable to germline and somatic deleterious mutations in the chromatin remodeling gene, SMARCA4. Since then, single case reports have been published on women affected with SCCOHT, but no large studies have been conducted on the effect of these mutations, germline or somatic, on the disease. Furthermore, while numerous publications have described the therapies used in single cases or small cohorts of SCCOHT patients, treatment remains varied, with no standardized protocols. In an attempt to clarify which factors have an effect on the course of the disease, we reviewed all published and a cohort of unpublished SCCOHT cases and collected information on the following factors: stage at diagnosis, age at diagnosis, treatment modality used (chemotherapy/radiotherapy/high dose chemotherapy), mutation type (germline/somatic), and length of survival. We were able to acquire information in at least 2 of these parameters in 267 cases and performed multiple analyses. Clinically, we found that the greatest influence on patient outcome was related to stage at diagnosis (p < 0.001) and treatment modality (p = 0.01), while age at diagnosis had no significant effect (p = 0.72). Molecularly, the presence of a germline mutation in the patient did not affect the age of onset (p = 0.86), nor did it have an effect on the length of survival of the patient (p = 0.63). Overall, this is the largest study conducted to date analyzing the influence of clinical factors on the outcome of patients diagnosed with SCCOHT, and is the first large study to analyze the effect of germline mutations on the outcome of these patients.

#3423

Overweight and obesity predict better overall survival rates in cancer patients with distant metastases.

Ngan M. Tsang. _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

Background: Recent studies conducted in patients with chronic diseases have reported an inverse association between body mass index (BMI) and mortality. However, the question as to whether BMI may predict prognosis in patients with metastatic cancer remains open. We therefore designed the current retrospective study to investigate the potential association between BMI and overall survival (OS) in patients with distant metastases (DM) and a favorable performance status.

Methods: Between 2000 and 2012, a total of 4,010 cancer patients with DM who required radiotherapy (RT) and had their BMI measured at the initiation of RT were identified. The relation between BMI and OS was examined by univariate and multivariable analysis.

Results: The median OS time was 3.23 months (range: 0.1−122.17) for underweight patients, 6.08 months (range: 0.03−149.46) for normal-weight patients, 7.99 months (range: 0.07−158.01) for overweight patients, and 12.49 months (range, 0.2−164.1) for obese patients (log-rank: P < 0.001). Compared with normal-weight patients, both obese (HR = 0.676; 95% P < 0.001) and overweight individuals (HR = 0.84; P < 0.001) had a reduced risk of all-cause mortality in multivariable analysis. Conversely, underweight patients had a significantly higher risk of death from all causes (HR = 1.41; P < 0.001).

Conclusions: Overweight and obesity are independent predictors of better OS in metastatic patients with a good performance status. Increased BMI may play a role to identify metastatic patients with superior survival outcome and exhibit a potential to encourage aggressive management in those patients even with metastases.

#3424

Longitudinal changes in volumetric breast density with adjuvant endocrine therapy among women with breast cancer.

Natalie J. Engmann,1 Celine M. Vachon,2 Christopher G. Scott,2 Matthew R. Jensen,2 Lin Ma,1 Kathleen R. Brandt,2 Amir P. Mahmoudzadeh,1 Serghei Malkov,1 Dana H. Whaley,2 Carrie B. Hruska,2 Fang F. Wu,2 Stacey J. Winham,2 Diana L. Miglioretti,3 Aaron D. Norman,2 John J. Heine,4 John Shepherd,1 V Shane Pankratz,5 Karla Kerlikowske1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _University of California Davis, Davis, CA;_ 4 _Moffitt Cancer Center, Tampa, FL;_ 5 _University of New Mexico Health Sciences Center, Albuquerque, NM_.

Breast density represents the stromal and epithelial tissue in the breast and is a strong risk factor for breast cancer. Reductions in breast density attributable to tamoxifen (TAM) and aromatase inhibitors (AI) may be associated with reduced risk of first primary and subsequent breast cancer. Studies assessing breast density changes have principally used two-dimensional measures. We assess changes in breast density following initiation of TAM and AI using two automated volumetric density measures that have shown strong associations to breast cancer risk.

Breast cancer cases with a full field digital mammogram (FFDM) prior to diagnosis (index mammogram) and after initiation of TAM (n=379) or AI (n=425) were identified from the Mayo Clinic Mammography Practice and the San Francisco Mammography Registry. Volumetric percent density (VPD) and dense volume (DV) were measured on 4-view FFDM using VolparaTM (Matakina Technology) and QuantraTM (Hologic) automated software. We used linear regression to assess the effect of treatment on mean annualized change in VPD and DV (change from index to last mammogram divided by time interval) for each software type, adjusting for age, body mass index (BMI) and density at index mammogram.

The median time between index mammogram and cancer diagnosis was 0.6 months (IQR: 0.2, 2.2) and median time between index and last mammogram was 3 years (IQR: 2.0, 3.9). Women on TAM were younger, had lower BMI and higher baseline VPD and DV relative to women on AI (Table). An annual decrease in VPD and DV was observed with both TAM and AI. Both Volpara and Quantra estimated a similar magnitude of change in VPD in women on TAM and AI, and a greater change in DV with TAM.

Our findings suggest that both Volpara and Quantra can assess volumetric changes in breast density among women on hormone therapy. If declines in volumetric density correlate with a reduction in breast cancer risk, these automated measures could be used in clinical practice to assess response to therapy.

Annualized changes in volumetric breast density estimated by linear regression.

---

|  | Tamoxifen (n=379) | Aromatase Inhibitors (n=425)

|  | Baseline Median (IQR) | Annualized Change (95% CI)* | Baseline Median (IQR) | Annualized Change (95% CI)*

Age at Diagnosis | |

50.0 (45.0, 60.0) | \-- | 63.0 (58.0, 71.0) | \--

Body Mass Index (BMI) | |

23.6 (21.5, 26.8) | \-- | 25.7 (22.7, 29.9) | \--

Time Interval¥ | |

3.0 (2.1, 3.9) | \-- | 3.0 (2.1, 3.9) | \--

Volpara | Percent Density (VPD, %) | 11.6 (6.8, 18.8) | -0.17 (-0.27, -0.10) | 7.2 (5.0, 11.0) | -0.19 (-0.29, -0.12)

Dense Volume (DV, cm³) | 64.7 (45.4, 90.9) | -0.90 (-1.45, -0.48) | 51.9 (38.9, 69.9) | -0.52 (-0.93, -0.23)

Quantra | Percent Density (VPD, %) | 14.5 (9.2, 20.2) | -0.42 (-0.59, -0.28) | 9.9 (7.1, 14.5) | -0.38 (-0.54, -0.25)

Dense Volume (DV, cm³) | 94.0 (58.0, 144.0) | -2.20 (-3.52, -1.19) | 80.0 (49.0, 128.0) | -0.95 (-1.85, -0.35)

IQR = Interquartile range

¥ Median number of years between index mammogram and last mammogram post-initiation of therapy.

*Annualized change estimated as change from index to last mammogram divided by time interval and adjusted for study site, age at diagnosis, BMI and density at index mammogram.

#3425

Prediagnostic alcohol consumption and colorectal cancer survival: the Colon Cancer Family Registry.

Amanda I. Phipps,1 Jamaica Robinson,1 Peter T. Campbell,2 Aung Ko Win,3 Jane Figueiredo,4 Noralane M. Lindor,5 Polly A. Newcomb6. 1 _University of Washington, Seattle, WA;_ 2 _American Cancer Society, Atlanta, GA;_ 3 _University of Melbourne, Melbourne, Australia;_ 4 _University of Southern California, Los Angeles, CA;_ 5 _Mayo Clinic, Scottsdale, AZ;_ 6 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Previous studies have shown an increased risk of colorectal cancer (CRC) among moderate to heavy alcohol consumers relative to non-drinkers; however, the relationship between alcohol and CRC survival remains unclear. Using data from the international Colon Cancer Family Registry (CCFR), we assessed the association between pre-diagnostic alcohol intake and survival outcomes after CRC diagnosis, overall and stratified by patient and tumor attributes. CRC cases were identified via population-based cancer registries at four CCFR study sites, with diagnoses of incident, invasive CRC from 1997 to 2006. Study participants completed a risk factor questionnaire at enrollment which included information on several pre-diagnostic behaviors, including consumption of wine, beer, and liquor. Prospective follow-up for survival outcomes was conducted for 4858 CRC cases with complete data on alcohol consumption. Using Cox proportional hazards regression models with delayed entry to account for the time between diagnosis and study enrollment, we compared non-drinkers (i.e., alcohol intake <1 drink per week) to individuals who consumed, on average, <1 serving of alcohol per day (but ≥1 per week), and those who consumed ≥1 serving per day in the years preceding CRC diagnosis. Separate analyses were carried out for overall and disease-specific survival. All models were adjusted for age at diagnosis, sex, study site, year of diagnosis, smoking history, CRC screening history, and body mass index. Over a mean follow-up of 7.5 years, 1872 (39%) study participants died, 1110 (59%) of whom died from CRC. Pre-diagnostic beer and liquor consumption were not associated with CRC survival; however, consumption of ≥1 serving of wine per day was modestly associated with more favorable overall [hazard ratio (HR) = 0.80, 95% confidence interval (CI): 0.63-1.00] and disease-specific survival (HR = 0.83, 95% CI: 0.62-1.12). In stratified analyses, having consumed ≥1 serving of wine per day was slightly, but not statistically significantly, more strongly associated with overall survival among men than among women (HR = 0.71 vs. 0.91, respectively, p-heterogeneity=0.12) and among those aged ≤50 than those aged >50 years (HR = 0.66 vs. 0.84, p-heterogeneity=0.35). Similar patterns were noted with respect to disease-specific survival. There were no differences in the relationships between wine consumption and survival outcomes stratified by tumor site, microsatellite instability, BRAF-mutation, or CpG island methylator phenotype status. These results suggest that wine consumption prior to CRC diagnosis is modestly associated with more favorable survival after CRC diagnosis. This modest inverse association does not appear to be limited to specific colorectal tumor subtypes and extends beyond overall survival to disease-specific outcomes.

#3426

Recurrent breast cancer risk and physical activity.

Teresa A. Lehman,1 Ramakrishna V. Modali,1 Luke D. Ratnasinghe2. 1 _BioServe Biotechnologies, Ltd., Beltsville, MD;_ 2 _Cytonix, Beltsville, MD_.

Introduction: Physical activity reduces the risk of breast cancer. However, the potential role of physical activity in recurrence of breast cancer is not well established. The aim of our study is to examine the association between physical activity and risk of recurrent breast cancer. Methods: Data was obtained from the Global Epidemiological Study (GES). The GES is an IRB approved multinational biorepository and database to assess cancer and other disease risk factors and biomarkers. In-person interviews of all subjects provided demographics, family-history and other disease related information including age, BMI, diet and physical activity. For statistical analyses, t-tests were used for continuous variables and chi-square tests for categorical variables. The association between recurrent breast cancer and physical activity was assessed using logistic regression in univariate and multivariate analyses. Results: From a total of 2435 breast cancer subjects 215 had recurrent breast cancer. The average age of subjects without recurrence was 55.62 years and those with breast cancer recurrence was 58.35 years. In univariate analysis, subjects in the highest tertile of physical activity were 39% less likely to have recurrent breast cancer compared to those who reported no physical activity [Odds Ratio: 0.61, 95% Confidence Interval: 0.40-0.93]. In multivariate analysis, subjects in the highest tertile of physical activity were 45% less likely to have recurrent breast cancer compared to those who reported no physical activity [Odds Ratio: 0.55, 95% Confidence Interval: 0.34-0.89] after adjusting for age, BMI and cancer-stage. A statistically significant dose-response for physical-activity and reduced risk of recurrent breast cancer was observed with a P-value for trend of 0.05. Conclusion: Our study suggests that physical activity reduces the risk of recurrence of breast cancer. Further studies with larger sample size are needed to confirm our findings.

### Exogenous Exposures and Cancer Risk

#3427

Chemical exposures and risk of acute myeloid leukemia and myelodysplastic syndromes in a population-based study.

Jenny N. Poynter,1 Michaela Richardson,1 Michelle Roesler,1 Cindy K. Blair,2 Betsy Hirsch,1 Phuong Nguyen,3 Adina Cioc,4 James R. Cerhan,3 Erica Warlick1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of New Mexico, Albuquerque, NM;_ 3 _Mayo Clinic, Rochester, MN;_ 4 _VA Medical Center, Minneapolis, MN_.

Background Myelodysplastic syndromes (MDS) are a group of clonal hematologic disorders that result in ineffective hematopoiesis. Individuals with MDS have a high risk of progressing to leukemia, with approximately 30% expected to develop acute myeloid leukemia (AML). Benzene exposure is one of the few well-established risk factors for AML, and recent studies suggest it is also a risk factor for MDS. Exposure to other occupational and residential chemicals has been inconsistently associated with hematologic malignancies. In this analysis, we evaluated occupational and residential chemical exposures as risk factors for AML and MDS using population-based data. Methods AML and MDS cases were identified by rapid case ascertainment through the Minnesota Cancer Surveillance System (MCSS). Centralized pathology and cytogenetics reviews were conducted to confirm diagnosis and classify by subtypes. Controls were identified through the Minnesota State driver's license/identification card list. Chemical exposures were measured by self-report and included occupational and residential exposure to a variety of chemicals. Unconditional logistic regression with adjustment for age, sex, previous cancer treatment, income, and farm or rural residence was used to calculate crude and adjusted odds ratios (ORs) and 95% confidence intervals (CI). Smoking, obesity and physical activity were considered as potential confounders but did not change effect estimates by 10% or more. Results We included 265 MDS cases, 420 AML cases, and 1388 controls in this analysis. As expected, we observed significant associations between benzene and both MDS and AML (OR=1.77, 95% CI 1.17, 2.67 and OR=2.03, 95% CI 1.45, 2.83, respectively). Exposure to vinyl chlorides was associated with MDS (OR=2.25, 95% CI 1.32, 3.81) but not with AML (OR=1.36, 95% CI 0.83, 2.21). Exposure to fertilizers (OR=1.70, 95% CI 1.11, 2.59), soot (OR=2.64, 95% CI 1.59, 4.39), creosote (OR=1.95, 95% CI 1.08, 3.51), inks, dyes and tanning solutions (OR=1.63, 95% 1.02, 2.60), and coal dust (2.64, 95% CI 1.30, 5.39) were associated with AML, while no association was seen between any of these exposures and MDS (range ORs=0.78-1.25). No significant associations were observed for occupational pesticide exposures in either group. Discussion These data confirm the importance of benzene as a risk factor for myeloid malignancy and provide risk estimates in a population-based sample. A number of other significant associations with occupational and residential chemicals were observed for AML; however, all exposures were reported by only a small percentage of cases (≤10%). While chemical exposures play a clear role in the etiology of myeloid malignancy, these exposures do not account for the majority of cases.

#3428

Domestic livestock exposure and lung cancer risk among non-smoking women in Xuanwei, China.

Wei Jie Seow,1 Wei Hu,1 Roel Vermeulen,2 George Downward,2 Jihua Li,3 Jun Xu,4 Jun He,3 Fusheng Wei,5 Guoping Wu,5 Nathaniel Rothman,1 Robert S. Chapman,6 Qing Lan1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Utrecht University, Utrecht, Netherlands;_ 3 _Qujing Centers for Disease Control and Prevention, Qujing, China;_ 4 _The University of Hong Kong, Hong Kong, Hong Kong;_ 5 _China National Environmental Monitoring Center, Beijing, China;_ 6 _Chulalongkorn University, Bangkok, Thailand_.

Background: Lung cancer rates of women in Xuanwei and Fuyuan, rural counties located in the Yunnan Province, are among the highest in China, even though most women there are non-smokers. The unusually high lung cancer rates are mainly attributed to smoky (i.e. bituminous) coal exposures. Many households there and in other rural parts of China have raised animals such as pigs, cows, horses, chickens and ducks in close proximity to their homes. It is hypothesized that contact with animals increases the risk of infection with potentially carcinogenic pathogens. However, findings on the association between livestock and poultry exposures and lung cancer risk, mostly from occupational studies, have been inconsistent.

Objective: To investigate the association between livestock and poultry exposures and lung cancer risk in a general population of non-smoking women who lived in close proximity to livestock and poultry in a rural area in China.

Methods: A hospital-based case-control study among non-smoking women was conducted in Xuanwei and Fuyuan counties in China between March 2006 and July 2013. A total of 1,074 lung cancer cases and 977 hospital-based cancer-free controls were enrolled. Exposure to livestock and poultry, including the animal type (pigs, cows/horses, chickens, ducks) and total number of years an individual was exposed to each animal type, was obtained by questionnaire. Reference groups for the analysis were either never-exposed, or never-exposed combined with those with less than the median years of exposure, depending on the prevalence of exposure to each animal. Logistic regression was used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) of the association between livestock and poultry exposures and lung cancer risk with adjustment for potential confounders, such as lifetime amount of smoky coal used and passive smoking.

Results: Individuals with above the median number of years of exposure to pigs (OR=1.61, 95% CI=1.19-2.17) and chickens (OR=1.49, 95% CI=1.12-1.99) had increased lung cancer risk, compared to those who had less than the median years of exposure. Compared to those who were never exposed, the risk of lung cancer increased with longer duration of exposure to cows/horses (<median years of exposure: OR=1.33, 95% CI=1.00-1.77; ≥median years of exposure: OR=1.38, 95% CI=1.03-1.86). The significant associations for pigs and cows/horses persisted after adjusting for other animal exposures in the same model.

Conclusions: Exposure to certain animals was significantly associated with increased lung cancer risk, suggesting that infectious agents may play a role in the etiology of lung cancer among non-smoking women in this region. However, due to the fact that this study was conducted in an area with a high risk of lung cancer due to the use of solid fuels for heating and cooking, we cannot rule out residual confounding.

#3429

Evidence chronic inflammation is a risk factor for histiocytic sarcoma in Bernese mountain dogs - implications for human histiocytic sarcoma.

Audrey Ruple-Czerniak,1 Paul S. Morley2. 1 _Purdue University, West Lafayette, IN;_ 2 _Colorado State University, Fort Collins, CO_.

The purpose of this study was to evaluate exposure variables associated with development of histiocytic sarcoma (HS), a rare but aggressive malignancy, using the canine spontaneous tumor model. The domestic dog has been shown to be an excellent model to investigate risk of occurrence of complex diseases in humans, especially cancers. This is due in part to the limited genetic heterogeneity within specific dog breeds. HS, though rare in most breeds of dogs, is a common malignancy in Bernese mountain dogs (BMD) with up to 25% of the population of dogs being diagnosed with HS during their lifetime. The frequency of HS diagnosis in BMDs is thought to be due to a heritable mutation in a single gene region analogous to the human chromosome 9p21. For this study, owners of BMDs registered with a non-profit breed registry, the Berner-Garde Foundation, were recruited for participation in an internet-based cross-sectional survey. Data were collected for a total of 216 BMDs. Of the total population of BMDs enrolled in the study 118 dogs were sibling-sets representing a case and a non-case that originated from the same litter. Mixed effects logistic regression (MELR) and conditional logistic regression (CLR) were used in parallel to examine associations between potential risk factors and the occurrence of HS. When controlling for litter of origin (as a marker of relatedness), dogs diagnosed with orthopedic conditions were found to be more likely to develop HS (MELR, OR: 2.5, 95% CI: 1.5, 5.2; CLR, OR: 2.81, 95% CI: 1.1, 7.3), while dogs receiving prescription anti-inflammatory medications were found to be at a considerably lower risk of developing this disease (MELR, OR: 0.43, 95% CI: 0.2, 0.8; CLR, OR: 0.32, 95% CI: 0.1, 0.8). These data suggest inflammation may be a modifiable risk factor for the development of HS. Therefore, identification and treatment of conditions that promote inflammatory mechanisms may reduce future risk of developing HS in dogs and people.

Results of logistic regression models examining risk factors associated with development of histiocy

---

Risk factor | Category | Odds ratio (MELR), n=216 | 95% CI | P-Value | Odds ratio (CLR), n=118 | 95% CI | P-Value

Orthopedic condition | Yes | 2.49 | 1.21, 5.14 | 0.014 | 2.81 | 1.08, 7.26 | 0.034

|

No | Reference | |  | Reference | |

Non-prescription medication use | Yes | 2.10 | 1.05, 4.18 | 0.036 | 2.88 | 1.04, 7.90 | 0.041

|

No | Reference | |  | Reference | |

Long-term prescription medication use | Yes | 0.43 | 0.22, 0.81 | 0.010 | 0.32 | 0.12, 0.83 | 0.020

|

No | Reference | |  | Reference | |

#3430

Association of PM2.5 and ozone exposure with mammographic breast density in the Breast Cancer Surveillance Consortium.

Lusine Yaghjyan,1 Robert Arao,2 Cole Brokamp,3 Ellen O'Meara,2 Brian Sprague,4 Gabriela Ghita,1 Patrick Ryan5. 1 _University of Florida, Gainesville, FL;_ 2 _Group Health Research Institute, Seattle, WA;_ 3 _University of Cincinnati, Cincinnati, OH;_ 4 _University of Vermont, Burlington, VT;_ 5 _Cincinnati Children's Hospital Medical Center, Cincinnati, OH_.

Background: Mammographic breast density (BD) is a well-established and strong breast cancer risk factor. Some studies suggest geographical disparities in BD. Environmental factors such as air pollution may contribute to geographic variation in BD because urban and rural areas have distinct air pollution patterns and some of the pollutants are known to have endocrine disrupting properties. We examined the association of BD with particles<2.5 μm in diameter (PM2.5) and ozone (O3) in a large population-based cohort.

Methods: Women were selected from participants of the Breast Cancer Surveillance Consortium (2001-2009). We included women aged ≥40 years with no history of breast cancer, with BD data available from screening mammography and with known residential zip code for at least 3 years prior to the index mammogram. Categorical BD was defined with BI-RADS breast density classification as predominantly fat, fat with some fibroglandular tissue (reference), heterogeneously dense, and extremely dense. PM2.5 and O3 estimates for grids across the United States from 2001-2008 were obtained from the US Environmental Protection Agency hierarchical Bayesian model (HBM), which combines monitoring data with numerical output from the Community Multi-scale Air Quality model. For both pollutants, the smallest grids available for each year were used. Daily concentrations of PM2.5 and O3 were used to calculate annual concentrations for each grid. Yearly mean exposures of PM2.5 and O3 for each subject were calculated using the inverse distance weighted method based on their zip code centroid and HBM grid centroids. PM2.5 and O3 exposures were modeled as continuous and as quartiles. Associations of PM2.5 and ozone with BD were estimated with polychotomous logistic regression adjusting for potential confounders.

Results: In this study of 368,302 women, participants with extremely dense breasts had mean PM2.5 and O3 exposures of 9.41ug/m3 and 33.76 ug/m3 as compared to 9.02 ug/m3 and 35.42 ug/m3 in women with fatty breasts, respectively. In multivariate regression, women with heterogeneously dense vs. scattered fibroglandular breasts were more likely to have been exposed to higher concentrations of PM2.5 (4th vs. 1st quartile Odds Ratio [OR]=1.21, 95% Confidence Interval [CI] 1.17-1.24; 3rd vs. 1st quartile: OR=1.27, 95% CI 1.23-1.30) and women with fatty breasts were less likely to have been exposed to high PM2.5 levels (4th vs. 1st quartile: OR=0.95, 95% CI 0.91-1.00; 3rd vs. 1st quartile: OR=0.75, 95% CI 0.73-0.78). Women with heterogeneously and extremely dense vs. scattered fibroglandular breasts were less likely to have been exposed to higher levels of ozone (4th vs. 1st quartile: OR=0.94, 95% CI 0.89-0.98 and OR=0.79, 95% CI 0.72-0.86, respectively).

Conclusion: Exposure to PM2.5 and ozone may in part explain geographical variation in BD. Further studies are warranted to determine the causal nature of these associations.

#3431

Occupation and the risk of early- and later-onset prostate cancer in five Nordic countries.

Kathryn Hughes Barry,1 Jan Ivar Martinsen,2 Michael C.R. Alavanja,1 Gabriella Andreotti,1 Aaron Blair,1 Johnni Hansen,3 Kristina Kjaerheim,2 Stella Koutros,1 Elsebeth Lynge,4 Par Sparen,5 Laufey Tryggvadottir,6 Elisabete Weiderpass,2 Sonja I. Berndt,1 Eero Pukkala7. 1 _NCI-DCEG, Bethesda, MD;_ 2 _Department of Research, Cancer Registry of Norway, Oslo, Norway;_ 3 _Danish Cancer Society Research Center, Copenhagen, Denmark;_ 4 _University of Copenhagen, Copenhagen, Denmark;_ 5 _Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden;_ 6 _Icelandic Cancer Registry, Icelandic Cancer Society, Reykjavik, Iceland;_ 7 _Finnish Cancer Registry, Institute for Statistical and Epidemiological Cancer Research, Helsinki, Finland_.

The etiology of prostate cancer remains largely unknown. Cases that occur late in life may have a different etiology than early-onset cases, and their inclusion in epidemiological studies may obscure associations with early-onset prostate cancer, which is often more aggressive and clinically relevant. We evaluated occupation in relation to the risk of prostate cancer separately for early-onset and later-onset disease in a large pooled study. We used census data to code occupations among census participants in five Nordic countries (Denmark, Finland, Iceland, Norway, and Sweden) from 1960-1990. We identified prostate cancer cases diagnosed from 1961-2005 by linkage to national cancer registries and calculated standardized incidence ratios (SIRs) by occupation separately for men aged 30-49 and those aged 50 or older. We also conducted separate analyses by period of cancer follow-up in two categories, 1961-1990 and 1991-2005, based on introduction of the prostate-specific antigen (PSA) screening test in the Nordic countries in 1990 or later. For early-onset prostate cancer, the highest SIRs were observed for public safety workers (e.g., firefighters) [SIR=1.71, 95% confidence interval (CI): 1.23-2.31] and military personnel (SIR=1.97, 95% CI: 1.31-2.85). These SIRs were significantly higher than those observed for later-onset disease (pheterogeneity=0.005 and 0.002), with public safety workers and military personnel demonstrating SIRs of 1.10 (95% CI: 1.07-1.14) and 1.09 (95% CI: 1.05-1.13), respectively. Administrators (SIR=1.41, 95% CI: 1.13-1.73) and technical workers (SIR=1.18, 95% CI: 1.01-1.37) also demonstrated significantly increased risk for early-onset prostate cancer, although the SIRs did not significantly differ from those for later-onset disease (for administrators, SIR=1.17, 95% CI: 1.15-1.19; pheterogeneity=0.08, and for technical workers, SIR=1.11, 95% CI: 1.09-1.12; pheterogeneity=0.40). With the exception of public safety workers, for whom the SIR for early-onset prostate cancer was higher in the later period (for 1961-1990, SIR=1.45, 95% CI: 0.79-2.43, and for 1991-2005, SIR=1.87, 95% CI: 1.25-2.71), the observed associations with early-onset disease tended to be restricted to the earlier, pre-PSA period. Our results suggest that occupational exposures among public safety workers and military personnel may contribute to the risk of prostate cancer, particularly for early-onset disease.

#3432

Outdoor air pollution and household coal combustion in a rural high lung cancer incidence area of China.

Wei Hu,1 George Downward,2 Boris Reiss,2 Nathaniel Rothman,3 Jihua Li,4 Jun He,4 K.F Morales-Collins,2 Jun Xu,5 Weijie Seow,1 Bryan Bassig,1 Dean Hosgood,1 Linlin Zhang,6 M Hoogeveen,2 Ingrid Rijk,2 Guoping Wu,6 Fusheng Wei,6 Roel Vermeulen,2 Qing Lan1. 1 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Rockville, MD;_ 2 _Institute for Risk Assessment Sciences, Division of Environmental Epidemiology, Utrecht University, Utrecht, Netherlands;_ 3 _NCI-DCEG, Rockville, MD;_ 4 _Qujing Center for Disease Control and Prevention, Qujing, China;_ 5 _The University of Hong Kong, Hong Kong, China;_ 6 _China National Environmental Monitoring Center, Beijing, China_.

Outdoor air pollution inside populated areas is a growing environmental health concern. Epidemiological data related to exposures to outdoor air pollution are limited in rural areas. We previously assessed indoor and personal exposures in two Chinese rural counties, Xuanwei and Fuyuan, where the domestic combustion of locally sourced "smoky" (i.e., bituminous) but not "smokeless" (i.e., anthracite) coal has been associated with the highest lung cancer rates in China. In addition, we have previously reported that improving home ventilation by installing stoves with chimneys was associated with a reduction in lung cancer rates in this region. However, outdoor air pollution from indoor burning of coal and the impact of improving household ventilation on outdoor air pollution have never been assessed in this area. Therefore, we measured outdoor fine particulate matter (PM2.5) and polycyclic aromatic hydrocarbons (PAHs) including the known carcinogen benzo-a-pyrene (BaP) over two consecutive 24-hour sampling periods in 29 villages. Half of the villages were revisited several months after the initial sampling period to repeat all measurements. The overall concentrations of outdoor pollutants in Xuanwei and Fuyuan were 53.1 µg/m3 (13.2-103.4 µg/m3) for PM2.5, and 11.1 ng/m3 (5.9-19.8 ng/m3) for BaP. PM2.5 levels were almost twice as high as the 24-hour mean World Health Organization guideline value of 25 µg/m3. Outdoor BaP concentrations in villages with homes using smoky coal were significantly higher than those with homes that used smokeless coal (11.6 vs 5.9 ng/m3, p < 0.05). Among villages using smoky coal, higher outdoor BaP levels were found in villages where >50% of households had a chimney compared to villages where ≤50% of homes had a chimney (12.3 vs 7.1 ng/m3, p = 0.024). Outdoor PM2.5 was moderately correlated with the indoor concentration (Spearman rs = 0.28, p = 0.011). These results show that outdoor air pollution in a rural region of China with a high incidence of lung cancer was associated with type of coal used for cooking and heating indoors and the presence of stove ventilation. These findings suggest that further reducing adverse health effects in rural villages from home burning of coal will likely require use of stoves that reduce environmental exhaust and/or the replacement of coal with cleaner fuel types.

#3433

Association between serum concentrations of polychlorinated biphenyls (PCBs) and increased risk of breast cancer among US women.

Marisa Morgan, Alok Deoraj, Deodutta Roy. _Environmental & Occupational Health, Florida International University, Miami, FL_.

The main objective of this study was to examine the cross-sectional relationship between exposure to PCBs or their congeners and breast cancer among US women. There is limited information on the association of specific PCB congener or their combination levels in blood serum with increased breast cancer risk. PCBs are weak estrogens which has been shown to act as endocrine disrupters by increasing or blocking estrogen like activities in animals and humans. We analyzed National Health and Nutrition Examination Survey (NHANES) data from 1999-2004 in the lipid adjusted blood samples of female participants (>20 years of age). Exposure assessment was based on lipid adjusted serum levels of 6 individual PCB congeners (PCB 074, 099, 118, 138, 153, and 180), the sum of dioxin-like PCBs (074 and 118), and the sum of non-dioxin-like PCBs (099 + 138 + 153+187) in conjunction with data obtained from the medical and reproductive health questionnaires. We calculated geometric means to compare PCB concentrations in women's blood samples who also self-reported a breast cancer diagnosis and women who were never diagnosed with cancer. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CI) for the association between PCB, their congener or their combinations and breast cancer. We evaluated age, race/ethnicity, age at menarche, body mass index (BMI; kg/m2), and lactation as potential confounders. Our results show a weighted geometric mean levels of 6 PCB congeners to be significantly higher in blood serum among women with breast cancer when compared to the rest of the study population. After adjusting for age, race, BMI, lactation, and age at menarche we found significant association of PCB 138 with breast cancer [odds ratios of 2.88; 95% confidence interval (CI): 1.14-7.30; 2.93, 95% CI: 1.04-8.26; and 3.43, 95% CI: 1.12-10.4] in women with higher body burdens of individual PCB congeners (> 50th percentile, 50th-75th percentile, and ≥75th percentile), respectively. After adjusting for age and race, it was also found that the sum of non-dioxin-like PCBs (074 and 118) in blood serum is significantly associated with breast cancer [OR of 1.14; 95% CI: 1.00-1.29]. In summary, our results suggest a possible link between environmental exposures to PCBs and increased risk of breast cancer among U.S. women.

#3434

Neighborhood obesogenic environment and the risk of prostate cancer: The Multiethnic Cohort.

Li Tao,1 Scarlett L. Gomez,1 Salma Shariff-Marco,1 Juan Yang,1 Song Yi Park,2 Cheryl Albright,3 Kristine Monroe,4 Brian Henderson,4 Laurence Kolonel,2 Loic Le Marchand,2 Lynne Wilkens,2 Iona Cheng1. 1 _Cancer Prevention Institute of California, Fremont, CA;_ 2 _University of Hawaii Cancer Center, Honolulu, HI;_ 3 _University of Hawaii School of Nursing, Honolulu, HI;_ 4 _University of Southern California, Los Angeles, CA_.

Background: Obesity is a potentially modifiable risk factor for advanced prostate cancer (PCa). However, the role of the neighborhood obesogenic environment, characterized by obesity-related built and social environment measures, has not been evaluated in relation to PCa risk.

Objective: To examine the association of neighborhood obesogenic environment with PCa risk in African American (AA), Japanese American (JA), Latino (LA), and White (WH) men in the California component of the Multiethnic Cohort (MEC) Study.

Method: Individual-level sociodemographic data and health behaviors were linked with neighborhood measures of built and social environment for 42,850 men in the MEC, who predominately live in Los Angeles County. A principal components factor analysis was used to create indices based on 10 measures—neighborhood SES, population density, total businesses, recreational facilities, retail food environment, restaurant environment, parks, street connectivity, commuting by car, and traffic density—resulting in 4 obesogenic environment indices: physically active, low SES urban, businesses density, and unhealthy food. During a mean follow-up of 15 years and through linkage to the California Cancer Registry, 4,775 incident PCa cases were identified. Multivariable Cox proportional hazards regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations between each index and PCa risk, overall and stratified by race/ethnicity and disease aggressiveness. For Latinos, additional stratified analyses were conducted by nativity.

Results: Associations between the obesogenic indices and PCa risk varied by race/ethnicity. WH residing in low business environments experienced lower PCa risk (quintile [Q] 1 [low] vs. Q5 [high]: HR, 0.56 [95% CI, 0.41-0.77], p-trend: 0.0002). Among AA, unhealthy food environments were associated with greater risk of aggressive disease (advance stage or Gleason score >=7) (more vs. no: HR, 1.36 [95% CI, 1.09-1.70], p-trend: 0.0026). Among LA, living in Hispanic enclaves was associated with reduced risk (Q3-5 [high] vs. Q1-2 [low]: HR, 0.86 [95% CI, 0.75-0.98]), but living in high business environments was associated with increased risk of PCa (Q1 [low] vs. Q5 [high]: HR, 1.35 [95% CI, 1.05-1.73], p-trend: 0.0002) for those born in the US (p-trend: 0.0059). Lower SES urban environments were associated with a decreased risk of non-aggressive disease among LA (Q5 [low] vs. Q1 [high]: HR, 0.78 [95% CI, 0.63-0.98], p-trend: 0.0026), and was more marked among foreign-born (p trend=0.0074) than US-born (p trend=0.1211).

Conclusions: Built and social environment factors are associated with PCa risk, but their associations varied by race/ethnicity, nativity (among LA), and disease aggressiveness. An unhealthy food environment was strongly associated with the risk of aggressive prostate cancer in AA men, warranting further investigation into the underlying mechanisms.

#3435

Prostate cancer in WTC respondents.

Dana Hashim, Paolo Boffetta, William Oh, Sylvain Wallenstein, Matthew Galsky, Emanuela Taioli. _Icahn School of Medicine at Mount Sinai, New York, NY_.

Background: Ten years following the 9/11/2001 attacks on the World Trade Center (WTC), an increased incidence of prostate cancer was reported in two separate cohorts of WTC rescue and recovery workers. Whether this increase is due to WTC-related exposures or increased surveillance is uncertain.

Methods: Prostate cancer cases and Gleason scores diagnosed after 9/11 from 2002 to 2014 were obtained from the WTC Health Program (n=180). Differences in proportions of Gleason scores 7+ (moderate to high-grade cancer) in WTC respondents and New York State were calculated based on the NYS Cancer Registry (NYSCR). An age-adjusted rate for Gleason scores of ≥7 was calculated using the indirect method based on NYSCR. Age- and race-matched prostate cancer non-WTC respondent controls (n=253) were obtained from Mount Sinai Hospital. We evaluated the effects of WTC-respondent status, exposure level, age, race, prostate-specific antigen (PSA) concentrations, and clinical stage on Gleason scores using log-linear regression. WTC exposure was analyzed by four categorical levels and continuously using a relative rank of potential carcinogen-related post-9/11 exposures. Differences in mean numbers of PSA screening visits were assessed using Student's t-test.

Results: The age-adjusted rate for a Gleason score ≥7 was 1.15 for WTC respondents (95% Confidence Interval (CI): 0.94, 1.36) compared to the NYSCR. WTC respondents < 40 years old had an 8-time higher proportion of aggressive cancers than expected based on NYSCR age-specific rates. The proportion of Gleason scores ≥7 was 0.33 (95% CI: -0.06, 0.12) higher in WTC respondents than NYSCR. WTC respondents had lower Gleason scores (Odds Ratio (OR): 0.90 (95% CI: 0.82, 0.99)), PSA values (OR: 0.79; 95% CI: 0.71, 0.89), but higher clinical stage (OR: 1.04; 95% CI: 0.92, 1.16) than controls. When Gleason score was adjusted for PSA and clinical stage, the OR for PSA testing was 1.04; 95% CI: 0.88, 1.23 and clinical stage was lower (OR: 0.82; 95% CI: 0.69, 0.98) in WTC-respondent cases. The mean numbers of PSA-screening visits were not different between WTC respondents and controls (t=0.64; P=0.52) and were not associated with Gleason scores ≥7 (OR: 1.0; 95% CI: 0.88, 1.13). Four-level exposure categories (OR: 0.99; 95% CI: 0.99, 1.00) and continuous rank (OR: 0.88; 95% CI: 0.73, 1.06) showed no associations with Gleason scores. Days spent at WTC (OR: 1.00; 95% CI: 1.00, 1.03) and working on the WTC debris pile (OR: 0.95; 95% CI: 0.86, 1.05) showed similar results.

Conclusions: Young WTC respondents had higher Gleason scores than the NYSCR. Number of PSA screening visits did not significantly differ between WTC respondents and controls and PSA values of WTC respondents were lower than controls. Aggressive prostate cancer was not associated with WTC exposure levels. Considering the high grade of prostate cancer in younger men and the long latency for prostate cancer development, continued monitoring is needed to determine alternative causes of elevated prostate cancer in the WTC population.

#3436

Exposure to long-term traffic-related air pollutants, NO2 and NOX, and breast cancer incidence: The Multiethnic Cohort.

Iona C. Cheng,1 Juan Yang,1 Chiuchen Tseng,2 Leo Lee,2 Jun Wu,3 Daniel Stram,2 Kristine Monroe,2 Loic Le Marchand,4 Scarlett Lin Gomez,1 Alice Whittemore,5 Salma Shariff-Marco,1 Beate Ritz,6 Anna Wu2. 1 _Cancer Prevention Institute of California, Fremont, CA;_ 2 _University of Southern California, Los Angeles, CA;_ 3 _University of California at Irvine, Irvine, CA;_ 4 _University of Hawaii Cancer Center, Honolulu, HI;_ 5 _Stanford University, Stanford, CA;_ 6 _University of California at Los Angeles, Los Angeles, CA_.

Introduction: California has one of the highest levels of air pollution in the nation. Vehicle exhaust contains a mixture of gases and particulate matter that are known to have mutagenic and carcinogenic effects. Our objective was to examine the effects of markers of traffic-related air pollutants on breast cancer risk among the California component of Multiethnic Cohort (MEC).

Methods: Residential addresses from baseline, 1993-1996, through 2010 for over 57,000 female California MEC participants were geocoded to latitude and longitude coordinates and used to estimate traffic-related air pollutant exposures of NO2 and NOX, using three complementary approaches: 1) land use regression (LUR) models based on passive diffusion measurements, land use patterns, roadway and traffic data, and satellite information (high spatial but low temporal resolution); 2) Bayesian kriging interpolation of state and national government air monitoring data (low spatial but high temporal resolution); 3) California line source dispersion model, version 4 (CALINE4) based on local traffic emissions, traffic volume, roadway geometry, meteorological conditions (high spatial and moderate temporal resolution). A total of 2,639 incident breast cancer cases (315 Japanese Americans, 830 Latinas, 974 African Americans and 511 whites) were identified by linkage to the California cancer registry. Cox proportional hazard regression was conducted to examine the long-term effects of NO2 and NOX (annual average exposure for up to 18 years), adjusting for age, race/ethnicity, education, health behaviors, established breast cancer risk factors, and neighborhood SES.

Results: For NO2, per unit (ppb) increase in the interquartile range, a non-statistically significant association with breast cancer risk was observed with the LUR model among all women (OR=1.04; 95% CI: 0.99-1.09). A significant positive association was seen in Japanese Americans (OR=1.20; 95% CI: 1.07-1.34; p=0.002) that met a conservative Bonferonni significance threshold (p=0.003), but no associations were seen in African Americans, Latinas, and Whites. For NOx, per unit (ppb) increase in the interquartile range, no significant increased risk of breast cancer was seen with the CALINE4 model among all women, but a positive association was seen in Japanese Americans (OR=1.10, 95% CI: 1.02-1.19; p=0.016). NO2 and NOx exposures based on the kriging model were not associated with breast cancer risk among all women or in specific racial/ethnic groups.

Conclusion: These preliminary findings suggest long-term NO2 and NOX exposure may influence breast cancer risk in Japanese Americans, using a LUR model of high spatial resolution and a CALINE4 model of high spatial and moderate temporal resolution. Future analyses will examine associations with other pollutants and differences in associations by neighborhood- and individual-level factors.

#3437

Cancer and all-cause mortality among white and blue collar workers in middle-aged and elderly chinese women in a prospective cohort study.

Bu-Tian Ji. _National Cancer Institute, Rockville, MD_.

Inequalities in health and mortality between white and blue collar workers have been well documented in western countries, particularly among men. Studies regarding occupation related mortality disparities in Asian populations including Chinese are limited. We included 74,941 40-70 years old Chinese women from the Shanghai Women's Health Study (SWHS) in an analysis of occupation and mortality. An in-person interview was conducted at enrolment to obtain lifetime job history as well as lifestyle and other potential risk factors for each participant. Each job title was coded in three digits according to the Chinese National Standard Occupation and Industry Codes Manual, and the categories of white and blue collar work were defined based on this coding. The information on mortality was provided by annual linkage to the Shanghai Vital Statistics Registry. Cox regression models were applied to evaluate the hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause and cause-specific mortality. In comparison with lifetime white collar workers, lifetime blue collar workers had 18% excess in all cause-mortality (HR=1.18, 95% CI=1.04-1.34) and 12% excess in cancer deaths (HR=1.12, 95% CI=0.93-1.35) after adjustment for lifestyle and other potential risk factors . A significantly increased risk was also associated with mortality from diabetes (HR=1.89, 95% CI=1.06-3.37). There were no statistically significant differences in mortality between white and blue collar workers for other causes of death including circulatory, respiratory, gastrointestinal or other diseases among this population. Our study provides suggestive evidence that blue collar workers may have a higher risk of total cancer mortality compared to white collar workers among women in Shanghai.

#3438

DNA methylation profiles of Helicobacter pylori strains from patients with gastric cancer and gastritis.

M. Constanza Camargo,1 Javier Torres,2 Jay V. Solnick,3 Castle Raley,4 Dylan Storey,3 Marina Becker,3 Roberto Torres,5 Allison Weis,3 Carol Huang,3 Nguyet Kong,3 Lori M. Hanson,3 Yongmei Zhao,4 Xiongfong Chen,4 Bao Tran,4 Bart Weimer,3 Charles S. Rabkin1. 1 _National Cancer Inst., Bethesda, MD;_ 2 _Instituto de Seguro Social, Mexico City, Mexico;_ 3 _Univ. California-Davis, Davis, CA;_ 4 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 5 _Univ. California-Davis and Escuela Nacional de Ciencias Biológicas, IPN, Mexico_.

BACKGROUND: Helicobacter pylori, a gram-negative bacterium associated with a spectrum of benign and malignant gastric conditions, is one of the most genetically variable pathogens. Its genome encodes a large number of DNA methyltransferases targeting specific motif sequences of ~4-15 bp. DNA base modifications epigenetically regulate gene expression and genome-wide methylation profiles (methylomes) have been hypothesized to be associated with virulence. A limited number of H. pylori methylomes have been published to date, precluding meaningful analyses of clinical correlates.

METHODS: We sequenced 30 H. pylori clinical isolates from Mexico City using Pacific Biosciences' Single Molecule Real-Time (SMRT) technology, a unique platform that simultaneously determines a wide range of DNA base modifications. The resulting methylome data were compared for the 15 strains from gastric cancer cases vs. the 15 from non-atrophic gastritis controls.

RESULTS: All 30 strains exhibited high levels of DNA methylation throughout their genomes. Three types of base modification were detected: N6-methyladenine (m6A), N4-methylcytosine (m4C) and 5-methylcytosine (m5C). m6A was more common than m4C, and m5C was rare. Over 100 different motifs were detected, including many novel motifs not found in the three reference strains. For most methylated motifs, >95% of all occurrences in a given genome were methylated. Individual strains had between 14 and 28 different motifs. Some of the methylated motifs were conserved across all (e.g., Cm6ATG) or almost all (e.g., Gm6ACT) strains, but the majority were found in only one or two strains. Two conserved motifs, GTm6AC and GTNNm6AC, were significantly (P<0.05) more common in cancer cases than controls. CONCLUSIONS: The H. pylori methylome varies widely across strains, in concert with the high plasticity of its genome. Epigenetic modifications may modulate bacterial pathogenicity. These intriguing observations warrant replication with larger numbers and multiple populations in different geographic areas. The inferred novel methyltransferases remain to be identified and functionally characterized. This report greatly increases the number of H. pylori strains with defined methylomes, and represents the first effort to associate these features with clinical outcome. Availability of these genomic and epigenomic data will enhance understanding of H. pylori pathogenesis.

#3439

Interaction between cutaneous human papillomavirus infection and telomere length in association with cutaneous squamous cell carcinoma.

Shalaka S. Hampras,1 Michael Pawlita,2 Massimo Tommasino,3 Jong Park,1 Pearlie K. Burnette,1 Neil A. Fenske,4 Basil A. Cherpelis,4 Dana E. Rollison1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _German Cancer Research Center, Heidelberg, Germany;_ 3 _International Agency for Research on Cancer-World Health Organization, Lyon, France;_ 4 _University of South Florida College of Medicine, Tampa, FL_.

Background: We previously reported that cutaneous human papillomavirus (HPV) infection was associated with significantly increased risk of cutaneous squamous cell carcinoma (SCC), while longer telomeres were associated with significantly reduced risk of SCC. We conducted further research to evaluate the interaction between cutaneous HPV and telomere length in association with SCC. Methods: Previously, a clinic based case-control (173 SCC cases and 300 controls) study was conducted, between 2007-2008, at the University of South Florida and Moffitt Cancer Center. HPV seropositivity (33 types) and DNA positivity (25 beta-HPV types) in eyebrow hairs (EB) and SCC tumors were measured using multiplex assays. Using quantitative PCR, relative telomere length was measured in peripheral blood leukocytes by determining the ratio of telomere repeat copy number to a single-copy gene copy number for each sample. For the present analyses, subjects with available data on telomere length and a) HPV serology (135 cases and 201 controls), b) HPV DNA in EB (130 SCC cases and 195 controls) and c) HPV DNA in SCC tumors (117 cases), were included. Association between telomere length and SCC was examined after stratification by HPV serostatus and HPV DNA status in EB, and odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using logistic regression, adjusting for age and gender. Results: Telomere length did not differ significantly between beta-HPV seronegative (mean=1.16, standard deviation (SD) =0.57) and seropositive controls [mean (SD) =1.24(0.75), P value=0.56], or by tumor HPV DNA status (P value=0.93). Longer telomere length was associated with significantly reduced risk of SCC among subjects seronegative to all HPV types or seropositive to a single beta1 HPV type (OR=0.01, 95% CI=0.001-0.10), while no association was observed among those seropositive to >1 beta1 HPV types (OR=1.01, 95% CI=0.95-1.07, Pinteraction=<0.0001). A significant interaction (Pinteraction=0.0004) was also observed between telomere length and DNA positivity for >=1 beta-HPV types in EB in association with SCC (OREB DNA negative=0.003, 95% CI=<0.001-0.09; OREB DNA positive=1.02, 95% CI=0.98-1.06). Further, longer telomere length was associated with 62% (OR=0.38, 95% CI=0.19-0.75, Pinteraction=0.004) and 55% (OR= 0.45, 95% CI=0.23-0.90, Pinteraction=0.01) reduced risk of SCC among subjects DNA negative for all beta-HPV types or positive for a single beta1 or beta2 type, respectively, while no associations were observed among those with EB DNA positivity for >1 beta1 or beta2 types (OR=1.02, 95% CI=0.98-1.07 for both groups). In summary, multiple beta-HPV infections showed significant statistical interaction with telomere length in association with SCC. Conclusion: Presence of cutaneous HPV infection may attenuate the protective effect of longer telomeres on the risk of SCC.

#3440

Time and regional incidence trends of human papillomavirus-associated oropharyngeal cancer in Ontario, Canada.

Wei Shi,1 Stephen F. Hall,2 Katrina Rey-McIntyre,1 Rebecca Griffiths,2 Wei Xu,3 Jie Su,3 Jeff Bruce,1 Shaohui Huang,4 Brian O'Sullivan,4 Kenneth W Yip,1 Fei-Fei Liu5. 1 _Ontario Cancer Institute, Toronto, Ontario, Canada;_ 2 _Surgical Oncology, Queen's University, Kingston, Ontario, Canada;_ 3 _Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada;_ 4 _Princess Margaret Cancer Centre and University of Toronto, Toronto, Ontario, Canada;_ 5 _Ontario Cancer Institute, Radiation Oncology, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada_.

Background: Despite the increasing incidence of human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) in the Western World, there are little data describing the change in HPV incidence within Ontario, Canada. We therefore assessed HPV-positivity in a retrospective study of 612 OPC patient samples.

Methods: Diagnostic formalin-fixed and paraffin embedded OPC patient samples belonging to two cohorts were retrieved from institutions across Ontario, Canada over two time periods: 1998-1999 (n=245) and 2003-2004 (n=367). In all samples, HPV status was assessed initially using p16 immunohistochemistry (IHC). For p16 negative and equivocal staining samples, HPV in situ hybridization (ISH; using an HPV subtype 16/18 probe) was performed as the final indicator for HPV positivity. HPV-positivity rate was then compared over time and geographic region.

Results: Overall, p16-positivity was observed in 52.3% (320 of 612) of the OPC patients. For the p16-negative (n=246) and p16-equivocal (n=46) samples, 25.3% (n=74) were positive by HPV16/18 ISH. The HPV infection incidence for the 1998-1999 cohort was 54.3% (133 of 245 samples; 95% CI: 53.9(47.4-60.2)). The HPV infection incidence increased significantly in the 2003-2004 cohort to 71.1% (261 of 367 samples; 95% CI: 71.4(66.5-76.0)) (p<0.0001). When divided based on geographical region, HPV incidence also increased significantly over time (Northern Ontario by 49%, p=0.04; Eastern Ontario by 23%, p=0.009; Southern Ontario by 17%, p=0.009; Central Ontario by 12%, p=0.05). There were no significant differences between urban and rural areas over the two cohort periods (urban p=0.8; rural, p=0.6).

Conclusions: HPV-associated OPC increased in incidence in Ontario, Canada, over a 6-year period from 1998 to 2004, which appeared to be applicable across the entire province, regardless of urban or rural regions. A composite HPV detection method using p16 in combination with HPV ISH identified an additional 25% of HPV-positive OPC cases.

#3441

Associations between lipopolysaccharide (LPS) and LPS pathway biomarkers and gallbladder cancer are modulated by markers of systemic inflammation.

Alison L. Van Dyke,1 Troy J. Kemp,2 Amanda F. Corbel,1 Bin Zhu,1 Yu-Tang Gao,3 Bingsheng Wang,4 Asif Rashid,5 Ming-Chang Sheng,6 Allan Hildesheim,1 Ann W. Hsing,7 Ligia A. Pinto,2 Jill Koshiol1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Leidos Biomedical Research, Inc., Frederick, MD;_ 3 _Shanghai Cancer Institute, Shanghai, China;_ 4 _Zhongshan Hospital, Department of General Surgery, Shanghai, China;_ 5 _MD Anderson Cancer Center, Houston, TX;_ 6 _Shanghai Cancer Center, Fudan University, Shanghai, China;_ 7 _Stanford University, Stanford, CA_.

Little is known about gallbladder cancer (GBC) etiology. Gallstones (GS) are a critical factor in GBC pathogenesis. Bacterial infections, such as Salmonella Typhi and Helicobacter pylori, also may contribute to the development of GBC. We previously observed strong associations between inflammation markers and GBC vs. GS patients and hypothesized that bacterial infections may explain in part associations between GBC and inflammation. In the current study of 41 GBC cases and 124 GS patients from Shanghai, China, we evaluated relationships between GBC and the bacterial infection-related proteins plasma lipopolysaccharide (LPS) and two LPS-pathway proteins, soluble CD14 (sCD14) and lipid binding protein (LBP), as measured by ELISA. Further, we explored the extent to which these associations were mediated through systemic inflammation. Logistic regression was conducted to assess associations of GBC with plasma LPS, sCD14, and LBP levels. To evaluate mediation through inflammation, an inflammation score was calculated by summing the value of 15 previously measured GBC-associated serum inflammation markers (β-coefficient multiplied by 1 if above the median or 0 if below the median) and included in the models. sCD14 and LBP levels (above vs. below the median) were strongly associated with GBC [Odds Ratio (95% Confidence Interval): 2.75 (1.27-6.29) for sCD14 and 2.83 (1.32-6.46) for LBP]. The highest vs. lowest tertiles of sCD14 and LBP were associated with a five-fold increased risk of GBC. These associations were attenuated when the inflammation score was entered into the model. Plasma LPS demonstrated a borderline association after adjustment for inflammation score [OR (95% CI): 2.46 (0.96-6.56)]. In contrast, the ORs for inflammation score remained >20 after adjusting for each bacteria-related marker. These findings suggest that bacterial infections may contribute to GBC etiopathogenesis through inflammatory pathways.

LPS, sCD14, and LBP and Gallbladder Cancer Relative to Gallstone Controls

---

LPS | Sex & Age

Adjusted OR (95% CI) | Sex, Age, &

Inflammation Score

Adjusted OR (95% CI)

Detectable Level | 1.00 | 1.00

Undetectable Level | 1.81 (0.85-3.80) | 2.46 (0.96-6.56)

sCD14 | Sex, Age, & Batch

Adjusted OR (95% CI) | Sex, Age, Batch,

& Inflammation Score

Adjusted OR (95% CI)

Below Median | 1.00 | 1.00

Above Median | 2.75 (1.27-6.29) | 1.05 (0.39-2.83)

1st Tertile | 1.00 | 1.00

2nd Tertile | 1.42 (0.45-4.71) | 0.44 (0.10-1.80)

3rd Tertile | 5.27 (1.98-16.06) | 1.37 (0.38-5.04)

p-trend | 0.0006 | 0.25

LBP | Sex, Age, & Batch

Adjusted OR (95% CI) | Sex, Age, Batch,

& Inflammation Score

Adjusted OR (95% CI)

Below Median | 1.00 | 1.00

Above Median | 2.83 (1.32-6.46) | 1.63 (0.64-4.28)

1st Tertile | 1.00 | 1.00

2nd Tertile | 1.95 (0.61-6.99) | 1.53 (0.37-7.01)

3rd Tertile | 5.01 (1.84-16.25) | 2.09 (0.58-8.71)

p-trend | 0.0008 | 0.25

#3443

Associations of immune-related conditions with squamous cell and basal cell skin cancer risk.

Elizabeth L. Yanik, Ruth M. Pfeiffer, D. Michal Freedman, Eric A. Engels. _National Cancer Institute, Bethesda, MD_.

Elevated risk of squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) of the skin has been identified in a number of clinical populations with conditions affecting the immune system, such as organ transplant recipients and non-Hodgkin lymphoma patients. However, because many other immune-related conditions are rare, their relationships with SCC and BCC have not been studied. We used Medicare claims to identify SCC and BCC cases in 2012 and to select controls in 2012 matched on sex and age with a 10:1 ratio. All subjects were of white race. For cases and controls, we used Medicare claims during 1999-2011 to identify 44 immune-related conditions (immunosuppressive conditions, autoimmune diseases, hematologic malignancies, and allergic diseases). The statistical significance of associations was determined after Bonferroni correction for multiple comparisons. In Medicare in 2012, there were a total of 470,756 SCC and 601,863 BCC cases. Of the 44 immune-related conditions included, SCC was associated with 41 (93%) and BCC was associated with 31 (70%). For most conditions, SCC was more strongly associated than BCC. We identified significant associations of SCC and BCC risk with well-characterized conditions, such as HIV, rheumatoid arthritis, and allergic rhinitis (SCC odds ratios (ORs) of 1.64, 1.29, and 1.29, respectively; BCC ORs of 1.60, 1.10, and 1.22; Table), as well as with poorly characterized or rare conditions, such as deficiency of cell-mediated immunity and polyarteritis nodosa (SCC ORs of 2.87 and 1.92; BCC ORs of 1.55 and 1.49). In conclusion, most immune-related conditions in our study indicated an elevated risk of non-melanoma skin cancer, particularly SCC. These findings provide evidence that immune dysfunction, as manifest across a wide range of conditions, may play an important role in development of SCC and BCC. Possible mechanisms could include direct effects of immune-related conditions or their treatments.

Results from a sample of the immune-related conditions assessed

---

|

Squamous cell carcinoma | Basal cell carcinoma

Immune-related conditions | N cases (%) | OR | 95% CI | p-value | N cases (%) | OR | 95% CI | p-value

Total | 470756 | |  | |

601863 | |

|

Immunosuppressive conditions | |  | |  | |  | |

Deficiency of humoral immunity | 1550 (0.33) | 2.55 | 2.41-2.69 | <1E-200* | 1426 (0.24) | 1.82 | 1.72-1.93 | 7.1E-99*

HIV | 276 (0.06) | 1.64 | 1.44-1.86 | 1.6E-14* | 366 (0.06) | 1.60 | 1.43-1.78 | 4.8E-17*

Deficiency of cell-mediated immunity | 53 (0.01) | 2.87 | 2.11-3.89 | 1.5E-12* | 37 (0.01) | 1.55 | 1.10-2.19 | 0.013

Autoimmune diseases | |  | |  | |  | |

Rheumatoid arthritis | 15754 (3.4) | 1.29 | 1.26-1.31 | 8.2E-188* | 16893 (2.8) | 1.10 | 1.09-1.12 | 3.7E-33*

Pernicious anemia | 10792 (2.3) | 1.13 | 1.11-1.15 | 9.8E-33* | 11218 (1.9) | 1.00 | 0.98-1.02 | 0.90

Psoriasis | 9765 (2.1) | 1.55 | 1.52-1.59 | <1E-200* | 9812 (1.6) | 1.23 | 1.20-1.25 | 2.0E-79*

Polymyalgia rheumatica | 8567 (1.8) | 1.28 | 1.25-1.31 | 6.4E-102* | 9623 (1.6) | 1.23 | 1.20-1.25 | 5.1E-78*

Ulcerative colitis | 3807 (0.81) | 1.30 | 1.26-1.35 | 1.5E-52* | 4524 (0.75) | 1.25 | 1.21-1.29 | 3.7E-44*

Crohn's disease | 3121 (0.66) | 1.64 | 1.58-1.71 | 4.8E-148* | 3226 (0.54) | 1.36 | 1.31-1.41 | 1.5E-61*

Hematologic malignancies and related conditions | |  | |  | |  | |

Non-Hodgkin lymphoma | 15165 (3.2) | 2.25 | 2.21-2.29 | <1E-200* | 13834 (2.3) | 1.65 | 1.62-1.68 | <1E-200*

Paraproteinemia & related disorders | 4876 (1.0) | 1.57 | 1.52-1.62 | 1.1E-189* | 5312 (0.88) | 1.42 | 1.38-1.46 | 6.5E-128*

Aplastic anemia | 3756 (0.80) | 1.51 | 1.46-1.56 | 9.2E-124* | 3433 (0.57) | 1.14 | 1.10-1.18 | 1.7E-13*

Multiple myeloma | 2050 (0.44) | 1.66 | 1.58-1.74 | 4.0E-102* | 1865 (0.31) | 1.22 | 1.17-1.28 | 2.4E-16*

Leukemia | 1370 (0.29) | 1.92 | 1.81-2.04 | 4.5E-112* | 1235 (0.21) | 1.37 | 1.29-1.45 | 3.5E-25*

Allergic diseases | |  | |  | |  | |

Allergic rhinitis | 53334 (11) | 1.29 | 1.28-1.30 | <1E-200* | 63566 (11) | 1.22 | 1.21-1.23 | <1E-200*

Asthma | 38871 (8.3) | 1.13 | 1.12-1.14 | 1.3E-106* | 44180 (7.3) | 1.02 | 1.01-1.03 | 2.3E-05*

Atopic dermatitis/eczema | 4338 (0.92) | 1.55 | 1.50-1.60 | 1.7E-160* | 4185 (0.70) | 1.22 | 1.18-1.26 | 1.7E-33*

*Associations were statistically significant at a Bonferroni adjusted alpha level of 0.000568 (=0.05/88 statistical tests).

#3444

Analysis of human papillomavirus 16 variants and risk for cervical cancer in Chinese population.

Dong Hang,1 Yin Yin,1 Jing Han,1 Jie Jiang,1 Hongxia Ma,1 Shuanghua Xie,2 Xiaoshuang Feng,2 Kai Zhang,3 Zhibin Hu,1 Shen Hongbing,1 Gary M. Clifford,4 Min Dai,2 Ni Li2. 1 _Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China;_ 2 _National Office for Cancer Prevention and Control, Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, China;_ 3 _Department of Cancer Prevention, Cancer Institute and Hospital, Chinese Academy of Medical Sciences, Beijing, China;_ 4 _Infections and Cancer Epidemiology, International Agency for Research on Cancer, Lyon, France_.

HPV16 is the most carcinogenic HPV type, but only a minority of HPV16 infections progress to cancer. Intratype genetic variants of HPV16 have been suggested to confer differential carcinogenicity. To investigate risk implications of HPV16 variants among Chinese women, a case-control study was conducted with 298 cervical cancer patients and 85 controls (all HPV16-positive). HPV16 isolates were predominantly of the A variant lineage, and variants of A4 (previously named "Asian") sublineage were common. A4/Asian variants were significantly associated with increased risk of cervical cancer compared to A1-3 (OR = 1.72, 95% CI = 1.04-2.85). Furthermore, a meta-analysis including 703 cases and 323 controls from East Asia confirmed the association (OR = 2.82, 95% CI = 1.44-5.52). In conclusion, A4 variants appear to predict higher risk of cervical cancer among HPV16-positive women, which may provide clues to the genetic basis of differences in the carcinogenicity of HPV16 variants.

#3445

C-reactive protein and lung cancer risk: a Mendelian randomization analysis.

Orestis A. Panagiotou,1 Neil E. Caporaso,1 Maria T. Landi,1 Nilanjan Chatterjee,2 NCI Lung Cancer GWAS Group. 1 _National Cancer Institute, Rockville, MD;_ 2 _Johns Hopkins University, Baltimore, MD_.

Background: Elevated levels of C-reactive protein (CRP) are associated with increased risk of lung cancer in observational studies. We investigated the causal relation between CRP and lung cancer using previously discovered single-nucleotide polymorphisms (SNPs) associated with CRP levels as genetic instruments.

Methods: We analyzed four lung cancer case-control studies (EAGE, PLCO, ATBC, CPSII) with available GWAS data (total 5,713 cases and 5,736 controls). We identified 21 SNPs associated with CRP levels at P<10-7 in genome-wide association studies (GWAS) and constructed a weighted polygenic score (PGS) multiplying the number of CRP-increasing alleles in each locus by the GWAS-reported effect of the respective allele on CRP levels; this is the largest number of CRP SNPs used in a Mendelian randomization for lung cancer to date. We first examined whether the PGS is associated with smoking phenotypes (smoking status, cigarettes per day, pack-years, smoking duration). Subsequently, we used this PGS as an instrumental variable and estimated the odds ratio (OR) and 95% confidence interval (CI) of lung cancer per standard deviation change in the PGS using logistic regression and adjusting for age, gender, and smoking. We conducted subgroup analyses for different types of lung cancer histologies. Analyses were performed separately for each study and study-specific results were combined through random-effect meta-analysis.

Results: The PGS was not significantly associated with the number of cigarettes smoked per day (P=0.621), pack-years (P=0.829), smoking status (0.739), or smoking duration (P=0.743). Overall, there was no association between PGS and lung cancer risk in EAGLE (OR=1.02, 95% CI 0.77-1.36; P=0.876), PLCO (OR=0.94, 95% CI 0.68-1.31; P=0.722), ATBC (OR=0.80, 95% CI 0.59-1.07; P=0.132), or CPSII (OR=0.61, 95% CI 0.37-1.02; P=0.059) as well as after meta-analysis (OR=0.88, 95% CI 0.73-1.05; P=0.149; I2=17.3%]. Meta-analysis results were similar for adenocarcinoma (OR=0.94, 95% CI 0.74-1.20; P=0.630; I2=9.3%), squamous cell carcinoma (OR=1.03, 95% CI 0.79-1.34; P=.844; I2=0%), small-cell lung cancer (OR=0.97, 95% CI 0.62-1.50; P=0.882; I2=34.9%), and other non-small cell lung cancer (OR=0.93 (0.53-1.65), P=0.815, I2=59.2%).

Conclusions: Genetically elevated CRP levels are not associated with the risk lung cancer. Hence, our results provide no evidence of causality between CRP and lung cancer.

#3446

Aspirin, non-steroidal anti-inflammatory drugs (NSAIDs) and the risk of glioma: Results from the Glioma International Case Control Study.

Rose K. Lai,1 Renke Zhou,2 E. Susan Amirian,2 Christoffer Johansen,3 Michael E. Scheurer,2 Georgina N. Armstrong,2 Ching C. Lau,2 Elizabeth B. Claus,4 Jill S. Barnholtz-Sloan,5 Dora Il'yasova,6 Joellen Schildkraut,7 Francis Ali-Osman,7 Siegal Sadetzki,8 Richard Houlston,9 Robert B. Jenkins,10 Daniel Lachance,10 Sara H. Olson,11 Jonine L. Bernstein,11 Ryan T. Merrell,12 Margaret R. Wrensch,13 Faith G. Davis,14 Sanjay Shete,15 Christopher I. Amos,16 Beatrice S. Melin,17 Melissa Bondy2. 1 _USC, CA;_ 2 _Baylor College of Medicine, Houston, TX;_ 3 _University of Copenhagen, Denmark;_ 4 _Yale University School of Medicine, CT;_ 5 _Case Western Reserve University, OH;_ 6 _Georgia State University, GA;_ 7 _Duke University, NC;_ 8 _Tel-Aviv University, Israel;_ 9 _Institute of Cancer Research, United Kingdom;_ 10 _Mayo Clinic, MN;_ 11 _Memorial Sloan Kettering, NY;_ 12 _Northshore University Health System, IL;_ 13 _UCSF, CA;_ 14 _University of Alberta, Alberta, Canada;_ 15 _MD Anderson Cancer Center, Houston, TX;_ 16 _Dartmouth University, Houston, TX;_ 17 _Umea University, Sweden_.

Background: Numerous epidemiologic studies have examined the association between aspirin (ASA), non-steroid anti-inflammatory drugs (NSAIDs) and the development of glioma, but the results have been inconsistent. The goal of this study was to evaluate the relationship between the intake of these drugs and glioma risk in a large, international case-control study.

Methods: Between 2010 and 2015, the Glioma International Case-Control Study (GICC) recruited newly diagnosed glioma cases and matched controls in 14 different sites across five countries. Each subject was interviewed using a standardized questionnaire to obtain NSAIDs and ASA use. We examined the associations between ever use (at least > 6 months), duration of drug use and glioma histology. Ever use data on 4533 glioma cases and 4171 controls was combined using maximum likelihood estimation/restricted maximum likelihood meta-analysis methods. Furthermore, based on a priori hypotheses, we performed subgroup analyses based on gender and glioma histological grades.

Results: Use of ASA for > 6 months was associated with a 33% lower glioma risk compared to those who never took it (adjusted Meta-OR 0.67, 95% CI 0.54-0.83). Duration of intake showed a significant trend test (p < 0.0001), with ORs became lower for increasing number of years of ASA use. In subgroup analyses, intake of ASA was significantly associated with glioma risk in both men and women (adjusted Meta-OR = 0.65, 95% CI 0.51-0.84 for men; adjusted Meta-OR = 0.74, 95% CI 0.58-0.93 for women). ASA intake was protective for grade IV glioma (glioblastoma) and grade II/III glioma (adjusted meta-OR 0.63, 95% CI 0.5-0.8 for glioblastoma; adjusted meta-OR 0.67, 95% CI 0.50 - 0.89 for grade II/III glioma). For NSAIDs intake, ever use > 6 months was not associated with glioma risk (adjusted meta-OR 0.87, 95% CI 0.71-1.07). However, NSAIDs use was protective for women (adjusted meta-OR 0.72, 95% CI 0.55-0.93) in subgroup analyses but not for men (adjusted meta-OR 1.03; 95% CI 0.86-1.23). The interaction between gender, NSAIDs and glioma risk was significant (p-value 0.0076).. Sensitivity analyses excluding those who took ASA or NSAIDs within the past 12 months for headache , and the removal of proxy respondents did not change our results.

Conclusion: ASA was associated with a significant protective effect for glioma, but NSAIDs were only associated with reduced glioma risk in women. Given the possibility of recall bias in case-control studies of brain tumors, we may verify dosage and duration of drug intake in those countries with electronic pharmacy records within the GICC.

#3447

Evaluation of NIH funding for metabolomics research in epidemiologic studies: opportunities and challenges.

Krista A. Zanetti,1 Mukesh Verma,1 Scott Rogers,1 Majda Haznadar,2 Padma Maruvada,3 Holly L. Nicastro,4 Mary C. Playdon,5 Sharon A. Ross6. 1 _National Cancer Institute, Division of Cancer Control and Population Sciences, Rockville, MD;_ 2 _National Cancer Institute, Center for Cancer Research, Bethesda, MD;_ 3 _National Institute of Diabetes and Digestive and Kidney Disorders, Division of Digestive Diseases and Nutrition, Bethesda, MD;_ 4 _National Heart, Lung, and Blood Institute, Division of Cardiovascular Sciences, Bethesda, MD;_ 5 _National Cancer Institute, Division of Cancer Epidemiology and Genetics, Rockville, MD;_ 6 _National Cancer Institute, Division of Cancer Prevention, Rockville, MD_.

Metabolomics, an emerging area of interest in the field of epidemiology, has significant potential to evaluate the effects of nutritional, environmental, and pharmaceutical exposures; conduct risk assessments; predict disease development; and diagnose diseases. To understand current funding and potential gaps for population-based metabolomics research, we cross-sectionally evaluated the National Institutes of Health (NIH)-supported research grant awards related to metabolomics and epidemiology that were active on February 9, 2015. The grant portfolio was analyzed using NIH's Query View Report (QVR) software, using relevant search terms, which included metabolome, metabonome, metabolomic(s), metabonomic(s), metabolic profile, metabolite profile, metabolic signature, glycomic(s), and lipidomic(s). Basic descriptive statistics on technology platform, approach, disease phenotype, biospecimen type, exposure, study design, and population were examined. The search terms initially identified 473 grants. After exclusion of grants with <100 cases and non-human subjects research, 102 grants remained. At the point in which the grants were coded, additional exclusions were made based on the same criteria (<100 cases and non-human subject research), and on more detailed review, 68 grants remained in the final analysis. We observed that the majority of currently funded NIH grants focus on cancer (22%), followed by diabetes (21%) and cardiovascular disease (13%), and primarily examine nutritional (24%) and drug/treatment (18%) exposures. Of the 15 funded cancer metabolomics studies, the most common organ sites investigated were breast (27%) and prostate (20%), followed by lung, colorectal, esophageal, oral cancer and lymphoma. Additionally, the primary epidemiologic study designs used in the funded grants were case-control (25%), cohort (21%), and nested case-control studies (19%), and intervention trials (19%); and they mainly use plasma (54%), serum (21%) and urine (19%) biospecimens. With regard to metabolite profiling, the majority of grants use both targeted (44%) and untargeted (44%) approaches, with the primary analytical technique being liquid chromatography-mass spectrometry (LC-MS) (74%). The least used analytical technique for these studies was NMR (9%). This descriptive analysis provides important insights into the current NIH funding patterns for metabolomics research with an epidemiologic study design component, which allowed us to identify several challenges and research opportunities pertaining to the field.

#3448

ERG-negative index tumor status combined with obesity predict prostate cancer progression in Caucasian American prostate cancer patients.

Jennifer Cullen,1 Denise Young,1 Yongmei Chen,1 Michael S. Degon,2 Wagner Baptiste,2 James S. Farrell,2 Jason S. Sedarsky,2 Huai-Ching Kuo,1 Jacob Kagan,3 Sudhir Srivastava,3 Inger L. Rosner,4 Gyorgy Petrovics,1 Albert Dobi,1 David G. McLeod,1 Shiv Srivastava,1 Isabell A. Sesterhenn5. 1 _Center for Prostate Disease Research, Uniformed Services University of the Health Sciences and Walter Reed National Military Medical Center, Rockville, MD;_ 2 _Walter Reed National Military Medical Center, Urology Service, Bethesda, MD;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Walter Reed National Military Medical Center, Bethesa, MD;_ 5 _Joint Pathology Center, Silver Spring, MD_.

Introduction: and Objectives: A significantly lower prevalence of ERG oncoprotein has been reported for African American (AA) compared to Caucasian American (CA) prostate cancer (PCa) patients. This difference has been observed when examining prostate cancer (PCa) index tumors as well as for comparisons of non-index tumor foci. To evaluate the potential of ERG status in predicting disease progression, this study examined a large cohort of AA and CA PCa patients within a racially diverse, equal access, military health care center.

Methods: Representative sections of whole mounted prostatectomy specimens from AA (n=373) and CA (n=684) patients were evaluated for ERG oncoprotein status by immunohistochemistry. Demographic, clinical, and pathologic patient features, including body mass index, were compared across ERG status (positive vs. negative). Time to event regression analysis was used to model ERG status as a putative predictor of biochemical recurrence (BCR) and distant metastasis.

Results: A significantly lower prevalence of ERG positivity was noted in both index and non-index tumors of AA patients as compared to CA patients. Among CA patients only, ERG-negative index tumors were associated with greater odds of developing BCR and metastasis. Similarly, among CA patients only, having an ERG negative index tumor and being obese at time of prostatectomy was strongly associated with developing biochemical recurrence and metastasis.

Conclusions: These provocative data warrant further investigation of the combined roles between race, obesity, and ERG status on PCa progression. Despite a robust sample size of AA men, the absence of statistically significant associations between ERG and obesity status on disease progression among AA patients could be attributed to insufficient statistical power.

Source of Funding: This research was supported by the National Cancer Institute R01CA162383 grant to SS, by the Center for Prostate Disease Research and by the EDRN/NCI ACN12011-001 to GP, AD, IAS, DGM and SS.

#3449

Placental characteristics and risk of cryptorchidism among populations at high and low risk of testicular germ cell tumors.

Armen A. Ghazarian,1 Britton Trabert,1 Barry Graubard,1 Matthew Longnecker,2 Mark Klebanoff,3 Katherine McGlynn1. 1 _DCEG, NCI, NIH, Bethesda, MD;_ 2 _NIEHS, NIH, Research Triangle Park, NC;_ 3 _Nationwide Children's Hospital, Columbus, OH_.

Background: Testicular germ cell tumors, which are significantly more common among white men than black men in the U.S., are thought to be part of a Testicular Dysgenesis Syndrome (TDS) that also includes impaired fertility, hypospadias and cryptorchidism. The etiology of the TDS conditions is not well understood, but the in-utero milieu is likely to be a significant factor. As the placenta is a critically important determinant of the in-utero milieu, placental characteristics may play a role in determining risk of the TDS conditions. Prior evidence suggests that placental weight, in particular, may be associated with cryptorchidism.

Methods: We examined placental characteristics (weight, thickness, placental weight/birth weight ratio, infarct size, disorders of membrane development, presence of macrophages in the amniotic fluid) and cryptorchidism in a nested case-control study within the Collaborative Perinatal Project (CPP). The CPP was a maternal-child cohort study conducted in 12 medical centers in the U.S. between 1959 and 1965. Diagnoses of cryptorchidism made during the first year of life were included in the case group. The analysis contrasted 413 (232 white, 181 black) boys with cryptorchidism to 23,799 (12,092 white, 11,707 black) boys without cryptorchidism. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression, stratified by race.

Results: Among white boys, cryptorchidism was significantly associated with circummarginate placenta (OR: 1.7, 95% CI: 1.1-2.7) or a combination of circummarginate/circumvallate (OR: 1.3, 95% CI: 1.0-1.8) placenta. Among black boys, cryptorchidism was significantly associated with having at least one placental infarct greater than 3 cm (OR: 2.3, 95% CI: 1.1-4.8) and with placental weight in the lower (OR: 1.7, 95% CI: 1.1-2.6) or higher (OR: 1.5, 95% CI: 1.0-2.3) tertile compared with the middle tertile. No significant associations were found between cryptorchidism and the other placental characteristics in either white or black boys.

Conclusion: These results suggest that there may be associations between cryptorchidism and certain placental characteristics which differ between white and black births. The race-specific findings suggest an area where future research may prove fruitful.

#3450

Epidemiological factors associated with breast cancer in young women.

Allyson L. Toro,1 Matthew T. Hueman,2 Craig D. Shriver,2 Rachel E. Ellsworth2. 1 _Windber Research Institute, Windber, PA;_ 2 _Murtha Cancer Center, Windber, PA_.

Background: Although breast cancer in young women (YW, <40 years) only accounts for ~7% of cases diagnosed annually, tumors in young patients have more aggressive characteristics and higher mortality rates. The cost of breast cancer increases significantly with more aggressive tumors in terms of treatment costs, physical and psychosocial effects on the patient and her family, and lost productivity. Improved understanding of factors associated with etiology of breast cancer in YW is critical to developing more effective prevention strategies and improved treatments, reducing the burden of breast cancer in this population.

Methods: The Clinical Breast Care Project database was queried to identify all YW with invasive breast cancer or benign breast conditions diagnosed after surgical evaluation between 2001 and 2014 that had questionnaire data available from the time of diagnosis. Epidemiological factors including reproductive and health history and lifestyle choices were using Student's t-tests and chi-square analysis with P<0.05 defining significance.

Results: Neither ethnicity nor family history differed significantly between YW with and without breast cancer. Reproductive factors that differed significantly between groups include longer use of oral contraceptives (P<0.001, 93 compared to 66 months), older age at first full-term pregnancy (FFTP, P=0.002, 24.5 compared to 22.7 years), and longer duration of breastfeeding in parous YW (P=0.024, 15.1 compared to 10.4 months) in YW with invasive breast cancer compared to those with benign conditions. YW with invasive breast cancer were also significantly more likely to be married (P=0.043, 78% compared to 68%) and less likely to exercise (P=0.003, 31% compared to 17%) compared to those with benign breasts.

Conclusions: Of the factors evaluated, including reproductive history, lifestyle choices, and co-morbidities, only five differed significantly between YW with and without invasive breast cancer. Paradoxically, breastfeeding, which has been associated with reduced the risk of breast cancer, was higher in YW who developed invasive breast cancer. In contrast, parity and use of oral contraceptives are associated with transient increases in breast cancer risk. The older age at FFTP of YW with invasive breast cancer combined with the significantly longer exposures to oral contraceptives may create an environment within the breast favorable to tumor initiation and progression.

#3451

Breast cancer risk factor associations by loss of E-cadherin tumor tissue expression: A pooled analysis of 5,896 cases in 12 studies from the Breast Cancer Association Consortium (BCAC).

Hisani N. Horne,1 Hannah Oh,1 Mark E. Sherman,1 Maya Palakal,1 Stephen H. Hewitt,1 Marjanka Schmidt,2 Javier Benitez,3 Roger Milne,4 Hermann Brenner,5 Heli Nevanlinna,6 Arto Mannermaa,7 Georgia Chenevix-Trench,8 Fergus Couch,9 Peter Devilee,10 Diana Eccles,11 Maartje Hooning,12 Anthony J. Swerdlow,13 Nick Orr,13 Melissa A. Troester,14 Renata Cora,15 Paul D. Pharoah,16 Montserrat Garcia-Closas,1 Jonine D. Figueroa17. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Netherlands Cancer Institute, Antoni Van Leeuwenhoek Hospital, Amsterdam, Netherlands;_ 3 _Spanish National Cancer Research Centre, Madrid, Spain;_ 4 _The University of Melbourne, Melbourne, Australia;_ 5 _German Cancer Research Center, Heidelberg, Germany;_ 6 _University of Helsinki and Helsinki University Hospital, Helsinki, Finland;_ 7 _University of Eastern Finland, Kuopio, Finland;_ 8 _QIMR Berghofer Medical Research Institute, Brisbane, Australia;_ 9 _Mayo Clinic Cancer Center, Rochester, MN;_ 10 _Leiden University Medical Center, Leiden, Netherlands;_ 11 _Southampton General Hospital, Southampton, United Kingdom;_ 12 _Erasmus MC Cancer Institute, Rotterdam, Netherlands;_ 13 _The Institute of Cancer Research, London, United Kingdom;_ 14 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 15 _independent contractor, CT(ASCP), MB(ASCP), Stamford, CT;_ 16 _University of Cambridge, Cambridge, United Kingdom;_ 17 _University of Edinburgh, Edinburgh, United Kingdom_.

Purpose: Expression of the tumor suppressor gene E-cadherin is diminished in lobular breast cancers and has been implicated in epithelial mesenchymal transition. We assessed risk factor associations for breast cancer stratified by low vs. high E-cadherin protein expression in a pooled analysis within the Breast Cancer Association Consortium (BCAC) studies.

Methods: E-cadherin tumor tissue staining was performed centrally at the NCI on formalin-fixed paraffin-embedded tissue microarray (TMA) sections representing 6,010 breast cancer patients from 12 US and European BCAC studies. TMAs were digitally scanned and scored using the SlidePath Digital Image Hub (Leica Biosystems, Wetzlar, Germany). For 5,896 cancers with evaluable tumors, E-cadherin was visually scored as estimated percent of positive cells times stain intensity (0, 1+, 2+, 3+) (score range 0-300). E-cadherin low was defined as tumors with a score < 100. Risk factor associations for low vs. high E-cadherin expressing tumors were evaluated by logistic regression, adjusted for age and study site, and stratified by ER status and histologic subtype. To assess the consistency in results by study, meta-analyses were performed using the random-effects model. All statistical tests were two-sided.

Results: E-cadherin low cancers comprised 20% of tumors and were associated with lobular histology, well/moderately differentiated cancers, > 2cm in size, and HER2-negative status (χ2, P < 0.003 for all factors). E-cadherin low status was associated with family history of breast cancer (FH) (OR=1.32, 95% CI=1.09-1.60, P-het=0.005) and ever use of menopausal hormones (OR=1.26, 95% CI=1.02-1.56, P-het=0.03). Study specific meta-plots showed consistent effects across studies for menopausal hormone therapy (I2=0.0%, p=0.64); however, E-cadherin loss by FH did show evidence of heterogeneity by study (I2=54%, P=0.03). Differences in E-cadherin expression remained significant for FH, and menopausal hormone use when further adjusted for ER (P < 0.05). We also found relationships with E-cadherin loss to vary by BMI, number of live births, age at first birth, and oral contraceptive use in stratified analysis by ER status and histologic subtype.

Conclusion: This large pooled analysis shows that breast cancer risk factor associations may differ by E-cadherin expression independent of ER status, suggesting that it may represent a marker of etiologic heterogeneity.

#3452

Antidepressant use and risk of central nervous system metastasis.

Megan Herr, Nimish Mohile, Edwin van Wijngaarden, Edward Brown, David Q. Rich. _University of Rochester, Rochester, NY_.

CNS metastasis is the spread of a primary cancer to the CNS and occurs in up to 25% of cancer patients. Antidepressant therapy, used in 15-30% of cancer patients, affects the blood-brain barrier, potentially making patients more susceptible to CNS metastasis. This hypothesis is supported by an experimental study reporting increased CNS metastasis from breast cancer in mice receiving fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRIs). However, no human studies have looked at this association. We conducted a case-control study to examine whether antidepressants, and specifically SSRIs, increased the relative odds of CNS metastasis.

We identified cancer patients diagnosed with breast cancer, melanoma, and non-Hodgkin lymphoma (NHL) at one academic cancer center in Rochester, NY between 2005 and 2013. We ascertained 189 patients with CNS metastasis (cases) and 945 patients without CNS metastasis (controls). Cases of CNS metastasis were identified by the ICD-9 code 198.3 secondary malignant neoplasm of brain and spinal cord. Cases and controls were classified with regard to their antidepressant use based on medical chart review. Using multivariable logistic regression, we estimated the relative odds of CNS metastasis associated with 'any antidepressant use' (any antidepressant irrespective of class), 'any SSRI use' (SSRI use, nonexclusively), and 'exclusive SSRI use' (only SSRI use and no other class of antidepressant), all compared to 'no antidepressant use'. Subset analyses were planned a priori for breast cancer and melanoma; subset analyses could not be conducted for NHL due to the small sample size.

Both cases and controls had a median age of 53 years (range: 25-94 years and 15-89 years, respectively). The prevalence of 'any antidepressant use' was 28.6% in cases and 25.7% in controls, whereas SSRIs were used in 16.9% of cases and 15.6% of controls. Among all patients, 'any SSRI use' was not associated with CNS metastasis (odds ratio (OR) = 1.21; 95% confidence interval (CI) = 0.74, 1.97). However, among breast cancer patients, 'any SSRI use' was associated with a 1.73 times greater odds of CNS metastasis (95% CI = 0.75, 4.04). No consistent patterns of association were observed in the analyses of other cancer subsets or exposure measures.

This was the first study to examine the association between antidepressant use and CNS metastasis. We did not observe clear patterns of association which may be due in part to the small sample size in many of our analyses. However, we believe that the increased odds in the breast cancer subset warrants further investigation as this is consistent with the animal research.

#3453

Medical history, lifestyle, and family history exposures and chronic lymphocytic leukemia outcome.

Sara Beiggi, James M. Foran, O'Byrne Megan, Celine M. Vachon, Timothy G. Call, Neil E. Kay, Tait D. Shanafelt, James R. Cerhan, Susan L. Slager. _Mayo Clinic College of Medicine, Rochester, MN_.

Background: Associations of medical history, lifestyle, and family history of hematological disorders with risk of chronic lymphocytic leukemia (CLL) have been previously established (1), but these exposures have not been well-studied as contributing factors to CLL prognosis. Here we investigate the association of these exposures with time to first treatment (TTFT) in 730 CLL patients.

Methods: Newly diagnosed CLL patients seen at the Mayo Clinic between 2002 and 2012 were enrolled. CLL was defined according to the 1996 NCI-Working Group criteria (2). Risk factor data (including body mass index, smoking, alcohol use, sun exposure, having lived on a farm, any allergy, asthma, eczema, blood transfusion, diabetes, rheumatoid arthritis, and family history of hematological malignancy) were collected from self-administered questionnaires. Clinical and prognostic characteristics for CLL were extracted from medical records. Analyses were performed using Cox regression with first treatment as the event. Hazard ratios (HR) and 95% confidence intervals were estimated. We used Bonferroni correction for multiple testing with statistical significance at 0.004.

Results: The median age at CLL diagnosis was 63 years (range 36 - 89); 65% were male, and 95% had a Rai stage< III. The median follow up time was 6.3 years, and 40% received treatment. In unadjusted analyses, patients with any history of allergies had significantly longer TTFT (HR=0.66, 95% CI: 0.51-0.85, P=0.001). We also observed borderline significance with history of asthma (HR=0.53, 95% CI: 0.34-0.85, P=0.008). These associations held after adjusting for important prognostic factors including age, sex, Rai stage, and IGVH mutational status. The other risk factors had non-significant associations (P>0.1).

Conclusions: We observed an improved CLL prognosis in patients with a history of any allergies or asthma. These results were independent of other prognostic markers and suggest that an overactive IgE level from environmental exposures may provide insight into the progression of CLL.

Literature Cited

1. Slager SL, Benavente Y, Blair A, Vermeulen R, Cerhan JR, Costantini AS, et al. Medical history, lifestyle, family history, and occupational risk factors for chronic lymphocytic leukemia/small lymphocytic lymphoma: the InterLymph Non-Hodgkin Lymphoma Subtypes Project. J Natl Cancer Inst Monogr. 2014 Aug;2014(48):41-51.

2. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S, et al. National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood. 1996 Jun 15;87(12):4990-7.

#3455

Association of oral microbiome with lung cancer risk: Results from the Southern Community Cohort Study.

Qiuyin Cai,1 Jirong Long,1 Hua Xie,2 Xiaofei Wang,3 Jie Wu,1 Regina Courtney,1 Xiao Ou Shu,1 Wei Zheng,1 William J. Blot1. 1 _Vanderbilt University Medical Center, Nashville, TN;_ 2 _Meharry Medical College, Nashville, TN;_ 3 _Tennessee State University, Nashville, TN_.

Lung cancer is the leading cause of cancer death in the United States. The lungs are inhabited by diverse microbial communities. Bacterial infection in the lungs, such as a history of pneumonia and tuberculosis, has been associated with increased risk of lung cancer. Recent studies have found that the oral microbiome is the primary source of the bacterial microbiota in the lungs and that the bacterial species detected in the lungs overlap those found in the mouth. Therefore, readily-accessible oral samples may be used to investigate the role of bacteria in lung cancer etiology. In this study, we evaluated the association of the oral microbiome with subsequent risk of developing lung cancer. We conducted a nested case-control study using resources of the Southern Community Cohort Study, a well-characterized prospective cohort study of approximately 86,000 adult men and women, two-thirds of whom are African American. DNA was isolated from pre-diagnostic mouth rinse samples of 177 lung cancer cases and 177 controls matched on age, race, sex, smoking, and date of sample collection. The V4 domain of microbial 16S rRNA gene was sequenced. Sequencing libraries were prepared using the NEXTflex 16S V4 Amplicon-Seq Kit and sequenced with paired-end 150 bp using Illumina MiSeq. Sequencing data were processed using QIIME, and sequence reads were clustered into Operational Taxonomic Units (OTUs). On average, 82,254 sequencing reads were obtained for each sample. Duplicated quality control samples (n=18; 2 samples each repeated for 9 times) showed very similar microbiome compositions, indicating high data quality. A total of 693 OTUs were observed and classified to 11 phyla. The observed composition was similar to the oral microbiome data from other studies, including the Human Microbiome Project. Multiple oral bacteria differed between lung cancer cases and controls. For example, Order CW040 was associated with increased lung cancer risk with OR of 3.64 (P=0.0098). Family Tissierellaceae and Genus Parvimonas were associated with decreased risk of lung cancer with ORs of 0.42 (P=0.0025) and 0.41 (P=0.0014), respectively. Some of the associations differed by race. Among the lung cancer-associated oral bacteria, Family Fusobacteriaceae, Family Neisseriaceae, and Order Bacteroidales were among the most abundant bacteria found in the lungs. In summary, our study suggests that certain bacteria in the mouth are associated with substantial development of lung cancer and that the oral microbiome plays an important role in lung cancer etiology. Further studies with a larger sample size and with more comprehensive investigation are needed to confirm these findings.

#3456

Polychlorinated biphenyls and relative telomere length in the Anniston Community Health Survey: A cross-sectional study.

Catherine Callahan,1 Marian Pavuk,2 Xuefeng Ren,1 James R. Olson,1 Matthew R. Bonner1. 1 _University of Buffalo, Buffalo, NY;_ 2 _Centers for Disease Control and Prevention, Atlanta, GA_.

Background: Polychlorinated biphenyls (PCBs) exposure is associated with increased risk of melanoma and non-Hodgkin lymphoma in some, but not all, epidemiologic studies. Although the carcinogenic mechanisms of specific PCB congeners remain to be fully explained, PCBs have been demonstrated to induce oxidative stress and inflammation and therefore may have deleterious impacts on telomere length. We investigated the association between serum concentrations of PCBs and telomere length in a highly exposed population.

Methods: The Anniston Community Health Survey (ACHS) is a cross-sectional study of 766 residents of Anniston, Alabama conducted between 2005 and 2007. We measured relative telomere length (RTL) with telomere/single copy gene ratio, in DNA extracted from peripheral blood samples in a subset of 57 African-American and 46 white participants, using monochrome multiplex quantitative polymerase chain reaction. We used multiple linear regression to estimate the association between 27 individual PCB congeners, and sum PCBs, adjusting for age, plate, and total lipids. All analyses were stratified by race. PCBs and RTL were log-transformed.

Results: ACHS participants had high serum concentrations of PCBs (mean sum PCBs among African-Americans = 14,687.69 ppt whole weight, SD =23,515.46; mean sum PCBs white participants 6,088.32 ppt whole weight, SD=8,750.57). RTL did not differ by race (mean RTL African-Americans= 0.94 SD=0.08; mean RTL whites=0.94, SD = 0.09). Among African-American participants, sum serum PCBs was not associated with RTL (0.70% per 100 ppt; 95% confidence interval= -9.43, 19.70). However, PCB 167 was weakly associated with higher RTL (2.35%, 95%CI= -8.56, 14.55), while PCB 157 was associated with shorter RTL (-4.36%; 95%CI= -15.63, 8.40). Among white participants, a 100 ppt increase in sum serum PCBs was associated with a 10.20% increase in RTL (95% CI= -7.10, 30.72). PCB 138, in particular, was associated with increased RTL among white participants (16.29%; 95% CI= 1.34, 33.46). Conversely, PCB 189 was associated with shorter RTL among white participants (-10.48%; 95%CI= -29.82, 14.19).

Conclusions: We did not observe consistent associations between serum concentrations of PCBs and RTL, suggesting that the association may vary by race and be congener specific. Our results are necessarily preliminary and are limited by the small number of participants. Additional measurements of RTL among ACHS participants are on-going. 

## PREVENTION RESEARCH:

### Behavioral and Social Science Studies across the Cancer Prevention Continuum

#3457

Social support as emotional adjustment determinant of breast cancer patients after surgery in selected hospitals in Nigeria.

Christiana Oluseun Oyewusi,1 Samuel Oyebode Oyewusi2. 1 _University College Hospital, Ibadan, Nigeria;_ 2 _University of Ibadan, Ibadan, Nigeria_.

Breast cancer is the most common invasive cancer and a leading cause of premature mortality for females worldwide. It accounts for 16% of all female cancers; 18.2% of all cancer deaths globally are from breast cancer. Social support plays a major role in modifying negative effect of this disease on affected individuals. It is an important psychosocial resource for coping with stressful life events. This study was carried out to examine social support as determinant of emotional adjustment of breast cancer patients after surgery in selected hospitals in Ibadan, Nigeria.

This cross-sectional descriptive survey utilized a quantitative approach. A sample of two hundred and eighteen patients was randomly selected from hospitals in Ibadan. The respondents were reassured of confidentiality of the data. The instrument used basically was self-structured questionnaire. Data were collected, coded and analyzed using Pearson correlation coefficient.

The respondents' ages were between 21- 51 years; 87.2% were females, majority of them (81.6%) were Christians, more than half (62.4%) were married, about one third (31.2%) had higher qualifications, 55.0% were civil servants and 74.3% were living with other family members. Result findings show that instrumental support had significant effect on emotional adjustment of the breast cancer patients after surgery. Family and friends helping the patients had a positive effect on their emotional adjustment. Therefore, it was revealed that instrumental support had a positive effect on emotional adjustment of the patients (r.cal = .217, r. critical = .195, P < 0.05). Also, emotional support had a significant effect on emotional adjustment of the patients after surgery. Having family members and friends to talk to and help with personal problems was helpful to emotional adjustment. Thus it was evident that emotional support had a positive effect on emotional adjustment (r. cal = .250, r. critical = .195, P < 0.05)

The patients admitted that they had friends and family members who helped them and were a source of comfort to them. This helped them to show a better psychological and emotional adjustment to breast cancer than those who did not have support. It is recommended that adequate attention be given to financial, material and emotional needs of the breast cancer patient.

Keywords: Social support, determinant, emotional adjustment, breast cancer patients.

#3458

UK lung cancer screening trial (UKLS) cost effectiveness: similarities with the NLST high-risk quintiles.

David Whynes,1 Stephen W. Duffy,2 David R. Baldwin,3 Anand Devaraj,4 John K. Field5. 1 _Univ. of Nottingham, Nottingham, United Kingdom;_ 2 _Queen Mary University of London, london, United Kingdom;_ 3 _Nottingham University Hospitals, Nottingham, United Kingdom;_ 4 _Royal Brompton & Harefield NHS Foundation Trust, London, United Kingdom; _5 _Univ. of Liverpool, Liverpool, United Kingdom_.

Introduction: The pilot UK lung cancer RCT screening trial recruited around 4,000 individuals, using the LLPv2 risk model (5% risk over 5 years). The cost effectiveness of the UKLS trial has been modelled and compared with that of the US National Lung Screening Trial (NLST), which has published an estimate of $81,000 per quality-adjusted life-year (QALY) as its mean incremental cost-effectiveness ratio (ICER).

Methodology: All UKLS cost estimates were based on 2011-12 NHS tariffs (Costs provided in $: £1=$1.5 on 30-11-15). Owing to the brief duration of the trial, observations relevant to economic evaluation were limited to cost-incurring events associated with screening and the initial management of screen-detected cancers. Expected outcomes of the cancers detected were simulated on the basis of both life tables and published survival data from other studies.

The costs incurred from UKLS are those of baseline and repeat screens ($424,072), diagnostic workup ($113,478), and treatment ($449,243), which totalled $1,036794 (95% CI, $719,332 to $1,350,766). Recruitment costs ($15) per person for invitation and selection) were modelled from the UK colorectal screening programme and we assumed a participation rate of 30% of those invited. The gross current costs of the programme amounted to $1,133,217 (CI $817,887 to $1,450,610).

Summary of findings: The ICER of screen-detection compared with symptomatic detection was estimated at $9495 per life-year gained. Using data from previous studies, we associated quality of life weights with the estimated survival gains, enabling us to report outcomes as QALYs. On this basis, the ICER equalled $12,709 per QALY gained (CI $ 8280 to $18966).

The difference in cost effectiveness between NLST and UKLS as suggested by the estimated ICERs is more apparent than real. Most of the discrepancy can be explained by differences between settings in (i) local unit costs, (ii) intensity of resource use, (iii) number of screening rounds and (iv) disease prevalence in the target population. Thus, UKLS selected high-risk subjects only whereas NLST screened a general population, yet the latter reported an ICER as low as $32,000 for its highest-risk quintile. Expected QALY gains from screen-detection were similar in both trials.

Conclusion: Other things remaining equal, ICERs will be higher in programmes where (i) unit costs of detection and management are higher, (ii) lower-risk subjects are invited to be screened, (iii) screens are repeated at frequent intervals. The convention for cost effectiveness acceptability in the UK is $30,000-45,000 per QALY gained, and we conclude that a lung cancer screening programme based on the UKLS protocol would be likely to offer acceptable value for money to the NHS.

#3459

Effects of Dehydroepiandrosterone (DHEA) treatment on serum methylation, oxidative and nitrosative stress biomarkers in an obesity model of mammary carcinogenesis.

Reza Hakkak,1 Soheila Korourian,2 Teresa Evans,3 Oleksandra Pavliv,3 Stepan Melnyk3. 1 _University of Arkansas for Medical Sciences and Arkansas Children's Hospital Research Institute, Little Rock, AR;_ 2 _University of Arkansas for Medical Sciences, Little Rock, AR;_ 3 _Arkansas Children's Hospital Research Institute, Little Rock, AR_.

Free oxygen and free nitrogen radicals, commonly named as reactive oxygen species (ROS) and reactive nitrogen species (RNS) play an important role in cancer promotion. The cumulative effect of ROS/RNS termed oxidative and nitrosative stress is commonly seen in the development of several cancers. Obesity has been also linked with the risk of development of various cancers, including breast cancer. DHEA is a dietary supplement used as an anti-cancer agent and anti-obesity supplement. Previously, we reported that obese rats fed DHEA had lower body weight gain and developed no mammary tumor in DMBA model. The effect of DHEA on development of oxidative/nitrosative stress is not known. The objectives of this study were to investigate the possible mechanism of DHEA tumor protection by determining the DHEA effects on serum concentration of methylation cycle metabolites, oxidative and nitrosative stress biomarkers. Twenty (20) six-week-old obese female Zucker rats were randomly assigned ad libitum to water and a diet of either chow as a control diet or chow with the addition of DHEA at a concentration of 6 g/kg of chow as a DHEA diet. All rats were orally gavaged at age 50 days with 65 mg DMBA/kg body weight and were sacrificed 155 days later. Serum concentration of Methionine, S-sdenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine, free reduced glutathione (fGSH), oxidized glutathione (GSSG), nitroglutathione (GSNO), free Cysteine, Cystine and 3-nitroTyrosine (3NT) were measured by HPLC method with electrochemical detection or LC-MS method. There were no significant differences in serum concentration of fGSH between control and DHEA fed-rats. However, DHEA-fed rats had lower (P<0.03) serum concentration of GSSG which led to higher (P<0.02) glutathione "intracellular oxidative ratio" (fGSH/GSSG) compared to control rats. Despite higher (P<0.004) serum concentration of free Cysteine in control rats, DHEA-fed rats had lower (P<0.04) serum concentration of Cystine (P<0.003) compared to control rats which led to a lower (P<0.04) Cystine/fCysteine "extacellular oxidative ratio". Concentration of biomarkers for "nitrosative stress" were lower for 3NT (P<0.04) and GSNO (P<0.02) in serum of DHEA-fed rats compared to control rats. Also, DHEA-fed rats had a lower (P<0.02) Methionine concentration which was accompanied with increased serum SAM concentration (P<0.005) and lower (P<0.02) concentration of SAH and homocysteine (P<0.03) compared to control rats. These changes lead to an increase (P<0.0001) in SAM/SAH "methylation ratio" in DHEA-fed rats compared to control rats. In summary, our results suggest that DHEA fed-rats are capable of reducing body weight gain and protecting mammary tumors formation by improving "methylation environment conditions" and decreasing damaging effect oxidative and nitrosative stress. Supported by ABI and ACHRI to RH and SM.

#3460

Breast and ovarian cancer prevention: is it time for population screening for BRCA mutations.

Ian G. Campbell, Ella Thompson, Gillian Mitchell, Alison Trainer, Mary-Anne Young, Paul James. _Peter MacCallum Cancer Ctr., E. Melbourne, Australia_.

Germline mutations in BRCA1 and BRCA2 confer high lifetime risk of breast and ovarian cancer but importantly these risks are not irreversible. Identification of asymptomatic carriers could significantly reduce the incidence of these diseases. The traditional model of familial breast and ovarian cancer practice involves ascertaining high-risk individuals based on family history. In general, the family is first identified because one member develops cancer and because of high-risk indicators, is referred to a Familial Cancer Centre. However, more than 50% of women who carry a BRCA1/2 mutation may not have a family history of cancer in a close relative. Momentum toward genetic screening of the asymptomatic population is growing but there remain some significant unknowns. For example it is unclear what is the true frequency of actionable mutations in the general western population and the extent to which the public would accept such screening, particularly for those individuals identified with an actionable mutation in the absence of an overt family history. As a first step toward population based BRCA gene screening, we are sequencing the entire coding region of 18 known and proposed HBOC genes in 4,000 cancer-free Australian women recruited from the lifepool study (www.lifepool.org), which is a cohort of women attending the Australian population, based mammographic screening program. To date, data from 1,997 women has identified 17 with actionable mutations in BRCA1 (4 mutations), BRCA2 (9 mutations) or PALB2 (4 mutations). All 17 women subsequently accepted an invitation to attend a Familial Cancer Centre and then proceeded to formal clinical genetic testing. In addition 4 women had pathogenic mutations in BRIP1. Our unique pilot data directly demonstrates a population carrier frequency of ~1% for pathogenic mutations in these recognized high risk breast and/or ovarian cancer genes and that such testing is well accepted by the screened population.

#3461

Smoking cessation following a limited 5A intervention in a cohort of patients with head and neck cancer attending for treatment at a regional cancer program in Ontario, Canada.

Michael S.C. Conlon, Deborah P. Saunders. _Northeast Cancer Ctr., Sudbury, Ontario, Canada_.

A smoking cessation intervention at the time of cancer treatment is an important opportunity to improve patient quality of life, treatment outcome, and survival in people with cancer. This prospective observational study was designed to assess intention to quit, motivation to quit, smoking characteristics and interest in smoking cessation, and assessment of cessation following a standardized smoking cessation intervention. The intervention was a limited clinical 5A intervention offered by trained health professionals and occurred within a dental oncology clinic that offered regular follow-up and any needed pharmacotherapy through the course of cancer treatment. The cohort was composed of head and neck cancer patients who were self-reported ever-smokers at enrolment and who attended the Northeast Cancer Centre, a regional cancer centre in Northeastern Ontario, Canada, for cancer treatment. There were 377 cancer patients who participated in study, with 35.8% (n=135/377) self-reporting as current smokers. Most current smokers had high nicotine dependence, with 82.2% (n=111/135) reporting a time to first cigarette (TTFC) of 30 minutes or less; 84.4% (n=114/135) were interested in quitting smoking. By 1 year post intervention 10 patients had died and 28 patients were lost to follow-up ; of the remainder 29 smokers had cessated yielding a cessation rate of 29.9% (n=29/97); or 23.2% (n=29/125) conservatively assuming those lost to follow-up remained current smokers. Offering comprehensive smoking cessation during the course of cancer treatment yields long term smoking cessation benefits in a cohort of patients with smoking associated cancers.

#3463

The use of HeLa cells as a cancer genetics education tool for middle and high schools.

Ayesha Umrigar, Mary Moore, Fern Tsien. _Louisiana State University Health Sciences Center, New Orleans, LA_.

Hands-on, inquiry based learning, and one-on-one mentoring are the most effective methods of disseminating scientific information pertaining to cancer. The Science Youth Initiative (SYI) is a K-12 science education partnership between the Louisiana State University Health Sciences Center in New Orleans (LSUHSC-NO) and local schools. The workshops are coordinated with LSUHSC-NO faculty and school teachers to maximize the level of student learning and complement their classroom curriculum and academic level. Since 2009, the SYI has reached more than 2100 K-12 students to date, of which about 77% were middle and high school students. A diverse group of trainees from the LSUHSC-NO Schools of Medicine, Graduate Studies, Public Health, Allied Health Professions, and Nursing help educate the K-12 students and serve as role models. The benefit of having a variety of health care professionals with a range of expertise is that students receive cancer information from multiple perspectives. The program utilizes the book, The Immortal Life of Henrietta Lacks by Rebecca Skloot, which is part of the curriculum in many schools. During these workshops, middle and high school students are able to stain and examine HeLa cell chromosomes, thereby learning about chromatin instability commonly associated with cancer. Students also learn about DNA isolation, PCR, restriction digestion, and gel electrophoresis as they engage in workshop discussions on science history, bioethics, the human papilloma virus (HPV), and cervical cancer. Pre and post workshop surveys were developed to evaluate the participants' learning and retention of the material covered, self-confidence in the academic material, awareness of science career options, and evaluation of the program. Results of the current academic year demonstrate: (1) a 51% increase in test scores based on the material covered (2) 59% of students were interested in participating in a medical or research related internship after the workshop (3) 54% of students were interested in working part-time in the medical sciences after the workshop. Participating schools included rural, urban, public/charter and private schools. These varied in socioeconomic and ethnic backgrounds: some which had almost 100% African American/Hispanic student body, while others had a diverse distribution of African American, Hispanic, Asian, and Caucasian students. The program's use of HeLa cells as a cancer genetics education tool has been successful in making health sciences and cancer research topics more interesting and accessible, motivating a variety of students to improve their grades, and promoting various health science related career opportunities.

#3464

Low concordance with CEA tumor marker monitoring in colorectal cancer survivors.

Guadalupe Palos, Katherine R. Gilmore, Patricia Chapman, Paula Lewis-Patterson, Weiqi Bi, Maria Alma Rodriguez. _University of Texas M. D. Anderson Cancer Center, Houston, TX_.

Purpose: To assess providers' concordance with surveillance and risk reduction recommendations for colorectal cancer (CRC) survivors after completion of their curative treatment.

Patients and Methods:

This was a longitudinal study of survivors who met the following eligibility criteria: diagnosed with a primary colon or rectal cancer before their first visit (V) to the CRC Survivorship Clinic, adult survivor ≥ 18 years old, no evidence of disease, alive at the time of data abstraction, and 1-3 clinic visits between 9/01/2011 and 8/31/2014. Data were collected at V1 scheduled between 9/1/2011 and 8/31/2012. V2 was scheduled 9-15 months after the first visit. V3 was also scheduled 9-15 months after V2. Data sources were survivorship care plans, electronic medical records, and CRC survivorship algorithms. For an annual visit, CRC algorithms recommended history/physical exams (H & PEs), carcinoembryonic antigen (CEA) testing when previously elevated, and colonoscopies for surveillance of cancer recurrence. Concordance rates (CR) were measured as the percent of yes/no responses to whether the providers followed minimum standards for the 3 procedures. Demographic and clinical characteristics were also collected. Descriptive statistics were used to summarize all data.

Results:

81 of 117 CRC survivors who met all eligibility criteria were included in this sub-analysis. The number of survivors visits varied across time, V1=81, V2=56, and V3=36. Most survivors were male (51.9%) and Caucasian (66.7%). 67.9% reported being 5-8 years post-treatment. 58% were diagnosed with colon cancer and of those 61.7% were Stage IIIA-IV compared to 55.8% of rectal cancer survivors with advanced disease. Table 1 summarizes the percentage of CR rates across the 3 visits.

Conclusion:

CRs for H & PEs and colonoscopies remained high across the 3 visits. CRs were lowest for CEA recommendations. These low rates suggest further work is needed to determine barriers in clinical practice that limit use of CEA tumor marker monitoring in CRC survivors.

Percent of Providers Concordance

---

Algorithm Recommendation | Visit 1 | Visit 2 | Visit 3

H & PE | 100 | 100 | 100

CEA | 53.3 | 38.3 | 22.2

Colonoscopy | 97.5 | 98.2 | 100

#3465

Dissemination and implementation of genomic cancer risk assessment in Latin America via innovative pairing of clinical training and genomic tools.

Jeffrey N. Weitzel,1 Cynthia Villarreal-Garza,2 Kathleen R. Blazer,1 Josef Herzog,1 Danielle Castillo,1 Tanya Chavez,1 Lenny Gallardo-Alvarado,3 Talia Wegman,3 Rosa Alvarez,3 Karla Unger,3 Maria Fernandez,4 Azucena del Toro-Valero,5 Adrian Daneri Navarro,5 Abelardo Meneses,3 Luis Herrera Montalvo,3 Alejandro Mohar Betancourt,3 Lily van Tongeren,1 Cancer Genomics Community Research Network. 1 _City of Hope Comprehensive Cancer Center, Duarte, CA;_ 2 _Instituto de Cancerología - Tec Salud, Monterrey, Mexico;_ 3 _Instituto Nacional de Cancerología, Mexico City, Mexico;_ 4 _University of Texas Science Center at Houston, Houston, TX;_ 5 _University of Guadalajara, Guadalajara, Mexico_.

Purpose:

Access to genomic cancer risk assessment (GCRA) is standard of care in most developed countries, but is not readily available in Latin America. Following needs assessment, a dissemination and implementation intervention, including clinical training and application of inexpensive genomic tools, was used in Mexico to develop a model for vertical integration of GCRA among underserved populations in Latin America.

Methods:

A roundtable forum with 15 Latin American physicians representing Brazil, Colombia, Mexico, Puerto Rico and Peru was conducted to assess the state of GCRA services and barriers to implementation in these countries. The roundtable was conducted in Spanish, moderated by bilingual cancer genetics clinicians, recorded, transcribed, translated and thematically analyzed. The training resources of the City of Hope Clinical Cancer Genomics Community of Practice (CCGCoP), a flexible, multi-modal program of distance didactics, face-to-face case-based workshops and continuing practice support, were deployed to promote practitioner level GCRA competence among clinicians in Latin America. A common prospective registry protocol was implemented via the Cancer Genetics Community Research Network (CCGCRN), and an inexpensive BRCA screening tool (HISPANEL) was introduced to support GCRA services. HISPANEL sensitivity compared to full sequencing was estimated; quality/content of GCRA process was assessed; and downstream barriers to follow up care were identified.

Results:

Roundtable findings pointed to the need for a multi-level approach with GCRA training, cost-effective genetic testing, and an evidence-based foundation to support the development of policy, infrastructure and resources to implement and sustain GCRA services in Latin America. Post-training assessments for the 41 Latin American clinicians indicated significant increases in professional self efficacy and skills, and demonstrate the value of CCGCoP training and practice support; 19 alumni (representing 7 sites in Mexico, Peru, Colombia, Brazil and Puerto Rico) joined the CCGCRN. To date 1,010 patients have been accrued to the Latin American component of the CCGCRN cohort. The HISPANEL genomic tool demonstrated a clinical sensitivity of 68-77%, at a cost of $20 per case in Mexico, where site assessments demonstrated successful initiation of GCRA services and facilitated identification of barriers to follow up care.

Conclusions:

Innovative pairing of multi-modal GCRA training and practice support, combined with access to affordable genomic screening tools, demonstrates the potential for dissemination and implementation of GCRA to improve cancer prevention and control in Latin America. Future directions: address scalability; adapt to other countries; and develop a Spanish-language version of the training program to facilitate broader dissemination.

#3466

Mental health-related quality of life assessment at diagnosis as a predictor for prognosis of colorectal cancer patients.

Monica E. Reyes, Yeling Zhou, Yuanqing Ye, Xifeng Wu, Michelle Hildebrandt. _UT M.D. Anderson Cancer Ctr., Houston, TX_.

Purpose: Health-related quality of life (HR-QoL) is emerging as an important metric for assessing and predicting outcomes in colorectal cancer (CRC) patients. While most studies focus on QoL in treatment response, few studies have investigated the impact of baseline mental health QoL variables on prognosis of CRC patients. The objective of our study was to explore mental health and vitality QoL measurements and association with prognosis in CRC patients.

Patients and Methods: QoL was assessed in a hospital-based cohort of 2,900 non-Hispanic white CRC patients that completed the SF-12 questionnaire within 1-year of diagnosis. Mental Component Summary (MCS) scores were dichotomized by the normalized scoring-based mean of 50. Hazard ratios were generated using univariate cox regression analysis and survival estimates generated using the Kaplan-Meier method. Sub-domain variables under mental health ("downhearted and blue" and "calm and peaceful") and vitality ("energy"), were scored and dichotomized before stratifying by patient demographics and statistically evaluated.

Results: We observed that CRC patients with poor mental QoL (MCS<50) had a 1.48-fold increased risk (95%CI:1.31-1.66; P=5.17x10-14) of a poorer prognosis compared to those with a high MCS score (≥50). This corresponded to a 61.9 month reduction in median survival time in this group (log-rank P=5.88x10-11). Patients that reported feeling downhearted and blue compared to those that did not, had a 65.7 month reduction in median survival time (log-rank P=1.13x10-5). Similarly, there was an increased survival time for patients that felt peaceful and calm (MST:113.1 months) compared to those that reported not feeling peaceful and calm (MST:59.4 months; log-rank P=1.36x10-5). The most striking finding was for the energy variable. Patients that reported having energy

compared to those that did not, were found to have significantly better survival with MSTs of >175 months and 42.3 months, respectively (log-rank P=2.91x10-27). Several patient characteristics (alcohol use, tobacco use, gender and marital status) were significantly associated with these sub-domain measurements. We found those that reported combined poor mental health and vitality had a dramatic reduction in survival by 102.7 months (log-rank P=8.39x10-18).

Conclusion: We conclude that baseline mental QoL assessment may identify CRC patients at increased risk of a poor prognosis and merits further investigation of these variables as independent CRC prognostic factors. Interestingly, a potential coping mechanism may underlie the protective effect observed among current users of alcohol, while smoking may serve as a risk factor for poor prognosis in those that reported poor mental QoL. Further studies are warranted to explore the relationship between these MCS sub-domain variables and survival in CRC patients.

#3467

Moderate-intensity exercise to reduce radiation therapy-related fatigue in black breast cancer patients: A feasibility trial.

Chiranjeev Dash,1 Mary Mills,1 Vivian Watkins,1 Pamela Randolph-Jackson,2 Claudine Isaacs,1 Kepher Makambi,1 Lucile L. Adams-Campbell1. 1 _Georgetown Lombardi Comp. Cancer Center, Washington, DC;_ 2 _Washington Cancer Insititute, MedStar Washington Hospital Center, Washington, DC_.

Background: Fatigue is an important side-effect of radiation therapy (RT) for treatment of early stage breast cancer. Evidence on the efficacy of physical activity (PA) interventions in reducing fatigue among Black cancer patients undergoing RT is lacking. In a randomized controlled trial we tested the efficacy of a structured PA intervention, coinciding with the start of RT, in reducing cancer-related fatigue among Black patients undergoing RT for breast cancer.

Methods: We randomly assigned 30 Black, sedentary, RT-naïve, non-pregnant patients diagnosed with stage 0-IIIA breast cancer who had completed adjuvant or neo-adjuvant chemotherapy and were scheduled for RT to the PA intervention (n=15) and control groups (n=15). PA intervention was an 8-week structured, moderate-intensity aerobic exercise regimen (75 minutes/week) using PEDLARS (portable stationary cycle ergometers) concurrent with RT. Fatigue was measured by using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) survey, a 13-item scale whose score ranges from 0 to 52 with a higher score denoting lower fatigue. We used ANCOVA to compare fatigue levels between the groups at T8 weeks after adjusting for baseline (T0) fatigue levels. We also compared change in fatigue scores (T8 weeks - T0) using 2-sample t-tests. All tests were double-sided with alpha=0.05.

Results: 27 women completed baseline and follow-up assessments. After adjusting for baseline fatigue scores, intervention group participants reported lower fatigue at T8 weeks than the control group (42.53 vs. 37.12; P = 0.17). Intervention group participants improved their fatigue scores at T8 weeks compared to T0 (mean change +1.3) but the control group did not (mean change -1.91; P = 0.42).

Conclusion: Although statistically not significant, moderate-intensity exercise regimen among Black breast cancer patients undergoing RT was associated with lower RT-associated fatigue. This trial demonstrated the feasibility and acceptability of conducting a randomized clinical trial of a moderate-intensity exercise program among women initiating RT for breast cancer.

#3468

High rates of informed consent for biospecimen sharing among hispanic women in a safety-net clinic.

Jesse Nodora,1 Maria E. Martinez,1 Richard Schwab,1 Kristen Wells,2 Hyeon-eui Kim,3 Claudiu Farcas,3 Marcia Bouton,4 Ian Komenaka4. 1 _Moores Cancer Center, La Jolla, CA;_ 2 _San Diego State University, San Diego, CA;_ 3 _UCSD School of Medicine, La Jolla, CA;_ 4 _Maricopa Medical Center, Phoenix, AZ_.

Introduction: Biospecimen sharing among low-income and undeserved individuals has been reported to low. Few data exist on participation in biospecimen sharing for research purposes and reasons for declining participation by individuals from underserved populations. To address this gap, we conducted a randomized study to compare the rate of informed consent for biospecimen collection and data sharing for research when participants were consented by their physician versus a research assistant.

Methods: Eligible participants included Hispanic English or Spanish-speaking patients 18 years of age or older who underwent breast biopsy at the Breast Center in Maricopa Integrated Health Services (MIHS), a safety-net facility in Phoenix, Arizona. Consecutive patients receiving care at the center were asked to participate in the study and individually randomized to be consented by their physician or a research assistant not working in the Breast Center. Informed consent was delivered vi an electronic tablet. Biospecimen sharing for research included a saliva sample for DNA extraction and formalin fixed paraffin embedded tissue. Data collection included sociodemographic variables, health literacy using Newest Vital Sign (NVS) instrument, and a trust questionnaire based on the Biobanking Attitudes and Knowledge Survey (BANKS-SP-Trust). Rate of participation was compared between the two randomization arms and reasons for participation were assessed.

Results: The study enrolled 140 women (70 to each randomization arm). Study participants had a mean + sd age of 46.7 +11.6 years and most (95%) were of Mexican descent and Spanish-speakers (85%). Mean (sd) education level was low (8.7 + 4.1) years. Eighty-five percent of the participants were found to have limited health literacy. Using a 1 to 10 Likert scale, the mean + sd range of responses to the BANKS-SP-Trust in relation to 10 different attributes was from 7.7 + 2.5 for "health insurance companies" to 9.2 + 1.5 for "doctors who do research." Results showed very high rates of consent with no significant difference between the two randomization arms: 97.1% for the physician and 92.9% for the research assistant. Participants who were current smokers were ~60% less likely to consent (p=0.045) and those who were unmarried were ~90% less likely to consents (p=0.043). All women who refused consent had limited health literacy whereas 86% of those who consented had this limitation but this was not statistically significant (p=0.346). No association between consent and trust was observed.

Conclusions: Consent for biospecimen and data sharing among low-income Hispanic women is very high, with only current smokers and unmarried females being significantly less likely to provide consent. More efforts should be made to reach out to patients in safety-net facilities for biospecimen sharing to overcome the scarcity of representation of underserved individuals in biospecimen repositories.

#3470

Obesity and metformin alter progression of triple negative breast cancer.

Laura Camacho, Lei Xue, Lacey Dobrolecki, Susan G. Hilsenbeck, Chandandeep Nagi, Nagireddy Putluri, Michael T. Lewis, Arun Sreekumar, Sao Jiralerspong. _Baylor College of Medicine, Houton, TX_.

Background: Epidemiological studies from us and others have reported an association of obesity with more aggressive breast tumors and >30% higher risk of recurrence and mortality compared to normal weight patients. This includes poorer outcomes for triple negative breast cancer (TNBC), an aggressive subtype with early recurrence and shorter survival. Metformin is a leading frontline therapy for type 2 diabetes and its safety profile is well documented. Laboratory and clinical studies including ours suggest that metformin may have anti-tumor effects in breast cancer. However, the nature and molecular mechanisms of these correlations are poorly understood.

Experimental design and methods: We used a PDX (patient-derived xenograft) breast cancer model in diet-induced obese mice to study how obesity promotes breast cancer growth and how metformin antagonizes these effects. After feeding the mice with a high-fat diet (HFD) vs. a control diet (CD), we transplanted a TNBC PDX into the mammary fat pads. When tumors reached about 50 mm3, mice in HFD and CD groups were randomly assigned to metformin treatment or no treatment. Tumor volumes were measured bi-weekly, and blood, tumors, and organs were collected after 4 weeks of treatment. Histological and immunohistochemical staining, serum adipokines array and metabolomics and lipidomics analyses were performed to examine potential mechanisms.

Results: HFD (obese) group compared to CD (lean) group was associated with larger tumors, whereas metformin compared to no treatment was associated with smaller tumors. A panel of adipokines and metabolites was identified to be differentially expressed between the groups.

Conclusion: Our results suggest a growth promoting effect of obesity in TNBC, while metformin appears to have the opposite effect. The identification of adipokines and metabolites involved in these effects will help us understand the mechanisms by which obesity and metformin alter the progression of TNBC.

#3471

Perceived role of family for Hispanic/Latino lung cancer patients: A qualitative study.

Ana Motta-Moss,1 William Alago,2 Lorna Lin,1 Jane Akhuetie2. 1 _CUNY Medical School, New York, NY;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Healthcare decisions are not made by the patients alone, but rather the result of collaborative efforts between healthcare professionals and, more importantly, family members who become significant because of the various roles they perform throughout the process. The purpose of this study was to examine the role of family in the care of Hispanic/Latinos patients with lung cancer. Inconsistent expectations of the role of family affect how patients view family support throughout the diagnosis and treatment process. Therefore, it is important to examine what patients perceive to be an appropriate role for family members that assist in their healthcare. Twenty-four patients self-identified as Hispanic/Latino and treated at Memorial Sloan Kettering Cancer Center (MSKCC) for lung cancer stage I-IV participated in semi-structured face-to-face interviews. The four major themes of the role of family identified in this study include: general support, navigation of healthcare system, communications, and lack of family support. It is important to understand that family roles change as the patients' needs change over the course of the disease. Nonetheless, identifying the various forms of support provided by family is essential to understanding the benefits of family presence in the diagnosis and treatment of lung cancer, particularly among Latinos.

#3472

Does being married independently predict survival in patients with head and neck cancer? Results from a single institution.

Nosayaba Osazuwa-Peters,1 Kara M. Christopher,1 Sean T. Massa,1 Lauren Cass,1 Ronald J. Walker,1 Mark A. Varvares2. 1 _Saint Louis University, Saint Louis, MO;_ 2 _Harvard Medical School, Boston, MA_.

Purpose/Objectives

An important factor in cancer survivorship is social support, often coming from a spouse. There is an emerging literature on the role of spousal support in head and neck cancer (HNC) outcomes; however most are based on results from national databases. While more generalizable, these studies may not always capture the unique variation in different patient populations. This single institution study aimed to describe the association between marital status and outcomes of HNC; and to determine if marital status independently predicts survival in a local patient population.

Materials/Methods

We identified 460 patients aged 20 to 91 (59.31 ± 11.42) years diagnosed with squamous cell carcinoma of the head and neck at an academic tertiary referral center between 1997 and 2012 in this retrospective cohort study. Cox proportional hazards model assessed the effect of marital status on survival. Results are based on the final model constructed after accounting for covariates in the data.

Results

Our study population was made up of 73% males, and 82.2% Whites. We found an association between marital status and HNC survival. Unmarried HNC patients had a 66% increase in the hazard of death compared to married HNC patients (HR = 1.66, 95% CI = 1.23 - 2.23). This was after controlling for covariates, which included sociodemographic variables (age, race, sex, and health insurance status), social habits (tobacco and alcohol), primary anatomical subsite (oral cavity, oropharyngeal, laryngeal, and others), stage at presentation (early vs. late stage), and treatment modality (surgery, surgery with adjuvant therapies, other single modality therapy, and palliative care). Other factors found to be associated with an increased hazard of death were age (≥ 50 years), current tobacco use, late stage of presentation, palliative care, and laryngeal subsite.

Conclusion

Marital status is associated with head and neck cancer outcomes, and being married is an independent predictor of survival among patients. This result, found in previous national studies, held true in our local patient population. This underscores the need for the multidisciplinary HNC team to recognize this aspect of survivorship and to emphasize the need for social support among unmarried HNC patients. It could be that it is necessary to add social support to the clinical practical guidelines for managing head and neck cancer beyond palliative care.

#3473

Psychosocial functioning in research participants at enrollment in a Li-Fraumeni syndrome study.

Jennifer T. Loud, Renee C. Bremer, Rosamma Decastro, June A. Peters, Phuong L. Mai, Sharon A. Savage. _National Cancer Institute, Rockville, MD_.

Background: Li Fraumeni Syndrome (LFS) is an autosomal dominant cancer predisposition syndrome in which about 90% of individuals develop cancer by age 60. Approximately 70% of individuals with LFS have germline TP53 mutation. We hypothesized that this high level of cancer risk may be associated with increased emotional distress, cancer risk perceptions and cancer worry in individuals from LFS families.

Methods: Data from 276 adult (≥18 years old) TP53 mutation carriers (TP53+), mutation negative (TP53-) and untested (UT) participants enrolled in an IRB-approved study at the Clinical Genetics Branch, NCI were obtained between January 2011 and June 2014.. Using questionnaires at baseline, we assessed emotional distress (global distress, somatization, anxiety and depression) as measured by the BSI-18, cancer risk perceptions (comparative: scores 1-5; 1=much less than other people your age to 5=much more than other people your age and absolute: 10-point scale; 1=no chance; 5=may or may not; 10=almost certainly) and cancer worry scales (frequency of cancer thoughts, interference with mood and daily activity, scores 1-4; =not at all or rarely to 4=several times a day).

Results: TP53+ estimated their comparative lifetime risk of cancer as higher than TP53- and UT; [4.9 (SD=0.5) vs. 3.8 (SD=1) and 3.2 (SD=1.3) p<0.01], respectively]. TP53+ also estimated their absolute cancer risk as significantly higher than TP53- and UT on a [8.6(SD=1.5) vs. 6.3 (SD=1.6) and 5.7 (SD=2.1), p<0.01].Cancer worry was higher in TP53+ than TP53-or UT. TP53+ also indicated they thought about their cancer risk more frequently thanTP53- and UT [2.3 (SD=0.9) vs. 1.3 (0.6) and 1.3 (SD=0.6), p<0.01]. There was a small but statistically significant difference between TP53+, TP53- and UT in their appraisal of whether cancer worry affected their mood over the past month [1.6 (SD=0.6) vs. 1.2 (SD=0.6) and 1.1 (SD=0.2), p=<0.01] and whether thoughts of cancer affected their ability to perform daily activities [1.2 (SD=0) vs. 1.1 (SD=0.4) and 1 (SD=0.2), p=<0.01]. No statistically significant differences were observed on measures of emotional distress between TP53+, TP53- or UT somatization [46.7 (SD=8.1) vs. 45.9 (SD=9.3) and 45.9 (SD=7.6); depression [50.1 (SD 9.7) vs. 49.6 (SD - 9.3) and 45.9 (SD= 7.6); anxiety [48.2 (SD=9.1) vs. 45.4 (SD=9.7) and 45.8 (SD=7.9) or global distress [47.9(SD=9.9); vs. 45.1(12.1) and 44.9 (SD=9.5)].

Conclusions: Participants in the LFSS do not demonstrate high levels of emotional distress. TP53+ appropriately identify their comparative and absolute cancer risk as high, but have levels of cancer worry that appears to be only slightly affecting their mood or ability to perform daily activities. Future research will focus on longitudinal changes in risk perception, cancer worry and emotional well-being while enrolled in a cancer screening study.

#3474

**Prevalence of** BRCA1 **and** BRCA2 **small mutation and large genomic rearrangements in breast cancer patients visiting a genetic counseling clinic.**

Ji Yeon Sohn,1 Boyoung Park,1 Kyong-Ah Yoon,1 Soo Jin Park,1 Moo Hyun Lee,1 Eun Hae Cho,2 Keun Seok Lee,1 Myong Cheol Lim,1 Sun-Young Kong,1 Eun Sook Lee1. 1 _National Cancer Center, Gyeonggi-do, Republic of Korea;_ 2 _Green Cross Genome, Yongin, Republic of Korea_.

BACKGROUND: This study was conducted to investigate mutation prevalence of the BRCA1 and BRCA2 genes in breast cancer patients, including both small mutations and large genomic rearrangements (LGRs) following the patients in the process of genetic counseling.

METHODS: A total of 358 breast cancer patients who visited genetic counseling clinic and screened for BRCA1/2 mutation at the National Cancer Center were included. Among the non-carriers by direct sequencing, a subset of patients who were agreed on participation of the research were screened for the presence of LGRs using a multiple ligation-dependent probe amplification (MLPA) assay. Clinico-pathologic characteristics of cancer were evaluated thorough medical record review and characteristics for those who found BRCA mutation through MLPA were described.

RESULTS: 53 (14.8%) mutation carriers were identified. The frequency of BRCA small mutations in specific subgroups was as follows: 18.0% of patients with a family history, 12.8% of early-onset breast cancer, and 20.6% of bilateral breast cancer patients. MLPA identified BRCA1 LGRs in 3 (1.5%) out of 204 patients with BRCA1 and BRCA2 small mutation-negative results. Those with LGRs showed higher probabilities of BRCA mutation carriers estimated by risk assessment models and receptor negatives of tumor immunohistochemistry.

CONCLUSIONS: The prevalence of BRCA1/2 mutation prevalence in breast cancer patients with family history or personal risk factors were comparable to previous Korean studies. MLPA to screen for mutations in the BRCA1 gene could be recommended for breast cancer patients negative for small mutations. To specify proper targets of MLPA, probabilities of carrying BRCA1/2 mutation and tumor receptor status could be considered.

#3475

Mechanistic implications of advanced glycation end-products to prostate cancer and racial disparity.

Danzell Smith,1 Dion Foster,1 Van Phan,1 Victoria Findlay,1 Lourdes Nogueira,1 Laura Spruill,1 Mahtabuddin Ahmed,2 Judith Salley,2 Marvella Ford,1 David Turner1. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _South Carolina State University, Orangeburg, SC_.

Poor diet, low income, obesity and a lack of exercise are established lifestyle factors that are known to increase cancer burden and are often more prevalent in African American communities. As our understanding of tumor biology advances it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biological make-up of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Sparse information exists about the genetic and biological factors that contribute to differential cancer survival and mortality rates observed in minority populations. A greater understanding of the interplay between risk factors and the molecular mechanisms associated with cancer disparity will significantly impact minority health.

Advanced glycation end products (AGEs) are reactive metabolites produced as a by-product of sugar metabolism. Failure to remove these highly reactive metabolites can lead to protein damage, aberrant cell signaling, increased stress responses, and decreased genetic fidelity. Critically, AGE accumulation is also directly affected by our lifestyle choices such as poor diet, low income, obesity and a lack of exercise. We recently reported a potential mechanistic link between AGEs and prostate cancer which may provide a molecular consequence of our lifestyle choices that can directly impact tumor biology and contribute to cancer disparity. We examined circulating and intra-tumoral AGE metabolite levels in clinical specimens and identified a race specific, tumor dependent pattern of accumulation in prostate cancer serum and tumor.

Further mechanistic studies in immortalized prostate cancer cell lines show that AGE treatment increases the expression of the receptor for AGEs (RAGE) to activate cancer-associated signaling cascades. Loss of function studies show that AGE mediated increases in cancer associated processes was dependent upon RAGE expression. Significantly, we show that AGEs are secreted into the tumor microenvironment by cancer cells and may function as signaling molecules to promote immune cell recruitment.

These data implicate the AGE-RAGE signaling axis as a potential biological mechanism promoting prostate cancer and may represent a biological mechanism promoting prostate cancer disparity. AGE metabolites may have high potential impact as prognostic/diagnostic markers and/or as a novel area of potential therapeutic intervention to reduce cancer disparity.

#3476

Factors related to cervical cancer screening differences between regions around Bagamoyo, Tanzania.

Brianna Rooney,1 Autumn Cummings,1 Amr Soliman,1 Crispin Kahesa2. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Ocean Road Cancer Institute, Dar Es Salaam, United Republic of Tanzania_.

Background: With cervical cancer being among the most common cancers in East Africa among women, it is important to look at the factors related to referral differences in Tanzania, especially in rural areas where there is little to no access to cervical cancer screening clinics (CCS). Cervical cancer is related to HIV because a woman is 13.3 times more likely to develop cervical abnormalities if she is HIV positive. There are more HIV-positive women that attend CCS than HIV-negative women. This leads to more research to discover the variation in screening rates between HIV-positive and HIV-negative patients within the same village, as well as the variation in the screening rates between different villages.

Purpose: This study aims to determine the multi-level factors related to differences in cervical cancer screening frequencies between villages in Bagamoyo, Tanzania, and providers' perception of barriers to screening at the Bagamoyo District Hospital (BDH). Factors identified that influence CCS attendance are HIV prevalence, distance to BDH, number of clinics, and female population.

Methods/Design: This mixed method design includes data obtained from the Tanzania Ministry of Health, the Bagamoyo District Medical Officer, and BDH medical records, as well as additional data from questionnaires conducted with health care providers, doctors or nurses, employed at BDH. The questionnaires included written informed consent. Data obtained from CCS medical records were used to estimate the frequency of women screened from 42 villages. ANOVA analysis was used to compare the percent of women screened from each village to the identified risk factors, HIV prevalence, distance, access to clinics, and population. Chi-square analysis was conducted in order to determine associations between type of health care provider and previously identified barriers to screening.

Discussion: This is the first study to examine providers' perceived barriers to screening in relation to previously identified factors related to differences in cervical cancer screening rates. Further analysis of patient perceived barriers to screening should be conducted in order to better understand differences in screening rates.

Funding: Funding was received from the National Cancer Institute through the Cancer Epidemiology Education in Special Populations program.

#3477

Graduate public health students as health educators: A pilot project to promote cancer health in Puerto Rico.

Laura Moreno,1 Himilce Vélez,2 Julio Jiménez,2 Susan Vadaparampil,1 Teresita Muñoz-Antonia,1 José Torres,2 Gwendolyn Quinn1. 1 _Moffitt Cancer Center, Tampa, FL;_ 2 _Ponce Health Sciences University, Ponce, PR_.

Introduction: Community health workers, known as promotores de salud in Spanish, have been recognized as an effective communication channel to promote health education among marginalized groups. However, cancer education programs often cease efforts when funding ends. This can be particularly problematic in Puerto Rico (PR), where cancer is the leading cause of death. In an effort to identify new ways to provide sustainable cancer health education programs, the NCI-funded PACHE partnership between Moffitt Cancer Center and Ponce Health Sciences University (PSHU) trained graduate public health students to disseminate cancer education to PR communities. This was achieved by developing a culturally adapted version of "Cancer 101: A Cancer Education and Training Program" (Cancer 101) to the PR community. A pilot study was conducted to test students' ability to: I) deliver the educational intervention, charlas, in the community; II) utilize a specific module from the curriculum, "Cancer in PR", which focused on current island data and statistics, and definitions of the cancer and its various types; and III) to measure community members' cancer knowledge before and after the charla.

Methods: Students enrolled in the class Principles of Health Education in Practice (n=15) participated in a two-day Cancer 101 training session. Upon completion, students were divided into 5 teams (n=3 per team), each delivering the "Cancer in PR" charla to a community group of their selection. Cancer knowledge was assessed before and after the charla via a 10-item true or false questionnaire. Items were summed to create an overall knowledge score (range: 0-10).

Results: A total of 201 participants attended the community educational intervention at 10 different locations in PR (Salinas Elderly, Guancha, Hogar San Jose, etc.). Participants pre and post-test mean score (SD) were 4.54 (2.31) and 7.65 (2.28) respectively, with a 3.1 (2.79) point increase in overall cancer knowledge (p<.000). Students reported the Cancer 101 toolkit was feasible and appropriate for each of the communities in which they delivered the intervention.

Conclusion: Results from this pilot project suggest public health students are an innovative and sustainable way to improve cancer health communication and a novel strategy for reducing cancer health disparities.

#3478

Management of chemotherapy induced alopecia using a topical botanical lotion via a proposed triple-action on apoptosis, inflammation and collagen.

Tadafumi Shiiba, Saad Harti, Angelo Mello, Geert Cauwenbergh, Jiawei Liu. _Legacy Healthcare, Lausanne, Switzerland_.

Background: Chemotherapy induced alopecia (CIA) is the most visible and emotionally distressing side effect of cancer therapies. No approved pharmacologic treatment is yet available for CIA. Hair follicular cells are damaged by chemoagents and prematurely undergo unwanted apoptosis, consequently resulting in massive hair loss. Such apoptosis-driven chemo toxicities generally cause dystrophic anagen and dystrophic catagen. Dystrophic anagen hair follicles recover much more slowly than dystrophic catagen hair. As anagen hair proportion represents >80%, hair recovery following chemotherapy may become a long term issue. Besides, shortened anagen will impede collagen production causing unhealthy scalp and hair. A previous study demonstrated that p53 knock-out mice did not undergo CIA, indicating the importance to modulate apoptosis pathways in CIA management. Secondary necrosis stimulates production of pro-inflammatory mediators, triggering or sustaining inflammatory conditions in the scalp. Unwanted apoptosis of more hair follicular cells will occur due to exacerbated inflammation. This vicious circle further contributes to excessive follicular cell death, consequently resulting in hair loss and delay of hair regrowth. Therefore, normalizing apoptosis process, dampening scalp inflammation, as well as improving scalp health by increasing collagen content and remodeling are essential to counteract the negative impact on hair by chemotherapies.

Objective: To test a novel topical botanical lotion for CIA management

Methods: Bcl-2 level in scalp biopsy of androgenetic alopecia (AGA) subjects was analyzed via immunohistochemistry before, after 3 months' application of the topical botanical lotion (n=20), and compared with Bcl-2 level in healthy volunteers (n=25). Expressions of E-selectin, ICAM-1 and il-8 were measured using HUVEC, +/- Product & TNF a. Type I collagen was analyzed using scalp biopsies from 11 AGA subjects before and after 4 months product application. Product's efficacy and tolerance were assessed on female cancer patients suffering from CIA (n = 30), Pictures were taken for comparison.

Results: Product restored Bcl-2 from 1.7 to 3.2, near normal level (4.73). Collagen content was increased by 79.93% after 4 months' product application. Biopsy analysis revealed collagen remodeling. Product inhibited TNFα-induced expressions of E-selectin, ICAM-1 & il-8. For patients undergoing chemotherapy, the product allowed faster hair recovery in CIA patients (5-16 weeks quicker than historical control). For "long-term CIA" patients, first improvement was observed in 33%, 52% and 76% of subjects after 1, 2 and 3 months, respectively. No side effect was reported.

Conclusion: We developed a novel topical product for CIA through local actions on apoptosis, inflammation and collagen.

#3479

Parental coping to retinoblastoma at the onset of diagnosis: A global study.

Trillium E. Chang,1 Yan Zhang,2 Chengyue Zhang,2 Junyang Zhao,3 Khairi Yi,1 Helen Dimaras4. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _Beijing New Century International Hospital for Children, Beijing, China;_ 3 _Capital Medical University, Beijing, China;_ 4 _The Hospital for Sick Children, Toronto, Ontario, Canada_.

Introduction:

Retinoblastoma is the most common ocular malignancy of childhood. At the onset of retinoblastoma diagnosis, parents must learn about retinoblastoma and its complex management, while managing concerns around their child's health and wellbeing. Coping methods are action-oriented efforts to manage, master, tolerate, reduce or minimize the demands of a stressful environment, such as a cancer diagnosis in the family. The purpose of this study was to assess parental coping to the diagnosis of retinoblastoma in their child. The study also aimed to identify factors that assist and/or detract from parental ability to cope.

Methodology:

Eligible study participants included parents or guardians of children diagnosed with retinoblastoma who were present during the child's retinoblastoma diagnosis and treatment.

Study participants completed a print (China, Mandarin language) or online (North America, English language) questionnaire that collected: 1) basic demographic information and information pertaining to the child's retinoblastoma diagnosis; 2) responses to the Mini Mental Adjustment to Cancer Scale (Mini-MAC), a validated scale that assesses coping by measuring levels of helplessness/hopelessness, anxious preoccupation, fighting spirit, avoidance and fatalism; 3) responses to the Hospital Anxiety and Depression Scale (HADS), a validated scale that determines the levels of anxiety and depression experienced by the participant; and 4) additional information on health literacy, self-determination and social support.

Coping strategies assessed by the Mini-MAC that correlated with low HADS scores were deemed adaptive (i.e. factors that aided in coping), while methods that correlated with high HADS scores were maladaptive (i.e. factors that detracted from coping). All models were analyzed with multivariate linear regression and significance was set at p<0.05.

Results:

A total of 153 retinoblastoma parents participated in the study; 30.7% (47/153) were from North America and 69.3% (106/153) were from China.

Chinese parents tended to have a lower correlation with anxiety and depression (R=0.51) than North American parents (R=0.71). For the North American cohort, high scores for helplessness/hopelessness and anxious preoccupation appeared to be maladaptive, as they correlated with increased anxiety and depression. In contrast, the Chinese cohort demonstrated that feelings of helplessness/hopelessness and anxious preoccupation were adaptive, as they correlated with reduced anxiety and depression. For the Chinese cohort, anxious preoccupation was also positively correlated higher parental health literacy.

Conclusions:

We observed significant differences in coping between North American and Chinese parents of children with retinoblastoma, which may be explained by cultural differences. The results of this study may help healthcare teams better care for the psychosocial needs of families affected by retinoblastoma. 

## ENDOCRINOLOGY:

### Clinical Endocrinology

#3480

Germline PARP4 mutations in patients with primary thyroid and breast cancers.

Yuji Ikeda,1 Kazuma Kiyotani,1 Poh Yin Yew,1 Taigo Kato,1 Kenji Tamura,1 Kai Lee Yap,1 Jessica L. Mester,2 Sarah H. Nielsen,1 Grogan H. Raymon,1 Charis Eng,3 Yusuke Nakamura1. 1 _University of Chicago, Chicago, IL;_ 2 _Cleveland Clinic, Chicago, OH;_ 3 _Cleveland Clinic, OH_.

Breast and thyroid cancers occur more frequently in the same individual than what is expected due to random occurrence, implying the presence of genetic susceptible factors. Germline mutations in the PTEN gene, which cause Cowden syndrome (CS), are known to be one of the genetic factors for primary thyroid and breast cancers, however, PTEN mutations are found in only a small subset of research participants with non-syndrome breast and thyroid cancers. In this study, we attempted to identify germline variants that may be related to genetic risk of primary thyroid and breast cancers by whole-exome sequencing. Genomic DNAs extracted from peripheral blood of 14 PTEN-wild-type female research participants with primary thyroid and breast cancers were analyzed. Among them, 7 (50%) and 5 (36%) participants had a family history of thyroid or breast cancer within 3 generations of the proband, respectively. No rare variants were found in SDHx/KLLN/PIK3CA/AKT1 genes previously known to be responsible for CS. We then performed a case-control association study using the information of 406 Europeans obtained from the 1000 Genomes Project database as controls. The predicted impact of amino acid substitutions was annotated using 5 algorithms of LRT score, MutationTaster, PolyPhen-2, HumDiv, PolyPhen-2 HumVar and SIFT. Gene-based association analysis identified 34 genes, including DNA repair-related genes, possibly associated with the phenotype with P<1.0×10-3. Among them, rare variants in the PARP4 gene were detected at significant high frequency (odds ratio = 5.2, P = 1.0×10-5). The variants, G496V and T1170I, were found in 6 of the 14 study participants (43%) while their frequencies were only 0.5% in controls. We subsequently performed functional analysis using HCC1143 cell line which showed the highest expression of PARP4 among 18 breast cancer cell lines examined, and found that knockdown of PARP4 with siRNA significantly enhanced the cell proliferation, compared with the cells transfected with siControl (P = 0.02). In addition, Kaplan-Meier analysis using GEO, EGA and TCGA datasets showed poor progression-free survival (P = 0.006, Hazard ratio 0.71) and overall survival (P < 0.0001, Hazard ratio 0.79) in a PARP4 low-expression group, suggesting that PARP4 may function as a tumor suppression. In conclusion, we identified PARP4 as a possible susceptibility gene of primary thyroid and breast cancer.

#3481

Kisspeptin regulates the invasiveness of endometrial cancer cells through FAK/ Src signaling-dependent activation of matrix metalloproteinase (MMP)-2.

Hsien-Ming Wu, Yung-Yu Liang, Wei-Jung Chiu, Hsin-Shih Wang, Hong-Yuan Huang, Chyong-Huey Lai, Yung-Kuei Soong. _Chang Gang Memorial Hospital, Taoyuan, Taiwan_.

Introduction:

More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastasis. The kisspeptin has an important role in reproduction. In mammals, kisspeptin may regulate tumor progression of endometrial cancer. FAK and Src signaling have been considered important components of tumor progression. MMPs are largely implicated in promoting angiogenesis and tumor metastasis. In the present study, we examined the action of kisspeptin-promoted motility of endometrial cancer cells and the mechanisms of the action in endometrial cancer.

Materials and methods:

Endometrial cancer cell line Ishikawa and ECC-1 were derived from an endometrial adenocarcinoma. Kisspeptin agonist and antagonist were synthetic peptides. Cell motility was estimated by invasion and migration assay. The activities of MMP-2 was assessed by gelatin zymography. Immunoblot analysis was done to study the expression of kisspeptin receptor and the effects of kisspeptin in the activation of FAK ,Src and MMP-2. Human FAK siRNA and Src siRNA were used to knock down the expression of FAK and Src to evaluate the effects of FAK and Src. MMP-2 inhibitor (OA-Hy) was pretreated for 30 min to evaluate the effects of MMP-2 in cell motility.

Results:

The kisspeptin regulated cell motility in a dose-dependent manner. Kisspeptin activated the phosphorylation of FAK and Src signaling and the phosphorylation was abolished by FAK siRNA and Src siRNA. Kisspeptin-regulated cell motility was suppressed in cells pretreated with FAK siRNA and Src siRNA. Moreover, FAK siRNA and Src siRNA abolished kisspeptin-induced activation of MMP-2. Inhibition of MMP-2 with 10μM OA-Hy suppressed cell motility in response to kisspeptin.

Conclusion:

Our study shows that the kisspeptin regulated the cell motility of endometrial cancer cells through the kisspeptin receptor, and the phosphorylation of FAK and Src-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanisms of kisspeptin-regulated cell motility in endometrial cancer cells, suggesting the possibility of kisspeptin analogues as a potential therapeutic intervention for the treatment of human endometrial cancer.

#3482

Inhibition of glucose metabolism in neuroendocrine tumors by lanreotide, sunitinib, and rapamycin.

Tami Rubinek, Ayelet Shabtay-Orbach, Arkadi Hesin, Tammi Rubinstein, Ido Wolf. _Tel Aviv Medical Center, Tel Aviv, Israel_.

Background: Neuroendocrine tumors (NETs) represent a heterogeneous family of neoplasms, which may develop from different endocrine glands or endocrine cells dispersed throughout the digestive and respiratory tracts. Lanreotide is a somatostatin analog approved for the treatment of NETs. While Lanreotide extends survival of patients and slows tumor growth, controversy exists regarding its mechanism of action. We hypothesized that lanreotide can interfere with the metabolic activity of NETs, and compared its effect to that of rapamycin and sunitinib.

Methods: The pancreatic carcinoid BON-1 cells and pulmonary carcinoid NCI-H727 cells were used. Viability was tested using colony formation assay. mRNA levels of the glycolytic enzymes hexokinase 2 (HK2), lactate dehydrogenase (LDH) and pyruvate kinase M2 (PKM2) was tested using real-time PCR. Phosphorylation levels of AMP-activated kinase (AMPK) and acetyl CoA carboxylase (ACC) were tested using Western blot. The activity of HK2 was tested using HK enzymatic assay. Lactate secretion was tested in the routine biochemistry laboratory of our institution.

Results: Lanreotide, rapamycin and sunitinib significantly decreased colony formation. They also reduced relative expression levels of HK2, LDH and PKM2 24 and 48h after treatment and decreased HK2 activity after 24h. An increase in the phosphorylation levels of AMPK and ACC was observed shortly after treating the cells with lanreotide and rapamycin. All three agents also decreased lactate concentration in the media.

Conclusions: These data indicate tumor metabolism as an attractive target for therapies against NET, and a novel mechanism of action of somatostatin analogs, namely, alteration of tumor metabolism.

#3483

ESR1 gene expression and expression ratio of ESR1 to ESR2 are associated with worse prognosis in papillary thyroid carcinoma.

Jin Wook Yi,1 Ra-Yeong Song,1 Hyeong Won Yu,1 Joon-Hyop Lee,2 Hyungju Kwon,1 Young Jun Chai,3 June Young Choi,2 Su-jin Kim,1 Kyu-Eun Lee1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Seoul National University Bundang Hospital, Seoul, Republic of Korea;_ 3 _Seoul National University Boramae Medical Center, Seoul, Republic of Korea_.

Introduction

Expression of estrogen receptor (ER) and progesterone receptor (PR) in breast cancer have been widely studied and used as prognostic markers or therapeutic targets in breast cancer patients. However, clinical and pathologic roles of these hormone receptor genes have not been clearly established in thyroid cancer.

Materials and methods

To evaluate the relationship between sex hormone receptor gene expressions and clinicopathologic variables, we analyzed ESR1, ESR2, ESR ratio (ESR1/ESR2) and PGR mRNA expression data from The Cancer Genome Atlas (TCGA) RNA sequencing experiment in papillary thyroid carcinoma (PTC). To validate results from TCGA analysis, we additionally analyzed a public microarray data (GSE 54598) from the gene expression omnibus (GEO) database.

Results

In TCGA data analysis, ESR1, ESR ratio and PGR expression were significantly higher in PTC tissue than in normal thyroid tissue (Mean value of normal versus tumor were 264.045 vs 659.427 in ESR1, 19.064 vs 92,017 in ESR ratio and 58.514 vs 119.792 in PGR, respectively). In female patients, ESR1 and ESR ratio was negatively correlated with advanced age (t-value was -2.257 and -2.949 in linear regression analysis). Expression of ESR1 and ESR ratio were higher in classic type PTC, presentation of lymphovascular invasion and BRAF V600E mutation. Higher expression group of ESR1 and ESR ratio showed worse overall survival in female PTC patients (Hazard ratio was 6.348 in ESR1 and 4.031 in ESR ratio, respectively). In microarray analysis, ESR1 and ESR ratio expressions were higher in tumor than normal, classic than follicular variant and BRAF V600E than wild type.

Conclusion

Expression of ESR1 and ESR ratio were associated with aggressive prognostic factors and worse overall survival in female PTC patients. These results suggest that higher expression of ESR1 and ESR ratio can be used as prognostic markers to predict survival in female patients.

#3484

**Analysis of** in situ **expression of hormone receptors and proliferation marker at a single cell level in breast cancer tissues.**

Takayuki Ueno,1 Hirotsugu Isaka,1 Hiroki Itoh,1 Kaisuke Miyamoto,1 Manami Kitamura,2 Shigeru Imoto1. 1 _Kyorin University, Tokyo, Japan;_ 2 _Showa General Hospital, Tokyo, Japan_.

Background: Hormone receptors and proliferation markers are critical parameters for treatment selection of breast cancer patients. The expression of different parameters are currently assessed separately in different tissue sections but it is unclear how different parameters are co-expressed in a single cell in breast cancer tissues.

Samples and Methods: Breast cancer tissues from fifty-one patients with ER-positive HER2-negative breast cancer were analyzed. Expressions of ER and PgR were assessed in association with Ki67 using dual fluorescence immunohistochemistry with specific antibodies: SP-1 (abcam, Tokyo), 1E2 (Roche Diagnostics GmBH, Germany), and MIB1 (Dako Japan, Tokyo), respectively. More than 500 cancer cells were assessed in each tissue. Expression levels of each marker in a single cell were semi-quantitatively assessed by MetaMorph image analyzer (Molecular Devices Japan, Tokyo). All statistical analyses were performed using JMP ver.8.01 (SAS Institute Japan, Tokyo).

Results: To validate the system, Ki67 LI in breast cancer tissues were compared between this system and the regular DAB system. The two systems showed a good concordance (p < 0.0001). All cancer tissues expressed ER. There were two distinct populations among Ki67-positive proliferating cells according to PgR expression status: PgR-positive proliferating cells and PgR-negative proliferating cells. Since cell proliferation is regulated in cancer cells by at least two different drivers including hormone receptor and growth factor, it is conceivable that these two populations depend on different driving systems. Indeed, tissues with dominantly PgR-positive proliferating cells showed mostly histological grade 1 (15/20, 75%), while most of tissues with PgR-negative proliferating cells showed grade 2 and 3 (22/31, 71%) (p = 0.0025 by chi square test). Moreover, the multivariate analysis using the ordered logistic regression analysis showed that PgR status in proliferating cells is an independent factor associated with histological grade (p = 0.003) while PgR expression rate and Ki67 LI were not (p = 0.3 and 0.25, respectively).

Conclusion: PgR status in Ki67-positive proliferating cells is associated with histological grade in ER positive HER2-negative breast cancers. Our results suggest that different driving systems give different expression patterns of PgR and Ki67 at a single cell level, which may distinguish between luminal A and luminal B cancers.

#3485

Estrogen receptor ligands and their responses in de novo and tamoxifen resistant cell models.

Lauren M. Gutgesell, Gregory R. J. Thatcher, Hitisha Patel, Rui Xiong. _University of Illinois at Chicago, Chicago, IL_.

Approximately 75% of breast cancer incidences are Estrogen Receptor positive (ER+). Tamoxifen, a selective estrogen receptor modulator (SERM), is the standard of care for many of these ER+ breast cancer patients. Unfortunately, tamoxifen resistance occurs in almost 50% of patients within 5 years of treatment, and endocrine-independence accompanying resistance negates the effects of aromatase inhibitors. Paradoxically, estradiol (E2) has shown clinical efficacy in patients with resistant breast cancer. Understanding the antiproliferative role of E2 and ER signaling in resistant ER+ cell lines is essential to gain a better understanding of paradoxical clinical efficacy and for the appropriate biomarker-assisted selection of endocrine therapy for various stages of ER+ breast cancer. We have discovered ER ligands, based upon a single chemical scaffold with a diverse set of pharmacological responses, which can be used to better understand the role of ER signaling in resistance and therapy: selective ER modulators (SERMs), selective ER downregulators (SERDs), selective estrogen mimics (SEMs), and selective human ER partial agonists (ShERPAs). These compounds were initially classified using an ERE luciferase reporter assay and affinity for ER confirmed by biochemical assays. The effects of these novel ER-directed chemical probes on cell viability were further examined in multiple 2D and 3D spheroid models of tamoxifen resistance. Finally, ERα localization upon administration of these ligands in tamoxifen-sensitive and tamoxifen-resistant cells was studied. While SERDs and SEMs showed growth inhibition in tamoxifen resistant cell lines, both molecules had different responses and mechanisms of growth inhibition. ERα was localized to extranuclear sites upon administration of E2, SEMs, and ShERPAs, an observation specific to the resistant phenotype and mechanistically associated with spheroid disintegration. SERDs inhibited the antiproliferative actions of E2, but were antiproliferative in resistant cell lines. Further dissection of the role of ER in resistance and survival is needed to define the appropriate ER-directed, endocrine therapy in ER+ breast cancer.

#3486

Dissecting the mechanism of AR-v7 nuclear translocation in prostate cancer.

Seaho Kim, Mohd Azrin Jamalruddin, Luigi Portella, Paraskevi Giannakakou. _Weill Cornell Medicine, New York, NY_.

Continuous androgen receptor (AR) signaling is the key driver of castration-resistant prostate cancer (CRPC), despite prior androgen deprivation therapy (ADT). Potent, next-generation AR signaling inhibitors, such as abiraterone and enzalutamide have been recently added to the standard of care in the treatment of prostate cancer. However, resistance to these drugs inevitably emerges and is mediated by adaptive mechanisms that restore AR function, in the form of constitutively active, ligand-independent AR-variant (AR-V) overexpression. The microtubule-targeting drugs Docetaxel (DTX) and Cabazitaxel (CTX) are the only chemotherapy that significantly improves survival of CRPC patients. We have shown that full-length AR (AR-fl) binds microtubules (MTs) via its hinge domain, and utilizes them as tracks to facilitate its nuclear translocation. Taxanes keep AR-fl inactive in the cytoplasm as a result of MT stabilization. Of the two most prevalent AR-Vs, ARv567 contains the hinge region and is sensitive to taxane treatment while AR-v7, expressed in 60% of CRPC patients, lacks the hinge region and does not depend on MTs for its nuclear translocation. Therefore, ARv7 nuclear translocation is MT-independent and neither taxane treatment nor the disruption of dynein motor complex inhibited its nuclear localization. Thus, AR-v7 expression confers taxane resistance in vivo, in addition to next-generation AR inhibitors. Hence, inhibition of AR-v7 nuclear translocation and activity is critical to overcome drug resistance to current therapeutic modalities. However, the mechanism of ARv7 nuclear translocation is not clearly understood. Previously, we showed that ARv7 exhibited faster kinetics of nuclear translocation compared to AR-fl. Additionally, AR-v7 nuclear import is independent of the importin-α/β pathway, which is utilized by the MT-dependent, AR-fl and ARv567. Upon disruption of the Ran-GTPase nuclear import pathway, by overexpression of the catalytic mutant Q69L form of Ran, we observed that the nuclear import of both AR-fl and AR-V567 was significantly impaired, while ARv7 nuclear import was only partially affected. In this study, we hypothesized that molecular dynamics of AR nuclear import and export determines overall spatiotemporal localization of AR and its activity. To test this hypothesis, we generated photo-convertible AR-fl and AR-Vs (AR-v567 and AR-v7) fused to mEos4b construct, which enables us to track protein mobilization in the region of interest after photo-conversion in live cells. We are currently studying the nuclear import and export kinetics of both AR-fl and AR variants (AR-v567 and AR-v7) alone and in combination using the photo-conversion methodology. Elucidation of the distinct pathway(s) that mediate ARv7 nuclear import and/or export will allow the rationale design of effective ARv7 inhibitors or the development of co-targeting strategies in combination with taxanes and AR signaling inhibitors.

#3487

Alcohol programs the pituitary to produce aggressive prolactin-producing tumors.

Shaima Jabbar, Jinhee Park, William Belden, Dipak K. Sarkar. _Rutgers University, New Brunswick, NJ_.

Chronic alcohol consumption is known to increase prolactin levels in blood and cell proliferation in pituitary prolactin-producing cells known as lactotropes, resulting in hyperprolactinemia in human and animals. We have recently shown that alcohol abuse during pregnancy result in fetal programming that alters lactotropic cell production of prolactin in the offspring during the adulthood. We determined whether fetal alcohol exposure increases the susceptibility to estrogen-induced pituitary prolactin-secreting tumors (prolactinomas). Pregnant Fischer 344 rats were fed between gestational days 7 and 21 with a liquid diet containing alcohol (AF), pair-fed with isocaloric liquid diet (PF), or fed ad libitum with rat chow (AD). At 90 days of age, female offspring rats were ovariectomized and received a subcutaneous estradiol implant. These rats were sacrificed at 3 moths after the estradiol implants. At the time of sacrifice, pituitaries of these animals were inspected for tumor and whole body were inspected for any tumor metastasis. Estradiol treatment increased pituitary weight about 5-folds in AD and PF treated groups and increased about 30-folds in the AF treated group. Most tumors in the AF group were hemorrhagic. About 30% AF rats had some tumors in the non-pituitary sites. AD and PF rats did not show any non-pituitary site tumor. Histopathological evaluation revealed that tumors in AF group were more densely packed cells as compare to the PF and AD groups who showed uniform cells with abundant cytoplasm. Pituitary tumor from AF animals showed strong nuclear p53, Ki67 and prolactin immunoreactivity. When AF rat pituitaries were grown in cultures, cells rapidly grew and formed colonies. Colony formation and rapid growth rate were not observed in cells of the pituitary from PF and AD rats. When pituitary tumor cells of AF-treated pituitary, but not AD- or PF-treated pituitaries were grown in ultra-low attachment plate, spheres developed. These spheres expressed genes and proteins related to multipotency (OCT4, NANOG, KLF4, SOX2, Nestin, CD34 and CD133). Differentiated cells from pituitary tumorspheres of AF animals successfully induce tumor in scid mice after three weeks from cells transplantation. In addition, pituitary tumor from AF animals showed strong immunoreactivity for N-cadherin and Snail, two epithelial-mesenchymal transitions (EMT) factors. Furthermore, RNA-seq of genes in tumor pituitaries of AF and AD rats revealed 8782 genes are significantly changed in AF compared to AD P<0.01. Many of these genes were involved in the cell migration, actin cytoskeletal regulation and cell-cell junction formations which are the most important phenotypes occur during EMT. These data provide evidence for the possible development of aggressive prolactinoma development in the pituitary after estrogen treatment in fetal alcohol exposed female rats. (This work is supported by a National Institute of Health grant R01 AA11591).

#3488

External validation of lysyl oxidase-like 2 (LOXL2) as a novel prognostic marker for gastro-entero-pancreatic neuroendocrine tumors (GEP-NET).

Jorge Barriuso,1 Marta Benavent,2 Angela Lamarca,3 Elsa Bernal,4 Laura Guerra Pastrian,4 Victoria Heredia,4 Maria Miguel,4 Clara Beatriz Garcia-Calderon,2 Cristina Alvarez-Escola,4 Jose Castell,4 Ana Custodio,4 Emilio Burgos,4 Jaime Feliu,4 Rocio Garcia-Carbonero,5 Marta Mendiola4. 1 _University of Manchester, Manchester, United Kingdom;_ 2 _Hospital Universitario Virgen del Rocio, Seville, Spain;_ 3 _The Christie NHS Trust, Manchester, United Kingdom;_ 4 _IdIPAZ - Hospital Universitario La Paz, Madrid, Spain;_ 5 _Hospital Universitario Doce de Octubre, Madrid, Spain_.

Aims: LOXL2 is a protein with a key role in epithelial-to-mesenchymal transition (EMT). EMT was established as an early event in GEP-NET. LOXL2 emerged as a new prognostic marker in the analysis of a 115 GEP-NET cases (training cohort (TC); Barriuso et al, ASCO 2014). Our main objective was to validate LOXL2 expression by immunohistochemistry (IHC) in an independent cohort.

Methods: Formalin-fixed paraffin-embedded samples (FFPEs) of consecutive GEP-NET patients from 1999 to 2010 who underwent surgery and their clinical data were collected from a different Spanish institution (validation cohort (VC)). Tissue microarrays were constructed from two non-necrotic areas of tumour foci. LOXL2 expression was studied by IHC and classified as presence (P) vs absence (A). Log rank test and cox regression were used to study Disease Free Survival (DFS) and Overall Survival (OS) in the VC and the combined series (TC+VC; n=206). Univariate (UVA) and multivariable analysis (MVA) were performed.

Results: A total of 91 FFPE samples were included in the VC. Median follow up was 77 months. Tumor grade was differently distributed between the TC and the VC (p<0.001) while stage was not (p=0.195). LOXL2 P was associated with better OS (p=0.023) and showed a trend for better DFS (p=0.066) in the VC. DFS at 3 years was 85% in LOXL2-P group vs 45% in LOXL2-A group. OS at 5 years was 82% vs 51% respectively. LOXL2 P was associated with better DFS and OS (p<0.001) when the combined series was analysed. LOXL2 remained as an independent prognostic factor for OS adjusted for grade and stage in the MVA in both settings.

Conclusion: Our results validated LOXL2 as a novel prognostic biomarker candidate for GEP-NETs in an independent cohort. Further testing in prospective studies to depict its potential value in the clinic is warranted. LOXL2 could also represent an actionable target in this scenario.  | |  | |

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

|

UVA for DFS | MVA for DFS | UVA for OS | MVA for OS

Training cohort (TC) (n=115) | 0.3 (0.1-0.7)

P=0.012* | 0.5 (0.1-1.8)

P=0.28 | 0.2 (0.1-0.5)

P=0.001* | 0.2 (0.04-0.8)

P=0.024*

Validation cohort (VC) (n=91) | 0.5 (0.2-1.1)

P=0.079 | 0.3 (0.1-0.7)

P=0.011* | 0.4 (0.1-0.9)

P=0.029* | 0.2 (0.1-0.6)

P=0.01*

Combined series (n=206) | 0.3 (0.2-0.6)

P<0.001* | 0.3 (0.1-0.6)

P=0.002* | 0.2 (0.1-0.4)

P<0.001* | 0.2 (0.1-0.5)

P<0.001*

Cox regression for LOXL2. MVA adjusted for grade and stage. Hazard ratios, 95% confidence intervals and p values. *statistically significant differences.

#3489

Elucidation of taxane resistance in prostate cancer through RNA-Seq analysis of circulating tumor cells.

Jiaren Zhang, Ada Gjyrezi, Prashant Thakkar, Giuseppe Galletti, Akanksha Verma, Olivier Elemento, Paraskevi Giannakakou. _Weill Cornell Medicine, New York, NY_.

Prostate cancer is the most commonly diagnosed male cancer in the United States. Taxanes are the only established chemotherapy drugs proven to be effective in improving survival of men with advanced prostate cancer through disruption of the AR-signaling axis downstream of microtubule stabilization. However, there is significant heterogeneity in how patients respond to taxanes and most patients ultimately become refractory due to the development of drug resistance. Currently, the molecular basis of clinical taxane resistance in PC is poorly understood. Prostate cancer circulating tumor cells (P-CTCs) are often found in the peripheral blood of patients suffering from metastatic prostate cancer and have been clinically used as prognostic biomarker for metastatic progression and treatment outcome. The objective of this study is to identify clinically relevant mechanisms of taxane resistance through conducting RNA-Seq analysis in P-CTCs isolated from patients before, during and after they become refractory to taxane chemotherapy.

To show feasibility of RNA-Seq experiments with limiting samples such as CTCs and given the presence of contaminating leucocytes, a pilot experiment was performed in which limiting numbers of prostate cancer cells (LNCaP) either pure or enriched following spiking into healthy donor blood, were analyzed by RNA-Seq. Matching healthy donor blood processed with the same enrichment protocol was used as germline control as well as control for the presence of contaminating leucocytes following CTC enrichment. Trimmed reads were aligned to human reference genome (hg38) using STAR. Determination of Fragments Per Kilobase of exon per Million mapped fragments (FPKM) was performed using Cufflinks and heat map was built based on the value of log10(FPKM+1). Gene expression analysis showed that markers of prostate (such as AR, PSMA, KLK3, KLK2, and AMACR) or epithelial lineage (such as EpCAM, CDH1, KRT8 and KRT18) were detected in both pure LNCaP cells-regardless of amount- as well as limited number of captured LNCaP cells in the presence of contaminating leucocytes. In contrast, healthy donor blood was negative for the prostate and epithelial lineage markers and positive for the leucocyte specific markers (such as CD45 and CD16). Gene set enrichment analysis (GSEA) indicated significant enrichment for Andorgen response, MYC, MTOR and RB related pathways in pure and captured LNCap cells compared with healthy donor blood. These data clearly show that by using RNA-Seq we can detect the prostate and epithelial specific gene signatures of limited number of spiked prostate cancer cells using the microfluidic device. Ongoing work includes RNA-Seq analysis of P-CTCs isolated from patients before and after taxane treatment, in order to detect differentially expressed genes, pathways, and potentially driver somatic mutations associated with clinical taxane resistance.

#3490

The effect of novel CYP17 inhibitor galeterone on gonadal and tumor progestogen and androgen levels in SCID mice bearing LNCaP prostate cancer xenografts.

Amanda J. Schech,1 Gauri J. Sabnis,1 Stephen Yu,1 Vincent C.O. Njar,1 Douglas B. Jacoby,2 Peter Nelson,3 Ruth Dumpit,3 Angela M.H. Brodie1. 1 _Univ. of Maryland School of Medicine, Baltimore, MD;_ 2 _Tokai Pharmaceuticals, Boston, MA;_ 3 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Prostate cancer growth is driven by androgen-dependent activation of the androgen receptor (AR). Inhibition of CYP17A1, a cytochrome P450 enzyme responsible for the conversion of progestogens to androgens, is a key strategy in the treatment of prostate cancer. CYP17A1 has both 17α-hydroxylase and 17,20-lyase function. The 17α-hydroxylase catalyzes the conversion of progesterone/pregnenolone to 17α-hydroxyprogesterone/17α-hydroxypregnenolone and the 17,20-lyase converts 17α-hydroxyprogesterone/17α-hydroxypregnenolone to androstenedione (AD)/dehydroepiandrosterone (DHEA). Galeterone, a novel AR degrader and antagonist, is also an inhibitor of CYP17A1 and has been shown to inhibit the growth of prostate cancer cells and tumors. However, the effects of galeterone on steroid levels affected by CYP17A1 inhibition have not been examined in vivo. Male SCID mice bearing LNCaP tumors were randomized to receive vehicle (30% beta-cyclodextran in saline) or 0.15mmol/kg galeterone (either po or sc). Mice were treated twice daily, seven days a week. Route of administration did not alter the efficacy of galeterone, which significantly reduced tumor volume (p = 0.044 and p = 0.049, sc, po). Upon completion of the experiment, tumors, testes, and plasma were collected for analysis of steroid levels. Analysis of intratumoral steroids showed an increase in progesterone (1.7- and 2-fold, sc, po) and a decrease in AD (89% and 77% reduction, sc, po). Similar results were observed in levels of pregnenolone which increased (1.4- and 3.0-fold, sc, po) while DHEA decreased (72% and 17% reduction, sc, po). Interestingly, levels of 17α-hydroxyprenenolone were increased in the sc treatment arm, suggesting selectivity of galeterone for the lyase over the hydroxylase catalytic function. Both routes of administration reduced intratumoral testosterone (97% and 77% reduction, sc, po). Intratesticular androgen levels showed similar trends compared to those in tumors. Following galeterone treatment, the levels of androgen precursors were higher in the testes than in the tumor (pregnenolone increased 2.3- and 1.5-fold sc, po; progesterone elevated 6.4- and 12.8-fold sc, po). Plasma androgen levels varied from those observed in the tumor and testes. However, galeterone treatment reduced the levels of AD (98% and 68% reduction, sc, po) and testosterone (99.6% and 99% reduction, sc, po). Together, these results show for the first time that galeterone specifically targets CYP17A1 in vivo as demonstrated by reduction of its enzymatic products DHEA, AD and testosterone. In addition, a selectivity for the lyase function was observed, as evidenced by greater decreases in DHEA and AD than in 17α-hydroxypregenelone. This is consistent with findings that patients treated with galeterone do not require cortisol replacement therapy and do not show symptoms of mineralocorticoid excess.

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Death 1

#3491

Rational combination of targeted agents and BH3 mimetics to improve cancer treatment.

Joan Montero, Dorota E. Sadowicz, Jeremy Ryan, Charles H. Yoon, Rizwan Haq, Anthony Letai. _Dana-Farber Cancer Institute, Boston, MA_.

Background: In the clinic is often observed that melanoma cancer patients first respond to targeted therapeutic agents like dabrafenib showing tumor burden shrinkage, but subsequently relapse with tumors that are more aggressive, no longer responsive to therapy and showing acquired metastatic capacity. Most chemotherapeutic agents kill via the mitochondrial pathway of apoptosis, but unfortunately tumors frequently are able to survive, often adapting their antiapoptotic strategy. We used a variant of the functional predictive test dynamic BH3 profiling (DBP) to determine the tumor's antiapoptotic defense adaptation over time, and rationally identify combination of therapies to restore programmed cell death.

Hypothesis: By using different BH3 peptides in DBP we can determine the adaptive tumor's antiapoptotic defense and adjust therapy to improve cancer cell death.

Results: In BRAF mutant melanoma, cKIT mutant GIST and HER2 overexpressing breast cancer cell lines, we performed DBP over time using different BH3 peptides: BIM, BAD, PUMA, MS1 and NOXA. We observed that when they are treated with specific targeted agents against their oncogenic drivers, there is an increase in MCL-1 dependence over time. This observation points to a possible tumor defense mechanism conferring resistance against anticancer drugs. Based on these observations, we exploited these altered dependencies by pretreating the cancer cells with these targeted agents and combining them with novel BH3 mimetics targeting MCL-1 to synergistically enhance cell death.

Exploiting DBP's capacity to measure changes in priming without the requirement for prolonged ex vivo culture, we assessed if this antiapoptotic evolution was also present in primary melanoma samples. Using FACS-based DBP to select for specific tumor populations, we tested the possible use of BH3 mimetics in combination with targeted agents to improve cancer treatment in the clinic.

Conclusions: Our preliminary results reveal the use of dynamic BH3 profiling to be used not only as a powerful real-time tool to predict chemotherapy response but also to evaluate the rational combination of targeted agents with BH3 mimetics in vitro and in the clinic.

[R.H. and A.L. contributed equally to this work.]

#3492

Differential sensitivity of normal liver and hepatocarcinoma tissues to tBid induced apoptosis is determined by the abundance of VDAC2.

Shamim Naghdi, Soumya Sinha Roy, Ludivine Walter, Gyorgy Hajnoczky. _Thomas Jefferson University, Philadelphia, PA_.

A conserved step in extrinsic apoptosis is the outer mitochondrial membrane (OMM) permeabilization by truncated Bid (tBid) that is generated from Bid upon cleavage by Caspase-8 activated at death receptors. tBid directly or indirectly activate proapoptotic proteins i.e. Bak in OMM and/ or Bax in cytosol which is followed by a change in their conformation and oligomerization, leading to a pore formation in the OMM. To better understand the mitochondrial phase of this pathway we applied tBid to semi-permeabilized cells and isolated mitochondria. We monitored cytochrome c release, and the ensuing mitochondrial membrane potential collapse, using immunoblotting and fluorimetry as the markers for mitochondrial cell death. We found that normal liver tissue and normal hepatocytes (Rat, Mouse or Human) are unexpectedly resistant to tBid, whereas hepatocellular carcinoma (HCC) tissues and cell lines (e.g. PLC, Huh7) are greatly sensitized. Considering that the effect of tBid is most effectively mediated through Bak, its abundance in the mitochondria was tested. Bak level is significantly higher in tumor tissue/cell lines, whereas OMM-bound tBid and Bax levels are similar in both cancerous and normal tissue. Since we have recently found that voltage dependent anion channel 2 (V2), a pore forming OMM protein, is specifically required for Bak targeting to OMM, the abundance of V2 was also measured. V2 showed significant upregulation in HCC tissue/cell lines. Rescue studies using V2 sensitized normal hepatocytes and liver mitochondria to tBid. We propose that V2 might be the main regulator for the differential tBid sensitivity in normal liver and hepatocellular carcinoma.

#3493

New, highly potent antibodies to death receptors having Fc mutations to increase antitumor activity.

Lihong Wang, Yi Ding, Hangil Park, April Zhang, Zhengran Wang, Maximiliano Vasquez, Cary Queen, Jin Kim. _Galaxy Biotech, LLC, Sunnyvale, CA_.

Death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2 or Apo2) are TNF receptor superfamily members that are receptors for TRAIL (Apo2 ligand), an immunomodulatory cytokine. Binding of TRAIL to DR4 or DR5 can activate the extrinsic apoptotic pathway selectively in tumor cells. Agonist antibodies to death receptors DR4 and DR5 thus have potential for treatment of cancer and may be better than derivatives of TRAIL itself because of their superior pharmacokinetics and receptor selectivity. However, despite dramatic effects in vitro and in preclinical xenograft models, several agonist antibodies to DR4 and especially DR5 have not provided significant therapeutic benefit in clinical trials. While resistance mechanisms in cancer cells are undoubtedly one reason, another factor may be inadequate potency of the antibodies tested. We therefore used intense immunization and screening protocols to develop very potent anti-DR4 and anti-DR5 monoclonal antibodies (mAbs), which were humanized to make the mAbs denoted HuD114 and HuG4.2 respectively. These mAbs were substantially more effective at killing tumor cells in vitro than the mAbs that have been tested in clinical trials. In addition, we introduced one or two mutations into the Fc (constant) regions of these and other potent anti-DR4 and anti-DR5 mAbs in order to increase affinity for the Fc gamma receptor IIb. Cross-linking, as provided by binding of anti-DR4 and anti-DR5 mAbs to Fc gamma receptors on immune cells, is required for effective transmission of an apoptotic signal through the death receptors. Accordingly, introduction of these mutations greatly increased the ability of the mAbs to kill tumor cells in vitro in the presence of human peripheral blood mononuclear cells, and to inhibit the growth of tumor xenografts in mouse models, with two mutations generally more effective than a single mutation. As a complementary approach to increase cross-linking, we also developed bispecific antibodies containing two binding domains from an anti-DR4 mAb and two binding domains from an anti-DR5 mAb. These bispecific mAbs were made in the IgG-like Bs(scFv)4-IgG format consisting of a homodimer of two monomers, each monomer having a single chain Fv from an anti-DR4 mAb linked to a heavy chain constant region, and a single chain Fv from an anti-DR5 mAb linked to a light chain constant region. Such a bispecific mAb was more effective at killing tumor cells than an anti-DR4 or anti-DR5 mAb alone, or even a mixture of anti-DR4 and anti-DR5 mAbs. Based on their potency in vitro and in animal models, we believe that HuD114 and HuG4.2, enhanced by the Fc mutations and/or in bispecific form, have the potential for greater clinical efficacy than previously tested anti-DR4 and anti-DR5 mAbs and thus merit further investigation.

#3494

MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor agonist, induces apoptotic cell death in breast cancer cells.

Yoshimi Greer,1 Sam Gilbert,1 David Tice,2 Stanley Lipkowitz1. 1 _NCI, Bethesda, MD;_ 2 _MedImmune, Gaithersburg, MD_.

TRAIL receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors 4 and 5 (DR4 and DR5) with little toxicity against normal cells. We previously reported that GST-TRAIL efficiently induced cell death in breast cancer cells, particularly mesenchymal triple negative breast cancer (TNBC) - so called basal B breast cancer cells (Rahman et al., Breast Cancer Res Treat. 2009). Recently, a newly developed multivalent TRAIL receptor agonist designed to activate DR5, has been shown to be a TRAIL super-agonist with significantly enhanced potency in multiple cancer cell lines (Swers et al., Mol Cancer Ther. 2013). We hypothesized that MEDI3039, developed by modification of this TRAIL super-agonist, is a potential new therapeutic agent to be used in human breast cancer treatment.

We used 19 human breast cancer cell lines that can be categorized into 4 groups: ER+, HER2 amplified, TNBC basal A and TNBC basal B. MEDI3039- or GST-TRAIL-induced cell death was analyzed by an MTS assay in a 96 well format after 72h of treatment. MEDI3039- or GST-TRAIL-induced caspase activation was measured by Caspase-glo 3/7 assay. Z-VAD-FMK was used as a pan-caspase inhibitor. To verify that MEDI3039 induced apoptosis via DR5, siRNA against DR4 and DR5 were transfected to cells and tested in MTS assay and Western blotting.

MEDI3039 induced cell death in MDA-MB231 (TNBC basal B), and the IC50 was 4.71pM. By contrast, GST-TRAIL induced cell death in this cell line with an IC50 of 624 pM (a 132 fold difference). MEDI3039-induced cell death was completely inhibited by Z-VAD-FMK, indicating that cell death was the result of caspase-mediated apoptotic pathway. Knockdown of DR5, but not DR4, inhibited MEDI3039-induced cell death, demonstrating that MEDI3039-mediated apoptosis requires DR5. MEDI3039 induced cell death in multiple breast cancer cell lines, but the sensitivity varied between cell lines from the four different subtypes. The TNBC basal B group was the most sensitive (avg IC50= 1.4 pM), the TNBC basal A group was next most sensitive (avg IC50 = 203 pM), the HER2 amplified group was less sensitive (avg IC50 = 314 pM), and the ER+ group was the least sensitive to MEDI3039 (avg IC50= 403 pM). This relative sensitivity of the different subtypes of breast cancer was similar to what was observed with GST-TRAIL. Importantly, MEDI3039 was at least 2 orders of magnitude more potent compared with GST-TRAIL in most cell lines tested. Drug combination experiments indicated that MEDI3039 has synergistic effect with multiple drugs, including cisplatin and the Wee1 inhibitor MK1775. Data from ongoing animal experiments will be presented. In conclusion, MEDI3039 is a potent TRAIL receptor agonist in breast cancer cells and has potential as a cancer drug in breast cancer patients, especially those with TNBC basal B.

#3495

Tracking reversal of apoptosis after caspase activation in cancer cells.

Ho Lam Tang,1 Ho Man Tang,2 Ming Chiu Fung,3 J. Marie Hardwick1. 1 _W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD;_ 2 _Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, Baltimore, MD;_ 3 _School of Life Sciences and Center for Soybean Research of the State Key Laboratory of Agrobiotechnology, Chinese University of Hong Kong, Shatin, Hong Kong_.

Using a novel caspase biosensor, we demonstrate that the late stages of apoptosis are reversible events in whole animals. Apoptosis is generally assumed to be a one-way process due to rapid activation of caspases that dismantle cellular components ending in cell suicide. Activation of apoptotic pathways is an important therapeutic strategy for treating cancer. Although some tumor types exhibit dramatic initial responses to such therapies, they inevitably recur, leading to treatment failure. One potential contributor to tumor recurrence is the failure to complete apoptotic cell death once the death pathway has been activated. We showed that cancer cells can reverse apoptosis at late stages (Anastasis, Greek for "Rising to Life"), even after passing through critical steps previously thought to be the "point of no return" such as cytochrome c release and caspases-3 activation. Simply by removing an apoptotic stimulus from cultured cells before they have completed the dying process, a high percent of cells can recover and continue to divide. We adopted the term anastasis to describe cell recovery from the brink of death. These in vitro observations suggest that anastasis may also occur in vivo, possibly allowing tumor cells to escape anti-cancer therapies. However, the major challenge to demonstrating anastasis in whole animals is that recovered cells appear morphologically indistinguishable from normal cells that never activated their cell death pathway. Therefore, we engineered the Drosophila CaspaseTracker, a novel caspase biosensor that permanently labels cells that have ever experienced caspase activation. This biosensor system is composed of 3 genetically encoded components, (1) a caspase-activatable transcription factor Gal4, and (2) G-TRACE, which is composed of Gal4-inducable red fluorescent protein (UAS-RFP), Gal4-inducable FLP recombinase (UAS-Flpase), and Flpase reporter (Ubi-LoxP-Stop-LoxP-nucGFP). Upon caspase activation, the linker tethering Gal4 in the cytoplasm is cleaved by caspases. Subsequently, Gal4 enters the nucleus where it drives the expression of FLP recombinase that removes the stop codon in nuclear-targeted GFP, robustly inducing biosensor activity in egg chambers and likely in the stem cell compartment. Following a recovery period after treatment with a death stimulus, normal healthy egg chambers reappear and continue to express biosensor activity, which is not observed in the absence of a death stimulus or with a control version of the same biosensor that has a single Asp to Ala point mutation abolishing cleavage and activation of the biosensor by caspases. Our study suggests that CaspaseTracker would be a useful tool to track reversal of apoptosis in cancer cells in live animals.

#3496

TRAF2 protects mammary epithelial and cancer cells from endoplasmic reticulum stress-induced apoptosis.

Hasem Habelhah, Laiqun Zhang, Ainiwaer Xialikaer, Ken Blackwell. _University of Iowa, Iowa City, IA_.

TRAF2 plays a key role in the immune response and cell survival by regulating the NF-κB and JNK pathways in response to most members of the TNF superfamily. TRAF2 has also been shown in cell culture systems to mediate IRE1α-induced activation of the ASK1-JNK pathway to promote apoptosis under conditions of acute endoplasmic reticulum (ER) stress. Gene knockout studies revealed that mice deficient for RANKL or NF-κB display defects in osteoclastogenesis and lactating mammary-gland development. Here, we report that TNFα and TRAF2 double knockout (DKO) mice develop with normal bone morphology and density, but the female mice display a severe lactation defect due to significantly increased apoptosis of cells in the lobuloalveolar tree. Unexpectedly, RANKL-induced activation of the canonical NF-κB pathway is not only normal in DKO mammary epithelial cells (MECs), but the expression of the NF-κB target genes is significantly enhanced due to constitutive activation of the non-canonical NF-κB pathway in the absence of TRAF2. On the other hand, cytotoxicity assays revealed that DKO MECs exhibit significantly increased susceptibility to apoptosis induced by chronic ER stress, but not by death receptor or DNA damage. In addition, siRNA-mediated knockdown of TRAF2 in breast cancer cells significantly sensitized the cells to chronic ER stress-induced apoptosis in a RIP1-dependent manner. These data suggest that the physiological function of TRAF2 is to protect the cells from apoptosis under conditions of pathologically relevant chronic ER stress. Given that the lactating MECs and rapidly growing cancer cells have high demands for protein syntheses and ER stress responses, our findings suggest that inhibition of the IRE1α-TRAF2 pathway could be used as a novel adjuvant therapy to treat rapidly proliferating cancer cells.

#3497

Metformin enhances TRAIL-induced apoptosis by Mcl-1 degradation via Mule in colorectal cancer cells.

Seong Hye Park,1 Dae-Hee Lee,1 Jung Lim Kim,1 Sun Il Lee,2 Bo Ram Kim,1 Yoo Jin Na,1 Suk Young Lee,2 Hong-Jun Kim,2 Sung Yup Joung,2 Sanghee Kang,2 Sang Cheul Oh2. 1 _Korea University, Seoul, Republic of Korea;_ 2 _Korea University guro hospital, Seoul, Republic of Korea_.

Recent studies have reported that metformin is an anti-diabetic drug, and is the potential a promising anti-cancer agent. The purpose of this study is to evaluate whether metformin effectively sensitize human colorectal cancer (CRC) cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Metformin alone did not induce apoptosis, but significantly potentiated TRAIL-induced apoptosis in CRC cells. CRC cells treated with metformin and TRAIL activated the intrinsic and extrinsic pathways of caspase activation. To elucidate the underlying mechanism, we found that metformin significantly reduced the protein levels of Mcl-1 (Myeloid cell leukemia 1) in CRC cells, and overexpression of Mcl-1 attenuated the cell death by metformin alone, or in combination with TRAIL. Further experiments revealed that metformin did not affect the mRNA level, but proteasomal degradation and protein stability of Mcl-1. The metformin-induced degradation of Mcl-1 required the E3 ligase Mule, which is responsible for the polyubiquitination of Mcl-1. Taken together, our study is the first report indicating that metformin enhances TRAIL-induced apoptosis through the degradation of Mcl-1 by the proteasome machinery.

#3498

Increased cellular stress and apoptosis induced by anti-SIAH2 therapy in human cancer cells with oncogenic K-RAS addiction.

Monicah M. Njogu, Caroline Lee, Jamie Eisner, Minglei Bian, Robert Vansciver, Amy H. Tang. _Eastern Virginia Medical School, Norfolk, VA_.

Hyperactivated K-RAS oncoproteins are known to drive cell proliferation, tumor growth, and metastasis. Oncogene addiction is often associated with increased ER and mitochondrial stress due to excessive reactive oxygen and nitrogen species (ROS & NROS) production. RING finger E3 ubiquitin ligase, Seven in Absentia homolog 2 (Siah2) is a highly conserved, critical signaling module downstream of the EGFR/HER2/K-RAS pathway. SIAH2 function is required for proper K-RAS signal transduction and has been shown to target key regulators of oxidative stress for ubiquitination and proteasome degradation. However, the mechanisms by which SIAH2 regulates cellular stress and protects against programmed cell death in oncogenic K-RAS-addicted cancer cells are not well understood. Since SIAH2 is upregulated in proliferating tumor cells, we hypothesized that SIAH2-deficient cancer cells may be compromised in their ability to adapt to increased cellular and oxidative stress induced by oncogenic K-RAS pathway blockade.

In this study, SIAH2 knockdown was carried out in cancer cells carrying oncogenic K-RAS mutations (A549, MDA-MB-231 and MiaPaCa cells). Triplicates of fresh cell lysates from SIAH2 proficient and SIAH2 deficient cells were then interrogated for altered expression of 19 signaling molecules with known functions in modulating stress response and apoptosis using the PathScan® Stress and Apoptosis Signaling Antibody Array kit. Differences in the protein expression were quantified and validated by Western blots, immunofluorescence (IF) and flow cytometry. We found that oxidative stress was markedly increased, evidenced by mitochondrial aggregations and dysfunction in a subset K-RAS-driven cancer cells, as we blocked SIAH2 function downstream of the oncogenic K-RAS signaling pathway. SIAH2 deficiency in breast cancer (MDA-MB-231) and non-small cell lung adenocarcinoma (A549) cells resulted in decreased ERK1/2 and AKT (S473) phosphorylation, increased ROS production, compromised mitochondria integrity and function, increased Caspase 3 and 7 activity, and PARP 1 cleavage that led to massive cell death observed in these cancer cells. In contrast, the prominent cell death was not observed in SIAH2 deficient pancreatic cancer (MiaPaCa) cells that expressed high levels of Survivin, which is an anti-apoptosis protein.

The anti-SIAH2 therapy is highly effective in inducing oxidative stress, altered mitochondrial bioenergetics, and apoptosis in a subset of invasive and metastatic cancer lines expressing insufficient levels of Survivin. Our results suggest that, anti-SIAH2 strategy reveals novel oncogenic K-RAS-dependent cancer cell vulnerability in stress response that can be exploited in future for anti-SIAH2 therapy alone, or in combination with other anti-proliferative treatment to effectively combat KRAS-dependent cancers.

#3499

Triptolide pro-drug decreases tumor burden and halts tumor progression in a murine model of acute myeloid leukemia.

Bhuwan Giri, Sulagna Banerjee, John George, Shrey Modi, Vineet Kumar Gupta, Mahendra K. Singh, Vikas Dudeja, Ashok K. Saluja. _University of Minnesota, Minneapolis, MN_.

Introduction

Standard treatment for acute myelogenous leukemia (AML) has not changed over the past few decades and relies primarily on the traditional "7 + 3″ regimen of daunorubicin, administered over 3 days and Cytarabine, administered over 7 days. This chemo-intensive regimen is poorly tolerated by patients and is characterized by a high rate of relapse and treatment failure. Triptolide is a diterpenoid tri-epoxide compound isolated from the Chinese herb Tripterygium wilfordii. The water-soluble pro-drug of triptolide, Minnelide has been shown by our group to be highly effective in a number of pre-clinical models of solid tumors and is currently undergoing Phase I Clinical trial for gastrointestinal tumors. Interim analysis of the Phase I data has shown that the equivalent mouse dose of 0.2 mg/kg/day is well tolerated by patients with no reported dose limiting toxicity.

Methods

Primary AML apheresis samples from patients with acute myeloid leukemia and multiple AML cell Lines (THP1, KG1, Kasumi1, HL-60) were treated with Triptolide at doses from of 2.5 nM to 50nM and cell viability was measured using a formazan based colorimetric assay. Apoptosis was measured using Annexin V via flowcytometry and colony forming ability of AML cell lines was measured using a methylcellulose based assay.

To generate an engraftment model of AML, NOD.Rag1-/-;Ƴcnull (NRG) animals expressing human interleukin-3 (IL-3) and human GM-CSF (NRGS) were injected luciferase expressing THP1 cells after sublethal irradiation of 250 cGy. These animals were then serially evaluated using In Vivo Imaging System (IVIS) and leukemic burden was calculated the total bioluminescent signal after intra-periotneal injection of luciferin. Treatment with vehicle or Minnelide at a dose of 0.1mg/kg/day and 0.15 mg/kg/day was started on the 10th day after confirming engraftment using IVIS.

Results

Triptolide at a dose range of 2.5 nM to 50 nM produced a dose and time dependent killing of leukemic cells in both cell lines and primary AML patient samples. The IC-50 for THP1, KG1, Kasumi1 and HL-60 cell lines with triptolide treatment at 48 hours were 5 ± 0.8 nM, 8 ± 0.7 nM, 7.2 ± 0.6 nM, 10 nM ± 2 nM respectively. Treatment with triptolide at a dose of 2.5 nM induced cell death and apoptosis as measured by Annexin V posiitvity in primary AML apheresis samples.

Colony formation by THP1 cells and KG1 cells was completely abrogated by treatment with Triptolide at 2.5 nM and 25 nM respectively.

Vehicle treated mice showed a rapid progression of disease burden when compared to Minnelide treated mice. At a dose of 0.1 mg/kg/day and 0.15 mg/kg/day, these mice had no appreciable increase in leukemic burden over normal mice when assessed by IVIS.

Conclusion

We show that Minnelide induces cell death at therapeutically relevant concentrations in vitro and decreased leukemic burden in a murine model of AML. Minnelide may emerge as a novel therapeutic strategy in treating patients with AML.

#3500

Mitochondrially targeted p53 domains as a stand alone or adjunct to paclitaxel for the treatment of ovarian cancer.

Phong Lu, Carol S. Lim. _University of Utah, Salt Lake City, UT_.

Although the main function of p53 is a nuclear transcription factor that has important roles in cell cycle arrest, DNA repair, and apoptosis, p53 can directly trigger the intrinsic apoptotic pathway through the mitochondria. p53 has been known to bind to mitochondrial anti-apoptotic proteins (Mcl-1, Bcl-2 and Bcl-XL) and pro-apoptotic proteins (Bak and Bax), which will cause the oligomerization of Bak and Bax. The result is the formation of permeable pores on the mitochondrial outer membrane, which in turn cause the release of cytochrome c and activation of caspase-3. Targeting p53 to the mitochondria is an attractive approach because it can cause a rapid apoptotic response. We have introduced the mitochondrial targeting signal (MTS) from Bak or Bax to the C-terminus of p53 and have shown the superior efficacy of p53-BakMTS and p53-BaxMTS over wtp53 in many human cancer cell lines. We have identified that the DNA binding domain (DBD) of p53 may be the minimal domain of p53 required for apoptosis. Our preliminary data in SKOV-3 ovarian cancer cells also suggests that DBD-BakMTS may work just as well as full length p53-BakMTS. In addition to BakMTS and BaxMTS, attaching pro-apoptotic factors such as Noxa or Bid to p53 has created a chimeric gene construct that show superior cell death activity than wide type p53. Our goal is to use a novel version of p53 directed to the mitochondria as a direct apoptogen to treat ovarian cancer, the most lethal gynecological malignancy with 69% of patients succumbing to this disease. Our next step will be to test these constructs in vivo using the syngeneic ID8 mouse model. To determine the activity of these mitochondrially targeted p53 constructs, TMRE, 7-AAD, and caspase 3/7 assays were performed in many human ovarian cancer cell lines including SKOV-3, OVCAR-3, Kuramochi, and mouse ovarian ID8 cells that have been transfected with our constructs. In all of our cell death and apoptotic assays, p53-Bax-MTS, p53-BakMTS, and p53-Noxa are always superior to wide type p53, regardless of the p53 status of the cells. Furthermore, we have also tested the possibility of combining our p53 constructs with paclitaxel, the current standard of care for ovarian cancer. Our p53 constructs showed a synergistic effect with paclitaxel and was able to reduce its IC5, indicating the possibility of dose-lowering of this highly toxic drug. Our ultimate goal is to use mitochondrially targeted p53 constructs that directly induce apoptosis alone or in combination with other chemotherapy drugs for the treatment of ovarian cancer.

Acknowledgements: NIH CA151847

#3501

Puma/pBcl-2 axis influences sensitivity of cancer cells to multiple stimuli.

Yubo Liu. _Dalian University of Technology, China, Dalian, China_.

Bcl-2 is a central regulator of cell survival that is overexpressed in the majority of cancers cells.Previous reports indicated that Bcl-2 phosphorylation (S70) may provide a switch by which cellscan regulate the pro-survival functions of Bcl-2. But there is still controversy over the function of phosphorylatedBcl-2 (pBcl-2).In the presentstudy, Puma/pBcl-2 axis was found to play a key role in regulating the sensitivity of cancer cells to multiple stimuli.Based on immunoprecipitation, weobserved that the pro-apoptoticBH3-only protein Puma which was induced by paclitaxel competitively bound pBcl-2 (S70) and displaced Bim and Bax from pBcl-2(S70) in Hela and HL60 cells.Further study showed thatdepletion of Puma (siRNA), but not other BH3 only proteins, significantlydecreased paclitaxel-induced apoptosis.Bim/pBcl-2 and Bax/pBcl-2 complexeswere largely maintained when Pumawas reduced. These data indicated that the phosphorylation of Bcl-2(S70) enhancedits anti-apoptotic function and the induction of Puma could neutralize this effect. Similarly,the induced Puma was also responsible for Bim and Bax displacement from pBcl-2 (S70) when Hela and HL60 cellswere treated with DNA damage agents(cisplatin and etoposide).However, Pumacannot displace Bax or Bim when pBcl-2 (S70) was redundant in the cells expressedphospho-mimetic S70E-Bcl-2. It demonstrated that DNA damage induced apoptosis could be inhibit by pBcl-2 (S70) and the induction of Puma could antagonize pBcl-2 (S70). In a cell free system, mitochondria from Hela and HL60 cells which expressing the S70E-Bcl-2 wereincubated with different BH3 peptides. Cytochrome c release couldbe induced by a low concentration of Puma-BH3 peptide, but a higher concentrationwas necessary during Bimengagement.By contrast, Bad-BH3 and Noxa-BH3cannot initiate the release of cytochrome c.In conclusion, multiple stimuli activates the mitochondrial apoptoticpathway in cancer cells, in which Puma/pBcl-2(S70) axisplay a key role in a novel mechanism involving the displacement of Bim and Bax from pBcl-2 (S70)by Puma. In contrast with the debatable function of Bcl-2 phosphorylation (S70), this study provided evidence that the phosphorylation of Bcl-2 at S70 enhanced cell survivalin multiplestimuli triggered apoptosis.

#3502

**Combination of BKM120 and erlotinib in squamous cell carcinoma of the head and neck: mechanism of** in vitro **and** in vivo **synergy.**

Abu Anisuzzaman, Abedul Haque, Zhuo Chen, Dong M. Shin, A.R.M. Ruhul Amin. _Winship Cancer Institute of Emory University, Atlanta, GA_.

Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. The EGFR-targeted monoclonal antibody cetuximab is currently the only FDA-approved targeted treatment for head and neck cancers (HNC) with a response rate of less than 15%. Recent large scale genomic studies including TCGA suggest that >50% of HNC cases have activation of the PI3K/AKT/mTOR

pathways, suggesting PI3K as an excellent target for HNC. The purpose of the current study is to investigate whether co-targeting EGFR and PI3K has synergistic antitumor effects and to understand the mechanism of synergy.

Methods: In a panel of 10 malignant and 1 premalignant HN cell lines, we evaluated cell growth inhibition (by SRB assay); IC50, combination index, and dose reduction index (by CalcuSyn), and apoptosis (by Annexin-V staining). mRNA and protein expression were measured by qPCR and Western blotting, respectively. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively.

Results: Single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K had in vitro synergy in all except one cell line (based on combination index and dose reduction index) and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (Tu686, 686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, 1483, UMSSC90) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. Only pan-PI3K inhibitors (BKM120 and BEZ235) induced effective apoptosis in combination with erlotinib, since the PI3K-α inhibitor BYL279 or allosteric mTORC1 inhibitor RAD001 failed to induce apoptosis in combination with erlotinib. Erlotinib strongly inhibited p-EGFR and p-ERK in vitro and in vivo, but only partially inhibited p-AKT. On the other hand, BKM120 completely inhibited p-AKT without affecting p-ERK. We also found that the combination of BKM120 and erlotinib strongly inhibited both axes of the AKT-mTORC1 pathway in sensitive cell lines, but failed to inhibit p-4EBP1 in one resistant cell line (UD-SCC2). In addition, the combination of BKM120 and erlotinib strongly inhibited Bcl-2, Bcl-xL and MCL-1 at the translational level in the sensitive cell lines but not the resistant one. siRNA-mediated knockdown of eIF4E (to inhibit 4EBP1-eIF4E-dependent translation) sensitized UD-SCC2 cells to the combination of erlotinib and BKM-induced apoptosis.

Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K is synergistic and induces apoptosis of HNC cell lines by inhibiting both axes of the AKT-mTOR pathway and translational regulation of anti-apoptotic Bcl-2 proteins.

#3504

Arginylated BiP/oxidized DJ-1 complex mediates crosstalk between the endoplasmic reticulum and mitochondria.

Dae-Hee Lee. _Korea University Medical Center, Seoul, Republic of Korea_.

In cell death induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although much is known about signal pathways involving external signals through cell surface receptors or internal signals through mitochondrial dysfunction, little is known about signal pathways activated by endoplasmic reticulum (ER) stress during TRAIL treatment. Here, we report that TRAIL-induced caspase activation is a prerequisite for TRAIL-induced ER stress response. During TRAIL treatment, ER chaperon BiP dissociates from ER stress sensors such as PERK and IRE1α and the activation of the PERK-eIF2α-ATF4-CHOP apoptotic signal transduction pathway occurs. Meanwhile ER-stress induces BiP release to the cytosol and ATE1-encoded Arg-tRNA transferase arginylates BiP. TRAIL also induces loss of ∆Ψm and redox sensor DJ-1 detects mitochondria-derived ROS. Oxidized DJ-1 binds to arginylated BiP (R-BiP) in the cytosol. DJ-1 binding activity was abrogated in C106A and C53A but not C46A mutant. R-BiP/oxidized DJ-1 complex may mediate crosstalk between the ER and mitochondria.

Key words: TRAIL; endoplasmic reticulum; apoptosis; colorectal cancer

#3505

A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis.

Kevin Kupcho,1 John Shultz,1 Andrew Niles,1 Wenhui Zhou,2 Robin Hurst,1 Jim Hartnett,1 Thomas Machleidt,1 Terry Riss,1 Dan Lazar,1 Jim Cali1. 1 _Promega Corporation, Madison, WI;_ 2 _Promega Biosciences LLC, San Luis Obispo, CA_.

The selective elimination of malignant cells via the apoptotic process continues to be the cornerstone of modern anti-cancer therapy regimens. Therefore, in vitro screening approaches aimed at identifying clinically useful apoptosis inducers remain critically important. Recently, phenotypic screening methods have enjoyed a resurgence due to more biologically complex and relevant cell models as well as advances in chemical proteomics which have allowed for more successful target identification. As a consequence, novel probes and tools with enabling attributes are required to fully realize this discovery potential. In an effort to address this unmet need, we have developed a bioluminescent and homogeneous annexin V binding assay for the assessment of apoptosis. Unlike traditional fluorescent annexin V methodology, the "no-wash" reagent employed in this new assay utilizes binary components of a novel luciferase separately fused to annexin V. The annexin V-luciferase subunit fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence until phosphatidylserine (PtdSer) exposure drives annexin-fusion pair oligimerization. Ultimately, this protein:protein interaction on or near the cell surface reconstitutes full luciferase activity causing an increase in luminescence in the presence of a luciferase substrate. A separate, pro-fluorescent, multiplexed component of the reagent further delineates differences in annexin positivity based on maintenance or loss of membrane integrity corresponding to apoptosis or necrosis, respectively. We validated this method using a panel of diverse cancer cell lines (U2-OS, DLD-1, HeLa, Jurkat, K562, A549, and PC-3), representing both attachment-dependent and -independent morphologies after dose-dependent challenge with intrinsic (bortezomib, panobinostat, staurosporine, and paclitaxel) and extrinsic (rhTRAIL) inducers of apoptosis as well as agents known to produce primary necrosis (ionomycin and digitonin). Caspase activation data was also collected in parallel plates at endpoint as a well-validated and sensitive orthogonal comparator. The bioluminescent annexin V method proved sufficiently robust in 384 well microplate formats to routinely produce Z' > 0.7 and rank-order potencies in good agreement with caspase activation values. In addition to this microplate functionality, the reagent allowed for sensitive, facile imaging of apoptotic induction in living cells using different imaging platforms. Taken together, the method and reagent should provide unparalleled flexibility with regard to live cell apoptosis detection in both conventional microplate and high content-like imaging formats and advance the pace of new chemical entity discovery.

#3506

Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models.

Mathew Larson,1 Jennifer H. Carlson,1 Yuliang Sun,1 Tonia Bucholz,2 Casey Williams,1 Nandini Dey,1 Brian Leyland-Jones,1 Pradip De1. 1 _Avera Research Institute, Sioux Falls, SD;_ 2 _Onyx Pharmaceuticals Inc., South San Francisco, CA_.

BACKGROUND: The proteosome is clinically validated as a target for cancer therapeutics. Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1/2 studies of CFZ reported signals of clinical activity in solid tumors. The aim of this study was to investigate the activity of CFZ in triple negative breast cancer models. METHODS: A diverse panel of human triple negative breast cancer cell lines was used to investigate the anti-tumor activity of CFZ. Cell viability following CFZ treatment was assessed using MTT assay. Proliferation was monitored both with IncuCyte Zoom real-time imaging system and 3D-ON-TOP clonogenic assay. Annexin-V flow cytometry was used to measure apoptosis. Apoptotic protein levels were measured with Western blot and proteome profiler array. MCL1 and Ki67 expression were also assessed using immunocytochemistry. RESULTS: CFZ had marked anti-proliferative activity in MDA-MD231, MDA-MB468, BT20, and SUM149 TNBC cell lines, with IC50 values following 96-hour exposure from 8 nM to 25 nM by MTT assay. The anti-proliferative activity of CFZ was observed by 3D-ON-TOP clonogenic assay. The anti-proliferative activity of CFZ was also demonstrated with IncuCyte proliferation assay. Proliferation was inhibited by low nanomolar concentrations in both MDA-MB468 and BT20 cells. Western blot analysis of CFZ-treated BT20, MDA-MB231 and MDA-MB468 cells showed cleavage of poly ADP ribose polymerase (PARP) and CASPASE-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. However, expression level of anti-apoptotic protein MCL1 was increased in RAS-RAF mutated cells (MDA-MB231) but not in PTEN-null MDA-MD468 cells following the treatment of CFZ. Double immune-fluorescence staining of MDA-MB231 cells following the treatment of CFZ showed higher expression of MCL1 but no change in Ki67 expression. Interestingly, a combination of CFZ and TOR kinase inhibitor (MLN0128) enhanced cleaved CASPASE3 and BIM expression with a concomitant increased induction of apoptosis (annexinV expression) in MDA-MB231 cells. CONCLUSIONS: CFZ demonstrated anti-proliferative activity in TNBC cell lines in vitro. Flow cytometric analysis of annexin V-positive cells and Western blot expression of cleaved CASPASE 3, cleaved PARP and BIM indicated that CRZ -induced TNBC cell death through an apoptosis-dependent manner.

#3507

Rhamnetin enhances anti-proliferative and apoptotic effects on prostate cancer cells.

Riddhi Patel, Rebecca Pakradooni, Christine Oak, Natarajan Bhaskaran, Sanjeev Shukla. _Case Western Reserve University, Cleveland, OH_.

Dysregulation of oxidative stress plays important role in both tumor development and response to anticancer drug treatment. Increased oxidative stress induces reactive oxygen species (ROS) which stimulate genes viz. FoxO3a and Klotho. Klotho modulates stress-induced senescence, whereas forkhead transcription factor; FoxO3a is known to involve in antioxidant, anti-proliferative and tumor suppressor functions. We have previously demonstrated that FoxO3a is deregulated in human prostate adenocarcinoma and in transgenic adenocarcinoma of the mouse prostate (TRAMP) model that mimics progressive forms of human disease. Here we demonstrate the potential involvement of Klotho protein in prostate cancer progression and using a natural agent; rhamnetin as anticancer in TRAMP mice modulated Klotho and FoxO3a signaling by altering ROS to inhibit prostate cancer. Per-oral feeding of rhamnetin at 10- and 20-μg/mouse/day (gavaged in 0.2 ml vehicle containing 0.5% methyl cellulose and 0.025% Tween 20) to TRAMP mice for six days per week for 16 weeks, starting at 8 weeks of age, exhibited marked reduction in tumor growth. Rhamnetin intake led to increased antioxidant potentials, which was evident from increased levels of klotho, Nrf2 and with significantly increased expression of Shc66 in the dorso-lateral prostate. Reports suggested that secretory klotho binds to multiple FGFRs (fibroblast growth factor receptors) with different affinity. However, TRAMP mice exhibited significantly increased FGFR1 expression in dorso-lateral prostates, which were reduced significantly after rhamnetin feeding to TRAMP mice. Furthermore, inhibited FGFR1 expression resulted in reduced phospho-Akt (Ser-473) and ERK1/2 phosphorylation in rhamnetin fed mice than control. This modulated signaling cascade decreased nuclear presence of p-Akt (Ser-473), which restricted FoxO3a phosphorylation and led to nuclear retention in dorso-lateral prostate. Increased nuclear presence of FoxO3a elevated transcriptional activity and expression of FoxO-dependent apoptotic protein, Bim. Moreover, rhamnetin feeding to TRAMP mice reduced Alix expression, which further helped in accelerating apoptotic machinery. Similarly rhamnetin treatment to human prostate cancer LNCaP and PC-3 cells also resulted in the restoration of Klotho and FoxO3a in the nucleus. Increase nuclear presence of Klotho and FoxO3a protein may be a decisive factor for increase resistance to oxidative stress and inhibited prostate cancer progression. Our finding suggests that rhamnetin may be developed as a preventive agent in prostate cancer.

#3508

The RNA-binding protein CELF1 regulates the tumor suppressive protein IL-24/mda-7 expression in oral squamous cell carcinoma.

Reniqua House, Viswanathan Palanisamy. _Medical University of South Carolina, Charleston, SC_.

The majority of oral squamous cell carcinoma (OSCC) patients are diagnosed at an advanced disease stage and the overall 5-year survival rate, surprisingly, has not changed dramatically over the past several decades. Therefore, identification and subsequent characterization of new molecular determinants of OSCC, may aid in the discovery of novel targets capable of predicting patient treatment response and disease prognosis. Co- and post-transcriptional gene expression plays a crucial role in disease development. RNA binding proteins (RBPs) are critical regulators of messenger RNA (mRNA) fate within mammalian cells. The RBP CELF1 associates with GU-rich element (GRE) containing mRNAs and post transcriptionally regulates mRNA stability, translation and pre-mRNA alternative splicing. Our previous studies demonstrated that the protein levels of CELF1, increased as HNSCC disease progressed from Stage I-IV. In addition, reduction of CELF1 in oral cancer cells promoted programmed cell death and decreased tumor burden in a xenograft mouse model. In an effort to understand the underlying mechanism employed by CELF1 to promote anti-tumor activity, we utilized genomic techniques to identify CELF1 mRNA targets in oral cancer cells. Interestingly, the gene IL-24/mda-7 was one of the top aberrantly expressed mRNAs identified as a function of CELF1 levels in oral cancer cells.

Interleukin -24 also known as melanoma differentiation associated gene-7 (IL-24/mda-7) is a member of the IL-10 related family of cytokines. IL-24 protein expression is absent in most cancers and forced expression of IL-24 using an adenoviral vector enhances tumor cell-specific apoptosis. Although the mechanism of IL-24 induced cell death has been studied for several cancers, the role of IL-24 in HNSCC is not well known. In addition, the regulatory mechanisms controlling IL-24 expression in cancer are not well studied. Here, we show that IL-24 mRNA is overexpressed in HNSCC tissues compared to normal specimens. Furthermore, utilizing a mouse model of oral carcinogenesis we determined that IL-24 mRNA levels are enhanced in hyperplastic/dysplastic and oral squamous cell carcinoma tissues compared to normal, which is coincident with increased protein levels of CELF1. Surprisingly, loss of CELF1 expression in oral cancer cells dramatically increased IL-24 protein from undetectable levels to robust expression. We are currently exploring the mechanism of the CELF-1/IL-24 axis in mediating apoptosis in oral cancer cells and are expanding our studies to include other cancer types. These results suggest a novel mechanism by which the RNA binding protein CELF1 promotes oral cancer cell survival, through controlling the expression of a potent tumor suppressive protein and supports CELF1 as a viable target for the development of cancer therapeutic interventions.

#3509

Knockdown of RNF2 induces apoptosis by regulating MDM2 and p53 stability.

Weihong Wen, Cong Peng, Myoung Ok Kim, Chul Ho Jeong, Feng Zhu, Ke Yao, Tatyana Zykova, Wei-Ya Ma, Andria Carper, Alyssa Langfald, Ann M. Bode, Zigang Dong. _University of Minnesota, Austin, MN_.

RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1 (PRC1). RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here we show that knockdown of RNF2 significantly inhibits both cell proliferation and colony formation in soft agar, and induces apoptosis of cancer cells. Knockdown of RNF2 in HCT116 p53+/+ cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53−/− cells, indicating that RNF2 knockdown-induced apoptosis is partially dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged and p53 ubiquitination was decreased. In contrast, cells overexpressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 and promotes MDM2-mediated p53 ubiquitination. RNF2 over-expression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.

Keywords: RNF2, p53, apoptosis

#3510

Tolfenamic acid induces apoptosis and growth inhibition in anaplastic thyroid cancer: involvement of nonsteroidal anti-inflammatory drug-activated gene-1 expression and intracellular reactive oxygen species generation.

Yeon Soo Kim,1 Jae Won Chang,2 SungUn Kang,1 JaeWon Choi,1 YooSeob Shin,1 Seung Joon Baek,3 Seong-Ho Lee,4 Kyoung Ho Oh,5 Chul-Ho Kim1. 1 _Ajou University, Suwon, Republic of Korea;_ 2 _Chungnam National University College of Medicine, Daejeon, Republic of Korea;_ 3 _College of Veterinary Medicine, University of Tennessee, Knoxville, TN;_ 4 _College of Agriculture and Natural Resources, University of Maryland, College Park, MD;_ 5 _Korea University College of Medicine, Seoul, Republic of Korea_.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are usually used for the treatment of inflammatory diseases. However, certain NSAIDs also have antitumor activities in various cancers, including head and neck cancer, through cyclooxygenase-dependent or independent pathways. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), a TGF-β superfamily protein, is induced by NSAIDs and has been shown to be induced by several antitumorigenic compounds and to exhibit proapoptotic and antitumorigenic activities. In this report, we demonstrate for the first time that tolfenamic acid (TA) transcriptionally induced the expression of NAG-1 during TA-induced apoptosis of anaplastic thyroid cancer (ATC) cells. TA reduced the viability of ATC cells in a dose-dependent manner and induced apoptosis, findings that were coincident with NAG-1 expression. Overexpression of the NAG-1 gene using cDNA enhanced the apoptotic effect of TA, whereas suppression of NAG-1 expression by small interfering RNA attenuated TA-induced apoptosis. Subsequently, we found that intracellular ROS generation plays an important role in activating the proapoptotic protein NAG-1. Then, we confirmed antitumorigenic effects of TA in a nude mouse orthotopic ATC model, and this result accompanied the augmentation of NAG-1 expression and ROS generation in tumor tissue. Taken together, these results demonstrate that TA induces apoptosis via NAG-1 expression and ROS generation in in vitro and in vivo ATC models, providing a novel mechanistic explanation and indicating a potential chemotherapeutic approach for treatment of ATC.

#3511

Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells.

Magdalena Circu,1 James Cardelli,1 Glenn Mills,1 Martin Barr,2 Hazem E. El-Osta1. 1 _Louisiana State University Health Sciences Center, Shreveport, LA;_ 2 _Institute of Molecular Medicine, Trinity Center for Health Sciences, St. James's Hospital & Trinity College, Dublin, Ireland_.

Introduction: Lung cancer is the leading cause of cancer-related death. Resistance to treatment contributes to poor outcome. A form of resistance to antineoplastic agents involves the sequestration of chemotherapy inside lysosomes followed by its delivery outside the cell through exocytosis. Lysosomal membrane permeabilization (LMP) not only releases the trapped drug, but also allows the translocation of lysosomal proteases into the cytosol, triggering cell demise. Since lysosomes in cancer cells are more susceptible to LMP than normal cells, we hypothesize that targeting LMP in cisplatin-resistant cells may provide a successful strategy to restore sensitivity to chemotherapy.

Materials and Methods: Cisplatin-resistant (cisR) A549 lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To detect LMP, lysosomes were loaded with FITC-dextran 40KDa and immunofluorescence images were obtained by confocal microscopy. To confirm LMP, cells were homogenized, cytosolic and lysosomal fractions were separated by ultracentrifugation, and cell lysates were immunoblotted with specific antibodies against cathepsin D, α-tubulin and LAMP-1. Cell viability was evaluated using CellTiter blue assay. To measure apoptosis, cells were stained for Annexin-V/Propidium iodide, or FITC-conjugated caspase 3 substrate, and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system.

Results: cisR A549 cells loaded with FITC-dextran, and incubated with different concentrations of chloroquine (25-100μM) for 24 hours demonstrated increased green haziness in the cytosol and loss of the punctate pattern of the dextran-loaded lysosomes, indicative of dextran leakage following LMP. Release of cathepsins into the cytosol due to LMP was confirmed by the increase in cathepsin D signal in the cytosolic extract on western blot. Next, cisR A549 cells were treated with chloroquine, cisplatin or the drug mix. Cell viability assay revealed a synergistic effect of the combination, with a combination index of 0.70 for cisplatin 25μM and chloroquine 100μM at 48 hours. Notably, there is significant increase in Annexin V stained apoptotic cells, accompanied by a corresponding increase in caspase 3 activity, in the combination. Interestingly, inhibition of lysosomal proteases using E64 was cytoprotective for cells treated with cisplatin and chloroquine, suggesting that chloroquine-induced cell death is LMP mediated.

Conclusion: Chloroquine re-sensitizes cisR A549 cells to cisplatin in an LMP-mediated manner. Our preliminary results support the concept that targeting LMP may provide a viable therapeutic strategy to restore sensitivity to chemotherapy in refractory lung cancer. Screening for other repurposed LMP inducer drugs is ongoing.

#3512

Cyclopamine enhances TRAIL-induced apoptosis by induction of DR5 via ER stress in gastric cancer cells.

Yoo Jin Na, Dae-Hee Lee, Jung Lim Kim, Bo Ram Kim, Seong Hye Park, Byung-Wook Min, Sun Il Lee, SangHee Kang, Sung Yup Joung, Suk Young Lee, Hong-Jun Kim, Sang Cheul Oh. _Korea University, Seoul, Republic of Korea_.

Activation of sonic hedgehog (Shh) signaling has been involved in progression of various cancers. Cyclopamine uses as effective treatment for cancers in which hedgehog signaling is overexpressed. In this study, we elucidated that cyclopamine sensitizes to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in gastric cancer cells. Single treatment with cyclopamine or TRAIL could not induce significant cytotoxicity in gastric cancer cells. However, combination treatment of TRAIL with cyclopamine effectively led to caspase dependent apoptosis. We found that cyclopamine increased ER (Endoplasmic Reticulum) stress level in gastric cancer cells, and using ER stress inhibitor attenuated the cell death by cyclopamine. As further underlying mechanism, induction of ER stress by cyclopamine caused upregulation of JNK (c-Jun N-terminal kinases) protein, and then accumulation of p53 protein. We conducted experiment using p53 wild-type and p53-mutated gastric cancer cells, and this of particular importance since p53 wild-type gastric cancer cells had more significant efficacy than p53-mutated cells by cyclopamine. Accumulation of p53 increased DR5 protein. Taken together, we showed that cyclopamine sensitizes to TRAIL-induced apoptosis in gastric cancer cells.

#3513

Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides.

Alan D. Brooks,1 Ya-ming Xu,2 E. M. Kithsiri Wijeratne,2 Poonam Tewary,1 Curtis J. Henrich,3 Cheryl L. Thomas,4 A. A. Leslie Gunatilaka,2 Thomas J. Sayers1. 1 _Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Cancer and Inflammation Program, Frederick, MD;_ 2 _Natural Products Center, University of Arizona, Tucson, AZ;_ 3 _Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, MTL, Frederick, MD;_ 4 _MTL, NCI Frederick, Frederick, MD_.

We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Treatment of cancer cells with WE induced a reduction in the levels of the antiapoptotic proteins cFLIPL and cFLIPS, resulting in an increased activation of caspase-8 on subsequent TRAIL binding to its death receptors DR4 or DR5. The reduction in cFLIPL and cFLIPS was due to their destabilization and increased degradation by the proteasome. Interestingly, WE (a 17-beta-hydoxywithanolide, 17-BHW) was a far superior TRAIL sensitizer than more widely studied withaferin A (WFA) and its analogues, which lack the 17-beta-hydroxy group and bear an opposite side chain orientation, exhibit more promiscuous reactivity and are much more directly toxic to cells. Therefore, over 30 natural and semi-synthetic 17-BHWs were evaluated for their ability to promote death ligand-mediated cancer cell death. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Our studies identified several 17-BHWs that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells ACHN to TRAIL-mediated apoptosis. These more active 17-BHWs were also more efficient at reducing cellular levels of cFLIPL and cFLIPS and enhancing caspase-8 activation. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition acetoxylation at C-18, an alpha orientation of the lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as TRAIL sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to TRAIL-mediated apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation.

Funded by FNLCR Contract HHSN261200800001E

#3514

Inhibition of PI-3 kinase signaling potentiates the apoptotic response to mitotic arrest.

Roaa M. Kassim, Charles B. Shuster. _New Mexico State University, Las Cruces, NM_.

Anti-mitotic drugs represent a common strategy for cancer chemotherapy by inducing a prolonged prometaphase delay and eventual cell death by apoptosis. Kinesin Spindle Protein (KSP) inhibitors arrest cells in mitosis by blocking bipolar spindle assembly, and these drugs have demonstrated limited side effects in clinical trials. However, they are not as effective as Taxol, whose efficacy is limited by the frequent development of resistance as well as the deleterious effects on non-dividing cells. Recent studies suggest that the ability of cells to maintain energy levels during mitosis could account for the differential responses of cancer cells to mitotic arrest. To determine if manipulating cellular metabolism could potentiate the efficacy of KSP inhibitors, we arrested cells in mitosis in the absence or presence of the PI3-kinase LY294002, and probed for the induction of apoptosis. Because tumor cells respond differently to mitotic arrest, we employed two cell lines that either survive mitotic arrest (MCF7) or undergo apoptosis shortly following mitotic slippage (HeLa). In both cases, simultaneous inhibition of KSP and PI3-kinase induced apoptosis more effectively than mitotic arrest or PI3-K inhibition alone. In the case of HeLa cells, there also appeared to be an acceleration of cell death such that arrested cells underwent apoptosis prior to mitotic slippage. Similar effects were observed using Akt inhibitors, providing additional evidence that PI3-kinase signaling was critical for surviving mitotic arrest. Lastly, we found that increased glucose had a protective effect, further supporting the notion that the ability to survive mitotic arrest depended on the ability to maintain energy levels. If confirmed, this may represent a novel approach for potentiating the anti-tumor activity of KSP inhibitors in the clinic.

#3515

MiR-320b-regulated TRIAP1 prevents mitochondria fragmentation and apoptosis by controlling cytochrome c release in nasopharyngeal carcinoma.

Ying-Qin Li, Na Liu, Jun Ma. _Sun Yat-sen University Cancer Center, Guangzhou, China_.

Mitochondria play critical roles in apoptosis. Imbalance of mitochondria fusion/fission leads to mitochondrial fragmentation and initiates intrinsic apoptosis, but the regulation of mitochondrial fragmentation in nasopharyngeal carcinoma (NPC) remains undefined. Here we show that TRIAP1 is aberrantly overexpressed and associated with poor survival in NPC patients. TRIAP1 overexpression promotes cell growth and suppresses NPC cell death in vitro and in vivo, whereas TRIAP1 knockdown inhibits cell tumorigenesis and enhances apoptosis through induction of mitochondrial fragmentation, membrane potential alteration and release of cytochrome c from mitochondria to cytosol. In intersection with our previous miRNA data and available bioinformatics algorithms, we identified miR-320b as a negative regulator of TRIAP1 expression. Further studies show that miR-320b suppresses NPC cell proliferation and enhances mitochondrial fragmentation and apoptosis by targeting TRIAP1. Moreover, loss of miRA-320b expression is conversely correlated with TRIAP1 overexpression and poor prognosis in NPC patients. This study reveals the regulation of miR-320b/TRIAP1 in mitochondrial fragmentation and apoptosis, and suggests potential therapeutic targets for NPC treatment.

#3516

**Tetrandrine promotes pancreatic cancer cell apoptosis** in vitro **and tumor regression** in vivo **.**

Karnika Singh,1 Prakash Srinivasan Timiri Shanmugam,1 Sweaty Koul,1 Qin Dong,1 Neil Koul,2 Hari K. Koul3. 1 _LSUHSC, Shreveport, LA;_ 2 _LSUHSC-FWCC, Shreveport, LA;_ 3 _FWCC-LSUHSC and Overton Brooks VA Medical Center, Shreveport, LA_.

Introduction: Resistance to apoptosis is a hallmark of tumor progression and therapeutic failure in cancer, including Pancreatic Cancer. Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related deaths in the United States with an overall five-year survival rate of less than five percent. The current standard treatment/s for PDAC are largely ineffective. There is an urgent yet unmet need for development of therapeutic agents for the treatment of PDAC. In the present study, we investigated the effects of Tetrandrine (a bis-benzylisoquinoline alkaloid) derivative- TET, on growth and viability of pancreatic cancer in vitro and in Xenograft models in vivo.

Methods: Pancreatic Cancer cell lines: PANC-1 (epitheloid carcinoma), BxPC3 (Pancreatic Ductal Adeno-Carcinoma) and MiaPaCa2 (pancreatic carcinoma) were used in this study. Cytotoxicity was evaluated using the Crystal violet and MTT survival assays. Apoptosis was monitored by Flow cytometry following Annexin V/PI staining. Nuclear morphology was visualized by Immunofluorescence. Western Blot analysis was used to measure protein expression. Human pancreatic cancer (BXPC3) derived xenografts were generated in NOD/ SCID mice and TET (40 mg/kg body weight) was orally administered daily for four weeks. Tumor growth (measured as tumor volume by Vernier Caliper) and body weights were measured on alternate days. Tumor weight was measured at the end of the experimental period, prior to xenograft tissue harvesting.

Results: TET inhibited growth and promoted cell death of pancreatic cancer cells in both dose and time dependent manner with an IC50 in the range of 5-10μM at 72 hr. The effects of TET were irreversible and there was progressive cell death with increasing time and at higher concentrations of TET. Treatment with TET resulted in nuclear condensation and apoptotic body formation, activation of caspase 3 and PARP cleavage, indicating apoptotic cell death. Moreover apoptosis was confirmed by flow cytometry after Annexin V and PI staining. TET administration not only halted the growth of BXPC3 derived xenografts, but also decreased tumor volume (TET treated vs PBS treated) over treatment period. Apoptosis (measured by TUNEL assay) was also observed in tumor tissues following TET treatment in vivo.

Conclusion: These results show for the first time, that TET inhibits pancreatic cancer cell growth in vitro and induces pancreatic tumor regression in vivo in part by apoptosis. These results highlight the potential use of TET in treatment of pancreatic cancer.

Acknowledgement: Financial support from following Sources is gratefully acknowledged: Carroll W. Feist endowed Chair Funds (Koul H), FWCC support and Chair commitment funds (Koul H) from the Chancellor and from the Dean School of Medicine- LSUHSC-Shreveport. All the members of Koul laboratory for their suggestions and help.

#3517

Expression studies of RBBP6 in breast and cervical cancer suggests a role in apoptosis activation.

Mpho Choene, Pontsho Moela, Lesetja Motadi. _Univ. of the Witwatersrand, Johannesburg, South Africa_.

Ubiquitin-like DWNN domain and RING finger-like domain presence on Retinoblastoma Binding protein 6 (RBBP6), is linked to its function as cancer promoting gene through p53 degradation via its p53-binding domain. RBBP6 bind and regulate the expression of p53 and pRB in the cell machinery implicates RBBP6 in cell proliferation during cancer development (Moela et al 2014). Studies indicate that overexpression of the mouse spliced variants led to apoptosis and cell cycle arrest whilst deregulation in mice led to embryonic lethality followed by p53 accumulation and a widespread apoptosis (Li et al., 2007). In this study, focuses on determining the expression pattern of RBBP6 in human tissue models of breast and cervical cancer. The study further aims to analyze apoptosis and cell cycle following either knockdown or overexpression of RBBP6 coupled to treatment with potential anticancer compounds, camptothecin and GABA. Quantitative analysis by qPCR in HeLa, SiHa, MCF-7and MDA-MB-231 cell lines shows that RBBP6 expression is fairly high in cancer cells when compared to normal lung fibroblast. Following co-treatment with GABA and cDNA or siRNA, in cDNA treated there was a reduction in both apoptosis as measured by flow cytometer and TP53 expression while in siRNA there was increased expression of TP53 and those of Bax and Bak1 which seemed to account for increased apoptosis. Cell cycle presented increase in G0/G1 and the G2/M following treatment with siRNA and GABA. GABA alone could not induce a significant increase in apoptotic cells whereas when combined with siRNA it was able to induce over 30% apoptosis. These results suggest that RBBP6 might be a cancer promoting gene.

#3518

Flow cytometry as a single platform tool to evaluate multiple mechanisms of actions of therapeutic antibodies.

Antony R. Chadderton,1 Shane Harvey,1 Brandy Strake,2 Jill Giles-Komar,2 Renold J. Capocasale,1 T. Shantha Raju2. 1 _FlowMetric, Inc., Doylestown, PA;_ 2 _Janssen Research and Development, LLC, Spring House, PA_.

The unique ability of flow cytometry to simultaneously examine intricate details of multiple cell subsets is unparalleled and the platform is now a dominant tool for measuring antibody mediated effector functions on cancer cells. The objective of this study is to illustrate the utility of flow cytometry as a single platform for concurrently measuring the multiple mechanisms of actions of therapeutic antibodies such as ADCC, ADCP, Apoptosis, CDC and Trogocytosis on human cancer cells and immune functioning cells. Rituximab® was tested with the Burkett's Lymphoma cell line (Daudi) for all assays between the ranges of 0.01 and 100ug/mL. Apoptosis was evaluated by measuring Caspase 3/7 staining. ADCC assays were established with Rituximab®/CFSE labeled Daudi cells and human PBMC's. ADCP was assessed with Rituximab®/ CFSE labeled Daudi cells and CD14+/CD11b+ in vitro generated phagocytes using a 4 hour exposure. CDC assays were established with Daudi cells/ 10% pooled human serum using 7-AAD as the cytotoxic indicator in a 4 hour assay. Trogocytosis was measured up to 4 hours with Rituximab®/ Daudi and PBMC's and CD19/20 and CD14. All assays were analyzed using a FACS ARIA III flow cytometer and replicates of 3 were evaluated for statistical relevance. Results demonstrated that Rituximab® induced effects were observed in all 5 assays using flow cytometry. Apoptosis was induced when 0.01ug/mL Rituximab® was present. ADCP occurred at levels as low as 0.01ug/mL Rituximab®. ADCC and CDC occurred at levels as low as 0.1ug/mL Rituximab®. Trogocytosis, indicated by the transfer of CD19+ B cells to CD14+ monocytes, occurred at 0.1ug/ml Rituximab®. In summary, we demonstrated the clinical applicability of flow cytometry as a single platform to simultaneously evaluate five different mechanisms of actions that can occur when therapeutic antibodies are used to treat cancer cells.

#3519

Paroxetine induces apoptosis in human breast cancer MCF-7 cells through mitochondrial dysfunction and modulation of K+ channels.

Kee R. Kang, Young-Woo Cho, Ji Hyun Ryu, Eun-Jin Kim, Jaehee Han, Dawon Kang. _Gyeongsang National Univ., Jinju, Republic of Korea_.

Purpose: Selective serotonin reuptake inhibitors (SSRIs) are widely used for the treatment of patients with depression. Approximately 60% of women with breast cancer suffer from depression. This study was performed to investigate the effect of SSRIs on cell viability of human breast cancer MCF-7 cells.

Methods: Cell viability, ROS generation, and Ca2+ increase were quantified using MTT, H2DCFDA, and Fluo-3 AM. Apoptotic signaling pathways were analyzed by immuno-blotting and -staining. Plasma and mitochondrial membrane potentials were determined by DiO PMP and JC-1 dyes, respectively. K+ channel activity was analyzed by whole-cell and single-channel recordings.

Results: Of the antidepressants tested in this study, paroxetine most reduced the viability of MCF-7 cells. Paroxetine (10 and 30 µM) treatment resulted in a significant increase in the number of apoptotic cells, an increase in Bax/Bcl-2 ratio, an activation of caspase-8, 9, and PARP, and a release of cytochrome c from mitochondria. In addition, paroxetine markedly increased ROS generation and intracellular Ca2+ levels. Moreover, paroxetine blocked K+ channels, such as large conductance Ca2+-activated K+ channel and TASK-3 channel, which are present in plasma membrane and mitochondria. The paroxetine-induced apoptosis was reduced by antioxidants, in particular NAC. Combination with SB202190 (a p38 MAPK inhibitor) prevented cells from undergoing apoptosis.

Conclusion: Our findings show that apoptotic effect of paroxetine is mediated by mitochondrial dysfunction and modulation of K+ channels in MCF-7 cells. We suggest that paroxetine could be an agent for anti-tumor therapy in breast cancer.

#3520

Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression.

Haesung Kim,1 Chea Ha Kim,2 Jeong-Hyeon Kim,3 Jeong-Min Lee,2 Jeong-Ho Jeon,2 Jae-Yong Lee2. 1 _Hallym Univ. College of Medicine, Chuncheon, Republic of Korea;_ 2 _Department of Biochemistry, College of Medicine, Hallym University, Chuncheon, Republic of Korea;_ 3 _Institute of Natural Medicine, College of Medicine, Hallym University, Chuncheon, Republic of Korea_.

Solidago virga-aurea (European goldenrod or woundwort) is an herbaceous perennial plant of the family Asteraceae. It is used for medical system with astringent, diuretic, antiseptic and homeopathic properties. And the studies about Solidago virga-aurea are in progress for the other functions.

Recently, we found that Solidago virga-aurea extract induces apoptosis of breast cancer cells. MTT assay showed that cellular viability was decreased in Solidago virga-aurea extract treated MDA-MB-231 and MCF-7 breast cancer cell lines by dose and time dependent manner. And apoptotic protein, especially bim, was increased in Solidago virga-aurea extract treated MCF-7 cell line. However, anti-apoptotic protein, Bcl-xL, does not changed by Solidago virga-aurea extract. In order to find out positive regulator of bim expression, we tested whether FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5' AMP-activated protein kinase) protein expression. In immunoblot analysis showed that FOXO3a expression level was dramatically increased in Solidago virga-aurea treated MCF-7 cells without changes of AMPK expression. Moreover, immunostaining showed that Solidago virga-aurea induces co-localization of FOXO3a and bim proteins in MCF-7 and MB-231 cells.

Taken together, Solidago virga-aurea induces apoptosis by FOXO3a mediated up-regulation of bim protein. The mechanisms of Solidago virga-aurea mediated FOXO3a regulation is not fully elucidated yet, but further studies about this molecular mechanism between two proteins will give some ideas for cancer therapies for certain breast cancer patients.

### Cell Death 2

#3521

The effect of MazF, Escherichia coli ribonuclease, on gastric adenocarcinoma (AGS cells).

Maryam Saffarian, Jeremy Tzeng. _Clemson University, Clemson, SC_.

Apoptosis is a gene-directed program that could be inhibited by several factors such as the disruption of the balance between pro-apoptotic and anti-apoptotic proteins, the impaired death receptor signalling or the reduced caspase functions. Several studies have shown that ribonucleases or antibiotics can induce apoptosis in mammalian cells through the shutoff of the protein synthesises. MazF produced by E. coli is a ribonuclease protein that cleaves mRNAs at ACA sequence sites and inhibits their translation. Thus, we hypothesized that the overexpression of MazF proteins in cancer cells could lead to the induction of apoptosis. In the present study, the ACA-less mazF gene was inserted into pSF-T7-EMCV T7 IRES expression plasmid. The mRNAs of mazF gene were synthesized in in vitro conditions, and one µg/ml of the mRNA was transferred into AGS (Gastric adenocarcinoma) cell line. The incidence of apoptosis was detected by conducting caspase 3/7 and Annexin-V assays. The results showed that 99.83% of cells were detached after 18 hours, 0.0086% of cells remained attached without apparent apoptosis, but 0.101% of attached cells were under apoptotic conditions. The results suggest that MazF protein has an ability to induce apoptosis in AGS cells. Since this protein can cleave mRNAs at ACA sites, it could inhibit protein synthesis, reduce the growth rate, and induce apoptosis in cancer cells. This finding is the first report of the application of MazF as an anti-cancer agent for the induction of apoptosis in AGS cell line.

#3522

Cyclins and CDKs regulation and caspase cascade activation by cucurbitacin D induced cell cycle arrest and apoptosis in pancreatic tumor.

Myeong-Sun Kim, Ji Hye Kim, Jin Mo Ku, Se Hyang Hong, Kangwook Lee, Hyeong Sim Choi, Sang Mi Woo, Jee Yun Chang, Tai Young Kim, Seong Gyu Ko Ko. _Kyung hee University, Seoul, Republic of Korea_.

Pancreatic cancer has a poor prognosis and very low survival rate over the world. Because pancreatic cancer probably is diagnosed at a late stage, aggressive local invasion, and poor response to chemotherapy. Gemcitabine was the standard treatment for advanced and metastatic pancreatic cancer patients, but it is associated with multiple adverse effects-fever, fatigue, nausea, and drug resistance. Whether cucurbitacin D has any efficacy against human pancreatic cancer was examined in cell culture system. In vitro, cell viability was measured by MTT assay to recognize of cell cytotoxicity. Consequently, cytotoxicity was observed at a low concentration of cucurbitacin D. Wound healing assay and clonogenic assay indicated that cucurubitacin D inhibited the growth of cell growth through cyclins and CDKs regulation, and decreased colony-forming ability. Also, this compound down-regulated expression level of anti-apoptotic protein, Bcl-2, up-regulated of pro-apoptotic molecule Bax, and activated caspase-8, caspase-3 cascade extrinsic pathway. Additionally, PARP, caspase-3 substrate, protein was cleaved by cucurbitacin D treatment. Overall, our study suggest that cucurbitacin D could be a clinical medicine for the treatment of pancreatic cancers.

#3523

N-acetylcysteine(NAC) chemoprotection with decreased cisplatin and RT toxic efficiancy in medullablastoma cells.

Dilek Cakir,1 Merve Alpay,2 Zekiye Altun,3 Gorem Kismali,4 Ayça Pamukoglu,5 Funda Kosova,6 Tevhide Sel,7 Ogunç Meral,8 Safiye Aktas,9 Nur Olgun10. 1 _Çanakkale Onsekiz Mart University, Canakkale, Turkey;_ 2 _Düzce Üniversity, Düzce, Turkey;_ 3 _Dokuz Eylül Üniversity, İzmir, Turkey;_ 4 _Ankara University, Ankara, Turkey;_ 5 _Dokuzeylül University, İzmir, Turkey;_ 6 _Celal Bayar University, Manisa, Turkey;_ 7 _Ankara University, Ankara, Turkey;_ 8 _Ankara University, Ankara, Turkey;_ 9 _Dokuz Eylül University, İzmir, Turkey;_ 10 _Dokuzeylül University, İzmir, Turkey_.

Introduction: Medulloblastoma are central nervous system (CNS) tumors that occur from degeneration of brain cells. The malignant solid tumors are commonly seen in children and young people, and they constitute 16% of all childhood malignant tumors. These cells spread to the central nervous system through cerebrospinal fluid, which may cause tumor metastasis. NAC is known as an antioxidant which scavenges reactive oxygen radicals. It provides limited protection against neuronal damage. NAC can easily penetrate cell membranes and the synthesis of glutathione is different from cysteine speed limiter and also causes very low toxicity. Cisplatin is used to treat neoplastic diseases, and the effect is due to an interaction with DNA.The aim of this study was to investigate the protective effects of NAC application aganist ototoxicity of cisplatin and radiation. Method: We tested the potential for interactions of high-dose NAC despite a minimally effective cisplatin chemotherapy in cancer models of medullablastoma and cochea cells. For this purpose, reactive oxygen species formation, Nrf2 gene expression, lipid peroxidation, apoptosis, and caspase activities were evaluated. HEI-OC1 and HTB-186 cells were used with optimal culture conditions. Different concentrations of NAC (100uL-1uM) were applied on 5gy RT and cisplatin combination therapied cells. After 72h detection, cell death flow cytometry analysis, caspase3, caspase 8, caspase 9 activities, mitochondrial membrane potential, Nrf2 expression, intracellular ROS measurement, protein analysis and RT-PCR were performed in experimental groups. Results: Cisplatin has increased apoptotic effect on HEI-OC1 cochea cells by activated intrinsic caspase-9 pathway. Mitocondrial membrane potential and ROS were significantly higher using RT and Cisplatin therapy. NAC (%100-18,9) protected membranes and (%100-27) cell viability to RT toxicity. This protective effect showed by antioxidant speciality gene expression, such as Nrf2. Conclusion: Radiotherapy and cisplatin-induced autotoxicity have developed DNA damage via both oxidative stress and apoptosis pathways. NAC reveals a scavenging effect by stimulating Nrf2 and (SOD, catalase, HO-1 Gpx, NQO1) target apoptotic genes. Our obtained data showed apoptotic and biochemical mechanism during radiation autotoxicity and cisplatin agent administration concominantly.

#3524

D-glucose exposure induced DNA damage and apoptosis in MCF-7 cells.

Ibrahim O. Farah, Christine K. Tchounwou, Clement G. Yedjou, Paul B. Tchounwou. _Jackson State University, Jackson, MS_.

D-glucose is the simple carbohydrate sugar that our bodies rely on to produce ATP energy. It has been shown that sustained high glucose burden is related to the promotion of many long-term health problems to various vital target organs including the kidneys, nervous system, eyes, heart (failure and stroke), erectile dysfunction in men and pregnancy complications in women. Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. Furthermore, the D-glucose paradox as cancer-promoting energy source as well as being toxic to cancer cells is puzzling. Therefore in the present study, we will investigate the mechanisms of glucose-induced toxicity in MCF-7 cells as an in vitro cellular model to simulate diabetic complications in tumor cells using trypan blue exclusion and MTT assays. Mechanisms of DNA damage and apoptosis were tested by alkaline single cell gel electrophoresis (Comet) assay, and flow cytometry analysis using Annexin V FITC/PI and caspase-3 analysis. The MTT assay indicated that low dose (5 mg/mL) of D-glucose slightly increase cell viability upon 2 h of exposure. On the other hand, high doses (10-80 mg/mL) of D-glucose significantly reduced the viability of MCF-7 cells in a dose and time-dependent manner. Similar trends were seen with the trypan blue exclusion test. Data obtained from the comet assay indicated that D-glucose caused DNA damage in MCF-7 cells in a dose-dependent manner. The flow cytometry assessment (Annexin V FITC/PI) showed a strong dose-response relationship between high glucose exposure and Annexin V positive MCF-7 cells undergoing early stage apoptosis. Similarly, a statistically significant and concentration-dependent increase (p <0.05) were recorded for caspase-3 activity in MCF-7 cells undergoing late apoptosis. Based on these in vitro findings, our study provides clear evidence that elevated level of D-glucose induced cytotoxicity to MCF-7 cells via induction of DNA damage, externalization of phosphatidylserine and caspase-3 activation as indicators for the apoptotic mechanisms of D-glucose exposure.

This research is supported by a grant from the National Institutes of Health-NIH (Grant No. NIMHD-G12MD007581).

#3525

The effects of Berberis vulgaris on bacterial inhibition and the p53 gene in cancerous Caenorhabditis elegans.

Jesal P. Bhatt. _Toms River High School North, Toms River, NJ_.

Recently, there has been an interest in the use of natural products to inhibit bacterial growth rather than manufactured antibiotics due to the problem of bacterial resistance. This study aims at finding the most effective natural antibacterial remedy. The disk diffusion method was used to compare the antibacterial properties of manuka honey, barberry, aloe vera, and colloidal silver. After a 48-hour incubation period, the zone of inhibition was measured for the following bacteria: Bacillus megaterium, Serratia marcescens, Pseudomonas fluorescens, Rhodospirillum rubrum, and Escherichia coli. Barberry was the most efficient natural product (p=0.02). Further tests were performed to determine whether barberry contained strong anticancer properties. Barberry was tested on Caenorhabditis elegans (C.elegans), strain JR1279, exposed to ultraviolet-C radiation and the population was counted daily. Prior to this experiment, barberry had never before been tested on a model organism. The purpose of this experiment would determine if barberry could increase the lifespan of the C.elegans by the addition of this reagent. It was shown that the C.elegans exposed to barberry had a longer lifespan than those that were not exposed to barberry (p=0.01), indicating that barberry could be a potential anticancer supplement.

#3526

Novel function of NRP2 in therapy resistant metastatic cancer.

Samikshan Dutta,1 Sohini Roy,1 Navatha S. Polavaram,1 Marissa Stanton,1 Surinder Batra,1 Michael H. Muders,2 Kaustubh Datta1. 1 _UNMC, Omaha, NE;_ 2 _Institute of Pathology, Dresden, Germany_.

Neuropilins (NRPs) are cell surface glycoproteins known for their role as co-receptors for plexins and VEGF family members in neuronal development and vascular and lymphatic growth. Neuropilin-2 (NRP2), a family member of NRPs has been reported to enhance the survival of cancer cells following therapeutic stress. In a recent study, our group has shown that the expression of NRP2 can be a poor prognostic factor for invasive bladder cancer patients treated with radiochemotherapy. Its expression correlated with an increased risk of an early cancer specific death and significantly decreased cancer specific survival among these patients supporting its role as a promoter of therapy resistance. This function of NRP2 is distinctly different from its known function as a co-receptor. In this study, we have provided the molecular mechanism of how NRP2 imparts therapy resistance in cancer cells. Our results indicate that depletion of NRP2 inhibits autophagy, a process which promotes survival of cancer cells during therapeutic stress. Further, we observed a role of NRP2 in the maturation of early to late endosomes. Since late endosomes are required for the fusion of autophagosomes with lysosomes, the decrease in the formation of late endosomes following NRP2 depletion can explain the defect in autophagy observed during inhibition of the NRP2 axis. We have provided a detailed molecular mechanism of how the recruitment and functions of several endocytic proteins such as EHD4, Rubicon, Mon1 and PIKFYVE are affected due to the depletion of NRP2, which resulted in the defect in endosomal trafficking. Recent evidences indicate that cancer cells maintain a high endosomal trafficking activity and the defect in trafficking promotes aberrant cellular signaling cascades either by interfering with receptor recycling to the cell surface or by inhibiting their lysosomal degradation. In this respect our data demonstrate that depletion of the NRP2 axis leads to an increased accumulation of active EGFR in early endosomes, which induces aberrant signaling cascade to promote apoptosis in cancer cells. We were also able to identify a novel downstream target of NRP2 axis known as WD Repeat and FYVE Domain Containing 1 (WDFY1). Our data indicate that depletion of NRP2 transcriptionally upregulates the expression of WDFY1. This is achieved due to an inhibition of the nuclear localization of a transcriptional inhibitor FAC1. Overall, our data suggest that NRP2 axis maintains the expression of WDFY1 in cancer cells to promote enhanced level of endocytic acitivity and autophagy. NRP2 is therefore important for the progression and survival of metastatic cancer cells and targeting this axis is crucial to enhance therapeutic efficacy of aggressive malignancies.

#3527

Inhibition of autophagosome closure promotes iDISC-mediated apoptosis in AML cells.

Zhenyuan Tang, Yoshinori Takahashi, Hong-Gang Wang. _Pennsylvania State University Hershey College of Medicine, Hummelstown, PA_.

Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradation process whereby cellular components, such as damaged organelles, are enclosed in double-membrane structures called autophagosomes and delivered to lysosomes for degradation and recycling. Autophagy is known to be essential for cancer pathogenesis and progression through its intimate crosstalk with programed cell death machinery, apoptosis. However, the role of autophagy in apoptosis pathway remains highly controversial and context-dependent, which makes developing autophagy-targeted anti-cancer treatments hard to achieve. We have previously reported that autophagosomal membranes can serve as a platform for caspase-8 activation through the assembly of an intracellular Death-Inducing Signaling Complex (iDISC). In the present study, we demonstrate that inhibition of phagophore closure by depleting Atg2A/B stabilizes iDISCs and induces caspase-8-initiated apoptosis in acute myeloid leukemia (AML) cells. We found that knockout of Atg2A/B, but not Atg7, promotes the oligomerization of caspase-8 on Atg16L- and LC3-positive phagophore membranes upon nutrient starvation. Starvation-induced capase-8 activation and apoptosis induction in Atg2A/B-deficient cells were strongly suppressed by the inhibition of caspase-8. These results suggest that autophagy inhibition at the phagophore closure step can shift cytoprotective autophagy to a pro-death mechanism and thus may be a promising strategy to enhance the efficacy of anticancer therapy.

#3528

Induction of cytoprotective autophagy and senescence in response to chemotherapy and ionizing-radiation as possible mechanisms of tumor dormancy.

Theresa Thekkudan, Supriya Joshi, David Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Proliferative recovery in tumor cells subsequent to chemotherapy and/or radiation-induced prolonged growth arrest may prove to represent a state of autophagy and/or senescence. This may also be useful as an in vitro model of tumor dormancy. To explore this hypothesis, studies were performed in primary mouse mammary carcinoma cells (MMC) treated with Adriamycin (ADR) (0.25uM - 1uM) for 3 days (2h each) and ionizing radiation (IR) (6Gy). Promotion of autophagy was confirmed based on detection of autophagosomes based on acridine orange staining, the appearance of the autophagy marker, LC3.B, associated with autophagosome, degradation of p62/SQSTM1, a hallmark of autophagic flux (i.e. the completion of autophagy and degradation of autophagosome content). Autophagosome formation was observed 24h post ADR treatment and 72h post IR treatment. Furthermore, to understand the function of autophagy (cytoprotective, nonprotective or cytotoxic) autophagy was blocked either pharmacologically using chloroquine (CQ) or genetically by silencing of the key autophagy gene, ATG5. LC3.B appearance and p62/SQSTM1 degradation were more pronounced in wt (autophagy proficient) cells compared to ATG5 silenced cells, confirming that autophagy was inhibited by these pharmacological and genetic strategies. Furthermore, interference with autophagy sensitized the MMC cells to ADR and radiation. These observations indicated that ADR and IR induced autophagy was cytoprotective in the MMC cells. Unexpectedly, apoptosis (as measured by PI/ Annexin V assay) was not increased in response to either ADR alone or the combination of ADR and CQ, suggesting that sensitization was likely occurring through alternative pathways. An evaluation of select markers of senescence indicated enhanced expression of p21 as well as enhanced secretion of the autophagy/senescence associated marker, HMGB1, in response to Adriamycin and IR. Overall, these data suggest that both autophagy and senescence are induced in breast cancer cells by conventional therapies and could contribute to tumor dormancy. Inhibition of autophagy and/or senescence associated signaling pathways could lead to therapeutic strategies for interfering with proliferative recovery of tumor cells after standard modes of therapy. Studies are currently in progress to determine whether the senescence associated secretory phenotype (SASP) accompanies the induction of senescence. SASP has been associated with promotion of tumor growth and alternatively, with activation of an immune response to therapy.

#3529

Immunotherapy targeting folate receptor induces autophagy in ovarian cancer.

Yun-Fei Wen, Anil Sood. _UT MD Anderson Cancer Center, Houston, TX_.

Cancer cells are highly dependent on folate metabolism mediated by the folate receptor (FRa). Gene encoding FRa (FOLR1) is overexpressed in endometrial and ovarian cancers. We aim to investigate the functional mechanisms of blocking FRa with MORAb-003 antibody, a humanized monoclonal antibody directly against FRa. We first analyzed clinical data from the Cancer Genome Atlas (TCGA) for correlation between copy numbers of FOLR1 gene and disease-free survival in patients with high-grade serous ovarian carcinoma (HGSC). Next, we determined the surface−expression of FRa on HGSC cells using flow cytometry (FACS). We then used xenograft mouse models to test tumor inhibition by MORAb-003 antibody-induced FRa blockade. We performed reverse-phase protein array (RPPA) analysis in FRa-positive and FRa-negative HGSC tumors to compare their responses to MORAb-003 treatment. To recapitulate the in vivo environment, we cultured FRa-positive and -negative epithelial ovarian cancer cells in three-dimensional (3D) condition and targeted the specific factors identified by RPPA using shRNAs knockdown. MORAb-003 monotherapy significantly reduced tumor weights and numbers of tumor nodules in both FRa-positive IGROV1 and SKOV3 models; combination of MORAb-003 and docetaxel significantly reduced both tumor weights and numbers of tumor nodules compared to docetaxel monotherapy. As expected, we did not observe these effects in the A2780 model, which has low FRa expression. Immunohistochemistry using PCNA and TUNEL staining in resulting tumors revealed that MORAb-003 reduced proliferation, but had no significant effect on apoptosis. Systematic analysis from RPPA identified a number of autophagy factors and cell-death initiators as being significantly upregulated by MORAb-003 treatment in FRa-positive tumors including Beclin-1 and PEA-15 (a factor involved in activation of autophagic cell death). cDNA microarrays further showed that MORAb-003 treatment up-regulated the expression of BECN1, ATG8, ATG3 and LC3B in FRa-positive tumors. Using FACS analysis with acridine orange (AO) staining, we observed significantly more AO positive population in 3D cultured SKOV3 cells treated with MORAb-003 alone (16.9%) or MORAb-003+docetaxel (21.2%) than in cells treated with control IgG (5.24%). Such differences were not observed in A2780 cells. We also observed significant increase of autophagy flux in SKOV3 cells but not in A2780 cells. We also found that blocking autophagy with hydroxychloroquine or bafilomycin A1, or knockdown Beclin-1 or PEA-15 with shRNAs reversed the growth inhibition induced by MORAB-003 in 3D cultured SKOV3 cells. We reported autophagy-associated cell death is a previously unrecognized mechanism for targeting of FRa in addition to the known mechanisms including antibody-dependent cellular cytotoxicity. Our studies also suggested that Beclin-1 and PEA-15 could be further explored as prognostic markers for FRa-targeted therapy.

#3530

Hsp90-Tom70 interaction facilitates ATM localization to mitochondria and induces mitochondrial autophagy.

Aloke K. Sarkar,1 Tej K. Pandita,2 Varsha Gandhi1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _The Houston Methodist Research Institute, Houston, TX_.

The serine/threonine protein kinase Ataxia telangiectasia mutated (ATM), plays a critical role in DNA damage response specifically to agents that induce DNA double strand breaks. Recently, mitochondrial autophagy (mitophagy)-associated non-nuclear functions have been attributed to ATM in AT fibroblast (Valentin-Vega YA et al., Blood 119: 1490; 2012). Our investigations suggested similar role of ATM in mantle cell lymphoma (MCL) cell lines (Sarkar, A et al., Proc. AACR, 74: Abstract # 313; 2014). This is clinically important, since ATM is lost in >50% of primary malignant B-cells in MCL patients. To identify the role of ATM in mitophagy, using MCL cell lines, we established that a fraction of ATM protein is located within mitochondria. The proportion of mitochondrial ATM increases following mitophagy induced by mitochondrial uncoupler CCCP or FCCP in ATM proficient cell lines. This process is compromised in MCL cells lacking ATM. However how ATM enters mitochondria is not known. Using quantitative liquid chromatography mass spectrometry analysis (LC/MS), we identified new ATM interacting proteins in HEK293 cells that facilitate mitochondrial transport of ATM. These new ATM interacting proteins (Hsp90, Hsp70 and the outer mitochondrial translocation (TOM) complex, including Tom70) are unrelated to DNA damage response. The physical association of ATM with Hsp90 and Tom70 was confirmed by co-immunoprecipitation experiments while ATM-Hsp70 interaction was mostly nonspecific. Additionally, using kinase dead FLAG-ATM construct in HEK293 cells or pharmacologic (KU55933) inhibition of ATM kinase activity in ATM proficient Jeko-1 and Mino MCL cell lines, we demonstrate that ATM phosphorylation is dispensable to enforce mitophagic exclusion of nonfunctional mitochondria since non-phosphorylated ATM can still bound to Hsp90 and induce mitophagy. These MCL cell lines when treated with an Hsp90 inhibitor; 17-AAG either alone or in combination with CCCP; abolished mitochondrial ATM localization and are defective in CCCP-induced mitochondrial clearance suggesting extra-mitochondrial ATM enters mitochondria via Hsp90 interaction. Further analysis revealed ATM-Hsp90 loosely associate with outer mitochondrial import receptor Tom70 via Hsp90 interaction and this targeting preference between Hsp90-Tom70 is abolished following deletion of the cytosolic tricopeptide repeat (TPR) clamp domain of Tom70 entailing that ATM-Hsp90-Tom70 complex may be required for ATM entry inside mitochondria. Moreover, trypsin digested purified mitochondrial fraction from HeLa cells revealed the entry of C terminal ATM protein within mitochondria while N terminal part remained in the cytosolic fraction. The molecular model of ATM's role in mitophagy and its biological consequence in ATM deficient MCL will be discussed.

#3531

Autophagy facilitates leukemogenesis in a murine model of MLL-AF9-driven AML.

Qiang Liu, Longgui Chen, Hong-Gang Wang. _Penn State Hershey Medical Center, Hershey, PA_.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the clonal expansion of immature myeloid cells. There is an urgent and unmet need for the development of novel therapeutics against AML. Autophagy is a lysosomal catabolic process that has been implicated in leukemia cell survival and chemotherapy response. However, the biological role of autophagy in AML leukemogenesis remains poorly defined. In this study, we genetically ablated ATG5, a gene that is essential for autophagy, in AML cells during leukemogenesis in a murine model of MLL-AF9-driven AML. The homozygous deletion of ATG5 in AML cells significantly delayed MLL-AF9-induced leukemogenesis and progression in vivo. Likewise, the in vitro deletion of ATG5 in MLL-AF9 driven AML cells demonstrated decreased growth as well as reduced colony forming units. Interestingly, ATG5 deficiency demonstrated an altered differentiation hierarchy of MLL-AF9-induced AML cells in vivo that could be linked to decreased proliferation and suppressed leukemogenesis. Overall, this study provides insight at how autophagy can facilitate AML progression. A more comprehensive understanding on autophagy in AML is needed prior to the deployment of autophagy modulators for the treatment of AML.

#3532

Compound Tc inhibits ATG4 to attenuate autophagic flux and sensitize cancer cells to chemotherapy.

Chih-Wen Shu. _Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan_.

Autophagy is induced to protect cancer cells against stresses, including starvation, hypoxia and chemotherapy. Antimalarial drugs chloroquine and its derivative hydroxychloroquine are used to inhibit autophagy and being investigated with clinical trials for certain types of cancer. However, the tumor suppressive effects of the drugs might be autophagy independent, which require more specific inhibitors target autophagy components for clinical uses. Here, we screened FDA-approved drug library with both biochemical and cellular ATG4 reporter assays and found compound Tc blocked proteolysis activity of ATG4, essential proteases in autophagy machinery. Docking and MD simulation results appeared compound Tc was directly associated with active site of ATG4. Further, compound Tc obstructed the binding between ATG4 and MAP1LC3 and accumulated autophagosomes, which in turn reduce autophagic flux in cells. Moreover, compound Tc suppressed tumor viability and sensitized chemotherapy efficacy in two-dimensional and sphere culture model. Silencing ATG4 or overexpression of ATG4 catalytic mutant had no additional effects on tumor suppression compared to compound Tc alone, indicating compound Tc inhibited ATG4s activity to diminish tumor viability. In addition, compound Tc enhanced genotoxic stress-induced apoptosis to synergize chemotherapy efficacy in vitro and in vivo. Thus, we report that the clinical drug compound Tc is a potent ATG4 inhibitor, which could interrogate the role of autophagy in clinical setting and provide compound Tc as an anticancer drug or chemosensitizer.

#3533

Role of autophagy-related protein expression in patients with rectal cancer treated with neoadjuvant chemoradiotherapy.

Yoon Ho Ko,1 Soon Uk Hong,2 Der Sheng Sun,1 Hye Sung Won,1 Myung Ah Lee,3 Byoung Young Shim4. 1 _Division of Oncology, Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea, Uijeongbu- si, Republic of Korea;_ 2 _Department of Pathology, Chung-Ang University College of Medicine, Uijeongbu- si, Republic of Korea;_ 3 _Division of Oncology, Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea, Seoul, Republic of Korea;_ 4 _Division of Oncology, Department of Internal Medicine, The Catholic University of Korea, Seoul, Republic of Korea, Suwon, Republic of Korea_.

Background: Autophagy, a cellular degradation process, has complex roles in tumourigenesis and resistance to cancer treatment in humans. The aim of this study was to explore the expression levels of autophagy-related proteins in patients with rectal cancer and evaluate their clinical role in the neoadjuvant chemoradiotherapy setting.

Methods: All specimens evaluated were obtained from 101 patients with colorectal cancer who had undergone neoadjuvant chemoradiotherapy and curative surgery. The primary outcomes measured were the expression levels of two autophagy-related proteins (microtubule-associated protein 1 light chain 3 beta (LC3Β) and beclin-1) by immunohistochemistry and their association with clinicopathological parameters and patient survival.

Results: Among the 101 patients, the frequency of high expression of beclin-1 was 31.7% (32/101) and that of LC3Β was 46.5% (47/101). A pathologic complete response was inversely associated with LC3Β expression (P = 0.003) and alterations in the expression of autophagy-related proteins (P = 0.046). In the multivariate analysis, however, autophagy-related protein expression did not show prognostic significance for relapse-free survival or overall survival.

Conclusions: High expression of autophagy-related proteins shows a strong negative association with the efficacy of neoadjuvant chemoradiotherapy in patients with rectal cancer. Autophagy has clear implications as a therapeutic target with which to improve the efficacy of neoadjuvant chemoradiotherapy.

#3534

Inhibition of melanoma cell survival through p62/LC3B autophagic signaling.

Heather G. Hambright, Addanki P. Kumar, Rita Ghosh. _UTHSCSA, San Antonio, TX_.

The primary objectives of this study were i) systematic evaluation of the differences in basal autophagic activity between melanocytes and malignant melanoma cells to determine whether these differences may be selectively targeted using the ROS inducer, Nexrutine (NX); ii) determination of NX selectivity in inhibiting melanoma cell survival and iii) specific involvement of autophagy signaling protein, SQSTM1/p62 in NX-mediated autophagic cell survival. NX is obtained from the bark of the cork tree, Phellodendron amurense. A panel of melanocyte and melanoma cells was evaluated for basal autophagy level and effect of NX treatment, including protein levels and turnover of p62 and LC3B (+/- CQ), and number of autophagic puncta per cell. Trypan blue, MTT, and Annexin V-APC were used to evaluate the effect of NX on cell death, proliferation and apoptosis. Genetic approach using siRNA for p62 and overexpression using HA-p62 followed by survival, proliferation, and autophagy measurements were used to further evaluate the possibility of p62 as a molecular target for inhibition of autophagy seen using NX. We found that i) melanoma cells displayed high autophagic flux compared with melanocytes, ii) NX treatment a) inhibited autophagy in melanoma cells, b) inhibited melanoma cell survival, and c) induced apoptosis in melanoma cells, iii) knockdown of p62 resulted in a lesser inhibition of cell viability and autophagy after NX treatment in high p62-expressing melanoma cells, and iv) overexpression of p62 sensitized low p62-expressing normal melanocytes and melanoma cells to NX-mediated cell death and autophagy inhibition, suggesting that p62 is a bonafide molecular target of NX. Overall, we show that Nexrutine is able to inhibit the inherent high level of pro-survival autophagy in melanoma cells, leading to loss of cell viability and induction of apoptosis, while melanocytes remain unaffected by NX at the same doses. Further, SQSTM1/p62 was found to be a critical mediator of the NX-mediated inhibition of autophagy and cell viability. This study demonstrates that NX maybe a promising novel, non-toxic agent for melanoma.

Supported by R21 CA125719 & ACRCF (RG); NIDCR T32 DE14318/COSTAR (HGH)

#3535

Induction of MAPK- and ROS-dependent autophagic cell death in gastric carcinoma by combination of bortezomib and romidepsin.

Alan K. Chiang, Pauline L. Yeung, Kwai Fung Hui. _Univ. of Hong Kong, Pokfulam, Hong Kong_.

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors can synergistically induce apoptotic cell death in certain cancer cell types but their combinatorial effect on the induction of autophagy remains unknown. Here, we investigated the combinatorial effects of a proteasome inhibitor, bortezomib, and an HDAC inhibitor, romidepsin, on the induction of apoptotic and autophagic cell death in gastric carcinoma (GC) cells. Isobologram analysis showed that low nanomolar concentrations of bortezomib/romidepsin could synergistically induce killing of GC cells. The synergistic killing was due to the summative effect of caspase-dependent intrinsic apoptosis and caspase-independent autophagy. The autophagic cell death was dependent on the activation of MAPK family members (ERK1/2 and JNK), and generation of reactive oxygen species (ROS), but was independent of Epstein-Barr virus infection. In vivo, bortezomib/romidepsin also significantly induced apoptosis and autophagy in GC xenografts in nude mice. This is the first report demonstrating the potent effect of combination of HDAC and proteasome inhibitors on the induction of MAPK- and ROS-dependent autophagy in addition to caspase-dependent apoptosis in a cancer type.

This project is funded by CRCG (#104003676) grant of KFH, CRCG (#104002845) and Epstein-Barr virus research (# 200004525) grants of AKSC.

#3536

**Rapamycin in combination with gemcitabine reduced viability, growth and metastasis of the murine osteosarcoma cell line LM8** in vitro **and** in vivo **.**

Takashi Ando,1 Jiro Ichikawa,1 Nobutaka Sato,1 Tetsuro Ohba,1 Kensuke Koyama,1 Tetsuo Hagino,2 Hirotaka Haro1. 1 _University of Yamanashi, Chuo, Yamanashi, Japan;_ 2 _Kofu National Hospital, Kofu, Yamanashi, Japan_.

Introduction

Rapamycin is an mTOR inhibitor with anti-tumor activity in several human cancers. However, the efficacy of this compound in osteosarcoma has not been fully elucidated. It acts downstream in the PI3K pathway and impedes cell growth, cell proliferation, cell motility, cell survival, protein synthesis and transcription. Here, we assessed the anti-tumor activity of rapamycin. We had previously reported that gemcitabine reduced viability, growth and metastasis of osteosarcoma cell lines. In this study, we investigated whether gemcitabine could enhance the anti-tumor effects of rapamycin in the murine osteosarcoma cell line LM8 in vitro and in vivo.

Results

In the LM8 cell line, rapamycin synergized with gemcitabine to reduce cell proliferation as determined by the WST assay and to induce apoptosis as analyzed by flow cytometry using the annexin V and 7AAD staining method. Rapamycin at doses >10 μM showed significant cytotoxicity. By combining with gemcitabine, the amount of rapamycin required for apoptosis was reduced to 1 μM. Autophagy was evaluated with the Cyto-ID® Autophagy detection kit. Not only rapamycin but also gemcitabine induced autophagy in the LM8 cell line. However, no synergistic effects were observed. Cell mobility was evaluated by migration assay using a trans-well system (LM8 cells in the upper layer and conditioned medium in the lower chamber). Rapamycin synergized with gemcitabine to reduce LM8 cell mobility.

In C3H mice inoculated subcutaneously with LM8, we observed frequent metastasis to the lung. Treatment of the mice with rapamycin reduced the size of the primary tumor; elevated levels of apoptosis were observed. Moreover, rapamycin in combination with gemcitabine had additive effects on the primary tumor and almost no metastatic lesions in the lung were seen. Consequently, combination therapy prolonged the overall survival of tumor-bearing mice.

Conclusions

Rapamycin synergized with gemcitabine to reduce cell proliferation, and to induce apoptosis, autophagy and cell mobility of the LM8 murine osteosarcoma cell line in vitro. Rapamycin in combination with gemcitabine exhibited synergistic antitumor effects. The combination prolonged overall survival and reduced the number of lung metastases in osteosarcoma-bearing mice. Rapamycin has been used to treat osteosarcoma in clinical trials and gemcitabine combined with docetaxel chemotherapy has shown good clinical results. Rapamycin combined with gemcitabine chemotherapy might be a novel and promising therapeutic approach to the treatment of osteosarcoma.

#3537

Cisplatin refractory lung cancer cells exhibit an increased autophagic flux at baseline and following exposure to cisplatin.

Magdalena Circu,1 James Cardelli,1 Martin Barr,2 Hazem E. El-Osta1. 1 _Louisiana State University Health Sciences Center, Shreveport, LA;_ 2 _Institute of Molecular Medicine, Trinity Center for Health Sciences, St. James's Hospital & Trinity College, Dublin, Ireland_.

Introduction: Lung cancer is the leading cause of cancer-related death. Platinum-based therapy is a standard treatment for advanced lung cancer. However, most patients develop resistance to treatment within few months. Autophagy is a catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, degraded in lysosomes, and recycled to sustain cellular metabolism. Activation of autophagy in response to cellular stress enables cancer cell survival, which can lead to tumor growth and therapeutic resistance.

Materials and Methods: Cisplatin-resistant (cisR A549) lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To measure autophagy, cells were stained for CytoID and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Autophagy was confirmed by immunoblotting for LC3II/I and p62 in cell lysates. Lysosomal membrane permeabilization (LMP) was detected after loading lysosomes with FITC-dextran 40KDa and detecting dextran leakage into the cytosol on confocal microscopy. Cell viability was evaluated using CellTiter blue assay.

Results: We observed an increase in baseline autophagic flux in cisR A549 lung cancer cells as compared to parental cells in full media condition. This was demonstrated by an increase in LC3II/I ratio, and reduction in p62 level in untreated cells. Compared with untreated cells, cisplatin induced a time-dependent increase in autophagosome formation in cisR A549 cells as manifested by the enhanced cytoID signal. To examine whether the increase in CytoID signal was due to autophagic flux stimulation or autophagosome accumulation secondary to blockage of the last step of autophagy, cells were treated with cisplatin in combination with chloroquine, a known lysosomal inhibitor. This had led to a significant increase in cytoID signal as compared to each drug alone, suggesting that autophagy was induced following exposure to cisplatin in cisR cells. Interestingly, increased CytoID signal following exposure to cisplatin was less pronounced in cisplatin-sensitive cells, suggesting that autophagy stimulation following exposure to the drug is inherent for resistant cells. Inhibition of autophagy using chloroquine sensitized resistant cells to cisplatin. Chloroquine was found to induce LMP. Indeed, neutralization of leaked lysosomal proteases with E64, a pan-protease inhibitor, attenuated the synergistic effect of chloroquine. Further works evaluating whether specific autophagy inhibition employing siRNA ATG3 or 3-MA can restore susceptibility to cisplatin are ongoing.

Conclusion: Our findings suggest that increased autophagy may be involved in acquired resistance to cisplatin in lung cancer. Targeting autophagy is a viable strategy to reverse chemoresistance.

#3538

Effects of oral chloroquine administration on a preclinical mouse model of PTEN/p53-deficient prostate cancer.

Marco A. De Velasco,1 Koichi Sugimoto,1 Yurie Kura,1 Yuji Hatanaka,1 Yutaka Yamamoto,1 Takashi Oki,1 Kazuhiro Yoshimura,1 Masahiro Nozawa,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kinki University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Nagakute, Japan_.

Autophagy is cellular process that can promote tumor survival during periods of metabolic and therapeutic stress. Inhibition of autophagy can promote senescence resulting in decreased tumor growth. Genetic alterations of PTEN and p53 are frequently present in advanced human prostate cancer and are associated with poor prognosis. In mouse models, the inactivation of PTEN leads to the development of prostate tumors that progress slowly, in part due to the induction of PTEN-(loss)-induced cellular senescence. Concomitant inactivation of both PTEN and P53 in prostate leads to the development of an aggressive cancer phenotype that can overcome PTEN-(loss)-induced cellular senescence and is characterized with accelerated growth, metastatic development and decreased survival. Published findings show that in some cancer models, the effects of autophagy inhibition are dependent on p53 status. In this study we assessed the effects of pharmacological inhibition of autophagy with chloroquine (CQ) on the growth and progression of mouse prostate tumors driven by the concomitant inactivation of PTEN and p53. CQ is a lysosomotropic agent that has widely used to impede autophagy and inhibit the growth of human tumor cells in vitro and in vivo. Genetically engineered mice with the conditional inactivation of PTEN and p53 were used to evaluate the antitumor effects and therapeutic benefit of CQ in a drug intervention model of prostate cancer. Twenty-seven-week-old PTEN/p53 double knockout mice bearing prostate tumors were randomized to control and oral CQ (100 mg/kg in drinking water) treatment groups. Progression-free survival was 145 days versus 161 days (P=0.703) for control and CQ treated mice, respectively. Median overall survival was 170 days versus 147 days (P=0.523) for control and CQ treated mice, respectively. Tumor burden assed by the median genitourinary tract weight was 2.42 grams versus 2.19 grams (P=0.645) for control and CQ treated mice, respectively. Our molecular assays revealed that CQ failed to consistently inhibit autophagy in tumors at the present dose. Overall, our findings show that CQ alone did not provide treatment benefit in mouse prostate cancer with loss of p53.

#3539

Vitamin D/EB 1089 mediated radio-sensitization of lung cancer cells and head and neck cancer cells.

Theresa Thekkudan, Khushboo Sharma, Tareq Saleh, David Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Lung cancer is a principal factor contributing to cancer-related mortality in the United States. Patients with lung cancer generally have a poor prognosis and resection is often not possible. Our previous work has demonstrated that Vitamin D and its analog, EB1089 (EB), enhance radio sensitivity of H460 and A549 non-small cell lung cancer (NSCLC) cells (Sharma et al; A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog, EB 1089. Autophagy; 2014;10(12):2346-61). In this study, we have extended these findings of Vitamin D/EB 1089 induced radio-sensitization in multiple tumor cell lines such as Lewis Lung mouse non-small cell lung cancer cells (LLC), head and neck cancer cells (HN30) and a primary cell line established from a lung tumor obtained from a cancer patient.

All cell lines were treated with multiple doses of radiation in the range of 2-6 Gy and EB1089 was used at concentration of 100nM. Sensitivity was determined based on viable cell number and clonogenic survival.

We observed enhanced radiation sensitivity of LLC and HN30 cells with the combination treatment of radiation and EB1089. However, as reported previously by our laboratory (Sharma et al., 2014; Autophagy), sensitization was dependent on the presence of functional p53 and was not evident in HN6 head and neck tumor cells that are mutant in p53. These data suggest that Vitamin D or a Vitamin D analog could be used to enhance radiation sensitivity of various malignancies but only where the tumors express wild type p53. Furthermore, we were able to demonstrate sensitization in a primary cell line established from lung tumor tissues utilizing conditioned medium from stromal cells treated with EB1089. These data suggest that stromal re-programming of cancer cells could influence radiation sensitivity.

In studies to define the signaling pathways associated with radiation sensitization, we also observed inactivation by EB 1089 of the NF-kB pathway that was activated by radiation alone in H460 and H1299 NSCLC cells. Silencing of AMPK attenuated radiation sensitization by EB 1089. Sensitization was accompanied by enhanced secretion of the autophagy/senescence related marker, HMGB1. Taken together, these studies indicate that Vitamin and/or its analogs enhance radiosensitivity by cellular pathways involving NF-kB and AMPK and that HMGB1 may be secreted to activate an immune response to the combination treatment.

#3540

Exploring novel treatment agent in GIST by using connectivity map.

Chueh-Chuan Yen,1 Li-Tzong Chen,2 Chien-Feng Li3. 1 _Taipei Veterans General Hospital, Taipei, Taiwan;_ 2 _National Health Research Institutes, Tainan, Taiwan;_ 3 _Chi Mei Medical Center, Tainan, Taiwan_.

Gastrointestinal stromal tumors (GIST) is gastrointestinal tract sarcoma which commonly contains a mutation in the KIT or PDGFRA. Imatinib mesylate, a specific tyrosine kinase inhibitor of the KIT and PDGFRA, causes regression or stabilization of disease in most of the patients with metastatic GIST. However, acquired resistance to imatinib is inevitable. Recently, E26 variant 1 (ETV1) pathway was found to be a key downstream effector of KIT. In this study, we explored the role of ETVI and its downstream effectors pathway in GIST by bioinformatics analysis, and identified potential novel agents that target ETV1 pathway of GISTs by using expression profile and a pattern-matching software "Connectivity Map" (CMAP), and confirmed the efficacy of novel agents by using GIST cell line in vitro assay. By using CMAP, four drugs with potential ETV1-inhibition effect were identified: suberanilohydroxamic acid (SAHA,Vorinostat) and trichostatin, two histone deacetylase inhibitors (HDACI), and trifluoperazine (TFP) and thioridazine (TDZ), two drugs of phenothiazine class. All four drugs were found to have ETV1-down regulating effect by immunoblotting. Phenothiazine could induce apoptosis and autophagy in GIST. Treatment of phenothiazine was found paradoxically up-regulate MEK activity. Combination of MEK inhibitor and phenothiazine showed great synergistic effect in exerting cytotoxicity against GIST cell lines. Our study established phenothiazine as a new class of agent with therapeutic effect in GIST, and combination of phenothiazine and MEK inhibitor showed great potential in GIST treatment.

#3541

Targeting autophagy to enhance paclitaxel sensitivity in esophageal carcinoma.

Jiajing Cai, Yan Cai, Qiang Ma, Jiang Zou, Binfeng He, Fan Chang, Lei Xu, Xiaolan Guo. _North Sichuan Medical College, Sichuan, P.R. China, Nanchong, Sichuan, China_.

Esophageal cancer is one of the malignancies threatening human health with high incidence and a very low 5-year survival rate with poor outcomes in the developing countries. Chemotherapy has been played indispensable roles while companied with more and more chemoresistance and side effects. Numerous studies have shown that autophagy may play specific role as one of the drug resistance mechanisms through promoting cancer cell survival via self-digestion. Therefore targeting autophagy might be another promising approach of therapeutics in cancer.

Paclitaxel(PTX) is widely used for malignant tumors treatment through inducing apoptosis and autophagy. More findings discovered that chemoresistance is still a major hurdle for PTX due to cytoprotective autophagy. In order to investigate whether targeting autophagy could confer the sensitivity of PTX in esophageal carcinoma, chloroquine (CQ) was applied as an autophagy inhibitor to study the esophageal carcinoma cells EC109 co-treated with PTX in vitro and explore the potential molecular mechanisms.

Firstly, CQ and PTX were used separately and also combined to treat EC109 cells at different dosages and time points, and the MTT results showed that CQ could contribute to the suppressive effect of PTX on the growth and proliferation of EC109 cells. Meanwhile the migration and invasion abilities of EC109 were significantly inhibited by co-treatment of PTX and CQ comparing with PTX only through scratch test and cell invasion Transwell chamber test, moreover the combination treatment dramatically suppressed the colony formation on soft agar.

Further investigation showed that mTOR pathway was activated in CQ or PTX exposure, which resulted in phosphorylated mTOR(p-mTOR) down-regulation, followed by the decline of phosphorylated p70S6K (p-p70S6K) and phosphorylated p-4EBP1, and the autophagy marker LC3I/II conversion and p62 elevated eventually, which indicated that autophagy was mediated in PTX or CQ treatment in EC109 cells. On the other hand, GFP-LC3 was transfected into EC109 cells to observe the LC3 translocation status after CQ or PTX treatment, which usually be regarded as the morphological maker of autophagy, and the results confirmed the findings in Western Blot. Most importantly, the combination treatment of CQ and PTX resulted in synergistic consequences. For further understanding the mTOR pathway was initiated in CQ or PTX mediated autophagy in this study, we used lentiviral shRNA embedding mTOR to knockdown the expression of mTOR first and then studied the signaling pathway changes after CQ and PTX co-treatment, and we found a decrease trend of LC3-II/ I ratio and p62 also compared with the competent mTOR control. From these results, we confirmed that autophagy would benefit the survival of EC109 cells, and our findings demonstrated that targeting of autophagy using CQ could be an effective and potential strategy to improve PTX treatment outcomes in the management of esophageal carcinoma.

#3542

Abnormal intracytoplasmic accumulation of autophagy-related protein p62/SQSTM1 characterizes giant cells of giant cell tumor of bone.

Toru Motoi,1 Akihiko Yoshida,2 Noriko Motoi,2 Ikuma Kato,1 Tomotake Okuma,3 Akiko Tonooka,1 Shinichiro Horiguchi,1 Takahiro Goto,3 Tsunekazu Hishima1. 1 _Department of Pathology, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;_ 2 _Deparment of Pathology, National Cancer Center Central Hospital, Tokyo, Japan;_ 3 _Department of Musculoskeletal Oncology, Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan_.

Background

Giant cell tumors of bone (GCTs) are locally aggressive neoplasms composed of osteoclast-like giant cells and mononuclear cells. Mononuclear cells are accepted as true neoplastic elements, while osteoclast-like giant cells are non-neoplastic. However, close interaction between two cellular populations is indispensable for tumor formation. Large giant cells with hypermultinucleation of GCT are distinct from normal osteoclasts; however, their abnormality has not yet been sufficiently investigated. Recently, a germline mutation of the gene encoding autophagy-related protein p62/SQSTM1 was reported in Paget disease of bone, which implicates a role of autophagy in the differentiation and function of osteoclasts. In order to understand the relationship between autophagy and osteoclastogenesis in giant cell lesions, we studied clinical samples of GCTs and related tumors for immunoexpression of p62 (an autophagy flux marker), LC3 (an autophagosome marker), and Cathepsin K (a known osteoclastic marker).

Design

Formalin-fixed paraffin-embedded specimens of 46 GCTs, 17 chondroblastomas (CBLs), 3 giant cell reparative granulomata (GRGs), 3 chondromyxoid fibromas (CMFs) and one each of aneurysmal bone cyst (ABC) and brown tumor (BRT) were retrieved. Immunohistochemistry was performed by using antibodies against p62, LC3, and Cathepsin K. Results were scored as 3+ (strongly positive in ≥50% of cells), 1+ (weakly positive in <10% of cells), 2+ (intermediate between 1+ and 2+), or 0 (negative). Giant cells and mononuclear cells were separately evaluated for nuclear and cytoplasmic staining.

Results

For giant cells, p62 was positive in a dot-like manner in all tumor types. Strong (3+) staining was found in nuclei and/or cytoplasms in GCTs (27/46, 59%), CBLs (14/17, 82%), GRGs (2/3, 67%), and other lesions (5/5, 100%). In addition, abnormal intracytoplasmic coarse aggregates were specifically noted in GCTs (21/46, 46%) and CBLs (3/17, 18%). In contrast, mononuclear cells showed a lower prevalence of 3+ p62 positivity in GCTs (2/46, 4%), CBLs (5/17, 29%), and an ABC (1/1), where positive cells were only presumable osteoclastic precursor cells around giant cells. LC3 showed a limited cytoplasmic 3+ positivity in giant cells of GCTs (6/46, 13%), CBLs (7/17, 41%), CMFs (1/3, 33%), and BRT (1/1). Cytoplasmic staining of Cathepsin K was constantly observed in giant cells.

Conclusion

Abnormal intracytoplasmic accumulation of p62 in a form of coarse staining aggregate characterizes giant cells of GCTs and CBLs. This finding may be a potential diagnostic marker for distinguishing GCTs and CBLs from other giant cell lesions. The presence of these p62 aggregates and low LC3 expression may indicate disturbance of autophagy-driven protein clearance. As p62 immunostain recognizes a wider osteoclastic population than Cathepsin K, p62 may be involved in abnormal osteoclastogenesis.

#3543

Alternating electric fields (TTFields) induce autophagy in human cancer cell lines.

Yaara Porat,1 Anna Shteingauz,1 Moshe Giladi,1 Rosa S. Schneiderman,1 Tali Voloshin,1 Mijal Munster,1 Roni Blat,1 Eilon D. Kirson,1 Uri Weinberg,2 Yoram Palti1. 1 _Novocure, Haifa, Israel;_ 2 _Novocure, Luzern, Switzerland_.

Tumor treating fields (TTFields) are an established anti-neoplastic treatment modality in patients with glioblastoma. TTFields are delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields to the region of the tumor. Previous studies have shown that TTFields lead to increased granularity in treated cells. Granular or vesicular structures can indicate the formation of autophagosomes which are key structures in autophagy. Autophagy has been shown to regulate cell survival and proliferation under stress conditions and, in certain cases, influence cellular response to cytotoxic drugs. The goal of this study was to evaluate the possible effect of TTFields on the induction of autophapgy in treated cells.

Different cancerous cell lines were treated with TTFields using the inovitro system. Cellular granularity was evaluated using flow cytometry. Autophagy was monitored by quantifying Lipidated Microtubule Associated Protein Light Chain 3 (LC3-II, a consensus marker for autophagosomes) levels using immunoblotting and immunofluorescence microscopy. Transmission Electron Microscopy (TEM) was used to visualize autophagosome-like structures.

Flow cytometry analysis demonstrated that TTFields application leads to a significant increase in cellular granularity in all tested cell lines (up to 40%, P<0.05). Significant elevation in LC3-II levels was observed in treated U-87 MG cells using immunoblotting analysis (37%, P=0.01). Evidence of increased autophagy following TTFields application was also detected using fluorescence microscopy, where punctate distribution of LC3-II was observed. TEM micrographs demonstrated the presence of autophagy typical, autophagosome-like structures in TTFields treated cells.

TTFields are known to exert anti-mitotic effects by disrupting highly dipolar structures which play critical roles in mitosis. Our results suggest that in addition, TTFields can also induce cellular autophagy. Future studies are warranted to examine to what extent TTFields-elicited- autophagy may affect treatment outcomes, and to investigate the therapeutic implications of combining TTFields with autophagy inhibitors.

#3544

Determining autophagic activity following cannabidiol treatment of melanoma: a potential new and novel treatment.

Erika L. Simmerman,1 Xu Qin,1 Kouros Motamed,2 Jack C. Yu,1 Babak Baban1. 1 _Georgia Regents University, Augusta, GA;_ 2 _Sorrento Therapeutics Inc, San Diego, CA_.

Introduction:

Autophagy is a catabolic pathway utilized for energy metabolism in nutrient-depleted tumors. Recent studies have reported autophagy to be a survival mechanism found in cancer during stress secondary to cancer progression or treatment. In this study we tested for autophagic activity in malignant melanoma tumors via detection of LC3 antibody following treatment with Cannabidiol (CBD) and cisplatin. The objective of this study was to investigate the effect of treatment with a cannabinoid derivative on autophagic activity in malignant melanoma.

Materials and Methods:

Murine B16F10 melanoma tumors were established subcutaneously in C57BL/6 mice. These mice were then subjected to treatment with local vehicle (PBS) injection (control - group 1, n=5), systemic Cisplatin injection (group 2; n=5), and local cannabidiol injection (group 3; n=5). Tumor cells were subsequently submitted to immunohistochemistry, Brightfield microscopy, and quantitative analysis (Bioquant) for LC3 expression.

Results:

The expression level of LC3 was significantly reduced in groups 2 and 3 when compared to the control group 1. Quantitative measurement of the slides using Bioquant software confirmed our immunohistochemistry and microscopic results.

Conclusions:

If CBD does indeed decrease autophagic activity, it may represent a new and novel therapeutic target in the treatment of malignant melanoma. Additional studies to clarify the specific relationship between CBD and autophagy are in progress and will be reported upon completion.

#3545

Gallotannin induces senescence and autophagy leading to cell death via SIRT1 inhibition and AMPK activation in hepatocellular carcinoma cells.

Hee Young Kwon, Sung-hoon Kim. _Kyung Hee University, Seoul, Republic of Korea_.

Hepatocellular carcinoma (HCC) is one of the most fatal malignancies with high mortality rate worldwide. In the present study, the underlying antitumor mechanism of gallotannin, a hydrolysable tannic acid, was elucidated in HepG2 and SKHep1 HCC cells. Gallotannin suppressed growth and colony formation and also induced senescence via upregulation of p21, G0 / G1 arrest and higher senescence-associated β-galactosidase (SA-β-gal) activity within 24 h culture in HepG2 and SKHep1 cells. Of note, gallotannin also induced autophagy by increasing the number of protein-cytosol-associated protein light chain 3 (LC3) punctae, LC3B-II conversion and SQSTM1/p62, autophagic vacuoles (AVOs) by transmission electron microscopy (TEM) and decreasing the of Beclin1 in HepG2 and SKHep1 cells. A tandem fluorescent-tagged LC3 reporter plasmid (GFP-mRFP-LC3) transfection revealed that gallotannin increased the number of yellow colored LC3 punctae through the co-localization of GFP and mRFP punctae and yellow colored LC3 punctae were increased by CQ or NH4Cl with accumulation of autophagosomes, LC3B-II and SQSTM1/p62 in HepG2 and SKHep1 cells. Additionally, gallotannin attenuated the of sirtuin1 (SIRT1) and mTOR and activated the phosphorylation of 5´-AMP activated protein kinase (AMPK) in two HCC cells. Furthermore, AMPK activator AICAR significantly enhanced SA-β-gal activity and antiproliferation induced by gallotannin, while AMPK inhibitor compound C did not in two HCC cells. Of note, gallotannin time dependently activated caspase 8 / 3 and time/concentration dependently increased sub G1 portion along with weak cleavages of PARP in two HCC cells. Collectively, gallotannin induces senescence and inhibits late autophagy flux, finally leading to apoptotic cell death in HepG2 and SKHep1 cells via inhibition of SIRT1 and activation of p-AMPK as a potent antitumor agent for HCC treatment.

#3546

DNA-damaging reagent -0404 effectively inhibits hepatocellular carcinoma through p53-dependent autophagy.

Min Zheng,1 Bingjue Ye,1 Guohua Lou,1 Caixia Xia,1 Weiyang Zheng,1 Yanning Yanning,1 Xiao Xu,2 Yongchao Zhao,3 Long Mao,2 Wanghong Xu2. 1 _Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases. State Key Labor, Hangzhou, China;_ 2 _Hangzhou ACEA Pharmaceutical Research Co., Ltd., Hangzhou, China;_ 3 _Institute of Translation Medicine, Zhejiang University, Hangzhou, China_.

Background

On a worldwide basis, the prevalence of hepatocellular carcinoma (HCC) is twice as much in the past two decades, which is the third most common cause in cancer deaths. Therefore there is still an urgent need for new drugs with greater validity against HCC. Autophagy, which is essential in the intracellular clearance of many proteins and organelles, has been closely related to cancer generation and development. Generally, p53 is recognized as a pro-autophagic factor in a transcription-dependent pattern and also an important factor in the progress of HCC. As a selective substrate for autophagy, p62 is a key factor in tumorigenesis on account of its crucial roles in the control of cellular events. Now that small molecule compound-0404 was defined as a DNA-damaging reagent by the p53 pathway, our work aims to examine whether 0404 can affect p53-dependent autophagy and its positive effect in the treatment of HCC.

Methods

The anti-cancer activity of 0404 was examined in vitro. In the HepG2 cells and Huh7 cells, the apoptosis was detected by Flow Cytometry, while cell viability was measured by the CCK8 Assay. The Hela eGFP-LC3 cells, which express fusion protein combined light chain 3 (LC3) with enhanced Green Fluorescent Protein (eGFP), were treated with 0404 at various concentrations for various time points. HepG2 cells were treated with 0404 at various concentrations (5.0 nM, 20 nM, and 50 nM) for 24h. Autophagy was identified by eGFP-labeled LC3. And the expression levels of autophagy-associated proteins, such as mammalian target of rapamycin (mTOR), phospho-mTOR (p-mTOR) , p62 and LC3B, were tested by Western Blotting.

Results

We confirmed that 0404 could inhibit the proliferation of HepG2 and Huh7 in a dose-dependent manner, and compared to Huh7 cells with mutant p53, HepG2 cells with wild-type p53 were more sensitive to 0404 treatment for apoptosis. We also found that 0404 promoted the development of autophagy in a dose- and time-dependent manner. Compared with untreated group, LC3 in 0404-treated groups resided primarily in the cytoplasm including cytoplasmic puncta indicative of autophagosomes. Simultaneously, the expression levels of mTOR and p62 were down-regulation, while the LC3B expression level was up-regulation by 0404-treatment. And the expression level of p-mTOR was down-regulation in a dose-dependent in HepG2 cells.

Conclusion

These results show clearly that DNA-damaging reagent-0404 can effectively inhibit HCC through p53-dependent autophagy.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (81272679) and the Major national S&T Projects (2013ZX09041003).

#3547

CEBPD induces apoptosis through autophagy in metformin-treated HCC.

I P. Lin. _The Institute of Bioinformatics and Biosignal Transduction, NCKU, Taiwan, Taiwan_.

The patients with hepatocellular carcinoma (HCC) face the risk of cancer relapse or liver dysfunction. Metformin (Met), a first-line drug of type 2 diabetes, has been proved that it could induce G1 arrest and prevent or inhibit HCC. Previously, we demonstrated that the transcription factor CCAAT/enhancer-binding protein delta (CEBPD) plays a tumor suppressor and is responsive to many anticancer drugs in HCC. In this study, we found that Met could induce autophagy, growth arrest and further apoptosis in Huh7 cells. Met enhanced CEBPD expression via stabilizing its protein stability. The loss of CEBPD attenuated the Met-induced autophagy, growth arrest and apoptosis. Importantly, in addition to increasing P27 and down-regulating cyclin D1, CEBPD also contributed the increases of LC3B and ATG3 and LC3 puncta formation. We revealed a novel involvement of CEBPD in Met-induced growth arrest, apoptosis and autophagy of liver cancer cells through regulating p27, cyclinD1, LC3B and ATG3.

#3548

In vivo **CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia.**

Júlia Aguadé-Gorgorió,1 Scott McComb,1 Gunnar Cario,2 Martin Stanulla,3 Cornelia Eckert,4 Jean-Pierre Bourquin,1 Beat C. Bornhauser1. 1 _Department of Oncology and Children's Research Centre, University Children's Hospital Zurich, Zurich, Switzerland;_ 2 _Department of General Pediatrics, University Hospital Schleswig-Holstein, Kiel, Germany;_ 3 _Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany;_ 4 _Department of Pediatric Oncology/Hematology, Charité Medical University Berlin, Berlin, Germany_.

Dysregulation of apoptosis constitutes a general mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death during chemotherapy. We have assessed the potential of SMAC mimetic compounds (SM) to eradicate drug resistant ALL by blocking the pro-survival cellular inhibitor of apoptosis proteins (cIAPs). On the basis of a large xenograft bank of primary ALL samples, obtained by xenotransplantation in immunodeficient NSG mice, we detected exquisite sensitivity to SM in vitro in a third of the cases tested, including drug-resistant and relapsed samples. Potent in vitro activity was highly predictive of in vivo efficacy in the xenograft model. Treatment of transplanted mice with SM not only delayed leukemia progression, but also completely eliminated leukemia burden in established disease for several weeks following treatment. SM activity required the presence of RIP1, since lentiCRISPR mediated gene knockout of RIP1 in patient-derived ALL cells blocked SM-induced cell death in vivo and in vitro. In contrast, RIP1 loss had no significant impact on the response to standard antileukemic therapies, supporting a view that RIP1-dependent death pathways are not likely to be selected against by front line chemotherapy. Furthermore, using a multicolor lentiCRISPR approach for simultaneous disruption of multiple genes, we found that downstream of RIP1, the response to SM was dependent on simultaneous execution of apoptosis and necroptosis, since genetic targeting of both cell death pathways was required to block SM-induced death. Upstream of RIP1, expression levels of TNF receptor II (TNFR2) in primary ALL correlated with sensitivity to SM, and genetic disruption of TNFR2 conferred resistance to SM treatment in vivo. Thus, SM treatment circumvents drug resistance through parallel activation of RIP1-dependent apoptosis and necroptosis, providing a strong rationale for application in salvage treatment of refractory childhood ALL.

#3549

The role of programed necrosis in oncolytic adenovirus-induced cell death in ovarian cancer.

Melanie Weigert,1 Alexander Binks,1 Stephen Tait,2 Iain McNeish1. 1 _University of Glasgow, Glasgow, United Kingdom;_ 2 _CRUK Beatson Institute, Glasgow, United Kingdom_.

Introduction.

Oncolytic viruses preferentially replicate within and kill cancer cells and are a promising novel cancer treatment. However, the mechanisms by which oncolytic adenoviruses induce death in cancer remains unclear. It was long thought that DNA viruses trigger classical apoptosis but there is now evidence that cell death induced by both adenovirus and vaccinia displays features of necrosis-like programmed cell death.

Methods and Results.

In order to investigate the role of necrosis in cell death following oncolytic adenovirus infection, a panel of ovarian cancer cells with varying sensitivities was infected with the E1A CR2-deleted adenoviral mutant dl922-947. By electron microscopy, dl922-947 infection induces key morphological features of necrotic death, including membrane rupture, nuclear swelling and cytoplasmic vacuolation. Using specific necrosis inhibitors (necrostatin-1, necrosulfonamide, GSK'840B, GSK'872B and GSK'843A) as well as RNAi-mediated knockdown of receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like (MLKL), we show that adenovirus-infected ovarian cancer cells undergo RIPK3-dependent necrosis and that blockage of the downstream effector MLKL significantly attenuates cell death. Co-immunoprecipitation of caspase 8 shows formation of a complex containing RIPK1, FADD and caspase 8 during adenovirus infection. However, unlike Tumour Necrosis Factor-α (TNF-α)-induced programmed necrosis, which relies on the RHIM-dependent interaction of RIPK1 and RIPK3, RIPK1 appears redundant in adenovirus-induced death. Furthermore, the addition of a TNF-α blocking antibody to virus-infected cells has no effect on either cell death or overall cell survival.

In a RIPK3 overexpression model, we show a direct correlation between adenovirus-induced cell death and the extent of RIPK3 expression. Moreover, RIPK3 expression has no effect on infectivity, viral protein expression or infectious virus production. Finally, expression of RIPK3 increases necrosis induction in vivo following direct intra-tumoural dl922-947 injection, and significantly improves anti-tumour efficacy.

Conclusions.

Our data suggest that cell death induced by oncolytic adenoviruses differs from TNF-induced programmed necrosis but still relies on the kinase RIPK3 and its downstream component MLKL, making these two proteins possible targets for future oncolytic virotherapies.

#3550

Activation of a novel form of regulated necrosis to eliminate extracellular matrix detached epithelial cells.

Mark A. Hawk, Cassandra L. Buchheit, Luqun Shen, Patrick Fagan, Zachary T. Schafer. _University of Notre Dame, Notre Dame, IN_.

The overwhelming majority of breast cancer deaths are caused by the metastasis of cancer cells from the primary tumor to distant sites in the body. For cancer cells to successfully metastasize, they must: detach from the primary tumor, move out of the original tissue into the circulatory or the lymphatic system, travel to a new site followed by arresting their movement, and then extravasate to begin colonizing a secondary site. When normal epithelial cells detach from the extracellular matrix (ECM), they induce caspase-dependent cell death which is known as anoikis. Anoikis can therefore serve as a critical barrier to metastasis as cancer cells are exposed to limited and variable matrix conditions during each step of the metastatic cascade. However, our previous studies suggest that anoikis evasion is not sufficient to protect ECM-detached cells from cell death. Using MCF10A mammary epithelial cells, we have previously shown that ECM-detachment induced metabolic changes can compromise the survival of detached cells, although the precise mechanism controlling cell death remains unclear. Here, we present data suggesting that ECM-detached cells can also be eliminated by regulated necrosis (RN), a genetically programmed, caspase-independent form of necrosis that is morphologically indistinguishable from classical necrosis. In general, the molecular mechanisms involved in RN are poorly understood. With this in mind, the current most appreciated subtype of RN, termed necroptosis, is dependent upon TNFα, RIP1K, RIP3K, MLKL, and PGAM5 to execute cell death. Our data in ECM-detached cells suggest that cells are being eliminated by a mechanism that is dependent on the kinase activity of RIP1K. Further studies have shown an increase in the expression of CYLD, a deubiquitinating enzyme that specifically removes ubiquitin chains from RIP1K, in turn stabilizing both RIP1K expression and activation. Upon analyzing other dependent executioners of necroptosis, we have strikingly found that TNFα, RIP3K, and MLKL are all dispensable for RN to occur in ECM-detachment. Furthermore, using a variety of molecular tools, such as 3D cell culture, we have found that PGAM5 is necessary for the execution of ECM-detachment induced RN. These findings uncover a novel and distinct RN pathway and highlight the need to more thoroughly understand RN as well as the mechanisms employed by ECM-detached cells to antagonize RN and promote their survival in detachment.

### Cell Death 3

#3551

Discovery of orally bioavailable novel Mcl-1 inhibitors that exhibit selective anti-proliferative activity in Mcl-1 sensitive cancer cell lines.

Taekyu Lee, Zhiguo Bian, Johannes Belmar, Subrata Shaw, James C. Tarr, Bin Zhao, Nick Pelz, DeMarco Camper, Craig M. Goodwin, Allison L. Arnold, John L. Sensintaffar, Carrie F. Browning, Olivia W. Rossanese, Edward T. Olejniczak, Stephen W. Fesik. _Vanderbilt University, Nashville, TN_.

Myeloid cell leukemia-1 (Mcl-1) is a member of the Bcl-2 family of proteins that regulate apoptosis. Amplification of Mcl-1 is found in various cancers, which causes the evasion of apoptosis and is one of the major resistance mechanisms for many chemotherapies. Mcl-1 mediates its effects primarily through high affinity interactions with pro-apoptotic BH3 containing proteins, Bak and Bax. Thus targeting Mcl-1 with small molecule inhibitors is a promising strategy but a very challenging task. Using fragment-based methods and structure-based design, we discovered a novel class of potent Mcl-1 inhibitors that exhibit selective anti-proliferative activity. New leads containing a tricyclic indole lactam scaffold exhibited dissociation constants of <0.5 nM with >1000-fold selectivity for Mcl-1 over Bcl-xL and Bcl-2. They also promoted apoptosis only in Mcl-1 sensitive cancer cell lines by activating caspases in a dose-dependent manner. These results provide a strong proof of concept for a selective inhibition of Mcl-1 function as an effective anti-cancer therapy. Finally, our leads also possess desirable pharmaceutical properties including in vivo oral bioavailability and represent an ideal starting point for developing clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

#3552

Regulation of immune homeostasis by direct activator BH3-only proteins.

Lindsey M. Ludwig,1 Lauren E. Roach,1 Jill K. Fisher,2 Loren D. Walensky,2 James L. LaBelle1. 1 _Department of Pediatrics and Committee on Cancer Biology, University of Chicago, Chicago, IL;_ 2 _Departments of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA_.

The BH3-only proteins BIM, BID, and PUMA have emerged as both inhibitors of anti-apoptotic BCL-2 family proteins and direct activators of the pro-apoptotic executioner proteins BAX and BAK, and have been implicated in regulating immune cell homeostasis. For example, BIM is a master regulator of B and T cells and its deletion in mice causes splenomegaly and lymphocytosis. Although deletion of Bid or Puma do not grossly impact murine hematopoietic organs, loss of each can protect lymphocytes from certain forms of cell death. Combined deletion of Puma and Bim causes increased lymphocytosis and spontaneous development of B-cell lymphoma, while loss of BID and BIM leads to delayed T cell contraction following antigen-mediated proliferation. To investigate the role of direct activator BH3-only proteins in regulating the adaptive immune response over time, we performed peripheral lymphocyte analyses on single and combinatorial knock-outs of Bim, Puma, and Bid. Select genotypes, such as Bim-/-, Bim-/-Puma-/-, and Bim-/-Puma-/-Bid-/- mice, show lymphocytosis early in life but, unexpectedly, counts were observed to gradually normalize by 40 weeks of age. Given the dominant role of BIM in immune cell homeostasis, we investigated whether this phenotype was dictated by global deletion of Bim or intrinsic to T or B cell-specific Bim deletion. In generating and analyzing Lck-cre and CD19-cre Bim floxed animals, we found that T cell-specific Bim deletion recapitulated early lymphocyte expansion followed by contraction, whereas the lymphocytosis of mice with B cell-specific Bim deletion showed no age-related changes. Further characterization of aged Lck-cre Bim floxed mice revealed spleen weights similar to wild type mice, and thymocytes with increased sensitivity to a range of apoptotic stimuli; these mice also have an increased percentage of regulatory T cells compared to similarly aged wild-type mice, as has been reported for globally-deleted Bim-/- animals. To determine the mechanism underlying the observed lymphocyte contraction, we performed qRT-PCR analysis for a panel of BCL-2 family proteins. We find that aged Lck-cre Bim floxed mice manifest upregulation of a series of BH3-only transcripts, including Puma and Bid, reflective of an intriguing compensatory mechanism to remedy, over time, the lymphocytosis that derives from Bim deletion in T cells. These studies provide new insight into the complexity and dynamism underlying BH3-only regulation of immune cell homeostasis during health, disease, and aging.

#3553

A novel allosteric mechanism for molecular inhibition of anti-apoptotic MCL-1.

Susan Lee,1 Thomas E. Wales,2 Catherine Gallagher,1 Daniel T. Cohen,1 Silvia Escudero,1 James Luccarelli,1 Annissa J. Huhn,1 Nicole A. Cohen,1 Gregory H. Bird,1 John R. Engen,2 Loren D. Walensky1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Northeastern University, Boston, MA_.

MCL-1 is an anti-apoptotic BCL-2 family protein that has emerged as a major pathogenic factor across a broad range of human cancers. Thus, pharmacologic inhibition of MCL-1 has been the focus of intensive drug development efforts. Like BCL-2, MCL-1 bears a C-terminal surface groove whose function is to bind and sequester the essential BH3 killer domains of pro-apoptotic BCL-2 family proteins, a mechanism harnessed by cancer cells to create formidable apoptotic blockades. Whereas drugging the BH3-binding groove has been successfully achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small molecule covalent modification of an interaction site that is distant from the BH3-binding groove. Biochemical and hydrogen-deuterium exchange mass spectrometry analyses revealed that both the BH3-binding capacity of MCL-1 and its functional suppression of BAX-mediated membrane poration is impaired by molecular engagement, a phenomenon recapitulated by mutagenic mimicry at the novel regulatory region. This allosteric mechanism for disrupting the anti-apoptotic, BH3-binding activity of MCL-1 could inform a new therapeutic strategy for disarming MCL-1 in cancer.

#3554

Functional disparity among BCL-2 members in tonsillar and leukemic B cell subsets probed by next generation BH3 mimetic profiling.

Victor Peperzak, Rachel Thijssen, Hanneke ter Burg, Erik Slinger, Arnon P. Kater, Eric Eldering. _Academic Medical Center, Amsterdam, Netherlands_.

Introduction and aim. Long-lived humoral immunity, in the form of plasma cells (PC) and memory B cells, as well as most B cell malignancies, originate in the germinal center (GC). For successful therapies against malignant B cells it is crucial to understand their survival requirements in relation to their normal counterparts. It was previously shown that murine GC B cells depend on pro-survival protein MCL-1, but not BCL-XL, although both are expressed. In contrast, naïve and memory B cells express BCL-2, and as a consequence, they are sensitive to the BH3 mimetic ABT-737, which blocks both BCL-2 and BCL-XL but not MCL-1. The divergent roles of MCL-1 and BCL-XL in GC B cells still remain unexplained, and it is unknown if this also holds for human B cells. We here dissect these aspects in human tonsillar B subsets and in chronic lymphocytic leukemia (CLL) cells, a B cell cancer where expression of BCL-2 members is influenced by signals that also occur in the normal GC.

Experimental procedures. We used FACS sorting of tonsil B subsets, mRNA and protein profiling, co-culture of CLL cells, co-IP of BCL-2 members, cell death assays with next generation BH3 mimetics specific for BCL-2, BCL-XL or MCL-1 (ABT-199, WEHI-539, A-1210477).

Results. Naive and memory B cells are mainly sensitive to specific inhibition of BCL-2 by ABT-199. In contrast, GC B cells and tonsil PC are insensitive to ABT-199, but undergo apoptosis when MCL-1 is inhibited. Tonsil PC display yet another profile and are remarkably sensitive to inhibition of BCL-XL. MCL-1 expression in GC B cells is regulated post-translationally and its physiological importance is highlighted by exclusive binding to pro-apoptotic BIM. In contrast, BCL-XL is transcriptionally induced in the GC-light zone, and is solely bound to the weak BH3-only sensitizer BIK, which may explain why BCL-XL is not required for GC B cell survival.

These approaches were extended to primary CLL cells either resting or stimulated via CD40, as model for circulating and lymph node resident cells. We showed recently that CD40 stimulation strongly induces MCL-1, BCL-XL and BFL-1, similar to the pattern in healthy GC B cells, and confers complete resistance to ABT-199, whereas untreated CLL cells are highly sensitive. We now demonstrate that dual or triple BH3 mimetic combinations are effective in killing CD40-stimulated CLL. In contrast to GC B cells, BIK is absent in CLL cells while NOXA is highly expressed and, like BIM, is bound to MCL-1. This may account for differential sensitivity to (combinations of) BH3 mimetics.

Conclusion. Using novel BH3 mimetics and interaction profiles, we were able to probe the contribution of individual BCL-2 members in survival of normal and malignant B cells. Our findings provide clues to determine therapeutic windows in novel treatment strategies in B cell and other malignancies.

#3555

Targeting the BCL-2 family in small cell lung caner.

Akane Inoue-Yamauchi,1 Paul Jeng,2 Kwanghee Kim,2 Hui-Chen Chen,2 Song Han,2 Yogesh Tengarai Ganesan,2 Yiyu Dong,2 Sylvia Jebiwott,2 James J. Hsieh,2 Emily H. Cheng2. 1 _Tokyo Women's Medical University, Tokyo, Japan;_ 2 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Small cell lung cancer (SCLC) represents 13% of all lung cancer cases, affecting approximately 30,000 people annually in the United States. The prognosis for patients with SCLC is poor, and cancer-specific mortality of this malignancy is 95% at five years. This dismal prognosis has been further marred by the absence of major improvements in its treatment: there have been no substantial changes in the standard of care for advanced SCLC over the last three decades. Interestingly, small molecule inhibitors of anti-apoptotic BCL-2 family members BCL-2 and BCL-XL, ABT-737 and its orally bioavailable analog ABT-263, were shown to effectively kill some SCLC cell lines in preclinical studies, suggesting that targeting the BCL-2 family proteins may hold promise for the treatment of this dreadful cancer. However, these inhibitors do not inhibit MCL-1, another anti-apoptotic BCL-2 family member, which may explain why multiple clinical trials have not led to meaningful results. Because there are no effective MCL-1 inhibitors developed yet, we propose to identify the best strategy that enhances the apoptotic effect of ABT-737/263. To this end, we have performed a high-throughput screen in ABT-737/263 resistant H196 cell lines using two libraries, a library of FDA-approved anti-cancer agents and a pathway inhibitor library including ~1000 compounds. As a result, doxorubicin and dinaciclib were identified as synergizers of ABT-737/263 in triggering apoptosis. These two drugs enhanced the death inducing activity of ABT-737/263 through downregulation of MCL-1 mRNA. Interestingly, some of the ABT-737/263 resistant cell lines rely on MCL-1 for survival and thereby were killed by dinaciclib or doxorubicin as a single agent, indicating that predictive biomarkers of cellular addiction to anti-apoptotic BCL-2 family proteins (BCL-2s) can guide treatment decisions and reduce unwanted toxicity. Importantly, we revealed that the expression ratio between BCL-2, BCL-XL, and MCL-1 could predict cellular addiction to BCL-2s. Surprisingly, we demonstrated that BCL-XL overexpression is an another potential mechanism behind ABT-263 resistance. ABT-263 failed to inhibit the interaction between BCL-XL and BAX/BAK in the absence of activator BH3-only molecules and thereby failed to kill one BCL-XL-addicted SCLC cell line with low expression of activator BH3-only molecules. Finally, we showed that BCL-2 selective inhibitor ABT-199 can also cooperate with doxorubicin or dinaciclib to kill ABT-263-resistant cell lines. The in vivo efficacy of combination therapy including ABT-199 and doxorubicin was demonstrated in a SCLC xenograft model. These data have direct translational implications for the treatment of SCLC.

#3556

CXCR4 controls BCL-2 expression and function by regulating miR-15a/16-1 expression in tumor cells.

Shiri Klein Silberman,1 Michal Abraham,2 Baruch Bulvik,2 Hanna Wald,2 Orly Eizenberg,2 Dvorah Olam,1 Lola Weiss,1 Katia Beider,3 Ori Wald,1 Shlomo Bulvik,4 Abraham Avigdor,3 Ohad Benjamini,3 Eithan Galun,1 Arnon Nagler,3 Yaron Pereg,5 Amnon Peled1. 1 _Hadassah Hebrew University Hospital, Jerusalem, Israel;_ 2 _Biokine Therapeutics, LTD, Rehovot, Israel;_ 3 _Chaim Sheba Medical Center and Tel Aviv University, Tel Hashomer, Israel;_ 4 _Laniado Hospital, Netanya, Israel;_ 5 _BioLineRX LTD, Tel Aviv, Israel_.

Background: CXCR4 is overexpressed in the majority of tumor cells and its degree of expression often correlates with disease severity. Binding of CXCL12 to the CXCR4 receptor activates signaling pathways which are crucial for the interaction of hematopoietic cells with the microenvironment and cell survival. Signaling activated through CXCR4 was shown to be detrimental by increasing survival of tumor cells and promoting resistance to therapy in many types of cancer. CXCR4-antagonists, such as BL-8040, currently in phase II trials, were shown to selectively inhibit tumor cell growth and to induce apoptosis in vitro and in vivo. However, the molecular mechanism by which CXCR4 overexpression triggers tumor cell survival and its inhibition leads to cell death is not fully understood.

Objective: To study the mechanism by which the CXCR4 pathway controls malignant cell survival and death through regulating miR-15a/16-1 expression.

Method: We assessed the effect of CXCR4 overexpression, its activation and inhibition, on the expression of miR-15a/16-1 and their target genes, BCL-2, MCL-1 and cyclin D1, in a variety of tumor cells in vitro and in vivo.

Results:

We found that overexpression of CXCR4 in tumor cells or stimulation of cells with its ligand, CXCL12, lead to up-regulation of miR-15a/16-1, resulting in down-regulation of their target genes BCL-2, MCL-1 and cyclin D1. Furthermore, overexpression of CXCR4 in these cells increases tumorgenesis and shifts their oncogenic dependency from the BCL-2 to the CXCR4/ERK signaling pathway. Antagonists of CXCR4 such as BL-8040 were shown to induce apoptotic cell death of malignant cells. BL-8040 was found to increase the expression of miR-15a/16-1 and reduce the expression of BCL2, MCL1 and cyclin D1. Importantly, CXCR4 inhibition using BL-8040 induced apoptosis in vitro and in vivo in AML and neuroblastoma tumors. This was mediated by inhibition of survival signals by ERK and down-regulation of BCL-2 expression. In support of these results overexpression of miR-15a/16-1 in AML and neuroblastoma cells was shown to induce their apoptosis.

Conclusions:

Our results demonstrate, for the first time to the best of our knowledge, that CXCR4 signaling regulates the expression of miR-15a/16-1 and their target genes. Our results suggest that overexpression of CXCR4 may override the survival dependency of tumor cells on BCL-2, MCL-1, and cyclin D1 leading to resistance of tumor cells to inhibition of these pathways. Furthermore, these results indicate that ligands of CXCR4 may tip the balance toward cell death by down- regulating survival signals through miR-15a/16-1 suppression of BCL2, MCL1 and cyclin D1 expression.

#3557

System-based BCL2 family protein signatures as predictive biomarkers in triple-negative breast cancer.

Federico Lucantoni,1 Andreas U. Lindner,1 Norma O'Donovan,2 Damir Vareslija,1 Lance Hudson,1 Arnold DK Hill,1 Michael Kerin,3 Roisin Dwyer,3 Leonie Young,1 Jochen HM Prehn1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _National Institute for Cellular Biotechnology, Dublin, Ireland;_ 3 _National University of Ireland Galway, Galway, Ireland_.

Breast cancer is a heterogeneous disease. More recently, the molecular basis for this heterogeneity has started to be analysed at the genomic, transcriptomic and proteomic level, allowing for the development of more personalized therapies. Cancer cells showing resistance to therapy frequently have an intrinsic deficiency in their ability to initiate or execute apoptosis [1]. The BCL2 (B-cell lymphoma 2) family of proteins controls the process of Mitochondrial Outer Membrane Permeabilization (MOMP), which is required for the activation of the mitochondrial apoptosis pathway [2]. We developed a deterministic systems model of BCL2 protein interactions, DR_MOMP that calculates the sensitivity of cells to undergo mitochondrial apoptosis [3]. Here we applied DR_MOMP in the context of triple negative breast cancer to determine whether this systems model can be used as a prognostic biomarker or patient stratification tool.

To analyze the contribution of BCL2 proteins in modulating apoptosis in breast cancer we validated DR_MOMP in a panel of Triple Negative Breast Cancer (TNBC) cell lines. Using quantitative Western blotting we determined the absolute protein levels of pro-apoptotic BAX and BAK and anti-apoptotic BCL2, BCL(X)L and MCL1. We found a significant correlation between cell survival and BCL(X)L levels (ρ = 0.76) after Cisplatin/Paclitaxel treatment, but protein levels of BCL2, MCL-1, BAX and BAK did not correlate with cell survival. Using absolute protein levels as input for DR_MOMP, we found a strongly improved correlation between model predictions and cells' responses to Cisplatin (ρ = 0.93) and Paclitaxel treatments (ρ = 0.97). Next we performed synergy studies using the selective BCL2 antagonist ABT-199 or the selective BCL(X)L antagonist WEHI-539 in combination with cisplatin. We observed differential effects of BCL(X)L and BCL-2 inhibition in individual cell lines that were remodelled by DR_MOMP. Modelling also suggested additional effects of BCL2 antagonists independent of MOMP regulation. BCL2 profiling was also performed in primary tumours of 16 TBNC patients in order to predict the patients' risk prospectively. DR_MOMP predicted a high risk in 37.5% of patients which is in line with 5-year recurrence rates in the literature [4].

Our findings provide evidence that DR_MOMP can be deployed to predict the response of TNBC breast cancer to genotoxic therapy, and can aid in the choice of the optimal BCL2 antagonists.

This work is supported by Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT

[1] Pillai et al. Am J Cancer Res. 2013 Jun 20;3(3):312-22. PubMed PMID: 23841030.

[2] Lee et al. BMC Cancer. 2007 Apr 12;7:63. PubMed PMID: 17430582.

[3] Lindner et al. Cancer Res. 2013 Jan 15;73(2):519-28. PubMed PMID: 23329644

[4] Haffty et al. J Clin Oncol. 2006 Dec 20;24(36):5652-7. PubMed PMID: 17116942

#3558

Predicting response to Mcl-1 targeting agents in NSCLC and multiple myeloma.

Kristen McEachern, Greg O'Connor, Justin Cidado, Matthew Belmonte, Evan Barry, Hannah Dry, Paul Secrist, Lisa Drew. _AstraZeneca, Waltham, MA_.

Mcl-1 is an anti-apoptotic member of the Bcl-2 family of proteins and is frequently amplified or over-expressed in both solid tumors and hematological malignancies, suggesting that its activity may be important for the survival of cancer cells. CDK9 inhibition results in the down regulation of Mcl-1 mRNA and subsequent protein levels by inhibiting transcription and represents an indirect approach to targeting Mcl-1. Mcl-1 can also be targeted directly using an inhibitor that disrupts the Mcl-1 complexes to induce apoptosis.

Using both molecular and pharmacological approaches, we sought to identify predictive biomarkers of Mcl1 dependency in sensitive NSCLC and multiple myeloma cell lines. Here we demonstrate that NSCLC cell lines lacking MCL1 gene copy number gains are not sensitive to siRNA mediated knockdown of Mcl-1 or Mcl-1 inhibition (cell line sensitivity to CDK9 or Mcl-1 inhibition is defined by potency and extent of caspase activation). However, the presence of a copy number alteration does not predict sensitivity to Mcl-1 inhibition. To better understand what the drivers of sensitivity are, we developed quantitative assays on the Peggy platform (a capillary based immunoassay platform by Protein Simple) to measure both Mcl-1 and Bcl-xL protein levels. Using these assays, we show a correlation between sensitivity to a CDK9 or Mcl1 inhibitor and Mcl-1 levels, as well as to the ratio of Mcl-1 to Bcl-xL protein in a NSCLC cell line panel.

These findings were then extended into a panel of multiple myeloma cell lines. While somewhat broad activity for CDK9 or Mcl-1 inhibition is seen across the cell lines tested, a subset of the sensitive lines have MCL1 amplification and express high levels of Mcl-1 protein. Mcl-1 levels alone, however, do not predict for sensitivity across the panel and, similar to NSCLC, the ratio of Mcl1 to Bcl-xL expression has greater positive predictive value.

These results provide the rationale for exploring Mcl-1 copy number alterations and Mcl-1 and Bcl-xL protein levels as predictive biomarkers for tumor response when treating with a CDK9 or Mcl-1 inhibitor in both NSCLC and multiple myeloma.

#3559

The rare BCL-2 isoform BCL-2β is associated with melanoma survival and the apoptotic response to UV and cisplatin.

Chloe Warren,1 Ricardo E. Vilain,2 Katie A. Ashton,1 Juhura G. Almazi,1 Stephen Braye,2 Pablo Moscato,1 Nikola A. Bowden3. 1 _Univ. of Newcastle, Newcastle, Australia;_ 2 _Hunter Area Pathology Service, Pathology North, New Lambton Heights, Australia;_ 3 _Univ. of Newcastle, New Lambton Heights, Australia_.

There are two isoforms of the anti-apoptotic protein BCL-2. BCL-2α (wild-type) is well characterised and is known predominantly for its role in apoptosis, BCL-2β lacks the C-terminal transmembrane (TM) domain and its function has not yet been characterised. The aim of this study was to quantify and determine the biological role of the BCL-2β isoform in melanoma.

BCL-2α and BCL-2β isoforms were quantified at the mRNA and protein level in a cohort of 141 FFPE melanoma tumours using qPCR and multiple reaction monitoring (MRM) tandem mass spectrometry and compared to clinical parameters including age at diagnosis, gender, solar elastosis, BRAF/NRAS mutation, Breslow thickness, disease free survival and overall survival. The role of the BCL-2α and BCL-2β isoforms in apoptosis were investigated by quantifying transcript expression and apoptosis before and up to 72 hours after UVB irradiation (650J/m2) and cisplatin treatment (10ug/mL). siRNA knockdown targeted to each individual isoform transcript was used to verify the apoptotic response.

Expression of the BCL-2β isoform in tumours was significantly associated with increased overall survival (686.4 weeks, 95% CI 462.5-910.3). BCL-2α response to UVB and cisplatin (i.e. downregulation prior to apoptosis) was the same in melanocyte and melanoma cell lines. However, BCL-2β response was varied across the melanoma cell lines, but was similar to that of BCL-2α in melanocytes. siRNA knock-down of BCL-2α resulted in increased apoptosis and cell death in melanoma cell lines, but knock-down of BCL-2β delayed onset of apoptosis, suggesting the BCL-2β is pro-apoptotic.

Our current understanding of the role of BCL-2β is based on the concept that it lack the C-terminal transmembrane domain, and is incapable of localising to target organelles. It is currently thought that the isoform is of null function. However, these observations are based on studies of non-representative synthetic versions of BCL-2β. This is the first time the naturally transcribed version of the rare isoform has been studied. We have demonstrated herein that BCL-2β performs an apoptotic role, and that its regulation in melanoma may be a biomarker of better overall survival.

#3560

Bcl-Xl over-expression is a poor prognostic marker in papillary thyroid cancer and can be therapeutically targeted to induce apoptosis and autophagy.

Azhar R. Hussain, Maqbool Ahmed, Shaham Beg, Rong Bu, Roxanne Melosantos, Zeeshan Qadri, Saif Al-Sobhi, Fouad Al-Dayel, Abdul Khalid Siraj, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia_.

Bcl-Xl is a member of the Bcl-2 family of proteins that are divided into either pro-apoptotic proteins or anti-apoptotic proteins on the basis of their functionality. All the members of the Bcl-2 family share common domains known as Bcl-2 Homology (BH1-4) domain. The pro-apoptotic family members include proteins that only contain the BH-3 domain and include Bax, Bak, Bid, PUMA, NOXA, Bim and Bad while the anti-apoptotic proteins include members such as Bcl-2, Bcl-Xl and Mcl-1. In normal conditions, there is a balance between the pro- and anti-apoptotic members of the Bcl-2 family. Because of their important role in cell survival, there has been lot of interest in utilizing dysregulation of Bcl-2 family members to counter cancer growth and induce apoptosis. Bcl-Xl is an anti-apoptotic protein that has found to be over-expressed in various cancers including lung cancer, DLBCL and breast cancer. However, the role of Bcl-Xl in papillary thyroid cancer (PTC) from Middle Eastern region has not been fully illustrated. In order to investigate the role of Bcl-Xl in PTC, we examined the expression of Bcl-Xl in a cohort of 1022 PTC clinical cases by immunohistochemistry in a tissue microarray format and found that Bcl-Xl was over-expressed in 51.7% of PTC cases. Bcl-Xl over-expression was significantly associated with aggressive high proliferative markers such as older age (p=0.0009), extra-thyroidal extension (p<0.0001), tumor size (p=0.0081), nodal involvement (p=0.0067), metastasis (p=0.0013) and showed a poor overall 5 year survival (0.0438). Bcl-Xl over-expression was was also found to be significantly associated with p-AKT (p<0.0001), XIAP (p<0.0001) and proliferative marker Ki67 (p=0.0041). In vitro, targeting Bcl-Xl expression in PTC cell lines using a small molecular inhibitor of Bcl-Xl; AB141657 (Z36) showed a dose dependent inhibition of cell viability and induction of apoptosis after 48 hours treatment. Using 5 and 10μM Z36, we found that PTC cells not only underwent apoptosis detected by flow cytometry, inactivation of AKT and activation of mitochondrial apoptotic pathway but also autophagy as detected by up-regulation of LC1-3, inactivation of p-mTOR1 and p-mTOR2 and their downstream targets. These data clearly indicate a role of Bcl-Xl in the pathogenesis of PTC as well as the importance of targeting Bcl-Xl using Z36 to induce both; apoptosis and autophagy in Bcl-Xl over-expressing PTC cells.

#3561

Role of bcl-2 family proteins in phorbol ester-induced apoptosis of acute myeloid leukemia cells.

Cassandra Holbert, Jeffrey Forrester, Michael Roberts. _Dickinson College, Carlisle, PA_.

Human acute myeloid cell lines undergo genetic re-programming in response to differentiation inducers. The M3 AML cell line, HL-60, has served as a model system for studying phorbol ester-induced myeloid differentiation into macrophage-like cells. Macrophage differentiation is preceded by cell cycle arrest and followed by apoptosis. Therefore, the system elucidates the changes in the leukemia cell gene expression program necessary to override the genetic alterations driving continuous cell division, preventing myeloid differentiation, and blocking programmed cell death. We hypothesize that defining the evolving transcriptome as AML cells reverse their proliferative, undifferentiated, and pro-survival phenotype will reveal genes regulating these processes and possibly identify new targets for AML treatment. Whole genome exon DNA microarray analyses identifies 1260 genes whose RNA expression levels change significantly during the reprogramming. Among these genes are members of the BCL-2 family that encode both pro- and anti-apoptotic proteins (BBC3, PMAIP1, BID and BCL2, MCL1, BCL2A1, respectively). Myeloid cell leukemia 1 (MCL1) is a BCL-2 family protein defined as an anti-apoptotic protein, however MCL1 increases in expression following phorbol ester treatment in HL-60 cells, as well as other AML lines including THP-1, Kasumi-1, and KG-1. The MCL1 gene can be alternatively spliced to generate a transcript encoding a BH3-only variant (MCL1S) known to exhibit pro-apoptotic function. Western blotting demonstrates the accumulation of MCL1S protein prior to the initiation of apoptosis in phorbol ester treated cells. Here we present evidence for splice variant switching and explore the possibility that reduction in the pro-survival form, MCL1L, and accumulation of MCL1S mediates the p53-independent initiation of apoptosis. Currently, we are evaluating the effects on the leukemic phenotype and apoptotic response to phorbol ester in HL-60 cell lines overexpressing MCL1L, MCL1S, and PMAIP1, as well as HL-60 lines containing CRISPR-Cas9 mediated knockouts of MCL1 and PMAIP1.

#3562

BCR/ABL increases EZH2 levels which regulates XIAP expression via miRNA-219 in chronic myeloid leukemia cells.

Takayuki Ikezoe,1 Chie Nishioka,1 Jing Yang,2 Akihito Yokoyama1. 1 _Kochi Medical School, Nankoku, Japan;_ 2 _Xuzhou Medical College, Xuzhou, China_.

In this study, we showed that the levels of enhancer of zeste homolog 2 (EZH2) in bone marrow mononuclear cells (BMMNCs) isolated from individuals with chronic myeloid leukemia (CML) (n = 12) were greater than those in BMMNCs isolated from healthy volunteers (n = 6). Lentiviral transduction of the BCR/ABL gene in Ba/F3 cells increased EZH2 levels in parallel with phosphorylation of STAT5. Notably, chromatin immunoprecipitation assays showed that STAT5A bound to a promoter region of the EZH2 gene, resulting in an increase in the transcriptional activity of EZH2 in leukemia cells. Importantly, downregulation of EZH2 by short hairpin RNAs (shRNAs) inhibited the expression of the antiapoptotic protein XIAP and increased the miR-219 levels associated with a decrease in hypermethylation of miR-219-1 CpG islands. Moreover, overexpression of miR-219 decreased the levels of XIAP in CML cells. Since the 3′-untranslated region (3′-UTR) of XIAP contains miR219-5p-complementary binding site, miR-219 might modulate the expression of XIAP through binding of miR-219 on the 3′-UTR of XIAP.

Taken together, BCR/ABL positively regulates the expression of EZH2 via STAT5 signaling. EZH2 modulates epigenetic changes at DNA methylated regions encoding miR-219 and downregulates the level of miR-219, resulting in upregulation of XIAP.

#3563

Ras-mediated regulation of cytochrome c-induced caspase activation is dependent on the status of extracellular matrix attachment.

Chelsea M. McCallister, Raju Rayavarapu, Nicholas Pagani, Brendan Heiden, Melissa Shaw, Sydeny Shuff, Zachary T. Schafer. _University of Notre Dame, Notre Dame, IN_.

Hyperactivating mutations in Ras are found in a significant percentage of cancers, with a particularly high frequency in pancreatic and colon carcinomas. The hyperactivation of Ras drives a vast number of distinct downstream signaling pathways that play an important role in disease progression. One outcome of overactive oncogenic Ras signaling is the inhibition of anoikis, a form of apoptotic cell death caused by detachment from the extracellular matrix (ECM). It is well-established that one mechanism of anoikis inhibition by Ras involves impaired mitochondrial cytochrome c release. In addition, here, we have found that Ras-mediated anoikis inhibition can also occur downstream of mitochondrial cytochrome c release. Our data suggest that anoikis inhibition following cytochrome c release is not dependent on changes in the abundance of the Apaf-1, pro-caspase-9, or pro-caspase-3. Instead, our data suggest that Ras signaling leads to an inhibition in the ability of caspase-9 to bind Apaf-1 which thereby inhibits proper formation of the apoptosome and caspase activation. Interestingly, in stark contrast to the inhibition of cytochrome c-induced apoptosis in ECM-detached cells, the overexpression of oncogenic Ras in ECM-attached cells results in enhanced sensitivity to exogenous cytochrome c. This sensitization was found to be due to upregulation of apoptosomal proteins (e.g. Apaf-1, pro-caspase-9) in an ERK/MAPK dependent manner. In aggregate, our data suggest that in addition to inhibiting the release of cytochrome c in both attachment and detachment, oncogenic Ras drives additional mechanisms that prevent apoptosome formation and caspase activation in detachment. Furthermore, our data support a model whereby ECM-attached cells containing oncogenic Ras mutations could be selectively eliminated by cytochrome c or agents that mimic its action.

#3564

MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target.

Yong H. Sheng,1 Yaowu He,1 sumaira Z. hasnain,1 Ran Wang,1 Hui Tong,1 Daniel T. Clarke,2 Rohan Lourie,1 Iulia Oancea,1 kuanyau wong,1 John W. Lumley,3 Timothy H. Florin,1 Philip Sutton,4 John. D. Hooper,1 Nigel A. Mcmillan,2 Michael A. Mcguckin1. 1 _Mater Research Institute-The University of Queensland, Brisbance, Australia;_ 2 _Griffith University, Gold Coast, Australia;_ 3 _Wesley Hospital, Brisbance, Australia;_ 4 _Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Australia_.

MUC13 is a transmembrane mucin glycoprotein that is overexpressed by many cancers, although its functions are not fully understood. NF-κB is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently up-regulating BCL-XL. MUC13 promoted TNF-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NEMO. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signalling in response to both TNF and DNA damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to death in response to cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

#3565

Dinaciclib, a CDK inhibitor promotes proteasomal degradation of Mcl-1 and enhances ABT-737 mediated cell death in malignant human glioma cell lines.

Esther P. Jane, Daniel Premkumar, Jonathon Cavaleri, Thatchana Rajasekar, Philip Sutera, Ian Pollack. _University of Pittsburgh, Pittsburgh, PA_.

The prognosis of malignant glioma, the most common brain tumor, is still poor, thus underscoring the need to develop novel treatment strategies. Because glioma cells commonly exhibit genomic alterations involving genes that regulate cell cycle control, there is a strong rationale to examine the potential efficacy of strategies to counteract this process. In this study, we examined the antiproliferative effects of the cyclin-dependent kinase inhibitor dinaciclib in malignant human glioma cell lines, with intact or deleted or mutated p53 or PTEN or p14ARF, or EGFR amplification status. Dinaciclib was found to inhibit cell proliferation and induce cell cycle arrest at the G2/M checkpoint, independent of p53 mutational status. In a standard 72 h MTS assay, at clinically relevant concentrations, dose-dependent antiproliferative effects were observed but cell death was not induced. Moreover, the combination of conventional chemotherapeutic agents and various growth signaling inhibitors with dinaciclib did not yield synergistic cytotoxicity. In contrast, combination of the Bcl-2/Bcl-xL inhibitor ABT-263 or ABT-737 with dinaciclib potentiated the apoptotic response induced by each single drug. The synergistic killing by ABT-737 with dinaciclib led to cell death accompanied by the hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c, smac/DIABLO and apoptosis-inducing factor (AIF), phosphatidylserine exposure on the plasma membrane surface and activation of caspases and PARP. Mechanistic studies revealed that dinaciclib promoted proteasomal degradation of Mcl-1.

#3566

Glycosylation of the TNFR1 death receptor controls cell fate.

Andrew Holdbrooks, Matthew J. Schultz, Zhongyu Liu, Daniel Bullard, Susan L. Bellis. _University of Alabama at Birmingham, Birmingham, AL_.

Activation of the TNFR1 death receptor by TNFα can induce cell survival or cell death signaling, however, the mechanisms regulating this TNFR1 signaling switch are poorly understood. TNFα-induced apoptosis is reported to require higher-order clustering and internalization of activated TNFR1, whereas surface retention of the activated receptor has been shown to promote survival signaling mediated by NFκB and MAPKs. Studies from our group have identified a novel glycosylation-dependent mechanism that controls this signaling switch. Specifically, we have found that TNFR1 localization and activity are regulated by the addition of a distinct sugar, an α2-6 linked sialic acid, by the enzyme ST6Gal-I, a Golgi sialyltransferase whose expression is upregulated in many cancer types. The sialylation effect on TNFR1 was examined in epithelial and monocytic cancer cell lines with forced overexpression or knockdown of ST6Gal-I, as well as in primary monocytes obtained from mice with ST6Gal-I knock-in or knockout. Data from these models indicate that α2-6 sialylation of TNFR1 blocks TNFα-induced apoptosis, and the suggested mechanism underlying this inhibition of apoptosis is the sialylation-driven inhibition of TNFR1 oligomerization and internalization, as observed through immunoblotting, immunocytochemistry and flow cytometry. Considering sialylated TNFR1 is retained on the plasma membrane following activation, we hypothesize that ST6Gal-I functions to not only block TNFR1-mediated cell death but also divert the cell's activity to favor survival. Supporting this hypothesis, cells with elevated levels of ST6Gal-I exhibit heightened expression and activation of many pro-survival factors, such as NFκB, MAPKs and AKT, in addition to decreased activation of caspase-3. Since levels of ST6Gal-I are elevated in multiple tumor cell populations, our group proposes that ST6Gal-I protects tumor cells against TNFα-induced apoptosis within the inflammatory tumor microenvironment. As preliminary support for this concept, peritoneal ascites fluid was collected from ovarian cancer (OC) patients, and the acellular, cytokine-rich fraction was incubated with OC cell lines with or without forced ST6Gal-I expression. Expression of ST6Gal-I conferred strong protection against cell death induced by soluble ascites. Based on these collective findings, we postulate that sialylation of TNFR1 by ST6Gal-I diverts the cellular response to TNFα from apoptosis to survival, providing a mechanism by which tumor cells can evade immune cell killing.

#3567

Metformin suppresses synthesis of pro-survival sphingolipid, sphingosine-1-phosphate, by inhibition of sphingosine kinase-1, in MCF-7 and SK-BR-3 breast cancer cell lines.

Ashley Rose, Daniel Wann, Janet Lightner, Marianne Brannon, William Stone, Victoria Palau, Koyamangalath Krishnan. _ETSU Quillen College of Medicine, Johnson City, TN_.

The antidiabetic drug, Metformin, may possess anti-cancer properties. Metformin has been shown to suppress proliferation of breast cancer cells primarily through activation of AMP-activated protein kinase (AMPK) and its suppression of downstream signaling pathways, such as mTOR, involved in cell replication. Other mechanisms may also play a role. Sphingolipids have a role in apoptosis and survival. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, promotes cell survival, proliferation, migration, angiogenesis, lymph angiogenesis, and immune response. S1P is involved in both intracellular and extracellular functions and regulates proliferation and survival. Blocking S1P synthesis inhibits cellular proliferation. Sphingosine kinase (SphK) is a lipid kinase that catalyzes formation of S1P from the precursor sphingosine. SphK is known to be upregulated in cancer cells, promoting tumor progression. S1P has a critical role in cancer progression and is considered a viable target for cancer therapeutics.

Our previous studies show that metformin has an effect on the synthesis of pro-apoptotic ceramides. We hypothesized that metformin induces cytotoxicity by reducing levels of the pro-survival sphingolipid, S1P. Firstly, MCF-7 and SK-BR-3 breast cancer cell lines were treated with increasing concentrations of metformin, and cytotoxicity was determined by MTT cell culture experiments after 24 hours of drug exposure. Metformin induces cytotoxicity in these breast cancer cells at a lowest concentration of 2.5mM, and percentage cytotoxicity increases in a dose-dependent manner. We utilized liquid chromatography and mass spectrometry and determined that cellular S1P levels are decreased in MCF-7 cells treated with 2.5mM metformin when compared with the control group. Finally, we treated MCF-7 and SK-BR-3 breast cancer cells with metformin, SK I/II (a known SphK inhibitor), and an untreated control group for 2, 4 and 6 hours. The dose of metformin was 10mM, which was chosen from a dose-response curve using MTT assay. The dose of SK I/II was 20uM, chosen based on the IC50 given. All treatments were done using low glucose media. Using the lysates from the harvested cells, gel electrophoresis and western blots using antibodies to SphK and S1P were run. Our results showed that metformin decreased the cellular levels of SphK and S1P. Thus, metformin exhibits anticancer properties via inhibiting the production of pro-survival lipid S1P. This data suggests that the pro-apoptotic effect of metformin may be partly mediated through its disruption of synthesis of S1P in breast cancer cells. Further work is necessary to characterize the sphingolipid content of MCF-7 and SK-BR-3 cancer cells before and after metformin treatment.

#3568

Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells.

Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. _East Tennessee State University, Johnson City, TN_.

Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident.

MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 µM), simvastatin (0-5 µM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 µM, simvastatin at a concentrations of 20, 10, 5 µM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA).

Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma.

#3570

Differential expression of the melanoma inhibitor of apoptosis protein (ML-IAP)/Livin in patients with ulcerated and non-ulcerated melanomas.

Nitin Chakravarti, Dawen Sui, Denái R. Milton, Wen-Jen Hwu, Elizabeth A. Grimm, Victor G. Prieto. _UT MD Anderson Cancer Center, Houston, TX_.

Background: Cutaneous malignant melanoma is one of the most aggressive forms of skin cancer with an extremely poor prognosis for the patients diagnosed with metastatic disease. Ulceration has been correlated with an increased risk of death within a given thickness range, however, the biologic basis for the development of ulceration is poorly understood. Livin, an inhibitor of apoptosis protein (IAP), is overexpressed in various cancers and possesses both the ability to protect from cell death and to promote it once it is cleaved. It is unknown whether there is a differential cellular expression of livin in ulcerated versus non-ulcerated melanomas. Methods: To explore the role of livin in relation to ulceration, other clinicopathological factors and survival of melanoma patients, we have performed semiquantitative immunohistochemical analysis of livin expression as measured by percentage and intensity (levels) of cells immunoreactive for livin in tumor specimens having both ulcerated and non-ulcerated areas from 50 melanoma lesions. Results: Superficial as well as deep melanoma cells located in non-ulcerated areas of tumor had higher percentage of cytoplasmic immunopositivity for livin when compared with melanocytes in the ulcerated areas of the melanoma (p<0.0001). We also observed nuclear expression of livin protein in this subset of melanoma patients. Compared to the ulcerated areas, the superficially located melanoma cells from the non-ulcerated areas showed significantly higher intensity of livin immunoreactivity in their nuclei (p=0.048) but not those from the deep non-ulcerated areas (p=0.32). Ulcerated melanomas with low tumor stage (I/II) had higher percentage of tumor cells with nuclear livin expression than those from higher tumor stage (III/IV) (p=0.002). Interestingly, we observed that patients with higher intensity of cytosolic expression of livin protein in the ulcerated area of tumor were more likely to have a brisk tumor-infiltrating lymphocytes (TILs) response (p=0.001). Patients with higher intensity of cytoplasmic livin protein of tumor cells in ulcerated areas had higher risk of death compared to those with lower levels (p=0.005). On the other hand, patients with higher intensity of cytoplasmic expression of livin in melanoma cells located superficially in the non-ulcerated areas of tumor had better prognosis (p=0.045). Conclusion: Our data indicate that there is a decrease of nuclear as well as cytoplasmic expression of livin protein in melanoma cells from ulcerated when compared to non-ulcerated areas. Also, cytoplasmic livin expression is an indicator of poor prognosis, but apparently only in patients with ulcerated lesions. Although IAPs regulate caspases, they also regulate signaling pathways that activate NF-κB modulating immunity. The prominent TILs response in livin-positive tumors suggests a possible involvement in tumor immunity in melanoma.

#3571

Targeted suppression of inhibitor of apoptosis proteins amplifies apoptosis and improves response to enzalutamide in prostate cancer.

Amanda Pilling, Ok Hwang, Clara Hwang. _Henry Ford Health System, Detroit, MI_.

Prostate cancer is the most common malignancy and the 2nd leading cause of cancer death in men worldwide. First-line treatment includes androgen deprivation therapy in addition to androgen receptor (AR) antagonists that work to block androgen signaling. Although most patients respond initially to hormone deprivation therapy, responses are not durable and the disease progresses to a lethal stage, termed castrate resistant prostate cancer (CRPC). Interestingly, most CRPCs are still dependent on AR signaling for growth and survival, therefore more potent AR-antagonists such as enzalutamide have recently been FDA approved for treatment of CRPC. However, response rates to enzalutamide can be limited by emergence of acquired resistance or intrinsic resistance. A universal mechanism of resistance to cytotoxic therapies is upregulation of anti-apoptotic programs that function to override death signals and permit survival of the tumor cell. In prostate cancer, overexpression of the Inhibitor of Apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP has been demonstrated in progression to CRPC. Additionally, aberrant expression of these proteins is reported to contribute to anti-androgen resistance. Therefore, to achieve more robust responses to AR inhibition with enzalutamide, we targeted IAP proteins in order to amplify the apoptotic signal and increase cell death in prostate cancer cells. Using a small-molecule IAP inhibitor, we evaluated response to enzalutamide upon IAP inhibition in prostate cancer cell lines LNCAP and C4-2. Here we demonstrate greater proliferation inhibition, reduced cell survival and synergistic induction of apoptosis signaling in response to combined AR antagonism and IAP inhibition. Furthermore, we demonstrate depletion of cIAP1 protein expression upon enzalutamide treatment indicating a possible role for AR in regulating anti-apoptotic pathways. Taken together these results demonstrate that IAP proteins may be a critical survival pathway in CRPC and antagonism of these proteins through small-molecule inhibition can amplify apoptosis and increase cell death in response to enzalutamide. Targeting a critical survival pathway can lower the apoptotic threshold providing a possible therapeutic opportunity to treat and prevent resistance to enzalutamide and increase benefit to patients with this lethal form of prostate cancer.

#3572

AZ5576, a novel potent and selective CDK9 inhibitor, induces rapid cell death and achieves efficacy in multiple preclinical hematological models.

Justin Cidado. _AstraZeneca, Waltham, MA_.

Cyclin-dependent kinase 9 (Cdk9) is a serine/threonine kinase that regulates elongation of transcription through phosphorylation of RNA polymerase II at serine 2

(pSer2-RNAPII). Mcl1, an anti-apoptotic protein that has been linked to increased survival and chemotherapy resistance in various cancers, can be indirectly modulated through transient inhibition of Cdk9 due to it having a short-lived transcript and being a labile protein. Transient inhibition of Cdk9, therefore, represents a potential therapeutic opportunity in tumors dependent on Mcl1 for survival, including various hematological malignancies. AZ5576 is a potent, highly selective, and orally bioavailable inhibitor of Cdk9 that inhibits Cdk9 enzyme activity with an IC50 <5nM and decreases phosphorlyation of Ser2-RNAPII in cells with an IC50 of 96nM. In the MLL-fusion containing acute myeloid leukemia (AML) line, MV411, short-term treatment with AZ5576 led to a rapid dose-dependent decrease in pSer2-RNAPII with concomitant loss of Mcl1 mRNA and protein, resulting in the cleavage and activation of caspase 3 and loss of cell viability. However, protein expression of other Bcl2 family members (e.g. Bcl2, BclxL, Bim) remained unchanged. Further, AZ5576 induced rapid caspase activation and loss of viability in a diverse set of hematological cancer cell lines, including those from acute myeloid leukemia (16/20), multiple myeloma (10/19), and diffuse large B-cell lymphoma (8/13). In vivo efficacy with intermittent dosing of AZ5576 was also demonstrated in multiple xenograft models from each hematological tumor type. Finally, AZ5576 was still able to induce loss of viability in various multiple myeloma lines cultured in the presence of cytokines or bone marrow stromal cells that represent potential protective effects of the tumor microenvironment. Together, these results highlight the therapeutic potential for selective CDK9 inhibition in the treatment of hematological malignancies.

#3573

TWIST1/E2A signaling axis suppresses apoptosis in oncogene driven non-small cell lung cancer.

Zachary A. Yochum, Susheel Khetarpal, Timothy F. Burns. _University of Pittsburgh Cancer Institute, Department of Medicine, Division of Hematology-Oncology, Pittsburgh, PA_.

Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. In addition, acquired resistance to currently available targeted therapies is inevitable. We previously demonstrated that inhibition of the basic helix-loop-helix transcription factor, TWIST1 in KRAS mutant, EGFR mutant, and MET amplified/mutant NSCLC can induce apoptosis, which suggests that a subset of oncogene dependent NSCLC are potentially "addicted" to TWIST1. Importantly, we have identified the harmala alkaloid, harmine, as a novel TWIST1 inhibitor which could inhibit growth in several oncogene driver defined NSCLC cell lines and decrease levels of TWIST1 and its dimerization partners, the E2A proteins, via degradation.

We examined the target genes and pathways required for suppression of apoptosis by TWIST1 and E2A. Genetic or pharmacological (harmine) inhibition of TWIST1 or E2A resulted in apoptosis in several oncogenic driver dependent cell lines. Additionally, treatment with a pan-caspase inhibitor resulted in rescue of growth inhibition following TWIST1 or E2A silencing or harmine treatment. This suggests that apoptosis is the mechanism of growth inhibition following TWIST1 inhibition. TWIST1 or E2A inhibition resulted in increased levels of Bid, Bim, and DR5, as well as, reduced c-FLIP and Bcl-2 levels. Conversely, we demonstrated that TWIST1 overexpression leads to increased levels of c-FLIP and anti-apoptotic Bcl-2 family members as well as decreased levels of Bim and Bid. c-FLIP appears to be a direct transcriptional target of TWIST1 as TWIST1 overexpression leads to transactivation of the c-FLIP promoter and is dependent on the ability of TWIST1 to bind DNA. Interestingly, the TWIST1-E2A heterodimer results in greater promoter transactivation when compared to the TWIST1 homodimer. Furthermore, knockdown of Bim, overexpression of Bcl-2, or overexpression of c-FLIPs resulted in partial rescue of growth inhibition and apoptosis following TWIST1 silencing. However, only knockdown of Bim or Bcl-2 overexpression was able to rescue apoptosis following harmine treatment. This suggests that apoptosis following harmine treatment only requires the intrinsic machinery, while specifically silencing TWIST1 also engages the extrinsic pathway.

In summary, we found that the apoptosis observed after TWIST1/E2A inhibition is dependent on the intrinsic and extrinsic pathways possibly mediated through its novel target genes, c-FLIP and Bim. Our studies will establish the target genes of TWIST1 that are required for suppression of apoptosis with the ultimate goal of identifying biomarkers of response to TWIST1 inhibitors. We also aim to determine if TWIST1, through its apoptotic target genes, modulates response to targeted therapies or standard chemotherapies.

#3574

Nicotine prevents chemotherapy-induced peripheral neuropathy in vivo, and fails to stimulate the growth of lung cancer cells or interfere with the effectiveness of chemotherapy in vitro.

Sarah L. Kyte, Wisam Toma, M I. Damaj, Xianjun Fang, David A. Gewirtz. _Virginia Commonwealth University, Richmond, VA_.

Chemotherapy has played a significant and unparalleled role in the treatment and survival of cancer patients. However, this pharmacological approach can lead to long-term symptoms of drug toxicity, including chemotherapy-induced peripheral neuropathy (CIPN), a result of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, tingling, cold sensitivity, or allodynia, and an overall decrease in quality of life. Paclitaxel (Taxol), a taxane that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59 to 78% of patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can present either during or after chemotherapy administration. Without the development of an efficacious treatment, CIPN can be a dose-limiting factor for chemotherapy or delay treatment, thereby influencing survival and quality of life. Our studies have revealed that the prototypical nicotinic acetylcholine receptor agonist, nicotine, is capable of reversing and preventing the development of paclitaxel-induced CIPN in vivo, and does not interfere with the cytotoxic properties of paclitaxel in vitro. Male C57BL/6J mice were infused with 24 mg/kg of nicotine via subcutaneous 7-day osmotic minipumps starting 48 hours prior to paclitaxel treatment, which consisted of 8 mg/kg intraperitoneal injections every other day for a total of four injections. The use of von Frey filament testing revealed that nicotine dose-dependently reverses and prevents paclitaxel-induced mechanical allodynia. In regards to the in vitro studies, MTT and MTS colorimetric assays showed that concentrations of nicotine ranging from 0.1 to 10 µM fail to significantly increase or decrease the viability of A549, H460, Lewis lung carcinoma, or human explant lung cancer cells. Similar results were obtained in ovarian cancer cell lines, where no significant difference in cell growth was observed following nicotine treatment of SKOV-3/DDP and OVCAR-3 cells. Most importantly, the paclitaxel-induced decreases in both H460 cell viability and growth were not significantly attenuated by nicotine. Moreover, 1 µM nicotine did not interfere with paclitaxel-induced sub-G1 DNA content or apoptosis of A549 cells. These findings suggest that nicotinic acetylcholine receptors may be promising drug targets for the prevention and treatment of CIPN.

#3575

Ferroptosis: A novel nonapoptotic cell death overcomes head and neck cancer resistance to cisplatin.

Hye Jin Jang,1 Jin Young Park,1 Eun Hye Kim,1 Ji Won Kim,1 Minsu Kwon,2 Jong-Lyel Roh1. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Gyeongsang National University School of Medicine and Gyeongsang National University Hospital, Jinju, Republic of Korea_.

Ferroptosis was recently identified as a novel form of iron-dependent cell death different from apoptosis or necrosis. Inhibition of key molecules related to ferroptosis, cystine-glutamate antiporter (xCT) and glutathione peroxidase (GPX4), may induce the eradication of cancer cells resistant to conventional chemotherapy or radiotherapy. The present study investigated whether ferroptosis overcome head and neck cancer (HNC) resistant to cisplatin treatment. Three cisplatin-resistant HNC cell lines (AMC-HN3R, -HN4R and -HN9R), their parental lines, and other human HNC lines were used. The effects of cysteine and glutamate alteration and pharmacological and genetic inhibition of xCT were assess by measuring viability, death, total and cytoplasmic lipid reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), protein expression, and preclinical mouse tumor xenograft models. Conditioned media with no cysteine or high glutamate supplement induced ferroptosis of both cisplatin-sensitive and -resistant HNC cells without apparent change of necrosis and apoptosis markers, RIP1 and RIP3 and casplase-3 expression, and ΔΨm. This was blocked by antioxidants trolox or N-acetyl cysteine pretreatment. Sulfasalazine, artensunate, and erastin inhibited the growth of HNC cells and accumulated cytoplasmic lipid ROS via induction of ferroptosis and inhibition of xCT protein expression. Genetic silencing of xCT with siRNA or shRNA treatment also induced the effective cell death (ferroptosis) of resistant HNC cells. These effects were blocked with trolox or ferrostatin-1 pretreatment. Pharmacological and genetic inhibition of xCT significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. Cystine deficiency or glutamate excess induces ferroptosis in HNC cells. Further, pharmacological and genetic inhibition of xCT overcomes the cisplatin resistance of HNC cells by inducing ferroptosis. 

### Genetic Instability in Cancer: Molecular Basis and Tools

#3576

Broad analysis of recurrent somatic mutations in cancer reveals a common novel non-coding mutation in the promoter of PMS2 associated with greatly increased tumor mutation load.

Zachary R. Chalmers,1 Franklin W. Huang,2 Laurie M. Gay,1 Siraj M. Ali,1 Roman Yelensky,1 Juliann Chmielecki,1 Jeffery S. Ross,1 Vincent A. Miller,1 Philip J. Stephens,1 Levi A. Garraway,2 Garrett M. Frampton1. 1 _Foundation Medicine Inc., Cambridge, MA;_ 2 _Dana–Farber Cancer Institute, Boston, MA_.

Purpose

Tumor mutation load is an emerging prognostic and diagnostic marker for many cancers. Sensitivity to immunotherapeutic agents, which stimulate an antitumor immune response by selectively inhibiting immunosuppressive cell surface ligands, is known to correlate with high mutation load in the tumor. Numerous somatic and germline defects can cause genomic instability, including alterations affecting the mismatch repair pathway, DNA polymerases, and cell cycle regulators. We describe here the discovery of previously unreported mutations in the promoter of the PMS2 gene that are associated with significantly increased tumor mutation load.

Methods

Comprehensive genomic profiling by hybridization capture of exonic regions from either 236 or 315 cancer-related genes and select introns from 19 genes commonly rearranged in cancer was used to characterize more than 60,000 clinical FFPE cancer specimens. At least 50 ng of extracted DNA was analyzed per sample and the constructed libraries were sequenced to high, uniform median coverage (>500x). Samples were assessed for base substitutions, short insertions and deletions, copy number alterations and gene fusions/rearrangements. Mutation load is assessed as the number of somatic coding point mutations per megabase of targeted territory.

Results

Mutation load from targeted cancer gene analysis recapitulates previous results evaluating whole exome and whole genome mutation load in tumors and cell lines. A novel mutation hotspot was identified in the promoter of PMS2, which codes for the PMS2 protein, a dimerization partner of MLH1 and integral to the DNA mismatch repair complex. Promoter mutations were found in 7.5% of melanoma specimens (n=101/1348) and 17% of skin squamous cell carcinomas (n=30/175). In both diseases, PMS2 promoter mutations are the most significant genomic correlate of high mutation load. In melanoma, PMS2 promoter mutant specimens have 4x the median mutation load of the general melanoma population. Skin squamous cell carcinomas show a 2.5-fold increase. Functional characterization is underway to support the hypothesis that these mutations lead to modified PMS2 transcriptional activity, which is known to cause hypermutation.

Conclusions

The growing corpus of cancer genome data continues to enable novel discoveries in cancer biology. Non-coding and regulatory mutations have not been the target of focused study, and our findings extend the small set of regulatory mutations thought to affect tumor development. Our discovery highlights the power of large-scale genomic analysis to uncover additional disease mechanisms, and furthers our understanding of how cells maintain genome integrity. With the documented association of mutation load and immunotherapy sensitivity, investigation into the response of PMS2 promoter mutant tumors is warranted.

#3577

Pan-cancer analysis of mutagenesis by APOBEC cytidine deaminases.

Dmitry A. Gordenin. _National Institute of Environmental Health Sciences, Durham, NC_.

The elucidation of mutagenic processes that shape cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic, and prevention strategies. We and others previously identified mutation clusters and genome-wide mutagenesis bearing the single-strand DNA (ssDNA)-specific APOBEC cytidine deaminase signature at 5′-tC-3′ motifs across many cancer types. Because of high abundance of APOBEC mutagenesis in several cancer types, we have developed a statistical approach capable of identifying cancer samples with APOBEC mutagenesis pattern based on WGS as well as on WES mutation data. This analysis, called Pattern of Mutagenesis by APOBEC Cytidine Deaminases (P-MACD), has been recently integrated into the Broad Institute GDAC Firehose and is currently available for Firehose standard and AWG-customized runs. The results of analysis using the P-MACD package will be discussed, including correlations with molecular and clinical features of TCGA cancer samples as well as relative roles of different APOBEC enzymes in cancer mutagenesis.

#3578

Novel insights into the cellular function and gene regulation of a master mutator, APOBEC3B.

Sudheer Kumar Gara, Yi Liu-Chittenden, Shweta Kotian, Dhaval Patel, Electron Kebebew. _National Cancer Institute, Bethesda, MD_.

Unequivocally, cumulative mutations are required for the development of cancer and many sources of mutagenic activity contribute to tumorigenesis. Recent evidence has implicated that a member of cytidine deaminase family, APOBEC3B (Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B or A3B) is enriched in the genomes of many human cancers such as cervical, bladder, lung, head and neck and breast cancers (Burns et al., Nature, 2013). APOBEC3B deaminate cytosines in the host genome to generate C-T mutations. Here we report for the first time that APOBEC3B is overexpressed with adrenocortical carcinoma (ACC), a very aggressive and lethal malignancy. We found that APOBEC3B is significantly overexpressed in primary and metastatic ACC as compared to normal adrenal and cortical adenomas by qPCR, immunofluorescence. We observed that γH2AX, a marker of DNA double-strand breaks co-localizes with the expression of APOBEC3B in patients with primary and metastatic ACC. Also ACC tumor samples with high APOBEC3B expression had high chromosomal gain/loss particularly in chromosome 4 and 8 compared to tumor samples with low APOBEC3B expression. To identify the cellular function of APOBEC3B, we performed transient knock-down studies in three ACC cell lines and found that it reduces cellular proliferation and induces cell cycle arrest. Furthermore, we observed that knockdown of APOBEC3B inhibits MAPK signaling by downregulating phospho ERK1/2 levels. Interestingly, we found that tumors with high APOBEC3B expression levels had twice as many TP53 inactivating mutations as compared to tumors with low levels of APOBEC3B. In order to understand the regulation of APOBEC3B expression, we analyzed the methylation status, copy number variation and potential targeting miRNAs of APOBEC3B from genome wide array platform but found no associations in human ACC samples. Therefore, we did a functional, knockdown screen of 90 cancer-associated transcription factors and found that GATA3 showed positive regulation of APOBEC3B. Our data provide novel insights into the function and regulation of APOBEC3B expression beyond its known mutagenic ability.

#3579

APOBEC3 activity sensitizes cells to ATR-Chk1 pathway inhibition.

Konstantin Budagyan, Matthew D. Weitzman, Abby M. Green. _The Children's Hospital of Philadelphia, Philadelphia, PA_.

There are six functional members of the APOBEC3 family of enzymes (A3A-A3C, A3F-A3H), which catalyze the conversion of cytosine to uracil in single-stranded DNA. APOBEC3 enzymes are best described for their ability to deaminate viral and LINE element DNA, thus restricting infection and retrotransposition. However, recent analyses of human tumor sequences reveal mutational signatures consistent with APOBEC3 activity. These signatures indicate the capacity for APOBEC3 enzymes to cause mutations and breaks in cellular DNA and further, their potential to contribute to cellular transformation or clonal evolution of cancer. We have previously shown that APOBEC3 enzymes are capable of causing cellular DNA damage and activation of the DNA damage checkpoint via Ataxia-Telangiectasia Mutated (ATM) kinase signaling. To investigate additional cellular mechanisms that respond to APOBEC3-induced DNA damage, we examined the impact of A3A expression on protein kinases involved in DNA replication checkpoint signaling. Here, we show that A3A expression can also cause cell cycle arrest through activation of the DNA replication checkpoint as indicated by ATM-Related (ATR) kinase phosphorylation of Checkpoint Kinase 1 (Chk1) on S345 and S317. Based on these results, we hypothesized that inhibition of DNA replication checkpoint kinases in tumors with upregulated APOBEC3 enzymes would lead to uncontrolled replication, accumulation of DNA damage, and ultimately cell death. To study the impact of ATR inhibition on cells in which A3A is active, we generated cell lines with inducible A3A expression. We introduced a doxycycline-inducible A3A gene into a human fibroblast cell line and treated the cells with a selective ATR inhibitor (VE822). Using a colony formation assay, we show that ATR inhibition restricts cell growth in the context of A3A expression. We also found that A3A expression led to decreased cell viability following treatment with an ATR inhibitor. We examined the mechanism of cell death and found that ATR inhibition in cells induced to express A3A resulted in increased Annexin V staining, indicative of apoptosis. To confirm that these findings represent effects specific to the DNA replication checkpoint mediated by ATR activation and phosphorylation of Chk1, we employed siRNA knockdown of Chk1. We found that inhibition of Chk1 in the context of A3A expression also resulted in reduced cell growth, decreased viability, and apoptosis compared to uninduced cells. These observations suggest that DNA replication checkpoint signaling is critical for protection of the cellular genome from A3A-induced DNA damage. Upregulation of APOBEC3 activity in tumor cells provides an opportunity for targeted therapy. Thus, the ATR-Chk1 pathway may represent a target for therapeutic intervention in tumors in which APOBEC3 enzymes are active.

#3580

Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements.

Matthew Gilbertson, Radhika Patel, Karin C. Nitiss, John L. Nitiss. _University of Illinois College of Pharmacy, Rockford, IL_.

Topoisomerase 2 (Top2) is the target of active anti-cancer agents such as etoposide and doxorubicin. These drugs interfere with the Top2 catalytic cycle and lead to trapping of the enzyme as a covalent protein: DNA complex. This trapped covalent complex is a unique DNA lesion that includes DNA strand breaks and covalent protein adducts at the site of the breaks. While Top2 mediated DNA damage is the major mechanism for tumor cell killing, it is also responsible for drug-induced translocations that can lead to secondary malignancies. We have developed several novel systems for assessing Top2-mediated genome instability using yeast as a model system. We developed repair proficient yeast strains that accumulate high levels of etoposide and other Top2 targeting drugs. We selected for loss-of-function mutations in the yeast CAN1 gene that arise following etoposide treatment, and examined the induced mutations by DNA sequencing. We identified a unique spectrum of mutations generated by etoposide in yeast. One novel class of mutational events induced by etoposide is relatively large deletions (>300 bp) and tandem duplications. Most of these etoposide-induced events are flanked by 4-8 nucleotide direct repeats. To further characterize the mechanism of Top2 induced genome instability, we took advantage of a novel Top2 allele isolated in our laboratory. This Top2 allele, termed top2AR, exhibits elevated levels of drug independent DNA cleavage in vitro, and inviability when combined with yeast mutants defective in DNA double strand break repair. We found a similar spectrum of large deletions and tandem duplications flanked by 4-8 nucleotide direct repeats. Interestingly, we also found that the recovery of large deletions and insertions was enhanced in yeast cells lacking Tdp1, a gene that participates in the repair of protein:DNA covalent complexes. Our results illustrate a novel type of genome rearrangement that is mediated by covalent Top2 DNA damage. It is noteworthy that the deletions and insertions flanked by short direct repeats have not been previously observed with other classes of DNA damaging agents. We suggest that the unique properties of these lesions may also contribute to oncogenic translocations induced by etoposide and other Top2 targeting drugs. We are currently using next-generation sequencing of etoposide treated yeast cells and cells carrying the top2AR allele to obtain a more complete picture of the types of alterations induced by Top2 mediated DNA damage.

#3581

How to deal with DNA damage during mitosis: a genetic interaction map of the PICH DNA translocase.

Inês Teles Alves, Anne-Magriet Heijink, Floris Foijer, Marcel van Vugt. _University Medical Center Groningen, Groningen, Netherlands_.

Genomic instability is one of the enabling characteristics of cancer, and is thought to be driven in large part by replication stress. It is generally believed that damaged DNA needs to be repaired before entry into mitosis. Recent evidence showed that DNA damage occasionally is present in mitosis, and can be repaired either through break-induced replication during early mitosis or during anaphase at so-called ultrafine DNA bridges (UFBs). These UBFs are characterized by the presence of the PICH DNA translocase, and Rif1, which we recently identified to be involved in the resolution of UFBs (Hengeveld et al, Dev Cell, 2015). However, detailed molecular composition of UFBs associated proteins and their mechanism of action are incompletely understood. Also, whether specific cancer types are dependent on this DNA repair pathway for their survival is not known. We hypothesise that cancer cells with high levels of replication stress may increasingly depend on these mechanisms for their survival. Here, we aimed to identify the genetic make-off of cancer cells that depend for their survival on repair of damage lesion during anaphase.

We used inactivation PICH or Rif1 as a model for defective resolution of anaphase bridges. To elucidate the consequences of loss of DNA repair at UFBs, we analysed the sensitivity to genotoxic agents and assessed genomic instability in HAP1 cells, in which the ERCC6L gene, which encodes the PICH DNA translocase, or the Rif1 gene were inactivated using Cas9/CRISPR-mediated mutation. ERCC6L as well as RIF1 knock-out cells showed increased levels of intrinsic DNA damage, as judged by elevated levels of H2AX phosphorylation and increased frequencies of 53BP1 bodies, known to represent unrepaired replication damage. ERCC6L as well as RIF1 knock-out cells also display increased sensitive to the topoisomerase II inhibitor ICRF193. Amplifications and loss of genomic areas was assessed using single strand sequencing of single cells. Subsequently, we wanted to determine which genetic mutations are not allowed or show competitive outgrowth in the absence of UFB resolution during anaphase. To this end, we exploited the haploid nature of the HAP1 cells, which allows insertional mutagenesis using retroviruses carrying a strong spice-acceptor to create random instantaneous knock-outs. The list of candidate genes of which depletion leads to a lethal phenotype specifically in cells lacking PICH is currently being analysed.

In conclusion, our studies have shown that the inactivation of PICH or RIF1 results in a marked increase in unresolved UFBs which ultimately lead to elevated numbers of 53BP1 bodies, and increased sensitivity to topoisomerase inhibitors. These data makes both RIF1 and PICH interesting therapeutic targets in tumours with high levels of replication stress, or targets to potentiate the effects of topoisomerases.

#3582

Chromothripsis in AML patients: A new mechanism of cancer initiation and progression.

Maria Chiara Fontana, Viviana Guadagnuolo, Cristina Papayannidis, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Marco Manfrini, Barbara Santacroce, Margherita Perricone, Silvia Lo Monaco, Emanuela Ottaviani, Simona Soverini, Michele Cavo, Giovanni Martinelli. _Inst. of Hematology 'Seragnoli', Bologna, Italy_.

Introduction: Genomic rearrangements can drive the development of cancer through different mechanisms: chromothripsis, a catastrophic mechanism of genomic instability, could be relevant for hematological disease.

Aim: To discover the mechanisms underlying the pathogenesis of Acute Myeloid Leukemia (AML), we studied chromothripsis in our cohort of patients (pts).

Methods: We perform SNP Array 6.0 or Cytoscan HD Array (Affymetrix) in a cohort of 104 AML pts at diagnosis. SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery).

Results: Seven/104 pts (6.7%) showed chromothripsis events involving different chromosomes (8, 17, 11, 5 and 16). These pts had median age of 68.5 years (range 56-76), complex karyotype and high risk disease according to ELN definition. Among the pts showing chromothripsis, we compared chromosomic abnormalities of pts with de novo (5/7) and secondary (2/7) AML. De novo AML showed a prevalence of trisomy of chromosome 8 in a non-statistical way (4/5 vs 0/2), due to the low number of cases. However, we identified significant differences in the pattern of genes altered in the two groups. De novo AML had copy number gain of PEX1, ANK1, NCOA2, ESRP1, TPD52, ESRP1, ZFPM2 and MITF (p <0.047). In the secondary AML group we detected amplification of APC and deletions of FOXO1 and APP (p <0.047)with enrichment of WNT signaling pathway proteins (p <0.001). The pathways recurrently targeted by chromosomal alteration in de novo AML pts were protein acetylation, transcription factor and histone acetiltrasferase (p <0.001). Moreover, by comparing pts with and without chromothripsis, we identified several genes differentially altered between the 2 groups (p<0.001), including deletion of CYFIP2, TBCA, CRHBP, FNDC9, IQGAP2, TUB3 and GAS8 and amplification of ZFPM2 in pts with chromothripsis, with 5q being the main altered chromosomic region. The most significantly affected pathways in the chromothripsis group were cytoskeleton, microtubules formation, histone acetyltransferase and immune and antigen presentation response pathways (p<0.001).

Conclusion: Our data suggest that different pathways and genomic alterations are involved in chromothripsis events in de novo and secondary AML, which could be explained by the repeated rounds of stress underwent by leukemic cells in secondary AML cases. Cytoskeleton and microtubules formation pathways appear to be the main cellular processes implicated in chromothripsis genesis, while alterations of histone acetyltransferase, immune response and antigen presentation pathways could sustain leukemic cells after chromothripsis.

Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project.

#3583

Thymocyte selection-associated HMG box protein (TOX) induces genomic instability in T-cell acute lymphoblastic leukemia.

Riadh Lobbardi,1 Jordan Pinder,2 Barbara Martinez-Pastor,3 Jessica Blackburn,1 Brian J. Abraham,4 Marc Mansour,5 Nouran S. Abdelfattah,1 Aleksey Molodtsov,1 Gabriela Alexe,6 Debra Toiber,7 Manon de Waard,8 Esha Jain,9 Deepak Bhere,10 Khalid Shah,10 Alejandro Gutierrez,11 Kimberly Stegmaier,6 Lewis B. Silverman,6 Ruslan Sadreyev,12 John Asara,13 A. Thomas Look,14 Richard A. Young,4 Raul Mostoslavsky,3 Graham Dellaire,15 David M. Langenau1. 1 _Massachusetts General Hospital, Charlestown, MA;_ 2 _Departments of Pathology and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada;_ 3 _Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA;_ 4 _Whitehead Institute for Biomedical Research, Cambridge, MA;_ 5 _Department of Haematology, UCL Cancer Institute, University College London, London, United Kingdom;_ 6 _Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA;_ 7 _Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel;_ 8 _University of Leiden, Leiden, Netherlands;_ 9 _Centre of Regenerative Medicine, Massachusetts General Hospital, Boston, MA;_ 10 _Molecular Neurotherapy and Imaging Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 11 _Division of Pediatric Hematology-Oncology, Boston Children's, Boston, MA;_ 12 _Department of Molecular Biology, Massachusetts General Hospital, Boston, MA;_ 13 _Division of Signal Transduction, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA;_ 14 _Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 15 _Departments of Pathology and Biochemistry and Molecular Biology; Dalhousie University, Halifax, Nova Scotia, Canada_.

MYC and NOTCH are major oncogenic drivers in T-cell Acute Lymphoblastic Leukemia (T-ALL), yet additional collaborating genetic lesions collaborate to induce frank malignancy. To identify these factors, a large-scale transgenic screen was completed where 28 amplified and over-expressed genes found in human T-ALL were assessed for accelerating leukemia onset in the zebrafish transgenic model. From this analysis, Thymocyte selection-associated HMG protein (TOX) synergized with both MYC and NOTCH to induce T-ALL. Here, we show that TOX is highly expressed in 95% of human primary and relapse T-ALL when compared with both normal T cells and B-ALL. TOX is highly and specifically expressed in human T-ALL due to both genomic amplification and transcriptional regulation by the master transcription factors MYB/LMO2. Characterization of zebrafish T-ALLs revealed that TOX promoted genomic instability as assessed by changes in DNA content and Whole Genome Sequencing. Effects on genomic instability were confirmed by metaphase spread following TOX expression in MEF cells, confirming roles for TOX in regulating genomic instability and elevated DNA translocation potential in a wider range of cell types. To identify TOX binding partners, Tandem Mass Spectrometry was performed in human T-ALL cells. TOX was found to interact with KU70/KU80 but not other DNA repair enzymes, a result verified by co-immunoprecipitation studies. Given that TOX elevated genomic instability in the zebrafish model, that Ku70 or Ku80 loss lead to genomic instability and T cell lymphoma in mice, and that TOX bound specifically to KU70/KU80 - the initiating factors required for Non-Homologous End Joining (NHEJ) repair - we hypothesized that TOX is a negative regulator of double-strand break repair. Fluorescent repair assays were completed in 3T3 fibroblasts and confirmed that TOX inhibits NHEJ. Dynamic real-time imaging studies showed that TOX suppresses recruitment of fluorescent-tagged KU80 to DNA breaks. Importantly, TOX loss of function increased NHEJ in human T-ALL cells and reduced time to DNA repair as assessed by fluorescent Traffic Light Reporter assays and quantitative assessment of 53BP1 and γH2A.X foci resolution following irradiation. Our results show that TOX is aberrantly re-activated in 95% of human T-ALL, thereby suppressing KU70/KU80 function to promote genomic instability and ultimately elevating rates at which acquired mutations and rearrangements are amassed in developing pre-malignant T cells. Our work shows that TOX is the major oncogenic driver of genomic instability human T-ALL and locks cells in a constant state of dampened repair.

#3584

Genomic analysis reveals a role for BCL9L in aneuploidy tolerance in colorectal cancer.

Carlos Lopez-Garcia,1 Nicholas McGranahan,1 Sebastjian Hobor,1 Nicolai Birkbak,1 Enric Domingo,2 Andrew Rowan,1 Ian Tomlinson,2 Charles Swanton1. 1 _The Francis Crick Institute, London, United Kingdom;_ 2 _The Wellcome Trust Centre for Human Genetics, London, United Kingdom_.

Cancer genomes are frequently characterized by changes in the number and structure of the normal diploid karyotype, normally denominated as chromosomal instability (CIN), that result in tumour cell populations with high karyotipic diversity. CIN plays a key role in tumour evolution by contributing to intratumour heterogeneity and is often associated with key clinical landmarks such as bad prognosis and drug resistance.

CIN originates from deficient segregation of chromosomes during anaphase, which generates sister cells with abnormal karyotypes. Additionally, mechanisms of tolerance need to be acquired by cancer cells to survive segregation errors.

In colorectal cancer (CRC), 20% of tumours present high levels of microsatellite instability (MSI) together with low levels of CIN. The remaining 80% present low or no MSI, together with a wide range of CIN.

In this project, we explored the mutational landscape of 25 MSI-negative CRC samples in order to extract somatic mutational patterns of aneuploid CRC associated with CIN tolerance or chromosomal missgregation. Notably, we found frequent truncating mutations in BCL9L and loss-of-heterozigosity in 5 aneuploid samples.

Further inspection of somatic alterations in the TCGA cohort showed BCL9L alterations in 14% of CIN CRC (mutations and deletions) and more importantly, bioinformatic analysis demonstrated that these tumours have significantly higher wGII scores (the fraction of the genome with alterations). We also compared wGII scores of samples with co-occurring BCL9L and TP53 mutations and found that tumours with alterations in both genes show higher levels of CIN than those with only one alteration.

Functional characterization of BCL9L showed that depletion in various diploid cell lines induces tolerance of drug-induced and naturally occurring segregation errors. Long-term BCL9L-depleted cells increased the fraction of aneuploid cells in a diploid cell line and also, and enhancement of engraftment efficiency when injected into immune deficient mice. Additionally, CRISPR mediated inactivation of BCL9L showed enrichment in monoallelic BCL9L mutant colonies when treated with a drug that induces segregation errora, which supports a role for BCL9L haploinsufficiency in CIN tolerance.

Finally, we observed that BCL9L regulates the expression of Caspase-2, a proteolytic enzyme that is activated in a p53-independent manner by segregation errors and cleaves several proteins important for cell survival such as the p53 regulator MDM2 and the mitochondrial apoptotic regulator protein BID.

#3585

KIF11 silencing or inhibition induces chromosome instability.

Yasamin Asbaghi,1 Zelda Lichtensztejn,2 Laura Thompson,1 Kirk McManus1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _Research Institute in Oncology and Hematology, CancerCare Manitoba, Winnipeg, Manitoba, Canada_.

Chromosome Instability (CIN) is defined as an increase in the rate at which whole chromosomes or large parts are gained or lost. CIN is not only associated with virtually all tumor types, but it is associated with aggressive tumors, the acquisition of multi-drug resistance and consequently poor patient prognosis. Despite these associations, the genes and molecular defects that contribute to CIN are only poorly understood. Recently, we performed a high content screen that identified KIF11, a microtubule associated motor protein, as a candidate CIN gene. Here, we couple RNAi-based gene silencing with biochemistry and cell biology to show that diminished KIF11 expression and/or function induce CIN. HCT116 cells were employed, as they are a karyotypically stable colorectal cancer cell line of epithelial origin that has been used for similar CIN studies. KIF11 was either silenced (both individual and pooled siRNA duplexes) or inhibited (Monostrol) and expression levels were determined by Western blots. To determine whether KIF11 silencing or inhibition affects DNA content, two phenotypes frequently associated with CIN, namely increases in nuclear area and micronucleus formation were evaluated. Fluorescence microscopy was employed on DAPI-counterstained samples and revealed statistically significant increases both nuclear area and micronucleus formation following KIF11 silencing and inhibition relative to controls. Next, flow cytometry was performed on propidium iodide labeled samples to assess whether increases in DNA content were associated with the changes in nuclear area. As predicted, increases in the proportion of cells with >G2/M DNA content occurred within the KIF11 silenced populations. Finally, mitotic chromosome spreads were generated and chromosomes were manually enumerated from 100 spreads per condition/control. Subsequent Kolmogorov-Smirnov tests identified statistically significant increases in the cumulative distribution frequencies of mitotic chromosome numbers within the spreads generated from the KIF11 silenced cells relative to controls. To extend our findings beyond the colorectal cancer cell context employed above, analogous studies were performed in hTERT cells (karyotypically stable fibroblast cell line) with very similar results. Collectively, these data indicate that KIF11 expression and function are normally required to maintain genome integrity. They further suggest that the loss of KIF11 expression and/or function may be a contributing factor in the etiology of tumorigenesis in colorectal cancer and perhaps other tumor types as well.

#3586

Overexpression of Emi1 causes chromosomal instability and cancer.

Srividya Vaidyanathan, Kathleen Cato, Sandra Pavey, Nikolas K. Haass, Brian G. Gabrielli, Pascal HG Duijf. _The University of Queensland Diamantina Institute, Brisbane, Australia_.

Chromosomal Instability (CIN) is a common occurrence in solid cancer associated with poor survival in patients. A significant percentage of these tumors show abnormal chromosomes with near-triploid or tetraploid numbers exhibiting numerical CIN (nCIN). nCIN is caused mainly due to deregulation of cell cycle proteins. Early Mitotic Inhibitor 1 (Emi1) is a cell cycle protein that regulates the activity of E3 ubiquitin ligase, Anaphase Promoting Complex/Cyclosome (APC/C), which targets many cell cycle proteins for degradation. Emi1 is overexpressed in many solid tumors but not blood cancers.

Unpublished data from our lab has shown that Emi1 overexpression in mouse models causes more penetrant and metastatic tumor than control tumors. Breast cancer tissues show significantly high expression of Emi1 at protein level as compared to control breast tissue samples, in agreement with the microarray data from The Cancer Genome Atlas (TCGA). In addition, Emi1 expression from breast cancer TCGA data strongly correlated with the chromosomal instability signature than many of the well-established genes known to promote CIN phenotype. To understand the mechanisms of CIN due to Emi1 overexpression, we overexpressed Emi1 in HeLa cells expressing GFP-tagged histone protein to monitor the cells during mitosis by live cell imaging.

HeLa cells overexpressing Emi1 show CIN following delays in both chromosome alignment at the metaphase plate and, subsequently, anaphase onset (p=0.0008 and p=0.0028). There is a significant increase in CIN phenotypes and abnormal mitoses such as anaphase bridges/lagging chromosomes, mitotic arrest and cell death. These results indicate that Emi1 overexpression actively drives tumorigenesis.

#3587

Multiplexed nuclear area and micronucleus screening identifies SKP1 as a human chromosome instability gene.

Laura Thompson,1 Allison Baergen,1 Zelda Lichtensztejn,2 Kirk McManus1. 1 _University of Manitoba, Winnipeg, Manitoba, Canada;_ 2 _Research Institute in Oncology and Hematology, Winnipeg, Manitoba, Canada_.

Chromosome instability (CIN) is defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost. It is hallmark of cancer that occurs frequently in both solid and liquid tumors. In addition, CIN is associated with highly aggressive tumors, the acquisition of multi-drug resistance, tumor recurrence and poor patient prognosis. Despite this, the majority of human CIN genes have yet to be elucidated, highlighting the need for studies aimed at identifying the defective genes that underlie CIN. In this study we utilized two complementary, image-based approaches capable of detecting CIN-associated phenotypes following RNAi-based silencing of candidate CIN genes. The first assay involves quantifying nuclear areas following silencing, where changes in mean nuclear area relative to controls act as a surrogate marker of CIN. The second approach monitors micronucleus (MN) formation where increases in the number of micronuclei are indicative of DNA damage or the mitotic defects that underlie CIN. These assays were employed in a high-content screen of 164 human candidate CIN genes in two unrelated cell lines, HT1080 and hTERT. In HT1080, the nuclear area and MN enumeration assays identified 88 and 96 putative CIN genes, respectively. In hTERT, the nuclear area and MN assays identified 112 and 19 putative CIN genes, respectively. Promising putative CIN genes such as SKP1 were identified and prioritized for subsequent validation based on the number of assays that identified the gene, and the strength of the CIN phenotype. Preliminary data collected through Western blotting, mitotic chromosome spreads and flow cytometry, provides evidence to support the validation of SKP1 as a bona fide human CIN gene. Identification and characterization of human CIN genes will provide critical insights into CIN and tumorigenesis, as well as identify potential targets that could be exploited in novel, precision medicine approaches for superior cancer treatment.

#3588

The spindle assembly checkpoint gene Bub1b is essential for the survival of some breast cancers.

Dilara Koyuncu, Erik T. Goka, Philip C. Miller, Marc E. Lippman. _University of Miami, Miami, FL_.

As normal breast epithelium evolves towards malignancy, cells accumulate genomic changes that give them a replicative advantage while at the same time increasing their genomic instability. Increased genomic instability results in accumulation of genomic aberrations that compromise the genomic integrity and, therefore, threaten cell viability, thus putting cancer cells under mitotic stress. As a consequence, cancer cells evolutionarily must adapt themselves to compete with the possible detrimental effects of genomic instability. Finding a balance between the instability that gives them a replicative advantage and the instability that could lead them to mitotic catastrophe is crucial. The mitotic stress caused by genomic instability may require overexpression of certain spindle assembly checkpoint (SAC) genes, which can prevent mitotic catastrophe that would occur if cancer cells undergo mitosis prematurely. Although the full mechanism of action of SAC is yet to be elucidated, Bub1b through its protein BubR1 is an important part of this checkpoint, and inhibits the onset of anaphase until all chromosomes are aligned correctly at the metaphase plate.

Our analysis of clinical datasets shows a significant increase in the expression of Bub1b in breast cancer as compared to normal epithelia. Furthermore, Bub1b overexpression correlates with decreased overall survival in patient samples. Our analyses also show a pattern of increasing Bub1b overexpression in more aggressive variants of breast cancer such as triple negative tumors and high-grade tumors, which also tend to be more resistant to current therapies. Expression analyses of breast cancer cell lines reveal that Bub1b overexpression is positively correlated with more aggressive behavior.

We postulated that the requirement for Bub1b expression might be a vulnerability of rapidly proliferating cancers; therefore, its inhibition will result in cell death through mitotic catastrophe. Using RNA interference with siRNAs, we reduced Bub1b levels in a variety of breast cancer cells. Our results showed significant decrease in cell viability and clonogenicity in soft agar upon Bub1b knockdown, especially in triple negative breast cancer cell lines. However, the viability of normal breast epithelium cells, MCF12A, was not affected.

Our data indicate that Bub1b is a critical player in breast cancer viability, and further investigation of the role of Bub1b in promoting successful proliferation of breast cancer cells with genomic instability could provide a new therapeutic strategy particularly in concert with standard genotoxic treatments such as alkylators, spindle poisons and radiation therapy.

#3589

Nucleotide excision repair is elevated in stage IV commonly used breast cancer cell lines as compared to stage I breast tumor explants.

Homood As Sobeai,1 Jennifer M. Johnson,2 Nancy Lalanne,3 Omar Ibrahim,1 Stephen G. Grant,1 Sharon L. Wenger,4 Jean J. Latimer1. 1 _Nova Southeastern University College of Pharmacy, Fort Lauderdale, FL;_ 2 _Thomas Jefferson University Hospital, Philadelphia, PA;_ 3 _Case Western Reserve University, Cleveland, OH;_ 4 _West Virginia University, Morgantown, WV_.

Genomic instability is a hallmark of human carcinogenesis. We have previously published that sporadic stage I breast tumors exhibit deficient Nucleotide Excision Repair (NER) capacity relative to non-diseased breast reduction primary cultures (Latimer et al., 2010). In the present work, we hypothesized that this feature of early human breast cancer would not be maintained in cell lines established from late stage tumors. Our objective was to determine functional NER capacity and relative NER gene expression in a series of commonly used stage IV breast cancer-derived cell lines relative to stage I breast cancer explants and cell lines. NER capacity was determined using the functional unscheduled DNA synthesis (UDS) assay in six established breast cancer cell lines (MCF-7, MCF-7LY2, MDA-MB-231, SK-BR-3, CAMA-1, BT-20). JL BTL-12 (Jean Latimer Breast Tumor Line-12), derived from a stage III breast tumor, was included to represent an advanced stage tumor cell line established using our culture system. The NER capacity of the cell lines was compared to that of 19 stage I breast tumor primary cultures. NER gene expression was determined by expression microarray using the Affymetrix HG U-133 plus 2.0 chip in five of these established cell lines (MCF-7, MDA-MB-231, SK-BR-3, CAMA-1, BT-20), JL BTL-12 and a representative stage I tumor-derived cell line (JL BTL-8, Breast Tumor Line-8). The stage IV cell lines and JL BTL-12 all manifested significantly higher NER capacity than explants of stage I breast cancer. Unsupervised as well as supervised analyses, based on expression of the canonical 20 NER genes, placed JL BTL-12 and the five stage IV commercial cell lines in one cluster, while JL BTL-8 clustered separately. Four NER genes were significantly upregulated in JL BTL-12 and all commercial cell lines relative to JL BTL-8. In conclusion, based on NER these five cell lines commonly used in breast cancer research are more representative of late stage disease than early stage breast cancer, regardless of the type of breast cancer they represent. The most commonly diagnosed stage of breast cancer in the U.S. is now stage I, and it is now possible to study stage I cell lines or explants.

#3590

A Neil1 DNA glycosylase germline variant induces genomic instability and cellular transformation.

Joann B. Sweasy,1 Heather Galick,2 Scott Kathe,2 Susan Wallace2. 1 _Yale Cancer Center, New Haven, CT;_ 2 _University of Vermont, Burlington, VT_.

NEIL1 DNA glycosylase is a bifunctional enzyme that recognizes oxidized pyrimidines and formamidopyrimidines, and appears to remove damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene has a minor allele frequency of 1% and is a G to A base substitution that encodes the G83D NEIL1 protein. Interestingly, this mutation is also present in cholangiocarcinomas. Unlike WT NEIL1, the G83D variant is not able to recognize oxidized bases efficiently due to a shift in the void filling residues that stabilize the DNA duplex once a damaged base has been extruded into the glycosylase substrate binding pocket. The goal of our study was to determine if the G83D NEIL1 variant exhibits a cancer-related phenotype in cells. We found that unlike wild-type (WT) NEIL1, expression of G83D in MCF10A immortal human breast epithelial cells induces genomic instability and cellular transformation. Expression of G83D but not WT NEIL1, in MCF10A cells leads to replication fork stalling and slow replication fork velocity. Our results suggest that G83D is unable to remove oxidative DNA damage at the replication fork, resulting in genomic instability and cellular transformation. Our results are consistent with the idea that individuals who harbor the G83D NEIL1 variant are at increased risk for cancer.

#3591

Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells.

Cecil J. Gomes, Michael Harman, Sara Centuori, Charles Wolgemuth, Jesse Martinez. _University of Arizona, Tucson, AZ_.

In normal lung tissue the expression levels of 14-3-3 gamma are very low and tightly regulated, however, in cancer it is evident that 14-3-3 gamma's expression patterns become deregulated and significantly elevated. In patients with advanced non-small cell lung cancer (NSCLC), increased expression of this isoform is associated with poorer survival and significantly correlates with lymph node and distant metastasis. These data prompted us to analyze TCGA's NSCLC gene expression dataset, and it is clear that 14-3-3 gamma's expression continues to increase in the progression from early to late stage cancers. Taken together, these data suggest that overexpression of 14-3-3 gamma is correlated with a more aggressive tumor phenotype, an observation also seen with breast and hepatocellular carcinoma. The focus of this study is to elucidate the mechanism(s) causing these more aggressive cancer phenotypes. We have previously shown that overexpressing 14-3-3 gamma in human lung cancer cells harboring very low levels of endogenous 14-3-3 gamma and no wildtype p53 results in a stable subpopulation of cells with polyploid DNA content. Interestingly, approximately 40% of lung adenocarcinomas present with hyper-triploid karyotypes, and even a higher percentage, 40-60%, have inactivation of p53. It is well established that polyploid tumors have the capacity to increase tumorigenicity by allowing resistance to conventional therapies and also permitting elevated tolerance to chromosomal instability (CIN). This leads us to hypothesize that in the absence of p53, NSCLC tumors overexpressing 14-3-3 gamma result in a polyploid population of cells that may be influencing the aggressiveness of the tumor. Further in vitro analysis of these 14-3-3 gamma induced tetraploid cells show that they have a prolonged mitosis with significantly more lagging chromosomes in anaphase than their diploid counterparts, indicating an increase in chromosomal instability (CIN). After isolating isogenic tetraploid clones with or without 14-3-3 gamma expression, the stability of the tetraploid cell state was assessed. Clones not expressing 14-3-3 gamma quickly reverted back to a pseudo-diploid state, whereas overexpression of 14-3-3 gamma significantly prolonged the tolerance of tetraploidy and eventually resulted in an aneuploid cell state. Our data suggests that overexpression of 14-3-3 gamma increases tumorigenicity through the stabilization of a polyploid cell state.

#3592

BaP induces chromosome missegregation through mitotic spindle defects in normal lung epithelial cells.

Jose T. Thaiparambil, Randa El-Zein. _Houston Methodist Research Institute, Houston, TX_.

Lung cancer remains the leading cause of cancer related deaths in the United States. Cancer results from an accumulation of multiple genetic events leading to genomic instability that is a crucial early event in carcinogenesis. Aneuploidy is the most frequently identified genomic abnormality in cancer. We have previously reported a significantly higher level of aneuploidy in smokers who developed lung cancer as compared to smoker controls. Benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon, is a pulmonary carcinogen and one of 60 different carcinogens found in tobacco smoke. In this study, we hypothesize that B(a)P disrupts mitotic spindle assembly that impairs the fidelity of mitosis leading to chromosome missegregation and aneuploidy.

To test our hypothesis, we use confocal imaging in combination with traditional molecular biology techniques to dissect the precise mechanism by which B[a]P induces chromosome missegregation and aneuploidy. Mitotic spindle defects in the form of chromosome lags and bridges, monopolar and multipolar spindles, were assessed in normal lung epithelial cells after treatment with 10µM B[a]P for 6 hrs. Spindle defects were assessed at 24, 48, 72 and 96 hours after treatment. Immunofluorescence revealed abnormal mitosis in the prometaphase through the telophase stage of mitosis. Our result show that B[a]P exposure induced abnormal spindles in the form of monopolar (14%) and multipolar (4%) spindles. High resolution confocal images further revealed that B[a]P exposure led to misoriented mitotic spindles with an abnormal spindle angle [-380] relative to normal plane [180]. Chromosome lagging at both metaphase (17%) and anaphase (9%) has also been recorded in response to B[a]P exposure, observations that result from defects in the mitotic apparatus and kinetochores. Furthermore, we assessed the extent of B[a]P induced genetic instability [in the form of double strand DNA breaks] using gama-H2AX in order to elucidate the underlying mechanism associated with accumulated genetic damage and chromosome missegregation. Our data indicate that B[a]P results in accumulation of mitotic abnormalities and gama-H2AX foci (22%) peaks at 48 hr compared to other time points.

Taken together, our data show that B[a]P potently induces mitotic abnormalities by disrupting kinetochore microtubules. The induction of mitotic abnormalities by B[a]P leading to chromosome missegregation and aneuploidy, would unravel a novel targetable molecular mechanism of cigarette smoke induced lung tumorigenesis.

#3593

Electronic cigarettes induce significant DNA damage and reduce cellular antioxidant levels.

Vengatesh Ganapathy,1 Jimmy Manyanga,1 Lacy Brame,1 David Rubenstein,2 Theodore L. Wagener,1 Ilangovan Ramachandran,1 Lurdes Queimado1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _Stony Brook University, New York, NY_.

Background: Cigarette smoking is the leading preventable cause of mortality in the world and the main risk factor for lung and head and neck cancer. E-cigarettes (ECs) are battery-operated devices that deliver nicotine via inhaled aerosols. Although ECs are marketed as a less harmful alternative to tobacco cigarettes and a smoking cessation aid, the health risks posed by exposure to EC aerosols are unknown. Nonetheless, the use of ECs has increased exponentially since 2003, with EC users reporting inhaling on average 200 puffs a day. EC aerosols have been reported to contain variable levels of genotoxins, including carcinogenic substances and reactive oxygen species (ROS). Some toxins in EC aerosols have been reported to reach the levels of similar to those in tobacco smoke. However, the genotoxicity of EC aerosols has not been characterized.

Aims: (1) To determine the cytotoxicity and genotoxicity of short and long-term exposure to EC aerosols on human epithelial normal and cancer cell lines. (2) To evaluate whether exposure of EC aerosols modifies the cellular total antioxidant capacity (TAC).

Methods: EC extracts were prepared from NJOY (12 or 18 mg/ml of nicotine) and Oakley eGo-T (0, 12 or 18 mg/ml of nicotine). Standard tobacco extracts were used for comparison. To assess the effects of short-term exposure, human epithelial normal (NuLi-1) and cancer (UD-SCC1) cell lines were exposed for 1 hour to various EC extract concentrations. To assess the effects of long-term exposure, cells lines were exposed every other day for 2 weeks to EC extracts. Cytotoxicity, DNA damage and TAC were evaluated at 1 h and 2 weeks. Cell viability was determined by MTT assay. DNA damage was quantified using the primer anchored DNA damage detection assay (PADDA). TAC was determined by the Antioxidant Assay Kit (Cayman). Data were analyzed by Student's t-test.

Results: At the range of EC extract concentrations used in this study and expected to occur in EC users (1 to 100 puffs/5 L of blood), no cytotoxicity was observed for either normal or cancer cells. However, significant levels of DNA damage were observed in cancer cells exposed to 10 or more puffs/5 L of EC extracts and in normal cells exposed to100 puffs/5 L. Long-term exposure to EC extracts resulted in a significant decrease in TAC, a measure of free radical scavengers.

Conclusion: Even short-term exposure to low levels of EC aerosols can cause significant DNA damage. Our study emphasizes the need to further investigate the carcinogenic potential of EC aerosols and highlights the importance of regulating EC use.

Grant support: This work was supported by the Oklahoma Tobacco Research Center (LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology.

#3594

The TGF-β effector β2SP depletion abrogates DNA damage repair.

Jian Chen,1 Vivek Shukla,1 Jiun-Sheng Chen,1 Raj K. Panditab,2 Yun Seong Jeong,1 Lior H Katz,1 Ji-Hyun Shin,1 YoungJin Gi,1 Lawrence N. Kwongc,1 Clayton R. Huntb,2 Patrizia Farci,3 Xiaoping Su,1 Jon White,4 Bibhuti Mishra,4 Asif Rashid,1 Milind Javle,1 Lei Li,1 Junjie Chen,1 John R, Stroehlein,1 Marta Davila,1 Rehan Akbani,1 Keigo Machidao,5 Hidekazu Tsukamoto,6 Tej K. Pandita,2 Lopa Mishra7. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Houston Methodist Research Institute, Houston, TX;_ 3 _National Institutes of Health, Bethesda, MD;_ 4 _Institute of Clinical Research, Veterans Affairs Medical Center, Washington DC, DC;_ 5 _Southern California Research Center for ALPD and Cirrhosis, University of Southern California, Houston, TX;_ 6 _Southern California Research Center for ALPD and Cirrhosis, University of Southern California, Los Angeles, CA;_ 7 _George Washington University, Washington DC, DC_.

Objective: Exposure to genotoxins, such as ethanol-derived acetaldehyde, leads to DNA damage, liver injury, and promotes the development of cancer. Alcohol-related genotoxicity, arising from DNA damage by metabolically generated reactive aldehydes, has recently been observed in models with genetic inactivation of members of the Fanconi anemia pathway. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Transforming growth factor β (TGF-β) is a critical protein in the regulation of several cancer phenotypes and also functions as an extracellular sensor of ionizing radiation-induced cell damage. Yet, how the TGF-β pathway contributes to toxin-induced DNA damage repair remains unclear. We utilized the TGF-β/β2SP mutant mouse model to investigate the mechanisms in relation to β2SP-mediated TGF-β modulation of the Fanconi anemia pathway for DNA damage repair, alcohol sensitivity, and liver tumorigenesis. Methods: (1) β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. (2) Genomic instability and sensitivity to DNA damaging agents in primary β2SP+/+, β2SP-/- MEFs were determined by clonogenic survival and metaphase chromosome aberrations analysis. (3) Defective S-phase specific DNA repair in β2SP-/- MEFs were determined by DNA replication restart assays. (4) ChIP assays were performed to determine the recruitment of β2SP/Smad3 at FancD2 promoter. (5) We investigated the clinical relevance of altered β2SP and FancD2 function using immunohistochemical analyses of 20 human liver specimens from alcoholic hepatitis (n=5), alcoholic cirrhosis (n=5), and alcohol-associated liver cancer (n=5), as well as normal controls (n=5). Results: (1) Sptbn1-deficient mice exhibit a phenotype similar to human fetal alcohol syndrome and are sensitive to ethanol exposure. (2) Sptbn1-deficient cells exhibit genomic instability and hypersensitivity to DNA damage (3) Sptbn1-deficiency delays DNA damage repair. (4) Furthermore, Sptbn1-deficient cells are defective in stalled DNA replication fork resolution and homologous recombination. (5) FancD2 ectopic expression rescues the DNA repair defect in Sptbn1 null cells. (6) β2SP and FancD2 are clinically correlated in alcoholic hepatitis and HCCs. Conclusions: Our model proposes that in response to liver toxins such as alcohol, the TGF-β/β2SP/Smad3 pathway prevents liver injury and cancer through its direct effects on DNA repair and genomic stability. Thus, characterizing the role of TGF-β in alcohol-induced injury could potentially enhance our mechanistic insight into the basis for therapeutics targeting toxin-induced DNA damage and tumorigenesis.

#3595

Improved therapeutics for hepatocellular carcinoma.

Jessica A. Zavadil, Christi A. Walter. _UTHSCSA, San Antonio, TX_.

Eighty percent of hepatocellular carcinoma (HCC) within the USA is diagnosed late when chemotherapy provides only 2 months increased survival. We fortuitously found that treatment with an alkylating agent, methylnitrosurea (MNU), prior to tumorigenesis reduced tumor prevalence later in life by ~50% in C3HeB/FeJ male mice, a model of spontaneous HCC. A similar effect was observed with temozolomide (TMZ), a glioblastoma therapy that induces the same DNA damage at similar proportions to MNU. O6-methylguanine-DNA methyltransferase (MGMT) repairs the highly mutagenic and cytotoxic O6-methylguanine lesions induced by TMZ, and MGMT abundance influences therapy response in glioblastoma. MGMT is reduced in 1/3 of human HCC, leading us to hypothesize that TMZ could be effective in treating HCC. We proposed to combine TMZ with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, to maximize efficacy, reasoning that SAHA would allow TMZ greater access to DNA to induce greater levels of damage. We tested this drug combination in two human HCC cell lines, Huh7, with reduced MGMT expression, and SNU398, with higher expression. TMZ and SAHA synergistically reduced cell viability in both cell lines with multiple dose combinations. Based on this outcome, we tested the drugs in tumor-bearing C3HeB/FeJ mice. Surprisingly, DMSO treated solvent control mice showed reduced tumor burden. The DMSO treatment was repeated adding a sham control group and confirmed that DMSO significantly reduces tumor burden. The TMZ cohort also displayed a significantly lower tumor burden compared to the sham cohort, but no significant difference was found with SAHA alone or SAHA+TMZ. We previously showed that a hMGMT transgene protects from hepatocarcinogenesis in C3HeB/FeJ mice, but surprisingly, there is not additional protection when transgenic mice are treated with MNU. These results suggest that the more prevalent cytotoxic lesions induced by MNU and TMZ, e.g. N7-methylguanine, may be more important in HCC than O6-methylguanine. These lesions are recognized by methylpurine-DNA glycosylase (MPG) and repaired through base excision repair. Therefore, we directly tested the role of MPG in TMZ therapy through altered expression of MPG in Huh7 and SNU398 cell lines and showed that it does affect sensitivity to TMZ.

#3596

KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin.

Madhav Sankunny,1 Emily Nizialek,2 Farshad Niazi,1 Charis Eng1. 1 _Cleveland Clinic Foundation, Cleveland, OH;_ 2 _Case Western Reserve University, Cleveland, OH_.

Dysregulation in the maintenance of chromatin organization resulting in genomic instability is a major driving force for inappropriate development and carcinogenesis. Tumor suppressor genes play a critical role in this regulation through the maintenance of epigenetic marks. Our study focuses on the role of KLLN in the maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN resulting in decreased KLLN expression has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN mutation negative patients. KLLN is a tumor suppressor gene necessary for p53-mediated apoptosis. The protein mediates S-phase arrest and is known to have DNA binding ability. Here, we first used chromatin immunoprecipitation-based sequencing (ChIP-seq) to investigate regions of KLLN binding on the genome and compared it to regions of H3K9me3 enrichment. We used H3K9 specific histone methyltransferase (HMT) activity assay and immunoblotting to measure levels of H3K9me3. Immunostaining was used to study the localization of KLLN, and micronuclei frequency and aberrations in chromosome number was used to assess chromosomal instability. Analysis of ChIP-seq shows enrichment of KLLN binding in regions of H3K9me3. Overexpression of KLLN using plasmid-based transfection correlates with increased H3K9 methyltransferase activity and increased global H3K9me3, while loss of KLLN expression through siRNA-mediated knockdown had an opposite effect. We also established that KLLN localizes to pericentric regions, and loss of KLLN results in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei frequency and numerical chromosomal aberrations. KLLN regulation of H3K9me3 could be correlated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SUV39H1, a H3K9 specific histone methyltransferase responsible for maintenance of pericentric heterochromatin. We hypothesize that KLLN sequesters DBC1 through their interaction, thereby abrogating DBC1 inhibition of SUV39H1, resulting in maintenance of pericentric heterochromatin and genomic stability. Our study therefore suggests a critical role for KLLN in the deterrence of tumor initiation and progression.

SIGNIFICANCE: Our study demonstrates a clear role for KLLN in maintenance of chromatin organization thereby maintaining genomic stability, which begins to uncover the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers.

#3597

RPS3 regulates melanoma growth and sensitivity to DNA damage and predicts a poor prognosis by targeting ADT3.

Yun Tian,1 Lijun Qin,2 Wei Guo,3 Changlin Zhang,1 Dingbo Shi,1 Tianze Liu,1 Wenbing Li,1 Jingshu Wang,1 Yixin Li,1 Ge Qin,1 Wendan Yu,3 Xiangsheng Xiao,1 Tiebang Kang,1 Wenlin Huang,1 Wuguo Deng1. 1 _Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center of Cancer Medicine; State Key Laboratory of Targeted Drug for Tumors of Guangdong Province, Guangzhou Double Bioproduct Inc, Guagzhou, China;_ 2 _Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guagzhou, China;_ 3 _Institute of Cancer Stem Cell, Dalian Medical University, Dalian, China_.

Melanoma, a malignant skin cancer, is resistant to DNA damage-mediated therapy. Discovering and identifying novel therapeutic targets for melanoma is urgently required. In this study, we found that ribosome protein S3 (RPS3) regulated melanoma growth and the sensitivity of melanoma to DNA damage by targeting ADP/ATP translocase 3 (ADT3). Knockdown of RPS3 inhibited cell proliferation and sensitized melanoma cells to DNA damage. RPS3 knockdown also promoted ADT3 translocation to mitochondrial of melanoma cells when exposed to DNA damage. RPS3 interacted with ADT3 in melanoma cells. Knockdown of ADT3 reduced its co-localization with RPS3. RPS3 could not obviously sensitize melanoma cells to DNA damage with ADT3 knockdown. In addition, we found that Lys18 in the death-induce-domain of RPS3 protein played a critical role in the interaction between RPS3 and ADT3. Mutation at Lys18 site could deadlock ADT3 and attenuate cell apoptosis in melanoma cells. Knockdown of RPS3 also inhibited tumor growth in a melanoma mouse model in vivo, but overexpression of the RPS3-18 mutation rescued the growth. Furthermore, we showed that the patients with higher expressed RPS3 had a much shorter median survival, whereas the patients with higher levels of ADT3 had a much longer median survival. Collectively, our results indicate that RPS3 cooperates with ADT3 to regulate the sensitivity of melanoma to DNA damage and suggest that the RPS3/ADT3 pathway is a potential therapeutic target for human melanoma. Grant support: This work was supported by the funds from the National Natural Science Foundation of China (81472178, 81272195) and the State "973 Program" of China (2014CB542005).

#3598

Laser micro-irradiations of large amounts of cells for high content applications.

Martin Mistrik,1 Eva Vesela,1 Tomas Furst,1 Ivo Frydrych,1 Jan Gursky,1 Jiri Bartek2. 1 _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky Univ., Olomouc, Czech Republic;_ 2 _Danish Cancer Society Research Center, Copenhagen, Denmark_.

A new methodical approach for the evaluation of photo-manipulated samples is presented and tested in various biological setups. The method is based on an atypical setting of a standard laser-scanning microscope (LSM) and image-processing software solution. Newly, a whole population of several dozen of cells is simultaneously micro-irradiated by a laser beam in a precisely defined pattern of collinear rays. Such laser induced striation pattern is automatically analyzed by specialized software routine providing quantitative assessment of various photo-manipulations, such as DNA-damage induced protein translocations or modifications, in both live and fixed cells. Automation of the process reduces workload and opens new possibilities for various dynamic studies and/or high content experiments. Results obtained during the testing of the method are presented and offer an insight into several DNA damage recognition and response pathways.

#3599

Implementation of a fully automated 3D foci counting algorithm to determine DNA damage in cells.

Alexander Katsis,1 Eric Abel,2 Raisa Pavlyuchkova,2 Shilpa Senapati,2 Swati Girdhani,2 Renate Parry2. 1 _Duke University, Durham, NC;_ 2 _Varian Medical Systems, Palo Alto, CA_.

Background: The number of DNA double stand breaks (DSBs) induced after ionizing radiation is strongly correlated with cell death, mutation and cancer. Although a wide variety of techniques has been used to quantify DNA damage, immunostaining of DNA repair proteins, like γ-H2AX or Rad 51, that localize to the site of DNA damage within seconds following IR exposure has become a method of choice due to its sensitivity. DNA damage foci are imaged using microscopy, and then counted either manually, or with some form of computer assistance. A number of open source or vendor provided focus counting programs are available as either stand-alone applications or as plugins to image processing utilities such as ImageJ. While counting efficiency is greatly enhanced through the use of these tools, they are limited in that the analysis is confined to a single plane, they are often not conducive to high throughput analysis, and finally, they suffer from user-variability. We therefore developed a fully automatic, MATLAB-based algorithm which can process and uniquely identify foci in a 3D image stack, with only limited user interaction.

Methods: We compared three different methods of quantifying foci for uniformly irradiated cell cultures: a) 2D counting on single plane with Nikon NIS elements software and b) 2D or c) 3D counting with the MATLAB-based algorithm. 3D image stacks of stained γ-H2AX and Rad51 foci, as well as DAPI stained nuclei were acquired at 100X magnification with 200 nm slice spacing. The automated image analysis process comprises three steps: (i) nuclei segmentation, (ii) dynamic background characterization for threshold determination and (iii) segmentation of separate DNA damage foci of the entire 3D stack to ensure the identification and counting of unique foci.

Results: The accuracy of the 3D counting algorithm was determined to be 99% using a numerical phantom containing a known number of randomly placed foci in a 3D volume. In cell culture, 2D counting on a single plane using NIS Elements identified a mean foci count 0.09 and 31.7 per cell for 0 Gy and 4 Gy respectively while our algorithm counted 0.24 and 26.7 foci/cell, demonstrating remarkable consensus. Counting in 3D yielded 0.39 (0 Gy) and 77.58 (4 Gy) per cell, demonstrating how single plane counting underestimates DNA damage. The algorithm yielded a six fold speedup in processing a single field of view over manual counting, with additional time savings realized by removing the need for user interaction.

Conclusions: We have successfully implemented a fully automated 3D foci counting algorithm which identifies both foci, as well as surrounding nuclei, with little to no human interaction. This algorithm allows for the fast processing of large quantities of immunofluorescence data, thereby providing greater confidence in our DNA damage experiments.

#3600

Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening.

Peter Sykora,1 Sandra Woodgate,2 Jay George,2 Robert W. Sobol1. 1 _University of South Alabama, Mobile, AL;_ 2 _Trevigen Inc., Gaithersburg, MD_.

The mechanism of action of many classes of chemotherapeutic agents involves either the repression of DNA repair or an increase in DNA damage. However the measurement of DNA damage levels within a cell has been notoriously difficult and current methods to asses DNA damage potential of new chemotherapeutics have major technical flaws. The single cell gel electrophoresis (SCGE) assay is a long-standing method for measuring levels of DNA damage within a cell. The principle of SCGE is that DNA damage can cause DNA strand breaks in cells. These breaks cause the relaxation of the compact highly supercoiled DNA. The application of an electric field while the cells are embedded in agarose allows damaged DNA to migrate faster than intact DNA. The more breaks in the DNA, the further it will travel in the agarose resulting in the formation of a "comet" tail, the size and length of which directly correlates to the amount of DNA damage. The SCGE method benefits from both technical simplicity and high sensitivity. The major drawback is the assay is extremely laborious, lacks appropriate controls and has poor reproducibility. We have recently overcome these drawbacks by developing a 96-well plate format of the SCGE, aptly named "CometChip". The CometChip uses micro-pillar technology to create an agarose 96-well chip where each well has approximately 300 micro-wells used to capture individual cells. Using this technology we can incorporate multiple treatments, controls and time points on a single CometChip which can then be rapidly analyzed using a fluorescence based imaging instrument. The utility of the new technology was tested using 75 different chemical compounds considered either genotoxic, non-genotoxic or unknown. The compounds were tested on two lymphocyte cell lines with different p53 status to compare the accumulation and repair of DNA damage. We report that the CometChip gives highly reproducible and accurate results without loss of sensitivity. In the high throughput screening approach using multiple CometChip apparatus, we estimate the throughput of the assay to be approximately 10,000% greater than doing traditional slide based comet analysis. The massive increase in processivity brings new opportunity for large-scale compound screening. The increased sensitivity coupled with large sample sizes will allow researchers the option to measure minor changes in DNA damage with unparalleled accuracy.

#3601

Evaluation and quantification of biomarkers of DNA damage in human plucked hair.

Aude-Marine Bonavita, Liam Walker, Adam Boanas, Nicola Tonge, Gregory Tudor, Cath Booth, Ben Reed. _Epistem Ltd, Manchester, United Kingdom_.

The hair follicle is a well vascularised structure that contains rapidly dividing epithelial cells. As a result it is a potentially useful surrogate biomarker for less accessible but highly similar tissues, such as rapidly proliferating tumours. We have developed an ex vivo culture method to validate target expression changes following exposure to DNA damage and/or potential therapeutics. Proof of concept studies performed ex vivo can then inform the design of clinical validation studies. Earlier work evaluated the response of targets such as pERK, pAKT and pSMAD2 to various kinase inhibitors, whilst recently we have used the model to measure the DNA damage response (DDR) following exposure to UV radiation (as a model damaging agent) and more clinically relevant chemotherapeutic agents.

Human plucked hair was placed in maintenance media in the presence of known chemotherapeutic agents (such as Gemcitabine, DNA topoisomerase inhibitor I, Irinotecan and Paclitaxel) or exposed to 0.2-0.5J/cm2 UVB radiation. Hairs were then fixed at a range of time points post-exposure and longitudinal sections labelled for various markers including pChk1, gamma-H2AX, p53, Ki67 and thymine dimers (TDM1). Quantitative image analysis to measure the level of labelling was performed using an Aperio® ScanScope®.

Following UVB exposure thymine dimer formation occurred immediately on the hair and was stronger on the side directly facing the source of radiation. It was followed rapidly by phosphorylation of Chk1 localised in the inner root sheath at first (10min to 1h after injury), then gamma-H2AX and p53 induction in the outer root sheath. Further studies are looking at DNA damage response in an UV dose dependent manner.

Chemotherapeutic agents known for their different mechanism of actions were tested at two concentrations with various responses depending on their mechanism of action. For example, Gemcitabine, which is linked to DNA polymerase inhibition, strongly induced p53 after 24h but no gamma-H2AX activity was seen above background level. We have profiled the large panel of responses for each chemotherapeutic agent, over an acute time course.

The ex vivo plucked hair follicle method is a rapid and convenient way of studying the DDR induced by exposure to various DNA damage agents. This model allows for screening of novel chemotherapeutic agents to test their efficacy in preventing or treating DNA damage.

#3601A

Concordance of genomic alterations between primary and metastatic matched breast tumors.

Anu G. Gaba,1 Steven F. Powell,2 Paul A. Thompson,2 Megan L. Landsverk,2 Chun-Hung Chan,2 Jennifer L. Weiss,2 Lora J. Black,2 James M. Ford3. 1 _Sanford Health, Fargo, ND;_ 2 _Sanford Health, Sioux Falls, SD;_ 3 _Stanford University School of Medicine, Stanford, CA_.

Cancer therapy exerts a strong selection pressure that shapes tumor evolution. Recent studies have demonstrated high rates of concordance (greater than 80%) between primary tumor and recurrences. The main objective of our study was to demonstrate the molecular evolution of breast cancer using next-generation sequencing (NGS) at initial diagnosis and after metastatic progression beyond at least 1 line of treatment.

Methods: We conducted a retrospective analysis of tumors in patients with metastatic breast cancer (MBC). Breast cancer specimens from metastatic sites following at least 1 line of treatment were obtained and analyzed using NGS from an existing prospective molecular profiling study (NCT02416518). Eligible participants from this study had their archival primary tumor specimens obtained and analyzing using the same NGS techniques. Samples were analyzed using Foundation One NGS, evaluating 343 genomic alterations. Concordance of genomic alterations from initial diagnosis to disease progression after metastatic recurrence was the primary endpoint. This study population was from an integrated, community-based healthcare system, serving rural populations primarily in the Dakotas and Minnesota.

Results: A total of 10 patients had evaluable primary tumors and matched metastatic specimens. For the 20 tumors evaluable for matched analysis, we found 126 unique gene alterations from the 343 genes assayed, of which 31 (24.6%) were actionable (defined as alterations with established or investigational therapeutics based on literature review). Each tumor had an average of 30.4 alterations (range 15-43) and 7 actionable alterations, (range 0-12). There was only 1 patient that had no actionable alteration in the primary or metastatic tumor. Taking the 10 individual patients into consideration, we found a concordance rate of 64% for all gene alterations and a 61.2% concordance rate for the actionable gene alterations between the two time points. The most common actionable alteration was the activating PIK3CA gene alteration, which had 100% concordance in the 6 patients that had the alteration. The other key actionable alterations with 100 % concordance involved the AR, BRCA2, CCND1, CDKN2B, ERBB2, PTEN, and ROS1.

Conclusion: Contrasting with prior studies, in our small sample there was moderate concordance of gene alterations between primary and metastatic tumor samples. Despite this, high concordance of key actionable alterations (PIK3CA, PTEN, CCND1, ERBB2) was maintained throughout the disease course. This suggests therapeutic potential early on in the disease course, as key actionable driver mutations are present at diagnosis and persist beyond metastatic disease progression.

### Genomic Technologies

#3602

Linked-Reads enable detailed, phased resolution of structural variation in the cancer genome.

Sophia Kyriazopoulou-Panagiotopoulou,1 Patrick Marks,1 Haynes Heaton,1 Heather Ordonez,1 Kristina Giorda,1 Cassandra Jabara,1 Billy Lau,2 John M. Bell,2 Michael Schnall-Levin,1 Hanlee P. Ji2. 1 _10X Genomics, Pleasanton, CA;_ 2 _Stanford University, Stanford, CA_.

Studies have shown that somatic structural variation (SV) plays a key role in the oncogenic process. Traditionally SVs in the cancer genome have been detected using low resolution cytogenetic approaches, such as FISH, or microarray-based techniques. More recently, next-generation sequencing (NGS)-based technologies have been employed to detect SVs, including indels and translocations. However, both short- and long-read NGS-based approaches are limited in their ability to accurately identify SV events and delineate their breakpoints due to the limitations inherent in assembly of billions of short-read sequences across a heterogeneous cancer sample, as well as the costly and burdensome laboratory infrastructure associated with long-read sequencers. We utilized a novel technology that combines microfluidics and molecular barcoding to generate libraries that are sequenced with an Illumina system. Open-source bioinformatics software produces linked-reads that maintain long-range information and single molecule sensitivity.

Cell lines and cancer samples were obtained from commercial sources, and genomic DNA was extracted. DNA sample indexing and partitioning was performed using the 10X Genomicx GemCode instrument. One ng of sample DNA was used as input for each reaction, and DNA molecules were partitioned into droplets to fragment the DNA and introduce molecular barcodes. Following barcoding, droplets were fractured, and library DNA was purified and sequenced on Illumina sequencers. The GemCode Long Ranger software suite was used to map sequencing reads back to original long molecules of DNA, generating reads linked to partition barcodes. Thus we can generate phased sequences covering many 10's to 100's of kilobases.

We first benchmarked the ability to call multiple SV types using a well-characterized germline HapMap sample (NA12878) as well as two recently characterized haploid hydatidiform moles (CHM1 and CHM13) that have been studied with multiple orthogonal technologies. Regions with evidence for structural variation were reassembled into distinct haplotypes. The barcode information allowed us to both phase the structural variants we detected and disambiguate calls within highly repetitive regions, such as segmental duplications. We demonstrated high concordance with alternative approaches across all major classes of SVs, including long insertions and deletions as well as copy-neutral events. In cancer cell lines, we detected well-annotated gene fusions, such as the EML4/ALK and ALK/PTPN3 fusions in the lung cancer cell line NCI-H2228, and the SLC26A/PRKAR2A fusion in the triple negative breast cancer cell line HCC38.

#3603

Megabase-scale determination of complex genetic aberrations of primary cancer genomes at individual DNA molecule resolution.

Billy Lau,1 John Bell,1 Stephanie Greer,1 Grace Zheng,2 Christina Wood,1 Hanlee Ji1. 1 _Stanford School of Medicine, Stanford, CA;_ 2 _10X Genomics, Pleasanton, CA_.

Cancer is a disease of the genome caused by genetic aberrations such as point mutations and rearrangements. To identify these genetic aberrations, cancer studies rely on DNA sequencing of short reads from highly fragmented DNA molecules that are typically less than 500 bases long. However, genomic structural alterations and context of genetic aberrations are much larger and cannot be effectively analyzed. To characterize such alterations with completeness, the contiguous relationship of variants from a given chromosome homologue must be determined; this analysis process is referred to as genome phasing. In this study, we demonstrate the significant advantages of genome phasing; we utilize a new technology called linked-read sequencing that enables tracking of individual DNA sequence reads to the input high molecular weight DNA at single-molecule resolution through the use of barcoded reads. We then reconstruct at megabase-scale the complex genomic somatic events derived from the original paternal or maternal chromosome homologues and demonstrate new insight into the undiscovered details of cancer genome alterations. We apply this analysis to primary gastrointestinal cancers from surgical resections; our study is a unique achievement given that this type of phased genome sequencing analysis has only been previously conducted on cancer cell lines.

For this study, we focus on specific categories of megabase-scale genetic aberrations: (1) copy number variants (CNV) derived from segmental duplications, (2) structural rearrangements, and (3) aneuploidy. As an example from gastric cancer metastases, we identified a set of unique and site-specific FGFR2 amplification events corresponding to segmental duplications. The structural details of this amplification were only evident using barcoded sequence reads and phased analysis. Secondly, in a primary colorectal adenocarcinoma we resolved a complex recombination event that covered the majority of the chromosome 5 q-arm. It contained a set of complex rearrangements that are difficult to determine by short read sequencing but can be identified using linked reads. This rearrangement event arose from a combination of a 80Mb homologous recombination that contained a small interval ~2.6Mb deletion. The allelic loss of two candidate cancer drivers, SMAD5 and TGFB, occurred in the affected interval. Finally, to assess aneuploidy we relied on the imbalanced ratios between paternal and maternal chromosomal homologues to generate contiguous blocks of variants on the order of tens and up to hundreds of megabases long. We performed this type of analysis on a number of cancer genomes derived from primary colorectal adenocarcinomas, and were able to robustly determine the phased single nucleotide variant content and large-scale genomic alterations involving entire chromosome arms.

#3604

Validated structural variant detection with prioritisation of known cancer related changes.

Miika Ahdesmäki,1 Brad Chapman,2 Sally Luke,1 Hedley Carr,1 Daniel Stetson,3 Oliver Hofmann,4 Justin Johnson3. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _AstraZeneca, Waltham, MA;_ 4 _University of Glasgow, Glasgow, United Kingdom_.

Reliable detection of structural variation (SV) is playing an increasingly important role in cancer diagnostics and treatments. We introduce a best practice integrative workflow for detecting large structural variations (SVs) such as deletions, duplications, inversions, fusions and translocations from standard DNA re-sequencing techniques, annotating and prioritising for clinically actionable events.

While established RNA-Seq fusion workflows exist to detect and filter fusion events in tumors detecting large structural changes from exome- and WGS data remains challenging. Current DNA-based SV callers predict a large percentage of false positive events, and the resulting event lists are not prioritised for validation. To streamline follow-up studies we automate running multiple structural variant callers (Manta, WHAM, Lumpy, MetaSV), identifying potentially disruptive large scale events. We evaluate the integrated workflow sensitivity and specificity against the ICGC-TCGA DREAM Mutation Calling Challenge data set, optimise filtering criteria based on quality and supporting information, and annotate the filtered calls using known cancer related alterations from CIViC and publicly available gene lists from AstraZeneca. This enables us to prioritise known and potentially interesting events over intergenic events or non-whole exon deletions.

We show validation results from test data and clinically actionable inversions, duplications and fusion events from cancer cell lines. Our approach is based on a two tier approach of first focusing on known events that yield fusion transcripts such as the TACC3-FGFR3 tandem duplication and ALK-EML4 inversion, and secondarily on events in oncogenes and tumor suppressors of interest. While whole genome sequencing is the preferred approach to detecting these events, we also show how by design or by the nature of the breakpoint being close enough to exons, hybrid capture data can also be surprisingly useful for SV inference.

#3605

ICGC in the cloud.

Christina K. Yung,1 Guillaume Bourque,2 Paul C. Boutros,3 Khaled El Emam,4 Vincent Ferretti,1 Bartha M. Knoppers,5 Brian O'Connor,1 B.F. Francis Ouellette,3 Cenk Sahinalp,6 Sohrab P. Shah,7 Lincoln D. Stein,3 Cancer Genome Collaboratory Consortium. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _McGill University, Montreal, Quebec, Canada;_ 3 _Ontario Institute for Cancer Research & University of Toronto, Toronto, Ontario, Canada; _4 _University of Ottawa, Ottawa, Ontario, Canada;_ 5 _Centre of Genomics and Policy, Montreal, Quebec, Canada;_ 6 _Simon Fraser University, Vancouver, British Columbia, Canada;_ 7 _BC Cancer Agency Research Centre, Vancouver, British Columbia, Canada_.

In November 2015 members of this consortium and the International Cancer Genome Consortium (ICGC) jointly announced the availability of more than 1,300 whole cancer genomes in the Amazon Web Services' elastic compute cloud (EC2). Another 480 whole cancer genomes are available in the Cancer Genome Collaboratory, an academic cloud being built by this consortium. By making the data available in cloud compute form, researchers benefit from the high availability, scalability and economy offered by cloud services, and to avoid the large investment in compute resources and the time needed to download the data. Over the next year, we will increase the number of ICGC genomes available in the cloud, with the goal of placing the entire ICGC data set of ~25,000 donors in academic and commercial clouds when the project is completed in 2018. For information and a getting-started guide, see https://dcc.icgc.org/icgc-in-the-cloud.

Cloud computing represents a fundamental shift in the way that cancer genomics is performed. Because of the large size of the ICGC data set, it can take many months to download the data across a typical university broadband connection, and it requires a substantial investment in hardware in order to analyze it. In practice, this has meant that only large computational groups could perform whole-genome analysis at scale. Using the cloud, research groups of any size can launch large analytic processes, pay only for the compute that they use, and avoid charges for data transfer and long-term data storage.

A practical demonstration of the power of working in compute clouds comes from our ongoing collaboration with the PanCancer Analysis of Whole Genomes Project (PCAWG; https://dcc.icgc.org/pcawg), which seeks to interpret patterns of variation in both coding and non-coding portions of cancer genomes. Upwards of 2,800 ICGC whole cancer genomes were subjected to a uniform data processing pipeline that included whole genome alignment, uniform quality control, and standardized germline and somatic variant calling using a large number of software packages that were adapted to run efficiently in the cloud. Using a series of 14 academic and commercial compute clouds, we were able to process this 800 terabyte data set in just over a year's time. Given the improvements in the software that occurred over this period, the whole project would take less than 4 months on just a single commercial cloud if we were to start over. When the project is completed later in 2016, we will again use academic and compute clouds to publish the PCAWG data, its major results, and all the software used during the analysis, thereby allowing the research community to integrate PCAWG with their own data sets, and apply the same analytic procedures.

#3606

Identification of oncogenic mutation hotspots via three-dimensional proximity.

Jianjiong Gao, Matthew T. Chang, Brooke E. Sylvester, Hannah C. Johnsen, Sizhi P. Gao, S. Onur Sumer, David B. Solit, Barry S. Taylor, Nikolaus Schultz, Chris Sander. _Memorial Sloan Kettering Cancer Center, New York, NY_.

One of the greatest challenges in studying the genomic basis of human cancer is distinguishing the few mutations that directly contribute to tumorigenesis ("drivers") from the many biologically neutral mutations ("passengers"). Existing methods can identify highly recurrent individual mutations or mutated genes, but capturing functional yet infrequent "long-tail" mutations remains challenging. While individually uncommon, these long-tail mutations are present in as many as one-third of cancer patients, and for many of them targeted therapies currently exist. Therefore, improved approaches to identify individual driver mutations in the long tail will facilitate our understanding of their potentially diverse biological and clinical significance. Some of these mutations can be identified as hotspots based on their recurrence on or around the same residue. Others may occur in different regions but are actually close in protein three-dimensional structures. We have developed a novel method that identifies mutations that significantly cluster together within three-dimensional proximity in protein structures, or 3D hotspots. We applied this method to a combined collection of mutation data of 21,000 sequenced tumor samples in 74 cancers.

Our analysis confirmed many well-studied 3D hotspots of functional mutations in cancer genes such as KRAS, BRAF, IDH2, SMAD4, FBXW7, SPOP, RHOA, and PTPN11. Most of these genes have well known individual hotspots, but our analysis suggests that additional mutations in other residues of the protein are adjacent in the folded protein structure and may also be oncogenic. We also identified novel 3D hotspots in known cancer genes such as EP300, MAP2K1, and KDR, as well as several other genes of unknown significance that harbor 3D hotspots (e.g., DCC). Most of these hotspots are present in multiple cancers.

To explore the functional consequences and potential translational significance of long-tail low-incidence mutations identified by our method, we assessed several MAP2K1 mutations in vitro. MAP2K1 (MEK1) is a critical effector of MAPK signaling, harbors conventional single-codon hotspots in several cancer types, and a number of selective MAPK pathway inhibitors are either FDA-approved or are being investigated in early-phase clinical trials. Our experiments confirmed that these mutations activate MAP2K1 and confer sensitivity to MAP2K1 inhibition.

We have provided an implementation of the algorithm via an interactive web resource connecting to cBioPortal for Cancer Genomics (http://cbioportal.org/) for easy interpretation of cancer genomics data in the context of three-dimensional structures. By adding to cBioPortal's already powerful capacity of clinical decision support, our method and analysis provide a useful approach of interpretation, prioritization and extension of biologically significant and clinically actionable mutations in cancer.

#3607

An evaluation of NGS to identify gene fusions using RNA from FFPE solid tumor samples.

Julianna Tdr Parks,1 Luo Byron,1 Brian Crain,1 Snedecor June,1 Zhao Chen,1 Tingting Du,1 Gabriel L. Sica,2 Taofee K. Owonikoko,2 Stewart G. Neill,2 Scott Newman,2 Debra F. Saxe,2 Jennifer S. LoCoco,1 Han-Yu Chuang,1 Charles Lin,1 Kathryn M. Stephens,1 Michael R. Rossi,2 Matthew C. Friedenberg1. 1 _Illumina, Inc., San Diego, CA;_ 2 _Emory University School of Medicine, Atlanta, GA_.

Gene fusions have long been considered strong drivers of cellular transformation, making the accurate and precise assessment of these variants a necessity for any tumor profiling assay. Recent studies have indicated the utility of next-generation sequencing (NGS) for tumor profiling due to increasing data output and decreasing costs of the technology. Unfortunately, because a critical facet of NGS is the evaluation of short DNA fragments, sufficiently covering all possible breakpoint regions (many of which are intronic) has proven difficult and costly. Recent studies have indicated that NGS may prove better at detecting gene fusions using RNA instead of DNA, given the higher probability of breakpoint-spanning reads. This allows for de-novo discovery of fusion partners without knowing the precise breakpoint and guarantees expression of the fusion transcript. To that end, Illumina is developing a novel method for simultaneous library preparation from low input amounts of degraded DNA and RNA from a single FFPE tumor sample. With a turnaround time from nucleic acid to data of less than 4 days, this enrichment-based assay surveys 170 genes for single nucleotide variants and small indels, 57 genes for gene amplifications, 55 genes for fusions and four genes for splice variants. To determine the limit of detection for gene fusions, a panel of different synthetic RNA transcripts were prepared in vitro, pooled at equal molar amounts, and spiked into 20ng of cell line RNA (MCF-7). Fusions were detected over several orders of magnitude down to 1×10-8 picomoles, equivalent to 3 to 15 fusion transcripts per cell. In addition, a similar range of fusion detection was observed when RNA from two different cell lines were mixed, as when RNA from a cell line with high expression of an FGFR2-COL14A1 fusion was mixed in proportional amounts with RNA from a different cell line where FGFR2 is minimally expressed. Importantly, our method allowed for fusion detection from as little as 100 picograms of cell line RNA. We then tested our new method on previously characterized FFPE solid tumor samples harboring known gene rearrangements identified by FISH and other methods. Not only was the NGS method able to detect the majority of previously characterized variants, including EML4-ALK and SDC4-ROS1, it also identified the gene fusions and their uncharacterized fusions partners by combining the non-targeted sequence information gained from using an enrichment-based assay with novel fusion calling algorithms. From this information, we were able to glean new insights into the structure of the rearrangements and how the gene fusions may be involved in tumorigenesis. These results indicate that NGS can identify fusions from the low amounts of degraded RNA from solid tumor samples, identify fusion partners not uncovered by current technologies, and further emphasizes the advantage of NGS in solid tumor profiling.

#3608

Detection of somatic structural variants using nanopore sequencing.

Alexis L. Norris, Rachael E. Workman, Yunfan Fan, James R. Eshleman, Winston Timp. _Johns Hopkins University, Baltimore, MD_.

Structural variants (SVs) are a hallmark of human cancer, but remain difficult to reliably and accurately detect with next generation sequencing (NGS). This is in part due to the difficult in mapping NGS's short reads (<300bp) to repetitive elements, which often flank the genomic rearrangements of SVs. Nanopore sequencing, a 3rd generation sequencing technology, offers long sequencing reads (up to 20kb), that should allow for more reliable mapping of SVs. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Here, we have tested the ability of nanopore sequencing to detect a series of well-characterized SVs (including large deletions, inversions, and translocations), that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we show that nanopore sequencing on the MinION platform can detect large deletions, translocations, and inversions at dilutions as low as 1:100. Given the speed, small footprint (USB size), and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of somatic SVs, with clinical applications of molecular relapse, early detection, or therapeutic monitoring.

#3609

A new innovative and robust targeted deep sequencing system: 36 hours turn round time from patient samples to the final mutation report.

Pedro Mendez,1 Jun-Hee Yoon,1 Sharon Lee,2 James Kim,2 Jenny Dang,2 Thomas Kim,2 David Jablons,1 Il Jin Kim1. 1 _Thoracic Oncology Laboratory, Department of Surgery, Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA;_ 2 _CureSeq Inc, Brisbane, CA_.

The emergence of next-generation sequencing (NGS) has changed the paradigm for genetic and genomic studies in many medical and life science fields. Despite its growing popularity and importance in many life science applications, several factors such as complicated sample preparation, high cost, and time-consuming data analyses may prevent NGS applications from being more widely used in clinical and research settings. Therefore, it is crucial that the current methods are improved or newly developed to become faster, more robust, and accurate in NGS applications. Targeted sequencing, focusing on small but important gene sets or genetic regions, is a very powerful approach to screen disease-related key genes. Reductions in cost and experimental time as well as availability of targeted sequencing are fueling the use of NGS for many genetic applications.

We have developed a new, simple, and robust sample preparation method called 'NextDay Seq,' enabling us to obtain targeted deep sequencing data within the next day of sample arrival. Researchers and clinicians can obtain sequencing data within 36 hours. Our method starts with a 15 minute DNA extraction kit followed by library preparation, sequencing, and then data analysis. The NextDay Seq library preparation method directly ligates adapters to targeted genes/amplicons. This new protocol does not require restriction enzyme digestion (i.e. Ion Torrent Ampliseq) or hybridization of the target region (i.e. Illumina TruSeq). It is faster and simpler than previously referenced library preparation kits. We have also developed a new data analysis tool called DanPA: a fast, accurate, and robust NGS data analysis tool. DanPA, yet developed mainly for targeted sequencing analysis, can also be used for whole exome or genome sequencing data analysis. DanPA detects any kind of reported mutations registered in the database such as Catalogue of Somatic Mutations in Cancer (COSMIC). We have so far analyzed 866 cancer FFPE tissue samples, 431 flash-frozen tissue samples, 18 cell lines samples, and 515 other biological or synthesized (i.e. plasmids) samples using these new inventions.

Our system will help clinicians in better deciding which therapeutic options (personalized medicine) or biological applications are optimal to treat patients with specific mutations. Cancer patients should have exact mutation information in a timely manner in order to select treatment options. By combining our new FFPE DNA extraction method, NextDay Seq library preparation method, and DanPA data analysis software, we are able to provide key mutation data to patients, medical doctors, and researchers within 36 hours (next day). We believe this new innovative NGS system can change the paradigm for mutation screening in clinical and research applications.

#3610

TRAC, a novel time and cost saving gene expression analysis technology with improved efficacy.

Laura Mattinen,1 Jani Salmivaara,1 Jussi Halleen,2 Hans Söderlund,3 Jari Rautio4. 1 _ValiRx Finland Oy, Helsinki, Finland;_ 2 _Pharmatest Services Ltd, Turku, Finland;_ 3 _Paras Biopharmaceuticals Finland Oy, Oulu, Finland;_ 4 _Biotech Start-Up Management, Helsinki, Finland_.

Gene expression analysis in the oncology space is largely performed with the classic and widely accepted methods real-time RT-PCR, microarray and next generation sequencing. These technologies are, however, too time consuming and expensive to be used in large scale screens with high sample numbers. TRAC (Transcription analysis with the aid of Affinity Capture) technology provides a solution for focused gene expression screening with unique combination of multiplex analysis, high-throughput sample processing, high precision and flexibility. Lysates of cells or tissues are directly applicable as sample material in TRAC assay without RNA extraction or cDNA conversion. The assay enables detection of up to 30 targets per sample and 96 samples per assay with 3-4h run time. In TRAC assay, each target mRNA of interest is recognized by a specific labeled ssDNA probe. Probes with different lengths and labels can be used for high-level of multiplexing. Hybridization of probes and targets takes place in solution, after which the probe-transcript complexes are captured by streptavidin coated magnetic beads. Unbound material is washed off and the probes are eluted for detection. The probes are then detected and quantified by capillary electrophoresis, which resolves the probes according to both size and fluorescence. The aim of this study was to apply TRAC assay to analyze the expression fingerprints of 20 gene markers related to angiogenesis, cell adhesion, protease activity and plasminogen activation, in cultured cells treated with different drug candidates. The expression of the target genes was analyzed from four different colon cancer cell lines (COLO, HT-29, CaCo2, DLD) to study culture age dependent effects and responses to drug candidates. Out of the 20 genes analyzed, curcumin caused consistent variation to cyclooxygenase-1 (COX-1) gene expression as a function of time in all tested cell lines. By comparison of expression fingerprints between non-treated cell line cultures, altogether four genes were identified as being expressed at higher level in one or two of the cell lines compared to the others. These genes were trypsinogen (PRSS1-3) in COLO, Serine protease inhibitor (SPINK) in HT-29, Protease Cathepsin B (CTSB) in COLO and cyclin D1 (CCDN1) in DLD and CaCo2 cell lines. The data showed good correlation with real-time RT-PCR results and reproducibility (CV <12%) and functioned as proof-of-concept for suitability of TRAC for screening purposes. Multiplex detection with TRAC from lysates offered high efficiency and significant cost and time savings compared to single-plex qPCR assay in large scale evaluation of drug candidates.

#3611

SMRT® sequencing of DNA samples extracted from formalin-fixed and paraffin embedded tissues.

Primo Baybayan,1 Michael Weiand,1 Kevin Eng,1 Guillaume Durin,2 Steve Kujawa1. 1 _Pacific Biosciences, Menlo Park, CA;_ 2 _Covaris, Inc, Boston, MA_.

Recent advances in next generation sequencing have led to the increased use of formalin-fixed and paraffin-embedded (FFPE) tissues for medical samples in disease and scientific research. Single Molecule Real-Time (SMRT®) sequencing offers a unique advantage in that it allows direct analysis of FFPE samples without amplification. However, obtaining ample long read information from FFPE samples has been a challenge due to the quality and quantity of the extracted DNA. DNA samples extracted from FFPE often contain damaged sites, including breaks in the backbone and missing or altered nucleotide bases, which directly impact sequencing and amplification. Additionally, the quality and quantity of the recovered DNA also vary depending on the extraction methods used.

We have evaluated the Adaptive Focused Acoustics (AFA™) system by Covaris® as a method for obtaining high molecular weight DNA suitable for SMRTbell template preparation and subsequent single molecule sequencing. Using this method, genomic DNA was extracted from normal kidney FFPE scrolls acquired from Cooperative Human Tissue Network (CHTN), University of Pennsylvania. Damaged sites present in the extracted DNA were repaired using a DNA Damage Repair step, and the treated DNA was constructed into SMRTbell libraries suitable for sequencing on the RSII System. Using the same repaired DNA, we also tested PCR efficiency of target gene regions of up to 5 kb. The resulting amplicons were constructed into SMRTbell templates for full-length sequencing on the RS II System.

We found the Adaptive Focused Acoustics (AFA™) system by Covaris® to be effective and efficient. This system is easy and simple to use, and the resulting DNA is compatible with SMRTbell™ library preparation for targeted and whole genome SMRT sequencing. The data presented here demonstrates single molecule sequencing of DNA samples extracted from tissues embedded in FFPE.

#3612

Challenges in variant searching and annotation for clinical cancer testing.

Jennifer Yen, Sarah Garcia, Michael Clark, Steve Chervitz, Brian Linebaugh, Aldrin Montana, John West, Richard Chen, Deanna Church. _Personalis, Menlo Park, CA_.

Cancer testing is undergoing a revolution. While small gene panels have previously dominated the landscape, whole exome (WES) based strategies have recently emerged as valuable tools for identifying mutations with clinical significance and are being rapidly adopted into routine clinical cancer care. Assessing the significance and actionability of these variants is largely dependent on the growth of public repositories of germline and somatic variants, such as ExAC, COSMIC, TCGA and ClinVar. However, efforts to integrate data from these various sources have revealed numerous challenges. One of these challenges is the generation and use of variant syntax in a standardized, unambiguous format, which is paramount for variant search for both the clinical and scientific community.

Variants can be represented in many different ways: in the VCF file format, which is the de facto standard for sequencing data; in genomic or transcript-based coordinates using HGVS nomenclature; as amino acid alterations in three- or single-letter codes according to transcript and protein definitions that can vary based on the database used. Analyses of dbSNP and COSMIC identified at least 350,000 and 27,000 variants respectively that, without "normalization", have ambiguous VCF representations (using software described by Tan et al., 2015). This indicates that one-to-one searches and annotations in VCF files may not identify an exact match in the absence of normalization.

At the coding and protein level, variants are typically reported according to syntax recommendations by the Human Genome Variation Society (HGVS). In evaluating a number of tools for generating HGVS nomenclature (snpEff, VEP, and Variation Reporter), we found challenges in reconciling syntax representation across these tools and databases. We demonstrate that variant annotation is dependent on both transcript and version, complicating comparisons between NCBI and Ensembl-based systems, such as ClinVar (NCBI) and COSMIC (Ensembl). Even given the same transcript, variants can be represented differently. Over 20% of variants output by the tools in our comparison reported different nomenclature for the same exact variant reported by ClinVar. Using a manually curated 'gold truth' set of variants, we found that as many as 75% of non-missense variants are called incorrectly by these tools. The results of our tests have significant implications for the search and annotation of variants during cancer analyses and interpretation, and serve to inform the ongoing adoption and refinement of available resources.

#3613

Complete phased sequencing of HLA genes using Linked-Reads.

Anton Valouev, David Jaffe, Neil Weisenfeld, Heather Ordonez, Adrian Fehr, Patrick Marks, Michael Schnall-Levin, Tarjei Mikkelsen. _10X Genomics, Inc., Pleasanton, CA_.

HLA/MHC genes are critically important for the efficacy of transplantation therapies, including T cell based immunotherapy. Traditionally, HLA allele typing has been performed using hybridization-based approaches, which have limited resolution and are prone to ambiguities due to highly polymorphic nature of HLA genes. More recently, next-generation sequencing (NGS)-based technologies have been employed to study the HLA region. However, short-read NGS-based approaches are limited in their ability to accurately resolve highly complex and polymorphic regions in the genome, such as HLA/MHC locus. These challenges include a lack of accurate variant phasing within and between HLA genes and limited sensitivity to structural rearrangements. We developed the GemCode Platform, which combines microfluidics and molecular barcoding with custom bioinformatics software to transform short-read sequencers into high-throughput systems providing phasing information across long (50-100 Kb) single DNA molecules. In this study, we assessed the feasibility of using the GemCode Platform to characterize the HLA region at high resolution and accuracy.

We initially analyzed HLA regions in 3 well-characterized cell lines (NA18555, NA18532, NA12878). DNA samples from these cell lines were partitioned and indexed using the 10X Genomics GemCode Platform. We used 1ng of sample DNA to partition molecules into droplets, introduce molecular barcodes and generate short-read NGS libraries for Illumina sequencing. We then used GemCode barcode-aware bioinformatics tools to recover Linked-Reads derived from single DNA molecules spanning 10's to 100's of Kb.

To obtain complete sequences of individual HLA haplotypes, we next performed local phased sequence assembly using a custom Linked Read assembler. Local assemblies of all major MHC class I genes (HLA-A, HLA-B, HLA-C) and MHC class II genes (HLA-DPA1, DPB1, DQA1, DQB1, DRB1) were performed. Assembled HLA haplotypes were compared to HLA typing results from traditional hybridization- and amplicon-based methods. Our results demonstrate that we can accurately assemble both haplotypes of each gene to identify known and novel alleles. Assembled sequences also enabled accurate phasing of SNPs throughout individual HLA genes and their adjacent regions, including homologous pseudogenes. Subsequent application of this method to the analysis of HLA regions from a larger cohort of clinically-relevant samples will also be reported.

In summary, our results demonstrate the feasibility of performing fully phased assembly of HLA gene sequences using the 10X Genomics GemCode Platform, and provide a path towards comprehensive identification and complete phasing of genic and intergenic variants across the entire HLA/MHC region using cost-efficient short read sequencers.

#3614

Comparison and evaluation of somatic mutation using PGM and proton platform in cell free DNA of non-small cell lung cancer patients.

Jae Sook Sung,1 Jong Won Lee,1 Boyeon Kim,1 Saet Byeol Lee,1 Nak-Jung Kwon,2 Won-Chul Lee,2 Hae Mi Kim,2 Won Jin Jang,3 Yun Ji Choi,3 Kyung Hwa Park,3 Yeul Hong Kim3. 1 _Cancer Research Institute, Korea university, Seoul, Republic of Korea;_ 2 _Macrogen, Seoul, Republic of Korea;_ 3 _Division of Oncology/Hematology, Department of Internal Medicine, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Republic of Korea_.

Non-small cell lung cancers (NSCLC) are characterized by a unique pattern of genetic driver mutations, and some of mutations may be used to predict prognosis and targeted treatment such as EGFR TKIs. Cell free (cfDNA) present in the blood stream shows much potential as a useful cancer maker for early diagnosis and cancer progression monitoring. Especially, analyzing the cfDNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. However, there are still no standardized methods to identify mutations in cfDNA. In this study, we examined the viability of PGM and Proton platforms. Ion AmpliSeq Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from NSCLC patients. The on target was 88% and mean depth was 643x using PGM platform. And, the on target was 92% and mean depth was 22,868x in Proton platform. To validate the results of two NGS platforms, we analyzed EGFR status by sanger sequencing in available 100 tumor tissues. EGFR mutations were identified in 34 (34%) by sanger sequencing. EGFR mutations were identified in 32 (25.6%) and 28 (22.4%) by PGM and Proton platform. Interestingly, out of 34 mutations of tumor tissue, EGFR mutations were matched to 11 and 26 in PGM and Proton platform, respectively. These results showed concordance of 76.5% between the tDNA (sanger sequencing) and cfDNA (Proton). In addition, KRAS (codon 12 and 13) mutations were 4 (3.2%) and 18 (14.4%), respectively. In our study, we demonstrated that Proton platform of high depth is a useful assay to identify somatic mutations of cfDNA in NSCLCs. [This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0066)]

#3615

Comparison of whole genome amplification methods on single and pooled cells for comparative genomic hybridization array analysis.

Rachel H.V. Needham,1 Arturo B. Ramirez,1 Iman Kishawi,2 Jackie L. Stilwell,1 Eric P. Kaldjian1. 1 _RareCyte, Inc, Seattle, WA;_ 2 _Agilent Laboratories, Santa Clara, CA_.

Background: Because blood can be easily sampled repeatedly over the course of disease, circulating tumor cells (CTCs) offer an opportunity to measure phenotypic changes during treatment permitting adjustments to therapy as cancer evolves and/or develops resistance. Since cancer therapeutics are often selected based upon molecular information, reliable, high quality methods for whole genome amplification (WGA) are required for single and small pools of CTCs. Here we compared REPLI-g (QIAGEN) and PicoPLEX (Rubicon) WGA methods using picked model CTCs (SKBR3 cells) employing the AccuCyte-CyteFinder-CytePicker system (RareCyte) and subsequent molecular analysis by array comparative genomic hybridization (aCGH).

Methods: We compared aCGH performed on WGA products from single and pooled SKBR3 cells processed with the typical AccuCyte protocol, or an alternative protocol using live cells in suspension. For the typical AccuCyte protocol, isolated buffy coat was spread and dried onto microscope slides, fixed and antibody stained (EpCAM, cytokeratin, CD45) on an automated instrument. Alternatively, pure, live SKBR3 cells were stained in suspension for EpCAM and Her2, and then placed into well slides. Slides were imaged using the CyteFinder digital fluorescence scanning microscope. Fixed SKBR cells were identified by positive nuclear, EpCAM, and CK staining, and negative CD45 staining. Live cells were identified by positive EpCAM/Her2 staining. Cells were picked into PCR tubes using the CytePicker module. DNA from individual or pools of cells (2, 3, 5, or 10) was amplified using either the REPLI-g or PicoPLEX WGA kits and processed for aCGH using Genetisure arrays (Agilent). WGA products were hybridized against matched WGA controls from normal male genomic DNA.

Results: aCGH profiles for pools of 10 cells using both WGA methods were very similar to unamplified SKBR3 genomic DNA. Signal to noise (S:N) was significantly higher with the REPLI-g amplified DNA compared to PicoPLEX, but single cells did not show similar profiles of genomic aberrations to unamplified genomic DNA until at least 3 cells were pooled before amplification. In contrast, single cells amplified using PicoPLEX demonstrated genomic aberrations consistent with unamplified genomic DNA. Fixation did not appear to adversely affect PicoPLEX WGA performance, but DNA from fixed cells could not be amplified with REPLI-g.

Conclusions: Diagnostic decisions can potentially be made from molecular data derived from small numbers of CTCs requiring WGA methods that provide accurate results in downstream analysis. WGA methods should be selected based upon the molecular data required. In this study, higher S:N indicate that REPLI-g may be more sensitive for finding smaller genomic changes than PicoPLEX, however PicoPLEX generates more consistent data with single fixed cells.

#3616

Fix the fixation: effect of formalin fixation on targeted sequencing, variant calling and gene expression.

Ravi Alla,1 Shujun Luo,1 Elena Helman,1 Sean M. Boyle,1 Michael J. Clark,1 Kirk Scott,1 Parin Sripakdeevong,1 Mirian Karbelashvili,1 Deanna M. Church,1 Michael Snyder,2 John West,1 Richard Chen1. 1 _Personalis Inc, Menlo Park, CA;_ 2 _Stanford University, Palo Alto, CA_.

Tumor biopsies are often Formalin-Fixed and Paraffin-Embedded (FFPE) for histological staining, genetic testing and archival purposes. Formalin treatment preserves tissue by crosslinking proteins, but can lead to mutation of the nucleic acid bases that can be detected by next-generation sequencing (NGS) methods despite being unrelated to the cancer. Studies have shown that certain fixation protocols are compatible with high quality nucleic acid isolation and variant calling by NGS. These studies used tissue fixed with 10% neutral buffered formalin for 24 hours, a standard protocol in the pathology field. In practice, however, FFPE sample handling varies from site-to-site. As we processed FFPE samples from various sites, we found that the quality of the isolated nucleic acids and subsequent sequencing results varied substantially. We hypothesized that this is due to deviations from the standard protocol such as improperly buffered reagents, variation in the fixation time and temperature, microwaving, and varied storage conditions of the samples.

To understand the role of formalin fixation on the quality of the isolated nucleic acids and subsequent NGS analyses, we subjected the widely studied cell line NA12878 to various formalin fixation conditions. We then performed an augmented target enrichment and sequencing assay on both the DNA and RNA isolated from fresh frozen (FF) and formalin fixed (FFPE) NA12878 samples. We assessed raw nucleic acid quality, library quality, alignment rate, duplication rate, on-target efficiency and variant concordance between FF and FFPE.

We noted substantial negative effects on sequencing library quality associated with sample incubation in unbuffered formalin, at high temperatures, and long periods of time (e.g. 3 days rather than 1 day). These harshly treated samples also showed poor alignment qualities, higher duplication rates, and lower mapping qualities. At the small variant level, they showed an increase in global C>T deamination (CG->TA, 50% FFPE vs 35% in FF) and oxidation (CG->AT, 15% FFPE vs 10% FF) rates. Variants caused by formalin fixation were most commonly detected at low (<5%) allele frequencies (AFs). When compared to gold set mutations in NA12878, we observed 80% false negative and 60% false positive variants in harshly treated samples. We are performing a similar analysis with RNA isolated from FF and FFPE NA12878 samples to understand the role of formalin on RNA variant and gene expression assays.

Using the results of this study, we intend to provide guidance on optimal fixation conditions for NGS applications. It is crucial to fix tissues in buffered formalin for less than 24hr at room temperature to preserve nucleic acid integrity and amenability for downstream NGS assays. We also demonstrate how a deeper understanding of the effects of formalin can improve variant calling from FFPE tissues, especially at lower AFs where formalin-related errors have the greatest impact.

#3617

Digital sorting rescues low-cellularity tumor FFPE samples for genome-wide copy-number profiling.

Claudio Forcato,1 Julie Lalibertè,2 Chiara Bolognesi,1 Ivana Cataldo,3 Genny Buson,1 Chiara Mangano,1 Cinzia Cantù,3 Francesca Fontana,1 Paola Tononi,1 Gianni Medoro,1 Tim Harkins,2 Rita T. Lawlor,3 Aldo Scarpa,3 Nicolò Manaresi1. 1 _Silicon Biosystems, Bologna, Italy;_ 2 _Swift Biosciences Inc, Ann Arbor, MI;_ 3 _ARC-NET Applied Research on Cancer Centre, University and Hospital Trust of Verona, Verona, Italy_.

Introduction: The analysis of formalin-fixed paraffin embedded (FFPE) cancer specimens is particularly challenging due to minute amount of tissue, low-tumor cellularity and heterogeneity, associated with low quality DNA. This results in an imprecise characterization of somatic variants and copy-number alterations (CNA). Here we show how digital sorting combined with low-pass whole genome sequencing (WGS) can resolve genome-wide copy-number profiling even for the more challenging FFPE samples.

Methods: An archival FFPE sample from one pancreatic cancer patient, rejected by the International Cancer Genome Consortium (ICGC) criteria due to low tumor content (<25%), was dissociated into a single cell suspension. Using the DEPArray™ digital sorter, tumor and normal stromal cell subpopulations (range=70-77) were recovered using Keratin/Vimentin immunofluorescence and DNA ploidy, from which 0.4-0.5ng DNA was obtained. After lysis, Illumina®-compatible libraries were prepared from one pool of pseudo-diploid sorted tumor cells, one pool of stromal cells and one unsorted sample (1500 cells, i.e. about 10ng gDNA) using the Accel-NGS® 2S DNA Library Kit from Swift Biosciences. Libraries were used for low-pass whole genome sequencing on Illumina® MiSeq. Paired-end (2x100) reads were aligned to hg19 human reference using BWA software and copy-number profiles were generated with Control-FREEC.

Results: A total of 11M paired-end reads and a mean coverage of 0.12x were obtained from low-pass WGS, enabling the detection of CNAs at good resolution. The resulting copy-number profiles clearly show the difference between the stromal and tumor populations, with the first characterized by a flat profile (0 gains, 0 losses) and the second presenting several somatic copy-number alterations (6 gains, 22 losses). As expected due to dilution by normal cells, CNA profiles of unsorted samples missed most of the gains and losses (only 5/28=18% of aberrant genomic regions were detected). At a single base level, the difference between unsorted and sorted samples was more significant as the unsorted fraction missed >99% of CNA bases found in the sorted tumor population.

Conclusions: DEPArray™ sorting combined with Accel-NGS® 2S kits and Illumina® low-pass whole genome sequencing enables high quality genome-wide profiling of pure tumor and stromal populations. The capacity of this method to deal with low DNA input (0.5ng) from archival FFPE samples characterized by low cancer cell content (<25%) makes it a valuable and easily accessible tool for studying tumor CNA profiles.

#3618

NGS-based detection of FLT3-ITDs with Anchored Multiplex PCR.

Marc Bessette, Benjamin Van Deusen, Michael Banos, Laura Johnson, Aaron Berlin, Erik Reckase, Joshua Stahl, Abel Licon, Brian Kudlow. _ArcherDX, Boulder, CO_.

FLT3 internal tandem duplications (ITDs) are found in > 20% of pediatric and adult acute myeloid leukemia (AML) cases and are generally associated with a poor prognosis. Nevertheless, detection of FLT3-ITDs presents a challenge to NGS-based approaches, as many variant callers fail to identify the highly variable repeated sequences associated with FLT3-ITDs.

We have developed a novel targeted assay, the Archer™ VariantPlex™ Core AML Panel, that utilizes Anchored Multiplex PCR (AMP™) with probes in multiple locations proximal to exons 13, 14, and 15 of FLT3. These exons encompass the commonly mutated juxtamembrane domain and tyrosine kinase domain 1. This panel yields high-complexity, dual-strand coverage of known FLT3-ITD locations. Because AMP probes, unlike traditional PCR probes, function independently of each other, we are able to produce multiple overlapping "snapshots" of the region of interest, thereby enhancing the ability to confidently identify complex mutation types. The sequenced reads are assembled using a novel de novo assembly algorithm in the Archer Analysis bioinformatics pipeline, in which the resulting consensus is annotated and events that involve FLT3 exons 13 14 or 15 are marked as FLT3 abnormalities.

In order to assess Archer Analysis and the VariantPlex Core AML panel, we tested it on >20 patient-derived samples with known FLT3-ITDs. In addition, we generated over two thousand in silico datasets, representing the spectrum of known ITDs, to further test our panel design and analysis algorithm. Each in silico dataset was constructed to simulate reads originating from probes in VariantPlex Core AML Panel. This methodology permitted rigorous optimization of both the VariantPlex Core AML Panel and the analysis algorithm.

The VariantPlex Core AML Panel, in conjunction with our novel detection algorithm, showed both exceptional sensitivity and specificity in the detection of FLT3-ITDs. FLT3-ITDs were successfully identified in all patient samples tested, and no false positives were detected. Our in silico datasets showed similarly high sensitivity and specificity.

These data indicate that AMP libraries targeting the FLT3-ITD region are ideal for the detection of this complex mutation type, largely because of the overlapping and anchored read structure. Therefore, the Archer VariantPlex Core AML panel, in conjunction with Archer Analysis represents a reliable platform for the detection of FLT3-ITDs, in addition to other mutations commonly found in AML.

#3619

Copy number estimation of cancer samples with genome, exome and targeted panel next generation sequencing.

Soheil Shams, Andrea O'Hara, Zhiwei Che. _BioDiscovery, Inc., Hawthorne, CA_.

Next-generation sequencing (NGS) is mainly used to obtain sequence variants (SNVs). However, obtaining copy number results from NGS has gained momentum in both research and clinical applications. While microarray has traditionally been the gold standard for copy number analysis, NGS is rapidly gaining momentum as the first line analysis for tumor samples. The genomic content captured may vary from batch to batch, where targeted regions may include the full genome, all exonic regions or may be limited to exonic regions in specific genes that have a clear diagnosis, are actionable with prescription drugs or compounds in clinical trials, or have known impacts on prognosis or outcome. Thus, obtaining copy number information from a range of NGS methods is needed. As a result, we have developed a method called BAM (pooled reference) which only requires the loading of BAM files and the NGS design file to generate copy number results. BAM (pooled reference) can derive copy number results from Whole Genome Sequencing (WGS), Whole Exome Sequencing (WES), and targeted panel NGS data. This method builds a reference file out of a pool of BAM files that are either from normal controls or from unrelated experimental samples, then generates copy number calls for each experimental sample. In combination with the VCF files that contain the sequence mutations, an integrated analysis of both events can be easily carried out. To test this algorithm, five colon adenocarcinoma whole exome sequencing samples were processed through the BAM (pooled reference) algorithm in Nexus Copy Number 8.0. GC correction schemes based on a range of window size and presence or absence of GC probe content were applied to the data and assessed for overall quality. Differences in overall read-depth resulted in variable sample quality across the cohorts, however most sample quality was adequate for copy number estimation and a quality threshold was assessed. Among the samples tested, the best quality after GC correction comes from the 50kb region size with or without the probes. Next, the copy number profiles of the TCGA COAD samples from WES and microarray were compared for accuracy. Using microarray results as a reference for assessing calls greater than 5 MB, no false positive and one false negative call were observed; the single false negative call was attributable to low-level mosaicism in the tumor sample. Results indicate that relative copy number can be estimated and is comparable to the results achieved with microarray for the same targeted regions. This analysis series was then repeated using a secondary cohort of unrelated samples subjected to microarray and targeted panel NGS to validate results. The BAM (pooled reference) method has been tested in a variety of cancer samples. This is an ideal tool for copy number estimation with NGS results in cancer samples because it provides a way for non-pair matched analysis with genome, exome and targeted NGS.

#3620

Enhancing clinical utility of NGS with reduced bias, low DNA input, library construction.

Lynne Apone, Pingfang Liu, Vaish Panchapakesa, Deyra Rodriguez, Karen Duggan, Krishnan Keerthana, Nicole Nichols, Yanxia Bei, Julie Menin, Brad Langhorst, Christine Sumner, Christine Chater, Joanna Bybee, Laurie Mazzola, Danielle Rivizzigno, Fiona Stewart, Eileen Dimalanta, Theodore Davis. _New England Biolabs, Inc., Ipswich, MA_.

Early detection and diagnosis of cancer substantially increases the likelihood for successful treatment. Tools that aid in detecting and diagnosing cancer early, therefore, have the potential to greatly impact the clinical outcome for cancer patients. Next Generation Sequencing (NGS) has emerged as an important tool in this area. The technology is sensitive, fast and high throughput to allow sequencing of many samples at once. Unfortunately, many clinical samples go unanalyzed because they do not yield sufficient quantities of DNA to generate NGS libraries or the libraries generated require so many rounds of PCR amplification that they display extreme sequence bias. Bias not only hampers data analysis, but also increases costs by requiring excess sequencing to obtain sufficient coverage over all relevant genomic regions.

To enable the increased use of NGS in the clinic and reduce the amount of sequence bias generated during library preparation, we have developed a PCR free library construction method that uses low quantities of DNA as input. As an initial test of the method, we generated PCR free libraries from 100ng, 50ng and 25ng of human genomic DNA. The libraries where pooled and sequenced on the Illumina NextSeq 500 instrument to approximately 10X coverage. All libraries, irrespective of input amount, showed minimal AT/GC bias and excellent coverage distributions, with most bases covered within 5X of the expected coverage depth. In addition, regions identified as difficult to sequence (Aird, D., et.al., 2011 and Ross, M. G., et.al., 2013) showed coverage at near expected levels for all libraries. This method can easily be adapted for use with extremely low DNA inputs by the introduction of a minimal number of PCR cycles. In fact, we have used this method to construct high quality NGS libraries with picogram quantities of DNA input.

Standard library construction methods require DNA inputs of 2ug to 500ng when PCR amplification is omitted. This new method utilizes inputs as low as 25ng to generate high-quality PCR free libraries and picogram quantities when amplification is performed. We are currently investigating the possibility of reducing input levels further and exploring the limits of the method with low quality DNA samples. Interestingly, we have observed substantial sample loss during DNA shearing and reaction cleanup. Samples that do not require fragmentation, such as DNA isolated from plasma (cfDNA) and low quality FFPE DNA, may reduce the input requirements even further. Finally, this new method utilizes low sample and reagent volumes, possibly paving the way for its use in microfluidic devices.

#3621

High efficiency detection of low frequency alleles in cell-free DNA.

Kamran Shazand, Jing Ning, Anthony Popkie, Egon Ranghini, John Paul Jerome. _Rubicon Genomics Inc., Ann Arbor, MI_.

Cancer is a genetic disease, caused by a variety of alterations to a cell's genome. One specific type of alteration associated with cancer is point mutations, and Next-generation sequencing (NGS) is a key tool to identify those mutations. Unlike inherited mutations, cancer mutations can be found at very low frequencies, mainly because of tumor heterogeneity and their dilution in the germline background. Cell-free DNA (cfDNA) derived from blood plasma, is a promising material for cancer research and diagnosis, and can be readily obtained via non-invasive procedures. However, the amount of tumor DNA and, hence, the amount of rare allele in the cfDNA are typically very low. Sensitive methods are therefore needed for the detection of somatic, cancer-associated mutations in cfDNA isolated from liquid biopsies to support both discovery and clinical decision making in oncology.

To overcome these challenges, we developed at Rubicon Genomics an NGS library preparation kit, namely ThruPLEX® Plasma-seq, optimized specifically for cfDNA at input levels from less than 1 ng to 30 ng. We designed experiments to determine the lower limit of SNV detection under these assay conditions. Six individual ThruPLEX® Plasma-seq libraries generated from 10 ng cfDNA were enriched with the Agilent SureSelect XT2 reagents and ClearSeq Comprehensive Cancer panel (151 genes). After sequencing to >500X coverage, SNP calling was performed using Agilent SureCall software to identify the SNPs specific to each sample. In a second step, the cfDNA isolated from two of these plasma samples with known SNP differences were mixed to generate various spike-in frequencies (10%, 5%, 2% and 0.5%) prior to preparing libraries and enrichment. This is to simulate low allele frequency, and to determine the sensitivity of ThruPLEX® Plasma-seq under the outlined assay conditions.

Our results showed that it was possible to detect specific alleles down to an actual 0.6% with the theoretical 0.5% spike-in ratio. The percent of ontarget bases was over 45%. The sensitivity (SNPs detected/total SNPs analyzed) of our test was above 97% for 10%, 5%, and 2% frequencies and 70% for 0.5%. This is a demonstration of ThruPLEX® Plasma-seq to produce libraries with high enough complexity to detect low frequency alleles from limited amounts of cfDNA from plasma.

#3622

Complete workflow for detection of low frequency somatic mutations from cell-free DNA using Ion Torrent™ platforms.

Jian Gu,1 Dumitru Brinza,2 Ann Mongan,2 Richard Chien,2 Dalia Dhingra,2 Fiona Hyland,2 Kelli Bramlett1. 1 _Thermo Fisher Scientific, Austin, TX;_ 2 _Thermo Fisher Scientific, South San Francisco, CA_.

Research detecting of somatic mutations in circulating cell-free DNA (cfDNA) using research blood samples from subjects previously diagnosed with cancer provides a potential non-invasive approach to monitor cancer status and evaluate cancer evolution in the future. However, most of the existing mutation detection methods show insufficient sensitivity to detect cfDNA mutations since only small amount of mutant gene fragments, derived from tumor cells, is present in a large amount of normal circulating DNA background.

We demonstrated a complete workflow that includes blood collection, cfDNA isolation, library preparation, sequencing, and data analysis to enable detection of rare DNA variants in blood plasma samples. Blood samples were collected using Streck™ DNA tubes followed by plasma preparation and cfDNA isolation using MagMAX™ Cell-Free DNA Isolation Kit. Library preparation was performed using Oncomine™ lung cfDNA kit. Barcoded libraries were pooled and sequenced on Ion Torrent™ Next Generation Sequencing Platforms. Sequencing data was analyzed in Torrent Suite™ using variantCaller-cfDNA plugin. ~150 biomarkers relevant to non-small cell lung cancer were interrogated in one sequencing run.

We demonstrated detection sensitivity at 0.1% frequency using engineered mutants that were spiked into control DNA samples. The workflow was tested on a set of research samples from matched tumor FFPE and blood plasma collected from research subjects with non-small cell lung cancer (NSCLC). About 1 mL of plasma was processed using the workflow described above. RecoverAll™ Multi-Sample RNA/DNA Isolation Workflow was used to isolate DNA from FFPE samples, followed by library preparation, sequencing and data analysis using the same workflow described above. Summary of variant calls from matched cfDNA and FFPE tumor samples are presented here. Results indicate high sensitivity of the workflow and expected levels of concordance between variants detected in the two types of research samples.

In this study, we developed a highly sensitive and reliable research workflow to detect rare somatic mutations in circulating cfDNA samples. Significant overlapping of mutations discovered in FFPE tumor and cfDNA samples suggests that this workflow may be used to monitor tumor dynamics in NSCLC and potentially other tumors in the future.

Disclaimer: For research use only. Not for use in diagnostic procedures.

#3623

Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay.

Jonathan Choi. _Roche Sequencing, Pleasanton, CA_.

Circulating tumor DNA can be found in the plasma of cancer patients, and can be utilized for non-invasive cancer detection and genotyping through Next Generation Sequencing. However, unique characteristics of ctDNA pose technical challenges that limit technical sensitivities and robustness. ctDNA are fragmented, short, rare in abundance, and found in high background of normal DNA from leukocytes. Cancer variants range as low as 0.1-1% minor allele fractions, within the noise of NGS methods. Furthermore, clinical interest includes large number of genes and mutation types including single nucleotide variants, copy number amplification, and fusions. We compared two popular target enrichment categories, digital PCR and a hybridization-based capture panel for target enrichment. We assessed the assay performance and detection limitations, using a diverse sample set to test the range of input material that reflects ctDNA found in plasma. We titrated important cancer mutations (BRAF, EGFR, KRAS, PI3KCA, etc.) testing minor allele fractions ranging from 33% down to 0.1%, MET amplification ranges of 14x to 5x, and CCDC6-RET fusions of 50% to 2%. We also assessed input DNA ranges of 1ng and 10ng, well below manufacturer recommend input of library prep methods. Testing the performance of a 29kb amplicon panel and a 114kb hybridization-probes panel, we found superior on-target rates with digital PCR compared to the hybridization capture method across all samples. However, despite higher depths of coverage with droplet PCR, hybridization based methods had superior read mapping, uniformity (1.4x better), error rate (0.16% vs 0.02%), and greater linearity of SNV variants. Hybridization methods had greater accuracy of variant detection and were able to accurately identify the RET fusion. We demonstrated the feasibility of utilizing target enrichment methods for next generation sequencing for ctDNA variant detection.

#3624

Development of a comprehensive and highly sensitive next-generation sequencing assay for detection of copy number variations.

Chia-Ling Hsieh, Clare Zlatkov, Byron Luo, Chen Zhao, Kathryn Stephens, Han-Yu Chuang, Lisa Kelly, Katherine Chang, Rachel Liang, Jianli Cao, Scott Lang, Ashley Adams, Naseem Ajili, Laurel Ball, Glorianna Caves, Danny Chou, Katie Clark, Brian Crain, Anthony Daulo, Sarah Dumm, Ridwana Ekram, Yonmee Han, Anne Jager, Suzanne Johansen, Li Teng, Jenn Lococo, Jaime McLean, Juli Parks, Jason Rostron, Jennifer Sayne, Jennifer Silhavy, June Snedecor, Mckenzi Toh, Stephanie Tong, Elizabeth Upsall, Paulina Walichiewicz, Xiao Chen, Amanda Young, Ali Kuraishy, Karen Gutekunst, Matt Friedenberg, Charles Lin. _Illumina Inc., San Diego, CA_.

Background: As our knowledge of how DNA alterations can drive cancer progression increases, assays that can simultaneously detect multiple types of variants in a simple and cost-effective manner are becoming increasingly crucial. This holds true of copy number variations (CNVs), where evaluation of this type of variant is an important and necessary feature of any solid tumor profiling assay. Conventional methods for detecting CNVs such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and quantitative PCR (qPCR) are limited to detecting only one gene amplification at a time. This can be a significant drawback with FFPE samples, where the DNA is of low abundance and often heavily degraded, while the number of gene amplifications known to be important in cancer continues to grow. Additionally, different tumor types may express amplifications at different rates and expression may be heterogeneous within the tumor, potentially with irregular staining patterns - all of which illustrate the need for new approaches to CNV detection.

Methods: Next-generation sequencing (NGS) offers the ability to assess variants in multiple genes using one sample. To that end, Illumina is developing a comprehensive, hybrid capture-based NGS assay targeting 170 key cancer genes for sequencing on the NextSeq1 platform. The assay consists of a DNA workflow for the identification of single-nucleotide variants (SNVs), small insertions and deletions (indels), as well as an RNA workflow for the identification of splice variants and gene fusions. In addition, using the DNA workflow, a novel analysis pipeline, and CNV caller, CNVs from 57 different genes can be simultaneously assessed all by sequencing of a single sample.

Results: Here we present data on both cell lines and FFPE samples of SNVs and indels down to 5% allele fraction and CNVs down to ~2-fold amplification, all from 40 ng of DNA. To demonstrate the accuracy and precision of our CNV detection method, we tested 7 samples for CNVs using orthogonal CNV detection methods. The Illumina NGS assay detected ERBB2 amplifications in 4 out of 7 samples. Of the 4 Illumina NGS positive samples, 3 samples were positive by FISH and all 4 were positive by droplet digital PCR (ddPCR) and the Illumina TruSight Tumor 15 panel. The 3 samples that were negative for ERBB2 amplifications by the Illumina NGS assay were also negative by both FISH and ddPCR. Within these samples we also found a previously unknown FGFR1 amplification.

Conclusions: The novel Illumina NGS library preparation method is an innovative and useful tool to find multiple CNVs, along with other variant types, within a single sample. The assay can detect multiple CNVs within a single FFPE sample and identify previously uncharacterized CNVs that could be important in finding the correct treatment for a cancer patient.

#3625

Beyond SNPs: Utilizing NGS methods to detect copy number alterations at high sensitivity in clinical cancer samples.

Lincoln D. Nadauld. _Intermountain Healthcare, St. George, UT_.

The emergence of novel technologies, including Next-Generation Sequencing, has enabled the identification of actionable variants in clinical specimens, which can then be targeted with molecular therapeutics in a precision medicine approach. In human cancers, Single Nucleotide Polymorphisms (SNPs) have become powerful predictors of response to certain targeted therapies. However, Copy Number Alterations (CNAs) also represent a major class of clinically actionable variants that are frequently identified in clinical cancer samples. CNAs account for more than one third of all actionable mutations. A major question in the clinical application of technologies such as NGS is whether CNAs are faithfully detected; a particularly important question given the prevalence of actionable CNAs in cancer samples. To address this question we prepared tumor samples from eleven distinct patients and compared CNA detection on two different NGS platforms. As an orthogonal method for validation, we compared the CNA detection results to Fluorescent In Situ Hybridization (FISH) performed on the same samples. We observed 20 copy number amplification events across the eleven patient samples according to FISH analysis. By comparison, our internal NGS assay, termed ICG100, detected 19/20 (94%) amplification events, whereas an external, commercially available NGS-based test detected only 12/20 (59%) amplification events. Of the seven amplification events that were not identified by the external NGS test, six were considered clinically actionable variants. These findings highlight the discrepancies that exist amongst NGS library preparation chemistries and their abilities to accurately detect CNAs. The relevance of these findings is particularly underscored by the clinically actionable nature of the variants that were discordantly detected, including a high-level ERBB2 amplification.

#3626

Establishing a robust NGS laboratory workflow and analysis pipeline for FFPE specimen RNAseq to support biopharmaceutical translational research.

Konstantinos Mavrommatis, Lauren Intagliata, Garth McGrath, Daniel Civello, Maureen Cronin. _Celgene Corporation, San Francisco, CA_.

Pharmaceutical R&D translational programs rely on analyzing DNA and RNA from patient specimens. High quality histopathology assessment is not possible from fresh and frozen tissues which are also difficult to store long term for follow up studies. FFPE preserves tissue architecture and allows easy archival storage and extracting genomic information from FFPE clinical trial samples is now commonly done, opening a variety of research possibilities. NGS is becoming the preferred approach to FFPE molecular profiling as it generates richer, more complete data than previously possible with microarray technologies.

Our translational research objective was to quantify RNA isoforms for a number of genes requiring that we control for NGS coverage and analysis pipeline biases using embedded controls and comparing multiple calling algorithms. To optimize RNAseq library construction performance using FFPE samples we compared five commercially available rRNA depletion/mRNA selection methods for reducing rRNA abundance: Ovation® (Nugen), Ribo-Zero® (Epicentre), Ribo-Zero GOLD® (Epicentre), RiboGone® (Clonetech) and TruSeq® RNA Access (Illumina). We also evaluated DV200 as a performance metric for predicting NGS library quality for FFPE samples using TruSeq® (Illumina) and SMARTer® (Clonetech) library preparation methods. Finally, we compared RecoverAll® (Life Technologies) and Allprep® (Qiagen) RNA extraction method effects on NGS library quality. By performing the experimental comparisons using two NGS service providers, we were able to assess the impact of laboratory performance on NGS RNAseq results. Ultimately, we were able to define an NGS workflow based on sample QC assessment at multiple points in the process resulting in a highly reliable RNAseq workflow for clinical NGS specimens.

Among the rRNA depletion methods, RiboGone and RNA Access outperformed alternative options. RiboGone virtually eliminated rRNA contamination from FFPE specimens with high reproducibility. RNA Access both eliminated rRNA and provided a high fraction of coding RNA sequences relative to the background of ncRNA allowing experimental designs with lower total read numbers. Depending on experimental requirements, either method is reliable; however, the two methods cannot be used interchangeably without computational normalization. The two library preparation and RNA extraction methods performed similarly in this experiment. DV200 was found to be an good metric for evaluating the quality of FFPE RNA and predicting success in generating RNAseq data.

Our experience across NGS vendors showed that to get optimal results, vendor performance must be managed by using layers of controls with agreed on laboratory performance metrics and explicitly setting NGS experimental parameters in advance of performing the work.

#3627

Automated, purification-free, Ion AmpliSeq™ library preparation from FFPE samples using the Ion Torrent™ Ion Chef™ System.

Tanya Biorac, Michael Allen, Francis B. Peters, Cristina Van Loy, Kate Rhodes, Mark Andersen. _Thermo Fisher Scientific, Carlsbad, CA_.

The ability to produce high quality libraries from FFPE samples is ever more important for next-generation sequencing. Quick turnaround time from sample to answer and simple protocols are critical components for this process. The Ion Chef and Ion AmpliSeq Kit for Chef DL8 is an automated system that has proven to reliably generate quality libraries from purified high molecular weight genomic DNA, in addition to formalin-fixed paraffin embedded (FFPE) DNA samples. We have further developed a simple and robust protocol for preparing DNA libraries from FFPE samples with no nucleic acid purification required. Our protocol is compatible with all Ion AmpliSeq panels and generates eight balanced libraries on the Ion Chef Instrument in under 8 hours, with less than 1 hour hands-on time. We observed equivalent or better sequencing performance using this method compared to a 16-hour manual FFPE DNA extraction protocol. This method has shown to work with low, medium, and high input FFPE samples from different tissue types on a single run. We evaluated a variety of FFPE samples using 1 or 2 pool Ion AmpliSeq DNA research panels containing assays designed to known oncogenes. We were able to generate and successfully sequence DNA libraries from a single FFPE slice as small as 2 mm X 2 mm. Minimal sample handling is an advantage for the NGS platform with this robust protocol.

For Research Use Only. Not for use in diagnostic procedures.

#3628

Improving sequencing quality of libraries prepared from FFPE DNA.

pingfang liu, Lixin Chen, Laurence Ettwiller, Christine Sumner, Fiona J. Stewart, Eileen T. Dimalanta, Theodore B. Davis, Evans C. Thomas. _New England Biolabs, Ipswich, MA_.

Targeted cancer therapy based on genomic alterations can be remarkably effective, and has made significant strides with the recent advances in next-generation sequencing (NGS) technology. Although samples of blood and other bodily fluids are being actively explored for early disease diagnosis and treatment monitoring, DNA isolated from FFPE samples is currently the main source for NGS-based cancer profiling in clinical settings. Unfortunately, sequencing DNA from FFPE samples is challenging due to limited quantities and poor quality, a result of DNA damage incurred during fixation and storage. Artifacts associated with FFPE DNA have limited the mutation detection sensitivity to ≥ 5% mutant allele frequency (Frampton et al, Nature Biotechnology 2013), which would unfortunately leave many low-abundance genetic variants of clinical significance undetected. For example, clinical resistance-causing KIT and EGFR mutations can be present in tumors at levels << 1% (Milbury et al, Clin. Chem. 2012).

In this study, we investigated the effects of DNA repair and different sample handling workflows on sequencing quality of libraries prepared from FFPE samples. Careful analysis of sequencing data showed that base calling qualities for all 4 bases are improved, and aberrant G:C to A:T mutations were significantly reduced upon DNA repair. Because the large majority of mutations encountered in human tumors are G:C to A:T mutations (Greenman, C. Nature 2007), we expect that lowering the damage induced background noise of FFPE DNA would allow more reliable detection of clinically important, actionable mutations at lower abundance. In addition, we observed specific sequencing artifacts associated with the method of handling FFPE samples and have since identified effective measurements to avoid such artifacts. We expect that these improvements in sequencing quality of FFPE samples would ultimately enable more sensitive and robust detection of many low level genetic variations in clinically and biologically relevant cancer genes.

#3629

Gene fusion detection in sarcoma using TRAC technology.

Laura Mattinen,1 Jari Rautio,2 Jani Salmivaara,1 jussi Halleen,3 Marko Sirkiä4. 1 _ValiRx Finland Oy, Helsinki, Finland;_ 2 _Biotech Start-Up Management, Helsinki, Finland;_ 3 _Pharmatest Services Ltd, Helsinki, Finland;_ 4 _GenoMill Health Oy, Turku, Finland_.

Genomic rearrangements are a defining feature of cancer, and can lead to gene fusions and chimeric transcripts with respective fusion proteins that have oncogenic properties. The fusion transcripts can have diagnostic, prognostic and therapeutic implications. However, the fusion partners and the exact breakpoints vary from patient to patient and from cancer type to another. This can result in significant sequence variation, and together with the large number of potential genes able to participate in fusion events makes reliable detection of fusion events challenging for many traditional methods. The TRAC (Transcript Analysis with aid of Affinity Capture) method solves a number of these issues by a combination of specific capture and detection probes. By capturing all known 5' fusion partners and detecting potential 3' partners for a given cancer type, only a very limited set of positive signals are detected from an individual sample, enabling high-level of multiplexing. In practice, dozens of different fusion events can be detected per sample by using an easily modifiable pool of capture/detection probes. This effectively enables detection of all known fusion types in sarcomas from one sample without the prerequisite of knowing the exact cancer type. TRAC is a direct hybridization based assay that enables rapid quantification of multiple mRNA targets simultaneously from large number of samples. Transcript levels can be measured directly from cell or tissue lysates without the need for RNA extraction, cDNA conversion or PCR-amplification. The TRAC protocol is straightforward and enables sample analysis in a high-throughput format with little hands-on time. In this analysis, TRAC was used to detect the fusion transcripts COL1A1-PDGFB, FUS-DDIT3, and SS18-SSX1/2 from three types of sarcoma tissue biopsies. All fusion types were detected for the respective targets. In addition, twenty Ewing sarcoma cell lines were analyzed for the EWSR1-FLI1 fusion, identifying 12 cell lines positive for the chimeric transcript. Sarcoma tissue lysate was found applicable for direct TRAC analysis. These results demonstrate that the TRAC Fusion Transcript Assay is functional and capable of discriminating sarcomas based on fusion transcript expression. The assay is easy to set up and it can be run using standard laboratory equipment with little hands-on time.

#3630

**Validation of the Archer FusionPlex solid tumor panel in the JAX cancer treatment profile** TM **.**

Samantha Helm, Aleksandra Ras, Vanessa Spotlow, Kevin Kelly, Susan Mockus, Cara Statz, Guruprasad Ananda, Joan Malcolm, Gregory J. Tsongalis. _The Jackson Laboratory, Farmington, CT_.

Introduction: A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the clinical utility of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment ProfileTM (JAX-CTPTM) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay.

Methods: AFPSTP was validated using 24 samples: 5 JAX Patient Derived Xenograft (PDX) cases, 4 translocation positive controls, 2 FFPE cancer samples, 1 normal tissue sample, and 12 cell lines. Nine of the cell lines were previously identified as positive for fusion transcripts and 3 lacked detectable fusion events. The validation was executed in 5 phases: (1) confirm that AFPSTP was able to detect known fusion or lack of fusion events in characterized specimens; (2) determine inter-assay concordance; (3) determine intra-assay concordance; (4) LOD and (5) sensitivity.

Results: The fusion detection results for this validation are listed in Table 1. All but one of these fusion events was previously identified. The one novel fusion was confirmed using TaqMan RT-PCR. In addition to the expected fusions, 4 false positive events were detected, 2 due to mispriming and 2 determined to be WT read through transcripts. The fusion detection inter and intra-assay concordance was found to be 100% and the sensitivity was calculated to be 91.67% at a LOD of 5%.

Conclusion: This analysis outlines the clinical validation of the incorporation of AFPSTP into the JAX-CTPTM test system. Once incorporated, the AFPSTP assay will accomplish the goal of making JAX-CTPTM a more comprehensive somatic tumor profiling assay without affecting the current acceptable turnaround time or required input material.

List of 15 samples that were found to be fusion positive and the corresponding detected fusion.

---

HorizonDx EML4/ALK Positive | EML4 → ALK variant 1

HorizonDx RET Positive | CCDC6 → RET

HorizonDx ROS Positive | SLC34A2 → ROS1

HorizonDx Triple Positive | EML4 → ALK variant 3b

HorizonDx Triple Positive | SLC34A2 → ROS1

HorizonDx Triple Positive | CCDC6 → RET

A673 Cell Line  | EWSR1 → FLI1

VCaP Cell Line  | TMPRSS2 → ERG

KM-12 Cell Line  | TPM3 → NTRK1

RPMI-2650 Cell Line  | BRD4 → NUTM1

NCI-H716 Cell Line  | FGFR2 → COL14A1

OCI-AML2 Cell Line  | MBNL1 → RAF1

REH Cell Line  | ETV6 → RUNX1

MDA-MB-175-VII Cell Line  | TENM4 → NRG1

ASPS-1 Cell Line | TFE3 → ASPSCR1

ASPS-1 Cell Line | ASPSCR1 → TFE3

PDX1 | EML4 → ALK 3b

PDX2 | SYN2 → PPARG

|

### Genomic Technologies and Analyses

#3631

Whole transcriptome and exome targeted RNA sequencing for FFPE tumor samples from clinical trials.

Erica B. Schleifman,1 Anna Kiialainen,2 Andreas Roller,3 Sabine Bader,3 Maipelo Motlhabi,1 Priti Hegde,1 Ian McCaffery,1 Garret Hampton,1 Michael Cannarile,4 Craig Cummings,1 Olivia Spleiss,2 Eric Peters1. 1 _Genentech, South San Francisco, CA;_ 2 _Roche, Basel, Switzerland;_ 3 _Roche, Penzburg, Germany;_ 4 _Roche, Penzburg, Georgia_.

Currently there are multiple approaches for measuring gene expression from human tissue samples on the market. These methods range from measuring the expression of a few genes using quantitative RT-PCR to measuring every gene in a sample by whole transcriptome RNA sequencing (RNA-Seq). Although many techniques and products for measuring gene expression are available, the highly degraded RNA isolated from formalin-fixed, paraffin embedded (FFPE) tumor tissues still poses a challenge for many of them. FFPE tumor samples are an abundant source of biomarker information and gene expression analysis in these samples has the potential to identify new signatures, biomarkers, or diagnostics that could predict patient response to treatment. Accurately measuring gene expression in a high throughput manner from this sample type has long been a struggle in the field.

To determine the ability of RNA-Seq to accurately measure gene expression from FFPE-derived RNA, we utilized a large collection of matched fresh frozen (FF) and FFPE samples from 12 different tumor indications. By comparing the results to the matching FF sample, we were able to determine the accuracy and sensitivity of each platform when using degraded FFPE-derived RNA. The FFPE samples in the collection had a wide range of RNA quality scores (RIN score: 0-6.8, DV200:0-78) representing what is typically isolated from clinical trial samples.

We further compared whole transcriptome RNA-Seq with a hybrid capture method, RNA Access, to determine the accuracy and feasibility of using this technology on degraded RNA. RNA Access selects for protein coding transcripts by hybridization, whereas whole transcriptome RNA-seq removes ribosomal RNA, but analyzes everything else i.e. the global transcriptome. All samples were sequenced to an average of 50 or 25 million paired-end reads for whole transcriptome RNA-Seq and RNA Access, respectively and 100 ng of RNA was used as input in both assays.

We conclude that both methods can be used for analyzing FFPE tumor samples. A similar dynamic range was observed for both methods and both show similar correlation between FF and FFPE samples within the method (0.76 vs 0.74). The overall concordance between methods was 0.69 and 0.63 for FF and FFPE, respectively. When looking specifically at the detection of 90 low expressing immune related genes, both assays were able to detect >94% with an inter-platform correlation of 0.66 and 0.56 in FF and FFPE, respectively.

Whole transcriptome RNA-seq provides the maximum amount of information from clinical samples including anti-sense and non-coding RNA detection while RNA Access can be applied more cost-efficiently when one is mostly interested in the protein coding transcriptome.

#3632

Adaptive operations and technology platform for nation-scale precision oncology.

Ogan Abaan, Amrita Basu, David Deal, Michael Hultner. _Health & Life Sciences, Information Systems & Global Solutions, Lockheed Martin Corporation, Rockville, MD_.

Precision oncology requires predictive models for therapy selection using variety of biomarkers and clinical features as input. Building and validating these models requires analysis of large numbers of diverse cases in order to relate markers and treatments to positive outcomes. The -omics technologies provide a rich source of genetic and epigenetic markers but demand large compute and storage systems to process the data. Thus, there is an urgent need for scalable and reliable information systems to support nation-scale research and delivery of precision oncology.

At Lockheed Martin, we deliver operational solutions to complex problems. Here, we present our vision for a precision oncology platform. This solution integrates best-in-class capabilities from multiple sources/vendors to support innovation, research and clinical care for a whole nation. We not only thought about the basic -omics based data collection, but also an infrastructure to collect and store data within a compliant privacy and security framework that also facilitates collaborative analytics and data sharing for deeper insight.

Taking a systems engineering approach, we have examined some of the challenges to implement such a platform. For instance, running the basic genomic data processing pipelines to yield variant calls, which in turn will feed the variant store, should be a single scalable workflow. Accounting for multiple data sources, various use cases and selections of tools are at the core of an adaptable workflow. A variant store design that can scale and support a national cohort with an overlaying cohort selection tool are all part of this intricate design.

It is our vision that a systems engineering and integration approach can deliver a unified solution for the national precision oncology roadmap. It is paramount that all the individual pieces should be well tuned to deliver scalability and reliability and simultaneously work in complete harmony. Only then we can process data at-scale needed for finding actionable mutations, designing effective treatments and implementing prevention strategies, affordably and reliably.

#3633

OncoGxOneTM - an advanced genomic test for precision cancer treatment.

Yang Han, Pengfei Yu, Qingxuan Song, Min Wei, Guanghui Hu. _Admera Health LLC, South Plainfield, NJ_.

The fundamental cause of cancer is the aberration of genes that control cell proliferation and apoptosis. With the rapid advancement of genomic technologies and increased arsenal of targeted therapies, genomic profiling has been largely accepted by physicians to assist cancer diagnosis and treatment. Developed by Admera Health, OncoGxOneTM is a CLIA certified and CAP accredited genomic profiling panel detecting all actionable mutations in 64 cancer genes. All the FDA approved targeted therapies and registered clinical trials are covered by this carefully designed panel. Utilizing cutting edge Next Generation Sequencing technology combined with accurate analysis and data interpretation, OncoGxOneTM detects all 4 types of genomic alterations including single nucleotide mutation, insertion/deletion, copy number variation and gene rearrangement. Using fresh frozen tissue or routine FFPE samples, OncoGxOne TM offers both high sensitivity and specificity greater than 99% from samples with as low as 10% tumor cell content.

#3634

Simultaneous isolation of genomic DNA and total RNA using the Qiagen AllPrep® method.

Aleksandra Ras, Samantha Helm, Kevin Kelly, Vanessa Spotlow, Guruprasad Ananda, Gregory Tsongalis. _The Jackson Laboratory, Farmington, CT_.

Introduction: The purification of genomic DNA (gDNA) and total RNA (RNA) from various specimen types using a single sample is currently in high demand in genomic settings due to the limitation of available biological material. There is an increased need for simultaneous isolation of nucleic acids in cancer genetics for use in downstream applications such as genotyping, microarrays or next generation sequencing. The Qiagen AllPrep® technique provides a comprehensive solution for extraction of gDNA and RNA from a single specimen. In this study, we tested the AllPrep® kits for concurrent isolation of gDNA and RNA from five different sample types.

Materials and Methods: Genomic DNA and RNA were extracted simultaneously according to Qiagen AllPrep® manufacturer's instructions from five specimen types: bone marrow mononuclear cells (BMMC), cryopreserved cultured cell lines, whole blood, fresh frozen and FFPE samples. Quantity and quality of both nucleic acids were assessed by the NanoDrop 2000 and Qubit fluorometer 2.0. Purified gDNA from BMMC, fresh frozen, whole blood and FFPE samples was further evaluated by library preparation, targeted hybridization capture and sequencing on an Illumina NextSeq500. FastQ files were submitted to a Clinical Genome Analytics (CGA) pipeline for quality metrics analysis.

Results: The AllPrep® technique resulted in effective isolation of high quality gDNA and RNA (Table1). All set wet lab and sequencing QC metrics were met for each sample type.

Conclusion: We have successfully verified the AllPrep® method can be used for simultaneous extraction of both gDNA and RNA using the same input material without compromising the yield or purity of both final products. This simple procedure based on spin column technology not only gave the advantage of generating high quality results using limited specimen resources, but also decreased the hands on time by 3.5 hours and cost when compared to extraction done with two separate kits.

Table 1. Represents average QC results and SD for each specimen type. **n=8 for sequencing metrics

---

|

genomic DNA | genomic DNA | total RNA | total RNA | |

Sample Type | OD260/280 | Total Yield (ng) | OD260/280 | Total Yield (ng) | Mean Target Coverage | % Duplication

BMMC (n=3) | 1.85 | 3033.3 | 1.94 | 1385 | 520 | 6.34%

Whole Blood (n=2) | 1.81 | 8936.5 | 1.88 | 1335 | 566.9 | 6.47%

Fresh Frozen (n=2) | 1.69 | 31275 | 2.04 | 32375 | 345.4 | 4.02%

FFPE Specimen (n=16) | 1.66 | 5133.4 | 1.94 | 10524.1 | **748.2 | **6.10%

Cultured Cell Lines (n=5) | 1.84 | 4298 | 1.98 | 10489 | N/A | N/A

Total Average (n=28) | 1.73 | 6898.14 | 1.95 | 10443 | 624.7 | 5.92%

Standard Deviation (SD) | .24 | 9180.9 | 0.1 | 10616.2 | 228.3 | 0.013%

#3635

Performance of the Agilent D5000 and high sensitivity D5000 ScreenTape assays for the Agilent 4200 TapeStation system.

Rainer Nitsche,1 Lidia Prieto-Lafuente,2 Claire MacDonald,2 Isabell Pechtl2. 1 _Agilent Technologies, Waldbronn, Germany;_ 2 _Agilent Technologies, Edinburgh, United Kingdom_.

The Agilent 4200 TapeStation system provides automated, fast, and reliable DNA and RNA electrophoresis for up to 96 samples using prepackaged reagents and minimal manual handling. The Agilent D5000 ScreenTape and High Sensitivity D5000 ScreenTape assays have been developed for the separation and analysis of DNA fragments from 100 bp to 5,000 bp, a size range that complements and resides between the Agilent D1000 ScreenTape and the Genomic DNA ScreenTape assays. The 4200 TapeStation system and the DNA ScreenTape assays can be used at several steps of the Next Generation Sequencing (NGS) workflow. With the emergence of methodologies, such as the use of Transposomes, NGS library sizes are tending to increase beyond 1,000 bp, even for the short-read NGS technologies. This Poster focuses on the performance of both D5000 ScreenTape assays with respect to the accuracy and precision of quantification and sizing, as well as the sensitivity of these assays. Data analysis for quantification and molarity determination was compared against the corresponding assay for the Agilent 2100 Bioanalyzer system. Additionally, performance of both the D5000 and High Sensitivity D5000 assays on the 4200 TapeStation was compared to the 2200 TapeStation system.

#3636

Improved clinical variant calling and HLA genotyping with GRCh38.

Brad Chapman,1 Alison Meynert,2 Deanna Church,3 Justin Johnson,4 Oliver Hofmann5. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _University of Edinburgh, Edinburgh, United Kingdom;_ 3 _Personalis, Inc, San Francisco, CA;_ 4 _AstraZeneca, Waltham, MA;_ 5 _University of Glasgow, Glasgow, United Kingdom_.

The Genome Reference Consortium released human genome build 38 (hg38, GRCh38) two years ago and it offers major improvements over the six year old previous build, 37 (hg19, GRCh37). The new build reflects our increased understanding of the heterogeneity within human sub-populations, contains a large number of alternative genomic loci that better capture our knowledge of genome structure, and includes highly diverse human leukocyte antigen (HLA) regions enabling HLA typing.

To help with the migration of clinical sequencing workflows we provide support for variant calling on build 38, validate methods using truth sets from the Genome in a Bottle consortium and Illumina's Platinum Genome and show that feature projection approaches are a viable intermediate approach to port existing annotation resources. We show how the refined genomic representation improves mapping and variant calling by reducing the number of false positive SNP calls and improving InDel detection, and how post-processing of sequence reads mapping to HLA regions enables accurate HLA genotyping from targeted panels, exome- and WGS data. Using Omixon HLA validation data, integrated OptiType HLA calling achieved 100% accuracy on 8 targeted panels and better than 90% accuracy on 1000 genome exome samples.

This work provides a freely available, validated, ready to run pipeline making use of the improved heterogeneity representation in build 38 and demonstrating the value of moving towards a more accurate, graph-based representation of human genomes.

#3637

Targeted exome and panel analysis from low input and FFPE DNA using hybridization capture for cancer genome studies.

Sukhinder K. Sandhu, Cassie Schumacher, Laurie Kurihara, Tim Harkins, Vladimir Makarov. _Swift Biosciences, Ann Arbor, MI_.

Introduction

Hybridization-based target enrichment techniques coupled with Next Generation Sequencing (NGS) provide a useful and cost-efficient means to study disease specific target regions including whole exomes and gene panels. More than 500 whole exome analyses are behind The Cancer Genome Atlas (TCGA), which is the largest knowledge base for cancer studies including research, prevention, treatment, and care. Clinical samples may be limited in input and of compromised quality due to formalin fixation, however most current NGS library preparation methods require 50-100 ng high quality DNA for such studies. Here we present an efficient method which enables high quality target enrichment and variant calling from inputs as low as 1-25 ng.

Method

NGS libraries for hybridization capture were made from 1 to 100 ng of Coriell Hapmap samples (NA12878, etc), clinical Formalin Fixed Paraffin Embedded (FFPE) and Horizon Discovery (HDx) reference DNA (HDx 701) using the Accel-NGS® 2S Hyb DNA Library Kit. Amplified libraries were then enriched using specific targeted panels (xGen® Pan-Cancer and xGen® AML) or SeqCap™ EZ MedExome using manufacturer's specifications (IDT™ and Roche NimbleGen™). Enriched libraries were captured using streptavidin beads. Captured libraries were then amplified according to the manufacturer's specifications. Targeted panels were sequenced on an Illumina® MiSeq® using V2 chemistry and MedExome on a HiSeq® using V4 chemistry. Sequence analysis was performed with custom pipelines using BWA for alignment and GATK, Samtools, and Freebayes for variant calling.

Results

Sequencing analyses yielded a minimum average coverage of 30x and more than thirty fold enrichment. Enriched libraries exhibited significant sequence complexity with minimal duplicates and without any base-composition bias. The percent on-target varied with type of input and ranged from 50-80%. Germline variant calling results had > 99% concordance with the NIST GIAB truth list with sensitivity and precision of > 98%, even from inputs as low as 1 ng of DNA. Somatic variant calling down to 1-5% allele frequency was also evaluated at various DNA input quantities and greater depth of sequencing; results for FFPE and DNA standards will be presented.

Conclusions

Accel-NGS 2S Hyb technology can be used for whole exome and targeted enrichment studies from low quantity and low quality FFPE clinical samples. High complexity libraries with minimal bias yield high quality sequence data which enables discovery and detection of somatic variants in tumor samples to identify molecular drivers associated with different cancer types. The technique facilitates better understanding of complex cancer genomes and guide precision medicine.

#3638

A rapid, multiplexed, and highly accurate next-generation sequencing RAS Panel for FFPE colorectal samples reporting on the absence or presence of low frequency somatic variants.

Tamsen Dunn, Anita Iyer, Margaret Porter, Robert Haigis, Shannon Smith, Desiree Lee, Nitin Udar. _Illumina, Inc., San Diego, CA_.

Introduction: Next generation sequencing (NGS) is a highly sensitive method for detecting somatic mutations. Mutations in NRAS and KRAS may affect up to 50% of patients with colorectal cancer (CRC). Recent data suggest the clinical benefit of panitumumab is restricted to patients who have no tumor mutations in RAS, defined as codons 12, 13, 59, 61, 117 and 146 of KRAS and NRAS genes. An Extended RAS panel* targets these codons as a single multiplex assay. A dual strand approach distinguishes true mutations from artifacts commonly found in DNA from Formalin Fixed Paraffin Embedded (FFPE) tissue. Analytical performance of this panel was characterized.

Methods: FFPE tissue was examined for tumor content and area. DNA was extracted from FFPE tissue, cell lines and plasmids. The DNA quality and quantity were simultaneously determined through a quantitative PCR measurement of amplifiability. TruSeq Custom Amplicon technology targeting each strand of DNA was used to construct NGS libraries. Sequencing was performed on the MiSeqDx , and the Illumina Somatic Variant Caller was used to call somatic variants.

Results: The dual-strand amplicon approach results in high accuracy by eliminating false positives observed on one strand of DNA typical of an FFPE artifact, by requiring every variant to be detected on both strands. We evaluated performance on 500 FFPE CRC samples processed with the Extended RAS panel. Concordance against Qiagen TheraScreen and Sanger Sequencing results was evaluated. The somatic variant caller yields a specificity of 0.98 and sensitivity of 0.72 for variants in the 5%-10% range, and a specificity of 0.96 and sensitivity of 0.98, overall, where we take the alternate methods as truth. Cumulative FFPE tissue area influences performance of the RAS panel. FFPE of a range of tissue area were tested using 1, 3, 5, and 8 sections per sample. 80mm2 minimum (240mm2 recommended) cumulative tissue area using 5uM slices was optimal for performance and accuracy. DNA quantity and quality also influence test accuracy and sensitivity. A study with 40 FFPE specimens assayed over multiple operators and sequencers produced average coverage of 32,000 reads per target per sample, and over 93% alignable reads per sample. Reproducibility of mutations detected over multiple operators and instruments was 100%. The Extended RAS Panel demonstrates robust performance across many sources of variation, including FFPE DNA extraction method, DNA storage time, recommended DNA input range, qPCR/PCR thermocyclers with little to no impact on sample pass rate and agreement with Sanger sequencing. We also demonstrate detection of 56 distinct mutations in a single run.

Conclusion: This multiplex assay achieves high accuracy for detection of somatic mutations from DNA extracted from FFPE colorectal tissue samples.

*In development For Research Use Only. Not for use in diagnostic procedures.

#3639

Analytical performance and validation of an enhanced TAm-Seq circulating tumor DNA sequencing assay.

Davina Gale, Vincent Plagnol, Andrew Lawson, Michelle Pugh, Sarah Smalley, Karen Howarth, Mikidache Madi, Bradley Durham, Vasudev Kumanduri, Kitty Lo, James Clark, Emma Green, Nitzan Rosenfeld, Tim Forshew. _Inivata Limited, Cambridge, United Kingdom_.

Circulating tumor DNA (ctDNA) is becoming established as a tool to supplement conventional biopsies for molecular characterization and monitoring of solid cancers, especially for cancers where tumor tissue is difficult to obtain or is only available at limiting quantity. This requires reliable identification, in patient plasma, of tumor-specific DNA alterations that in some cases may be present as a small fraction of the total cell-free DNA molecules. To overcome these technical challenges, we have developed an enhanced platform for tagged-amplicon deep sequencing (TAm-Seq™). Using a combination of efficient library preparation and statistically-based analysis algorithms, this platform can be used to sequence, identify and quantify cancer mutations across a gene panel including both cancer hotspots, as well as entire coding regions of selected genes.

This poster will present validated performance specifications of this multi-gene ctDNA sequencing assay. To perform analytical validation, we used reference standards and plasma DNA controls to demonstrate the sensitivity, specificity and quantitative accuracy of this ctDNA analysis platform. We found that our workflow, using 4 mL input plasma, yields very high sensitivity for variants that are present at allele fraction 0.25% or higher in plasma, and retains substantial sensitivity at allele fractions as low as 0.1%. Using dilution mixtures of well-characterised reference samples, we show that the assay accurately quantifies allele fractions with precision predominantly limited by stochastic sampling. Analysis of plasma samples from control individuals demonstrates a low false positive rate. The assay also detects DNA amplifications (including in ERBB2, MYC, KRAS, EGFR, MET, FGFR1, FGFR2) when the ctDNA are sufficiently high.

Together, these data demonstrate the analytical validity and robustness of the TAm-Seq assay and support its use as a basis for clinical applications.

#3640

Validation of qBiomarker as an accurate and efficient mutation detection method in a comprehensive analysis of patient-derived tumor xenografts.

Mariana Brait,1 Evgeny Izumchenko,1 Luciane Kagohara,1 Samuel Long,2 Piotr Wysocki,1 Brian Faherty,1 Elana Fertig,1 Tin Khor,3 Elizabeth Bruckheimer,3 Gilson Baia,3 Daniel Ciznadija,3 Ido Sloma,3 Ido Ben-Zvi,3 Keren Paz,3 David Sidransky1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 3 _Champions Oncology, Baltimore, MD_.

Screening of patients for cancer-driving mutations is now used for cancer prognosis, remission scoring and treatment selection. Although recently emerged targeted Next Generation Sequencing (NGS) - based approaches provide accurate results and offer promising diagnostic capabilities, there are still limitations which impede their adoption for large-scale population-based screening. There is a pressing clinical need for a well-validated, rapid, cost-effective and accurate mutation profiling system with optimal analytical performance in patient specimens. Given their speed, and cost-effectiveness, real-time, quantitative qPCR mutation detection techniques are well suited for the clinical environment. The novel qBiomarker somatic mutation PCR array has high sensitivity and shorter turnaround times compared to other methods. However a direct comparison to existing viable alternatives is required to assess its true potential and limitations. In this study, we extensively evaluated a panel of 117 patient-derived tumor xenografts (PDX) by the qBiomarker array and directly compared its efficacy with that of other routinely used methods for mutation detection, including Ion AmpliSeq sequencing, whole exome sequencing (WES), and ultra-sensitive droplet digital PCR (ddPCR) genotyping. Our comprehensive analysis demonstrates that qBiomarker's performance is on par with that of other routinely used but more complex, labor-intensive and expensive methods of cancer mutation detection, and provides a foundation for advancing the adoption of qBiomarker assay for cancer driving mutation profiling in clinical diagnostics. Furthermore, a large-scale direct comparison of different mutation detection approaches will lead to informed choice of screening methodologies, especially in lower budget conditions or timeframe limitations.

#3641

ReCapSeg: Validation of somatic copy number alterations for CLIA whole exome sequencing.

Lee Lichtenstein, Betty Woolf, Alyssa MacBeth, Ozge Birsoy, Niall Lennon. _Broad Institute, Cambridge, MA_.

The presence of specific somatic copy number alterations (SCNAs) in tumor genomes can be used to predict sensitivity to specific treatments and to predict outcomes. CapSeg Revised (ReCapSeg) detects SCNAs from tumor sequencing data. The Broad Institute is integrating ReCapSeg into the Clinical Research Sequencing Platform (CRSP), a CLIA-certified platform, to enable physicians to use detected SCNAs in treatment decisions. By using sequencing data CRSP can produce SCNA reports using the same data being generated for other reports (e.g. somatic single nucleotide variants). ReCapSeg produces copy ratio estimates for regions of the genome and includes a caller that labels regions as amplified, deleted, or neutral. ReCapSeg leverages a panel of normals (PoN), which obviates the need for the matched normal for a given tumor sample.

Validation for ReCapSeg, in CRSP, is repeatable and standardized with a process for creating a PoN and gathering performance metrics from case samples. Additional QC metrics and criteria on input sequencing data were identified and included, so that reports can indicate when reduced performance can be expected.

#3643

Flow cytometry multiplexing used to analyze metabolic activity and assess pharmaceutical compound toxicity as a high throughput screening tool.

Mike OGrady, Leticia A. Montoya, Michelle Yan, Scott T. Clarke, Quentin Low, Carolyn DeMarco, April Anderson, Kathy Kihn, Carmen Finnessy, Marcy Wickett, Veronica Calderon. _ThermoFisher, Eugene, OR_.

High throughput screening (HTS) is an extremely effective method for allowing researchers to identify putative active compounds for therapeutics. Recently, flow cytometry has emerged as a powerful HTS tool with the added benefit of cell-by-cell analysis. Flow cytometry allows a researcher to study protein-protein interactions, metabolic activities, as well as DNA content in a single or multiplexed assay format. For this study, we screened the Tocriscreen Total compound library and compared HTS using a plate reader with flow cytometer multiplexing.

Peripheral blood mononuclear (PBMCs) and Jurkat cells were used as cell models. Cells were cultured under standard conditions and either at normoxic (5%) or hyperoxic (19%) oxygen levels. We also varied length of time the cells were treated with compound library (24, 48, or 72 hrs). Membrane integrity and metabolic activity were measured as an output for evaluating cellular viability. To further assess cellular activity, post-screening analysis was implemented to establish EC50s of "hits" from compound library.

Screening analysis identified target drugs that were further analyzed using cell health readouts with different mechanisms of action to assess compound toxicity. Results indicate that compound "hits" and potency differed in the screen depending on cell type, the mechanistic readout, length of compound exposure, and mode of readout used to perform the HTS experiments.

#3644

Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification.

Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. _Dana-Farber Cancer Institute, Boston, MA_.

Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis.

Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged.

#3645

Low level somatic variant detection by Sanger sequencing of formalin-fixed paraffin-embedded (FFPE) samples.

Arpad Gerstner, Edgar Schreiber, Stephen Jackson, Kamini Varma. _Thermo-Fisher Scientific, South San Francisco, CA_.

Deleterious sequence variants play an important role in the initiation and progression of many different cancer types. The detection of germline variants by the gold standard Sanger sequencing has been well established, however, the detection of somatic mutations, especially in heterogeneous tumor samples where variants may be present at a lower level, has been more challenging.

To facilitate analysis of somatic mutations in tumor samples, we have developed Sanger sequencing panels that cover the entire coding regions of specific genes implicated in tumorigenesis (e.g. TP53, KRAS and NRAS). We have also developed companion software, Minor Variant Finder (MVF), that facilitates detection of low levels of somatic mutations in Sanger sequencing studies. To demonstrate the workflow of these panels with MVF, we analyzed DNA from lung cancer FFPE samples. We initially determined variants of TP53 and KRAS in these samples using Ion Torrent™ Personal Genome Machine (PGM™) next generation sequencing (NGS). We confirmed the identity and variant allele frequency of these variants by Sanger sequencing coupled with MVF. Furthermore, we were able to confirm these results in 1 ng, 0.5 ng or 0.1 ng of DNA from these samples. Finally, we made serial dilutions of one of these samples to establish limit of detection (LOD). We show that this workflow can detect as little as 3% of a minor variant in an FFPE sample.

Sanger sequencing is the gold standard for confirmation of minor variants detected by NGS. In this study, we show that Sanger sequencing of limited number of targets, in conjunction with the MVF software, can also be an ideal first line screening choice for tumor FFPE samples where limited amount of DNA is available.

For Research Use only - Not for use in diagnostic procedures.

#3646

Highly sensitive and cost-effective detection of somatic cancer variants using single-molecule, real-time sequencing.

Steve Kujawa,1 Anand Sethuraman,1 Kevin Eng,1 Primo Baybayan,1 Lien Heyrman,2 Jurgen Del Favero2. 1 _Pacific Biosciences, Menlo Park, CA;_ 2 _Multiplicom, Niel, Belgium_.

Next-Generation Sequencing (NGS) technologies allow for molecular profiling of cancer samples with high sensitivity and speed at reduced cost. For efficient profiling of cancer samples, it is important that the NGS methods used are not only robust but also capable of accurately detecting low-frequency somatic mutations. Single Molecule, Real-Time (SMRT®) Sequencing offers several advantages, including the ability to sequence single molecules with very high accuracy (>QV40) using the circular consensus sequencing (CCS) approach. The availability of genetically defined, human genomic reference standards provides an industry standard for the development and quality control of molecular assays for studying cancer variants. Here we characterize SMRT Sequencing for the detection of low-frequency somatic variants using the Quantitative Multiplex DNA Reference Standards from Horizon Diagnostics, combined with amplification of the variants using the Multiplicom Tumor Hotspot MASTR Plus assay.

First, we sequenced a reference standard containing precise allelic frequencies from 1% to 24.5% for major oncology targets verified using digital PCR. This reference material recapitulates the complexity of tumor composition and serves as a well-characterized control. The control sample was amplified using the Multiplicom Tumor Hotspot MASTR Plus assay that targets 252 amplicons (121-254 bp) from 26 relevant cancer genes, which includes all 11 variants in the control sample. We also sequenced a second sample containing a series of mixes, each with known mutations, at levels below 10% and down to 0.01%. PCR-amplified targets were sequenced and analyzed using SMRT Sequencing to identify the variants and determine the observed frequency. The random error profile and high-accuracy CCS reads make it possible to accurately detect low-frequency somatic variants.

#3647

Multiplex digital PCR analysis of EGFR mutations for lung cancer characterization and monitoring.

Michael L. Samuels, Christina Read, Jennifer Jackson, Damien Slater, David Chappell, Mark Consugar. _RainDance Technologies, Inc., Billerica, MA_.

Lung cancer is the most common cause of global cancer-related mortality, leading to over a million deaths each year, with adenocarcinoma the most common histological type. Recently, molecularly targeted therapies have dramatically improved the treatment of patients whose tumors are driven by somatically activated oncogenes such as mutated EGFR, BRAF or ERBB2, or translocated ALK, RET, or ROS1. In addition, circulating cell-free DNA in the blood is undergoing assessment as an accessible biomarker for dynamic monitoring of treatment efficacy and clonal evolution of the cancer patient's entire tumor burden. Thus a new paradigm for monitoring and resistance of solid tumors is currently under development: sequencing the initial tumor biopsy for driver mutations followed by ultrasensitive detection of circulating tumor DNA (ctDNA) using methods such as digital PCR (dPCR).

In recent years, dPCR has become the gold standard for precise quantification of nucleic acids, showing detection sensitivities unparalleled by other methodologies. Picodroplet-formatted dPCR compartmentalizes the sample (and one or more assay) into a large enough number of discrete reaction volumes (e.g. 10 million) such that there is at most one target molecule per reaction. Standard qPCR reagents (target-specific primers and fluorescent hydrolysis probes) and master mixes are used for endpoint PCR amplification, resulting in plateaued fluorescent signals only in target-containing compartments. This enables digital counting of the absolute number of target molecules present in the sample volume tested.

Here we report the use of the RainDrop dPCR system together with multiplexed assays measuring the most common cancer-relevant mutations in EGFR. Multiplexed primer and probe sets were designed for wild-type EGFR, T790M and L858R point mutations, and exon 19 deletions in EGFR. Using spike-in plasmid controls and genomic DNA from lung cancer cell lines (H1975,H1650), we verified simultaneous detection and discrimination of all mutations across a broad dynamic range. Finally, we demonstrated application of the multiplexed EGFR assays on matched tumor and ctDNA samples from lung cancer patients.

Sensitive quantification of EGFR mutations in lung cancer patients is critical for stratifying patients into different treatment regimens, and can potentially benefit EGFR mutation-positive patients by allowing dynamic non-invasive tracking of treatment efficacy and clonal evolution. Development of a multiplexed dPCR EGFR mutation assay should facilitate personalizing lung cancer therapy and treatment management, towards the goal of monitoring disease progression for better outcomes. The RainDrop system enables sensitive simultaneous dPCR detection of multiple mutations from the same sample, which is particularly important when available sample is limited, e.g. with tumor biopsies, FFPE samples, circulating tumor cells, and ctDNA in biological fluids.

#3648

Resolving copy number variations from existing exome SNP data without the need for sophisticated software.

Marcus C. Hansen,1 Laura L. Herborg,1 Anne S. Roug,1 Sadudee Chotirat,2 Chirayu U. Auewarakul,2 Eigil Kjeldsen,1 Peter Hokland1. 1 _Department of Hematology, Aarhus University Hospital, Aarhus, Denmark;_ 2 _Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand_.

Several tools exists to evaluate copy-number variation (CNV) from sequencing read data. Here, the aim is to investigate whether larger CNVs in leukemia can be extracted directly from already called variants - without specialized algorithms.

Methods: 12 cases of leukemia, with 6 paired and 6 unpaired, were included (Table 1). Following exome sequencing and reference alignment variants were called with GATK to produce recalibrated sets in variant call format (VCF). Passed single nucleotide polymorphisms (SNPs) were limited to depth of coverage > 29x. Each chromosome were visualized as frequency scatter plot and smoothened histograms of frequency distribution in order to inspect frequency bands and number of peaks in histograms. Kolmogorov-Smirnov test was employed to compare distributions between paired samples.

Results: None of the AML cases with normal karyotype displayed CNVs and no significant changes in frequency distributions were detected. In the relapse of paired T-ALL a Chr 4 trisomy and a 33x10-6 bp copy-number neutral loss of heterozygosity (CN-LOH) on Chr 9 were detected. Collectively, 7 aberrations confirmed standard cytogenetics, 8 new aberrations were added to the cytogenetical profile and 5 could not be detected as shown in Table 1. The aberrations not detected were, as expected, balanced translocations (3) and microdeletions (2).

Discussion: No previously found large aberrations escaped detection, when disregarding translocations. In addition, other CNVs not detected by conventional cytogenetics were found by SNP analysis. Moreover, the added information is in agreement with the literature: 5% of T-ALL patients display del(11)(p12p13) and +12 is one of the most common cytogenetic changes of CLL, e.g.

Thus, we suggest that CNV profiling can be initiated by directly evaluating frequencies from existing variant calls. This may ease implementation of "closed toolboxes", enable analysis of single unpaired samples and function as a direct complement to array-CGH or FISH to detect CN-LOH.  | |  | |

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

Table 1 | Samples | Paired | Cytogenetics | CNV detected

NK-AML | 5 | Yes | Normal karyotype | None

T-ALL

relapse | 1 | Yes | 4+, 3p26.2 and Xp22.2 micro-deletions | +4, partial del(9p)

AML M0 | 1 | Yes | Normal karyotype | Partial del(11p)

AML M3 | 1 | No | t(15;17) | None

CML | 1 | No | t(9;22) | None

T-ALL | |

No | del(5)

t(7;14) | Partial del(5q), +4q, +9p, del(11p), del(12q)

CLL | 1 | No | Not analyzed | +12

AML M0 | 1 | No | Normal karyotype | Partial +11

#3649

Targeted sequencing of 409 cancer-related genes for somatic mutations and copy number variations in human cancer using the semiconductor sequencers.

Yasushi Sasaki, Ryota Koyama, Takafumi Nakagaki, Miyuki Tamura, Masashi Idogawa, Takashi Tokino. _Sapporo Medical Univ. School of Medicine, Sapporo, Japan_.

Next-generation sequencing technologies have revolutionized cancer genomics research by providing a comprehensive method of detecting somatic cancer genome alterations. In this study, we describe a next-generation semiconductor sequencing protocol for rapid (2 days), standardized, and cost-effective gene analysis for human cancer specimens including FFPE samples. DNA was extracted from 19 human cancer cell lines and 61 human cancer specimens and their corresponding non-cancerous tissues, including oral squamous cell carcinomas and multiple myelomas. Using the Ion Ampliseq Comprehensive Cancer Panel, we sequenced 15992 loci from 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers (covered regions = 95.4% of total). Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 461x, and >95% of targeted bases were represented by at least 20 reads. We also detected copy number variations in which segments of the genome can be duplicated or deleted from sequencing data. We found several genetic alterations that may have been associated with the poor prognosis and poor response to chemotherapy of cancer patients. Pathway assessment has shown that somatic aberrations within myeloma genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. This study demonstrates the utility of using a semiconductor-based sequencing to efficiently identify human cancer mutations. The targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings.

#3650

Multi-pronged approach to establish control standards for somatic mutations in next generation sequencing (NGS) oncology test.

Christopher Johnson, Sharanya Raghunath, Jackie Wayne, Aimee Shamo, Patrick Bradley, Moises Hernandez, David Loughmiller, Jason Gillman, Derrick Haslem, Gary Stone, Lincoln Nadauld, Pravin J. Mishra. _Intermountain Precision Genomics, St George, UT_.

In recent years, somatic mutation testing via Next Generation Sequencing (NGS) has emerged as a powerful tool to detect targetable mutations in diseases such as cancer. These include mutations such as BRAF V600E, which can lead to targeted drug therapy; therefore, improving the standard of care in cancer diagnostics. To generate clinically meaningful results, we need to define standards that can be applied to laboratories performing somatic testing on tumor samples. With ongoing debate about the use of positive or negative controls in somatic testing assays, we designed an experiment to test the concordance of clinically actionable mutations commonly seen in tumor testing. We use cell line controls and commercially engineered DNA to validate concordance of mutations at specific allelic ratios. Our results depict an experiment design to determine if a genomic test can detect actionable mutations with high levels of accuracy and precision.

In this study, we designed a multi-pronged approach to evaluate the need for control standards by the ICG100 clinical NGS panel. To establish a baseline negative control, we performed concordance testing on the NA12878 cell line. We compared SNPs, insertions and deletions found using our methodology to the variants reports by NIST. We found our results to be concordant with NIST at a sensitivity of 92% and a specificity of 93%. To establish a baseline for positive controls, we utilized commercially engineered DNA which contains variants spiked-in at known allelic frequencies. This allowed us to determine if the ICG100 panel was able to retrieve specific somatic mutations. We were able to detect high confidence somatic mutations, such as BRAFV600E, as well as BRAF V600G which was spiked in at a lower expect allelic frequency of 4%. Moreover, we are able to detect with high confidence mutations such as ALK F1174L, EGFR G719S, PIK3CA H1047R, and MET Y1247D, and we were able to reproduce the results across multiple cell lines. Overall, our results show the need to utilize controls as standard protocol which can help assess the proficiency of a clinical assay. With rapid advances is NGS testing methodologies, there is a need for establishing standardized controls that asses the performance of a somatic tumor testing.

#3651

Normal versus tumor and subtype prediction in renal cell carcinoma TCGA data sets.

Konstantin Volyanskyy,1 Yong Mao,1 Yee Him Cheung,1 Balaji Santhanam,1 Vlado Menkovski,2 Zharko Aleksovski,2 Minghao Zhong,3 John T. Fallon,3 Nevenka Dimitrova1. 1 _Philips Research North America, NY;_ 2 _Philips Strategy & Innovation, Research, Eindhoven, Netherlands, Netherlands; _3 _Westchester Medical Center/New York Medical College, NY_.

Background: In clinical practice it is a common challenge to correctly classify disease against normal cases and to identify disease subtypes. In complex diseases such as cancer where patterns are heterogeneous, highly complex interaction of pathways are involved, and continuous multi-level genomic changes occur, this is a challenging task. Using genomics data, analyzed by un-supervised and supervised machine learning tools, we demonstrate the ability to quantitatively and accurately describe a patient's tumor.

Design: We used Level III RNASeq gene expression data from 20531 genes in 889 tumor samples of RCC across three subtypes - clear cell renal cell carcinoma (CCRCC), papillary renal cell carcinoma (PRCC), chromophobe carcinoma (ChRCC) and 129 normal samples from the Cancer Genome Atlas (TCGA). We developed a computational framework for feature (gene/transcript) selection and subtype predictive model construction. This framework relies on a well-known "random forest" (RF) method with iterative feature selection and 10-fold cross-validation. We performed a series of (1) tumor vs normal tissue experiments for each subtype; (2) pairwise subtype comparison, and finally (3) all three subtypes comparison and predictive genes identification.

Results: In each computational run our method detected 2054 (10%) top varying genes, and estimated the predictive power of each of the selected genes using RF. On average this method demonstrated 93-97% accuracy. We identified genes known to play a role in renal cell carcinoma for example, CA9, LOX, SFRP1, SLC4A1, CDKN2A, KISS1R, EGF and others. In addition, our analysis uncovered genes that may represent characteristic patterns for subtyping and differentiation from normal renal tissue cells, for example TCF21, IRX1, STC2, UMOD, AQP2, ANGPTL4, BSND and FABP7, genes not previously associated with renal cell carcinoma.

In three different experiments we differentiated each of the three subtypes from normal tissue, and performed enrichment analysis for the top most significant genes in each case. We observed that both CCRCC and PRCC have genes involved in the "glycosaminoglycan biosynthesis - heparan sulfate" (HS6ST2, HS6ST3) and "riboflavin metabolism" (ACPP) pathways. Whereas ChRCC is more strongly associated with the "glycosphingolipid biosynthesis - lacto and neolacto series" pathway (B3GNT3, FUT6) and have five genes (B3GNT3, CYP2B6, CYP2J2, FUT6, UGT2A3) involved in other metabolic pathways. Changes in glycosaminoglycan and glycolipid were also previously reported for associations with RCC.

Conclusions: We demonstrate the effectiveness of a computational framework and predictive power of gene expression data for tumor subtyping in RCC. Our framework is generic and can be applied in combination with other types of data such as different modalities of genomic data (copy number variations, methylation) as well as clinical data.

#3652

Identification of cancer-specific signaling networks: what is "normal" control.

Manjushree Anjanappa, Yangyang Hao, Howard J. Edenberg, Yunlong Liu, Harikrishna Nakshatri. _Indiana University School of Medicine, Indianapolis, IN_.

Success of precision medicine depends on definitive identification of cancer-specific alterations in signaling pathways. However, identifying cancer-specific signaling networks is challenging because of lack of proper control tissue for differential gene expression analyses. Most studies in breast cancer utilize tumor-adjacent normal tissue or reduction mammoplasty samples as "normal" controls. We recently reported that breast epithelial cells from healthy donors as well as tumor-adjacent normal are in different differentiation states compared with tumor cells and the differences in differentiation status alone could account for major transcriptome variations between normal and tumor. To overcome these limitations, we propagated breast epithelial cells from three healthy donors (healthy-normal), two high-risk patients, two tumor-adjacent normal (HR/AD-normal) and five tumor samples of different molecular subtypes. Phenotypically defined (CD49f+/EpCAM+) luminal progenitor cells were sorted from these cultures and subjected to RNA-seq analyses. Pathway analysis revealed activation of cell-intrinsic pro-inflammatory signaling in HR/AD-normal cells compared with healthy-normal cells. This signaling network was further amplified in tumor cells. The pro-inflammatory chemokine CCL2, which is overexpressed in highly aggressive breast cancer, and the cytokine TNFRSF11B were elevated in HR/AD-normal luminal progenitor cells. Despite using phenotypically defined cells in the transcriptome analyses, cancer-specific signaling network identification was directly influenced by the type of controls used; healthy-normal or HR/AD-normal. While cancer-enriched PI3K and NF-κB activation was observed when compared to any kind of control, SRC kinase activation was noted only when cells from healthy-normal were used as a control. In general, the number of tumor signaling networks identified using healthy-normal as a control was higher than when compared with HR/AD-normal as a control. These results suggest that considerable attention should be placed on the type of tissues used as control for definitive identification of cancer-specific signaling networks and therapies to target such pathways. Additionally, these data show that non-cancer tissues of breast cancer patients acquire a cell intrinsic pro-inflammatory phenotype, which may be prerequisite for cancer development and potentially an early-detection tool. 

### Tumor Suppressor Genes and Pathways

#3653

Mutational analysis of PARK2, a regulator of cyclin D1 stability, in sporadic parathyroid adenoma.

Kelly Brewer,1 Jessica Costa-Guda,2 Andrew Arnold3. 1 _UConn Health, Farmington, CT;_ 2 _University of Connecticut School of Dental Medicine, Farmington, CT;_ 3 _University of Connecticut School of Medicine, Farmington, CT_.

The molecular mechanisms underlying the pathogenesis of sporadic parathyroid adenomas are incompletely understood. The oncoprotein cyclin D1 is overexpressed in 20-40% of sporadic parathyroid adenomas; however, known aberrations in the gene that encodes cyclin D1, CCND1, account for less than half of these cases. Genetic alterations of PARK2, which encodes the E3 ubiquitin ligase responsible for targeting cyclin D1 for degradation, have been identified as a frequent finding in a variety of human tumors. Interestingly, genetic abnormalities of PARK2 were mutually exclusive of cyclin D1 gene alterations, suggesting a common pathway in tumorigenesis. We postulate that inactivating mutations in PARK2 lead to increased stability of cyclin D1 and thereby may contribute to the development of sporadic parathyroid adenoma. We therefore sought to identify PARK2 coding region mutations in a series of sporadic parathyroid adenomas by direct DNA sequencing. The entire coding region and intron-exon boundaries of PARK2 were examined in a cohort of eighty typically presenting, sporadic parathyroid adenomas. Six known polymorphisms were identified in several of the adenomas in this series; however, none of the polymorphisms are associated with cancer, and no other cancer-associated mutations were found. The absence of intragenic mutations in PARK2 suggests that such mutations do not commonly contribute to the development of sporadic parathyroid adenoma. However, because such intragenic mutations could be rare and limited to only those samples in which overexpression of cyclin D1 cannot be explained by genetic abnormalities in the CCND1 gene, a significant role for cyclin D1 accumulation in parathyroid tumor formation due intragenic inactivating mutations of PARK2 cannot be ruled out. Further research must be conducted in order to elucidate the mechanism(s) of cyclin D1 accumulation in parathyroid adenomas in which CCND1 aberrations are not present.

.

#3654

In vivo CRISPR screen identidied Nf1 as a new tumor suppressor recurrently inactivated in liver cancer.

Chunqing Song. _University of Massachusetts Medical School, RTI, Worcester, MA_.

Our CRISPR screen identified NF1, encoding a RasGAP, as a new tumor-suppressor gene. CRISPR mediated loss-of-function mutations in NF1 led to increase RAS activation and increased tumor formation in liver cells. NF1 expression was reduced in 60% (225/373) of human HCC and was associated with reduced patient survival. Point mutations of NF1 was found in 3% of HCC. These findings identify NF1 inactivation as a new tumor suppressor in the liver and highlight the importance of RasGAPs in liver cancer.

#3655

Suppression of Plk3 expression by nickel may contribute to lung carcinogenesis.

Cen Li,1 Soyoung Park,1 Wei Dai,2 Dazhong Xu1. 1 _New York Medical College, Vahalla, NY;_ 2 _New York University, Tuxedo, NY_.

Exposure to nickel (Ni) compounds is known to induce human lung malignancies. Research shows that Ni induces lung cancer mainly through epigenetic and metabolic pathways. Polo-like kinase 3 (Plk3) is a multifunctional protein kinase that involves in tumor initiation and progression. Plk3-/- mice tend to develop highly vascularized lung tumors at an advanced age. Reduced expression of Plk3 was reported in human lung cancers. In this study, we examined the effect of Ni on Plk3 expression in A549 lung carcinoma cells. Western blot showed that NiCl2 reduced the Plk3 protein level in a time- and dose-dependent manner. We then treated A549 cells with cycloheximide for various times to examine the effect of NiCl2 on the half-life of Plk3 protein. We observed a shortened half-life of Plk3 in the presence of NiCl2. A reduced level of Plk3 protein was also found in hypoxia-treated A549 cells. In addition, MG132, a specific proteasome inhibitor, could partially reverse the effect of NiCl2 or hypoxia on the Plk3 protein level. Our cell fractionation experiments showed that majority of the endogenous Plk3 protein is located in the cytoplasm where degradation of Plk3 likely occurs. Transfection of A549 cells with FLAG-tagged ubiquitin (FLAG-Ub) followed by treatment with NiCl2 and/or MG132, immunoprecipitation (IP) with an anti-FLAG antibody, and Western blot with an anti-Plk3 antibody showed that Plk3 was poly-ubiquitinated and that the ubiquitination was significantly enhanced by NiCl2 and/or MG132 treatments. The elevated ubiquitination of Plk3 in response to NiCl2 was further confirmed in 293T cells with co-transfection of Plk3 and FLAG-Ub. Furthermore, overexpression of USP28, a deubiquitinase known to be inhibited by Ni or hypoxia, partially restored the protein level of Plk3 suppressed by NiCl2. Moreover, real-time PCR analysis showed that NiCl2 also reduced the Plk3 mRNA level, in a time- and dose-dependent pattern. Taken together, our data show that NiCl2 suppresses Plk3 expressions by lowering the Plk3 mRNA level and promoting proteasome-mediated degradation of the Plk3 protein. Given the potential role of Plk3 as a tumor suppressor in the lung, suppression of Plk3 expression likely contributes to Ni-induced lung carcinogenesis and tumor progression.

#3656

Cooperation of Rab25 loss with oncogenes in breast cancer.

Krishna A. Rao, Pooja S. Joshi. _Southern Illinois University School of Medicine, Springfield, IL_.

Breast cancer is the most common type of cancer in females worldwide. A number of genes such as BRCA1, BRCA-2, Hras, and p53 undergo mutations and contribute to tumorigenesis of the mammary tissue. The RAB guanosine triphosphates (RAS-related in brain) belong to the Ras superfamily of GTPases. The human RAS family consists of 3 proto oncogenes - H-ras, K-ras, N-ras. Rab 25 belongs to the Rab11 subfamily and plays an important role in vesicular trafficking. The Rab proteins are ubiquitously expressed. Loss of Rab 25 expression has been reported in a number of breast cancer cases and in transformed mammary cell lines containing H-ras point mutations.

Human Mammary Epithelial Cells (HMEC) were obtained from healthy individuals undergoing reduction mammoplasty. The cells were immortalized by transducing with LXSN CDK4 R24C. This was followed by transduction with hTERT, catalytic subunit of the telomerase enzyme.

In this study, we are trying to understand if loss of Rab25 expression in HMEC co-operates with wild and mutant H-ras as well as other oncogenes and contributes to tumorigenesis. HMEC lines in which Rab25 has been knocked down were transduced with various oncogenes, including wild type and mutant H-ras, Her-2, and IGFR1. We examined the co-operativity between loss of Rab25 and these oncogenes in migration, anchorage independent growth, stem cell marker expression, and also in vivo tumorigenesis and metastasis in nude mice.

#3657

ZBTB7A is a tumor suppressor and novel therapeutic target in triple-negative breast cancer.

Mary Ellen Molloy,1 Monika Lewinska,1 Xue-Song Liu,2 Angela A. Wang,1 Zhi-Min Yuan1. 1 _Harvard School of Public Health, Boston, MA;_ 2 _Shanghai Tech University, Shanghai, China_.

Due to lack of targeted therapies triple-negative breast cancers (TNBC) are the deadliest form of breast cancer, raising a pressing need to develop new therapeutic strategies. Recent advances in tumor metabolism have created exciting therapeutic opportunities by unveiling many previously unrecognized therapeutic targets. Like other tumor types, TNBCs also feature elevated glucose and glutamine metabolism (Shen, L., et al. 2015; McCleland, M., et al. 2012). A better understanding of the underlying mechanisms may uncover novel therapeutic targets for TNBC. Our lab has recently discovered that ZBTB7A suppresses glycolysis by transcriptionally repressing key genes within the glycolytic pathway in various cancer types (Liu, X.S., et al. 2014). Currently, ZBTB7A is proposed as an oncogene in breast cancer (Zu, X., et al. 2011), however our data suggest that ZBTB7A acts as a tumor suppressor specifically in TNBC. The purpose of this study was to determine the potential therapeutic value of targeting ZBTB7A in TNBC. Using publicly available databases, we find that low ZBTB7A expression is associated with poor prognosis in patients with hormone receptor-negative breast cancers. Our studies corroborate with patient derived data by demonstrating that reduced expression of ZBTB7A in TNBC cell lines leads to increased proliferation and resistance to radiation and chemotherapy. Differential gene expression analysis across human breast cancer samples in TCGA database revealed an inverse correlation between ZBTB7A and glycolytic genes. Overexpression of ZBTB7A in TNBC cells leads to decreased MCT1, LDHB and GLUT3 expression at the transcript level, consistent with our published data. Modulation of ZBTB7A expression also leads to changes in lactate production and glucose consumption in TNBC cells. Results from this study support ZBTB7A as a novel tumor suppressor and perspective therapeutic target in TNBC.

#3658

A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers.

Panneerdoss Subbarayalu,1 Subapriya Rajamanickam,1 Suryavathi Viswanadhapalli,2 Benjamin C. Onyeagucha,1 Vijay K. Eedunuri,1 Nicholas Dybdal-Hargreaves,2 Santosh Timilsina,1 Hima Bansal,1 Sanjay Bansal,1 Tabrez Mohammad,1 Yidong Chen,1 John C. Herr,3 Susan L. Mooberry,2 Manjeet K. Rao1. 1 _Greehey Children Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 2 _University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 3 _University of Virginia, Charlottesville, VA_.

Microtubule-targeting drugs are widely used as cancer chemotherapeutic agents and they have been shown to inhibit mitotic progression and interphase signaling. Despite their use as a first line treatment for cancer, many patients develop resistance to microtubule-targeting drugs leading to early relapse and shorter survival. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with these drugs. Therefore, identification of new factors that determine the effectiveness of microtubule-targeting agents will not only facilitate a better understanding of the mechanisms of acquired drug resistance but will also be amenable to therapeutic interventions. We discovered a novel microtubule associated protein "Matrin 3 (MATR3)" that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Our results revealed that MATR3 acts as a potent tumor suppressor as it inhibits long-term growth, migration, invasion as well as tumor growth of triple negative breast cancer (TNBC) cells in vivo. We demonstrated that MATR3 overexpression induced cell cycle arrest and apoptosis in TNBC cells. Furthermore, analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Importantly, our RNA immunoprecipitation (RIP)-seq analysis showed that MATR3 controls expression of several mitotic spindle organizing proteins by binding to their RNA. Interestingly, we found that these MATR3 regulated proteins were highly altered in breast cancer patients. In conclusion, we identified a novel RNA-binding protein that inhibits breast cancer growth and progression suppressor by regulating microtubule dynamics.

#3659

DIRAS1 and DIRAS2 are novel ovarian cancer tumor suppressors that regulate cell growth, motility and autophagy.

Margie N. Sutton, Zhen Lu, Hailing Yang, Gilbert Huang, Yan Wang, Weiqun Mao, Robert C. Bast. _UT MD Anderson Cancer Center, Houston, TX_.

DIRAS3 (also known as ARHI; Aplasia Ras Homolog Member I) is a potent tumor suppressor that has been well characterized for its role in ovarian cancer and autophagy. DIRAS3 is a maternally imprinted tumor suppressor gene that encodes a 26kDa GTPase which shares 60% homology to Ras and Rap. DIRAS3 is downregulated in many tumor types including ovarian cancer. DIRAS3 is downregulated by multiple mechanisms including loss of heterozygosity, transcriptional regulation by E2F1 and E2F4, hypermethylation of the second allele, and regulation by miRNAs 221 and 222. Re-expression of DIRAS3 at physiologic levels inhibits cancer cell growth, reduces motility, induces autophagy and promotes tumor dormancy. The mechanisms by which DIRAS3 induce autophagic cell death in vitro and tumor dormancy in vivo have been well characterized by previous work in our laboratory demonstrating that DIRAS3 is required for the induction of autophagy, and that upon inhibition of autophagy you can prevent outgrowth of dormant ovarian cancer cells in vivo. Interestingly, mice do not have DIRAS3 as it was lost during evolutionary rearrangement of murine chromosomes 3 and 6 nearly 60 million years ago, yet murine cells can still undergo autophagy. The DIRAS family members, DIRAS1 and DIRAS2 share 50-60% homology with DIRAS3 and are found in the murine genome. These 22kDa GTPases differ from DIRAS3 by the truncation of their N-terminal extension. Although DIRAS1 and DIRAS2 have not previously been characterized in ovarian cancer, TCGA analysis suggests that higher mRNA expression of these genes results in a survival advantage for patients with high grade serous ovarian cancer. In this study we demonstrate that re-expression of DIRAS1 and DIRAS2 inhibit ovarian cancer cell growth in vitro and induce autophagy in both human and murine cells. DIRAS1 and DIRAS2 are required for murine autophagy induced by rapamycin or amino acid starvation. Overexpression of DIRAS1 and DIRAS2 inhibits cancer cell growth and motility, and results in the inhibition of both the PI3K and Ras/MAPK signaling pathways. Thus DIRAS1 and DIRAS2 provide many of the functions of DIRAS3 in normal and malignant murine cells.

#3660

ATF4 is a putative tumor suppressor gene in medullary thyroid cancer.

Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. _UT MD Anderson Cancer Center, Houston, TX_.

Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ~45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40 % of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR.

ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n=43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR=5.015, 95% CI: 1.38-10.64), P=0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r= -0.89, R square=0.799, p<0.0001). Second, we showed that targeted heterozygote and homozygous deletion of Atf4 in mice causes C cell hyperplasia, a precancerous lesion preceding MTC. Third, ATF4 overexpression inhibits proliferation and survival of MTC derived cell lines. ATF4 decreases RET protein levels by inducing ubiquitination and degradation of activated RET. In contrast, ATF4 depletion upregulates RET protein levels as well as RET autophosphorylation. Finally, combination treatment of tyrosine kinase inhibitor (sunitinib) and eeyarestatin that stimulates ATF4 transcription, induces apoptosis of the MTC cells in a synergistic manner through activation of ATF4 and its target genes NOXA and PUMA as well. These results suggest ATF4 as a potential tumor suppressor gene in MTC. We postulate that inactivation of ATF4 is a central impediment to induction of apoptosis in MTC tumors. Therefore, strategies to upregulate ATF4 during TKI therapy would render tumor cells exquisitely sensitive to stress induced apoptosis, offering a novel synergistic treatment.

#3661

ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers.

Krysta M. Coyle,1 Patrick Murphy,1 Dejan Vidovic,1 Cheryl A. Dean,1 Margaret L. Thomas,1 Derek Clements,1 Mohammad Sultan,1 Ahmad Vaghar-Kashani,2 Carman Giacomantonio,1 Lucy Helyer,1 Ian Weaver,1 Shashi Gujar,1 Patrick WK Lee,1 Paola Marcato1. 1 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 2 _Uppsala University, Uppsala, Sweden_.

The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general.

First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy.

Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum.

Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP.

We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer.

#3662

Analysis of the tumor suppressor function of B55alpha, the PP2A regulatory subunit encoded by PPP2R2A, in prostate cancer cells.

Ziran Zhao,1 Alison kurimchak,1 Mary Adeyemi,1 Patrick Woodruff,1 Vladimir Kolenko,2 Xavier Graña1. 1 _Temple University Lewis Katz School of Medicine, Philadelphia, PA;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Genome wide DNA copy number studies in prostate cancer (PCa) have identified deletions on chromosome 8p21.2 affecting the PPP2R2A gene, which encodes B55α, a B regulatory subunit of Protein Phosphatase 2A (PP2A). The frequency of PPP2R2A hemizygous and homozygous deletions in prostate tumors is 38-58% and 2-7%, respectively. Moreover, PPP2R2A hemizygous loss correlates with reduced mRNA expression. In contrast, no significant correlation is observed for NKX3.1, a tumor suppressor also located at 8p21.2. Bioinformatics analysis indicates that PPP2R2A may belong to the class of "mutator alterations" occurring early in tumorigenesis that promote driver gene mutations. Indeed, PCa patients with these alterations exhibit reduced disease free progression. PP2A consists of a collection of trimeric holoenzymes whose substrate specificity and function is determined by the regulatory B subunit. There are 14 B subunit genes. While it is well accepted that PP2A plays a key tumor suppressor function in human cells, the particular holoenzyme(s) implicated in different tumor types is unclear. We have found, that limited ectopic expression of B55α in PCa cell lines that naturally express low levels of B55α results in dramatic toxicity. We have linked this to G2/M arrest, without concomitant loss in DNA replication, which leads to euploidy, nuclear enlargement and cell death. Thus, early hemizygous loss of PPP2R2A may facilitate effective mitotic progression. We have also seen that increased expression of B55α in these cells dramatically reduces their tumorigenic potential in SCID mice. Moreover, we observe inhibition in the phosphorylation of PP2A/B55α substrates when B55α activity is reconstituted, including AKT and pRB, suggesting alterations in multiple cancer pathways when B55α is downregulated in tumor cells. Interestingly, treatment of PC3 cells with PP2A activating drugs mimic cell cycle effects seen with B55α reconstitution.

#3664

RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma.

Yi Liu-Chittenden, Kelli Gaskins, Sophie Wang, Electron Kebebew. _National Cancer Institute, Bethesda, MD_.

Tumor suppressors and the immune system are critical players in tumor suppression. However, little is known about whether a tumor suppressor can function through both immune-dependent and immune-independent mechanisms. RARRES2 is a small secreted protein that is down-regulated in multiple human cancer types such as adrenocortical carcinoma (ACC), hepatocellular carcinoma (HCC) and melanoma. It has been shown that RARRES2 can play an indirect tumor-suppressive role by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor CMKLR1 to sites of tumor. In this study, we show that epigenetic CpG hypermethylation of RARRES2 in vivo and in vitro in ACC causes RARRES2 down-regulation. We also found that RARRES2 can function as a direct tumor suppressor and that its tumor suppressive effect does not depend on the recruitment of CMKLR1-expressing immune cells. We show that although exogenous treatment of cultured cells with recombinant human RARRES2 protein did not show any tumor-suppressive effect, transient overexpression of RARRES2 significantly inhibited cellular invasion. Stable overexpression of RARRES2 in ACC cell line NCI-H295R led to significantly reduced cell proliferation, cell invasion, and tumorigenecity in a RARRES2 dose-dependent manner. Using xenografts of stable NCI-H295R cell lines in two immunodeficient mouse models (athymic nude mice and NSG mice), we found that in the absence of functional immune cells, stable RARRES2 overexpression can still significantly suppress tumor growth in vivo. RARRES2 overexpression promoted beta-catenin phosphorylation and degradation, reduced the phosphorylation of p38 MAP kinase and inhibited the secretion of matrix metalloprotease protein 10 (MMP10) from cancer cells in a RARRES2 dose-dependent manner. Taken into account that CMKLR1 expression is barely detectable in all the cell lines tested, our results present the first evidence that RARRES2 can function as a direct tumor suppressor independent of CMKLR1 and CMKLR1-mediated anti-tumor immune response.

#3665

WAC: A candidate tumor suppressor gene in colorectal cancer.

Christopher R. Clark,1 Caitlin Conboy,1 Makayla Maile,1 Callie Janik,1 Julia Hatler,1 Robert Cormier,2 David Largaespada,1 Timothy K. Starr1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Minnesota Duluth, Duluth, MN_.

Our lab recently performed a DNA transposon forward genetic screen in mice that was designed to identify low-frequency mutations that contribute to colorectal cancer (CRC) initiation and progression. Results from this screen identified the WW domain-containing adaptor with coiled-coil (WAC) gene as a top DNA transposon insertion site. WAC has previously been implicated in several cellular processes including amino acid starvation-induced autophagy, golgi biogenesis, and transcription associated histone modification but has never before been linked to tumorigenesis. Transposon mutagenesis screens performed by others (Takeda et al. Nature Genetics 2015) have also identified Wac as a common insertion site, a result that further implicates WAC as a candidate CRC driver gene. Analyses of transposon insertion patterns within Wac predict loss of gene function and a role as a tumor suppressor. Soft agar colony formation assays reveal that shRNA mediated silencing of Wac cooperates with Apc mutations in mouse colorectal cells to promote cellular transformation. Additional colony formation assays using immortalized human colonic epithelial cells and the adenoma derived AAC1 cell line also shows that silencing WAC is protumorigenic. Using a zebrafish model we demonstrated that overexpression of wild type but not cancer-associated mutant forms of WAC induce expression of the cell cycle inhibitor p21, which suggests that loss of WAC may lead to uncontrolled cellular proliferation. Finally, using publicly available mutation data we determined that WAC is somatically mutated in both breast and lung cancers; a finding that indicates WAC may serve a critical tumor suppressive role in several tissues. Currently we are developing a conditional knockout mouse to further investigate the role of WAC in CRC tumor formation.

#3666

Co-occurring mutations of tumors suppressor genes, NF2 and LATS2, in malignant pleural mesothelioma.

Robin Tranchant,1 Anne Tallet,2 Lisa Quetel,1 Annie Renier,1 Françoise Le Pimpec-Barthes,1 Jessica Zucman-Rossi,1 Marie-Claude Jaurand,1 Didier Jean1. 1 _INSERM U.1162, Paris, France;_ 2 _CHRU TOURS - Plateforme de Génétique Moléculaire des Cancers, Tours, France_.

Background: Malignant Pleural Mesothelioma (MPM), mainly due to asbestos exposure, is an aggressive thoracic tumor resistant to conventional anti-cancer therapies. MPM patients have poor prognosis and a median survival around 12 months. These tumors are heterogeneous at the clinical and molecular level. To better characterize MPM heterogeneity, we recently identified a robust MPM transcriptomic classification defining two subgroups (C1 and C2). Epithelioid MPM, the most frequent histologic subtype, was found in both tumor subgroups, with a worse survival prognosis in the C2 subgroup. MPM heterogeneity is also found at the genetic level. Literature data showed alteration of Hippo signaling pathway in MPM linked to a frequent inactivation of NF2 tumor suppressor gene (30-40%). LATS2 tumor suppressor gene, another member of the Hippo pathway, was also shown to be altered but the frequency is not well determined.

Methods: Sixty-one primary cultures of MPM established in our laboratory were screened by Sanger sequencing for genes involved in mesothelial carcinogenesis, including CDKN2A, CDKN2B, BAP1, TP53, NF2 and LATS2. Transcriptomic data were obtained by Affymetrix microarray. Functional studies were performed using siRNAs to knockdown gene expression in three different MPM in culture. Gene expression was analyzed by quantitative RT-PCR and protein expression by Western blot. Cell proliferation was determined by MTS assay.

Results: Alteration frequencies of NF2 and LATS2 genes were 39% (24 cases) and 11% (7 cases), respectively. Five of seven LATS2 mutations were found associated with NF2 mutations in MPM of the C2 tumor subgroup. Patients with a co-occurring mutations in NF2 and LATS2 in tumor cells showed a worse prognosis (Logrank p-value of 0.02). These double mutants shared a similar transcriptomic profile. We identified MOK gene, a member of MAP kinase family, which is specifically deregulated in these double mutants (P<0.01). Modeling NF2 and LATS2 co-inactivation by RNA interference resulted in a specific up-regulation of MOK expression (P<0.01). Knockdown also induced a significant increase of MPM proliferation when the cells reach confluence (P<0.01). In the MPM double mutants, MOK knockdown showed that MOK may be involved in the loss of contact inhibition.

Conclusions: In summary, we identified and characterized cell cultures of MPM tumors with co-occurring mutations of two members of the Hippo signaling pathway, NF2 and LATS2. Our findings demonstrate that NF2 and LATS2 inactivation leads to MOK overexpression and loss of contact inhibition. Novel target therapies are needed for MPM patients and MOK kinase could be a new potential therapeutic target.

#3667

Potential role of DUSP4 as a tumor suppressor in pancreatic cancer.

Chandra K. Singh, Jasmine George, Minakshi Nihal, Nihal Ahmad. _University of Wisconsin-Madison, Madison, WI_.

Pancreatic cancer remains one of the most lethal of all human malignancies, with 48,960 expected new cases and 40,560 deaths predicted in the USA in the year 2015. In order to develop novel strategies for the management of pancreatic cancer, it is of utmost importance to intensify our efforts to dissect the molecular mechanisms involved in the pathophysiology of this fatal neoplasm. Dual-specificity phosphatase-4 (DUSP4), a family member of dual-specificity phosphatases, is capable of dephosphorylating both phosphotyrosine and phosphoserine/phosphothreonine residues and possesses activity against key signaling components of the MAPK pathway that plays an important role in cancer growth. DUSP4 has been shown to be widely expressed in different tissues and implicated in cancer development, differentiation, apoptosis and inflammation. In a recent study from our laboratory that was aimed at defining the molecular targets of the anti-cancer plant-based alkaloid sanguinarine in pancreatic cancer, we identified DUSP4 as a key protein modulated by sanguinarine. Specifically, based on proteomics data and validation by qRT-PCR and immunoblot analysis, we found that sanguinarine significantly increases DUSP4 in BxPC-3 and MIA PaCa-2 pancreatic cancer cells. Indeed, an association of DUSP4 loss with cancer progression has been shown in certain cancers. However, the role of DUSP4 in pancreatic cancer is not known. This study was designed to determine the role and importance of DUSP4 in pancreatic cancer. We determined the expression profile of DUSP4 in pancreatic cancer using immunohistochemical analysis of a tissue microarray containing a variety of pancreatic cancers and normal pancreatic tissues. The DUSP4-immunostaining was microscopically imaged followed by quantification of DUSP4 protein by multispectral Vectra™ system coupled with inForm software. We found a significant downregulation of DUSP4 in human pancreatic cancer tissues, when compared to normal pancreatic tissues. Further, to understand the importance of DUSP4 in pancreatic cancer, we determined the effect of a force overexpression of DUSP4 in MIA PaCa-2 pancreatic cancer cells. We found that DUSP4 overexpression in MIA PaCa-2 cells resulted in a significant i) decrease in cell growth and proliferation, ii) clonogenic survival, and iii) induction of apoptosis. These preliminary findings suggest a potential tumor suppressive function of DUSP4 in pancreatic cancer. Further detailed studies are ongoing in our laboratory to validate the role and functional significance of DUSP4 in pancreatic cancer.

#3668

Investigating a tumor suppressor role for Parkinson's susceptibility gene LRRK2 in lung cancer.

Chandra Lebovitz, Norman Chow, Wan Lam, Sharon Gorski. _BC Cancer Agency, Vancouver, British Columbia, Canada_.

In this study we investigate a novel and previously untested research question: does a known disease susceptibility gene in Parkinson's disease (PD) also act as a tumor suppressor gene in lung cancer? Our screen of publicly available cancer patient sequence data for genome and transcriptome alterations of genes that modulate autophagy, an important tumor cell survival process, revealed a dramatic loss of gene expression of Leucine-rich repeat kinase 2 (LRRK2) in non-small cell lung tumors compared to matched normal tissue. We further confirmed reduced or absent LRRK2 protein expression in a panel of lung cancer cell lines. Activating kinase-domain mutations in LRRK2 and their effect on autophagy in neuronal cell types is under study in PD; however, the pathological role of altered LRRK2 in cancer and its effect on autophagy status and tumorigenicity is completely unexplored. Pharmacological inhibition of the LRRK2 kinase domain was recently shown to stimulate autophagy in PD cell lines, while many tumors are thought to exploit elevated autophagy to survive metabolic stress and chemotherapy. Ongoing clinical trials are testing combination autophagy inhibition with current standard of care treatments in multiple cancers. Therefore, a better understanding of pathogenic LRRK2-mediated autophagy in lung cancer could form the basis of a new approach to help identify lung cancer patients that may benefit from treatment strategies employing autophagy inhibitors. Our preliminary data indicates that LRRK2 knockdown in lung cancer cell lines leads to altered cellular phenotypes and increased protein levels of a known lung cancer gene. We are further testing whether genetic and/or pharmacological inhibition of LRRK2 facilitates tumorigenesis in standard murine models of lung cancer. Our investigation of the biological relevance and therapeutic potential of a surprising discovery linking neurodegenerative disease-associated LRRK2 and lung cancer may uncover a new tumor suppressor gene and provide a novel marker (i.e. LRRK2 loss) to aid in lung cancer detection.

#3669

"DNA binding region" of BRCA1 affects genetic stability through modulating the intra-S-phase checkpoint.

Takaaki Masuda,1 Koshi Mimori,1 Chuxia Deng2. 1 _Kyushu University Beppu Hospital, Beppu, Japan;_ 2 _Faculty of Health Sciences, University of Macau, Macau, China_.

Introduction. BRCA1, a tumor suppressor gene in breast or ovarian cancer, is a large multifunctional protein implicated in DNA double-strand break repair, centrosome duplication, transcription regulation, DNA damage response, and cell cycle control, all of which are important for maintaining genomic stability, BRCA1 contains 3 domains; a N-terminal RING domain, C-terminal BRCT domain and a central region (CR). RING and BRCT domains are well addressed, but the function of the CR remains unclear. The CR is reported to have DNA binding region (DBR). Our aim of this study is to identify DBR and to clarify the biological significance of BRCA1 binding to DNA.

Materials and Methods. 1) Identification of DBR of BRCA1. First, electrophoretic mobility shift assay (EMSA) was performed to identify an essential region responsible for BRCA1 binding to DNA using purified tagged full length BRCA1 protein or the fragments and DNA oligos, and to investigate features of the DNA binding using various structures or lengths of DNA. Next, atomic force microscopy (AFM) was performed to see how BRCA1 binds to DNA. 2) Analysis of the biological significance of BRCA1 binding to DNA using mouse ES cells which are deficient in the DBR of BRCA1(ΔDBR) by TALEN-mediated Gene Targeting. 1. MTT survival assay under PARP1 inhibitor or Hydroxyurea (HU) to see the ability to homologous recombination (HR) repair or the sensitivity to replication stress, respectively. 2. HR assay in vitro. 3. EdU incorporation assay in the presence of HU to see cell cycle under replication stress. 4. Western blot to evaluate the phosphorylation status of CHK1 which regulates intra-S-phase checkpoint. 5. Karyotyping analysis to check for the genetic instability.

Results. 1) We identified an essential DBR (421-701 amino acids) within the CR of human BRCA1 by EMSA or AFM, which preferably binds to splayed-arm DNA (mimic of replication fork) in a sequence-independent manner and brings DNA strands together. These in vitro data suggest that binding of BRCA1 to DNA may affect protection of the DNA replication fork. 2) 1. The ΔDBR cells exhibited decreased survival as compared to wild type (WT) cells treated with a PARP1 inhibitor or HU in MTT assay. 2. There was not statistical difference with a HR assay between WT cells and ΔDBR cells. 3. ΔDBR cells continued to incorporate more EdU in the presence of HU in EdU incorporation assay. 4. Phosphorylation of CHK1 was moderately decreased in ΔDBR cells in Western blot. 5. The ΔDBR cells exhibited an increase in abnormal chromosome structures in Karyotyping analysis.

Conclusion. In this study, we demonstrate that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint that is activated by replication stress. Thus, our finding refines a functional domain of BRCA1, DBR, deficiency of which results in moderately impaired DNA replication upon replicative stress, leading to accumulative DNA mutations and cancer development.

#3670

Matrix metalloproteinase 9 has a novel tumor suppressive role in inflammation associated colorectal cancer by modulating intrinsic tumor microenvironment.

Lewins Walter,1 Adani Pujada,1 Sayeed Mohammadian,1 Agnieszka Bialkowska,2 Vincent Yang,2 Pallavi Garg1. 1 _Georgia State University, Atlanta, GA;_ 2 _Stony Brook, Stony Brook, NY_.

Colitis associated cancer (CAC) is a deadly complication of inflammatory bowel disease, and a subtype of colon cancer. CAC malignancy progresses through enhanced DNA damage. Observance of microsatellite instability and chromosomal instability in non-cancer related inflammatory conditions, supports the fact that inflammation could contribute to genomic destabilization by generating reactive oxygen nitrogen species, producing double strand breaks leading to DNA damage. Thus chronic colitis can induce DNA damage and modulate DNA repair systems- components of the intrinsic pathway to modulate tumor microenvironment. Matrix metalloproteinases (MMPs) are the most prominent family of proteinases associated with almost all the junctures of inflammation and tumorigenesis. They mediate inflammation, tissue remodeling, invasion, metastasis and tumor growth. Among the 24 known human MMPs, MMP9 is very unique as it is undetectable in normal tissues but highly expressed in inflamed or ulcerated tissues. We have observed that MMP9 has a protective role in CAC, which is exclusive and contrary to its traditional role of a facilitator in acute inflammation and sporadic colon cancer. In this study we explore the molecular mechanism by which MMP9 prevents DNA damage by activating tumor suppressors and DNA repair pathways in CAC. We generated transgenic mice overexpressing MMP9 in intestinal epithelium (TgM9) and to mimic human CAC, 3 cycles of 3% DSS (dextran sodium sulfate, a colonic inflammation inducer) for 5 days, followed by two weeks of recovery was used for in vivo model. Stably-transfected HCT116 cells with/without p-EGFP-MMP9 plasmid were used for in vitro experiments. We have observed that in CAC, TgM9 mice had significantly decreased tumor incidence and dysplastic lesions and a lower histological score compared to their WT littermates. TgM9 mice exhibited increased apoptosis, protein expressions of active-Notch1, p19ARF, p53, p21WAF1/Cip1, caspase-3 and cyclinE in CAC compared to WTs. HCT116-cells overexpressing MMP9 indicated decreased cell proliferation. FACS analysis indicated S-phase cell-cycle arrest and WB analysis of phosphorylated gamma-H2AX expressions indicated less DSBs compared to vector. HCT116 cells overexpressing MMP9 indicated increased expression of MDC1 and mis-match repair proteins MLH1. Our study highlights two novel findings i) MMP9 being a secretory protein activates transcellular protein Notch1 which translocates to nucleus and activates wildtype p53 via ARF and ii) MMP9 suppresses DNA double strand breaks by activating MMR gene- MLH1 and removing the damaged cells by apoptosis and or cell cycle arrest. Our study highlights the paradox of using MMP9 inhibitors in current therapies to treat CAC patients, implying that MMP9 expression might be a natural/ biological way to suppress inflammation associated ulceration.

#3671

Polarity and tumor development: Regulation by adenomatous polyposis coli.

Alyssa C. Lesko,1 Madeline Chandra,1 Jenifer R. Prosperi2. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _IUSM-SB and University of Notre Dame, Notre Dame, IN_.

The process of epithelial polarity is tightly regulated by multiple complexes including aPKC, PAR, and Crumbs proteins. Our laboratory has recently investigated loss of Adenomatous Polyposis Coli (APC) in the MDCK 3D culture system. We have observed a lack of hollow lumens and mislocalization of podocalyxin (gp135) to the basal surface, both of which are independent of the canonical Wnt/β-catenin signaling pathway. We further showed that the c-terminus of APC, which contains binding domains for polarity proteins (Dlg and Scrib) and cytoskeletal proteins (microtubules and actin), is responsible for the observed phenotypes. In the current study, we have investigated the molecular mechanisms required for the inversion of polarity and the potential impact on tumorigenesis. We have made the novel observation that different mechanisms are responsible for the increased size versus altered polarity observed in cysts derived from APC-KD cells. Alterations in size, although independent of changes in proliferation and/or apoptosis, are regulated by the c-terminal domain, focal adhesion kinase (FAK), Src, and β1-integrin. Despite this, the mis-localization of gp135 is only rescued by the c-terminal domain of APC, suggesting a unique molecular mechanism. As loss of epithelial polarity is an early marker for tumor development, we investigated tumor-associated phenotypes. APC-KD cells were found to grow larger colonies in soft agar, and have increased migration in wound-filling assays, which appeared to be regulated through similar molecular mechanisms. These data suggest that APC regulates polarity and associated disease phenotypes (including kidney development, cardiovascular disease, and cancer) through pathways independent of Wnt signaling.

#3672

Coordinated roles of CHD1 and SPOP in prostate cancer pathogenesis.

Divya Vasudevan,1 Mirjam Blattner,1 Lesa D. Deonarine,1 Yu Chen,2 Mark A. Rubin,1 Christopher E. Barbieri1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Prostate cancer (PCa) is a clinically heterogeneous disease, consequently presenting challenges while designing efficacious therapeutic strategies across diverse patient cohorts. There is an increasing need to improve our understanding of the disease by classifying PCa into distinct molecular subtypes characterized by specific genomic alterations. To this end, loss of the gene encoding chromodomain helicase DNA binding protein 1 (CHD1) represents one of the largest classes of recurrent gene deletions detected exclusively in PCa, hallmarking up to 20% of characterized tumors. CHD1 is well known to facilitate the assembly of euchromatin during transcription. Therefore, we hypothesized that loss of CHD1 would induce a significant change in overall transcription and alter prostate cell fate, promoting tumorigenesis. To confer a high degree of physiological relevance to our studies, we utilized genetically engineered mouse (GEM) models harboring prostate-specific CHD1 deletions. Since loss of CHD1 has a significant tendency to co-occur with mutations in the SPOP gene, we generated GEM models that also co-express the SPOP F133V transgene. We derived organoid lines from GEM prostates to recapitulate prostate histology in a three dimensional matrix, which allowed us to track changes at the molecular level and overall phenotype as they relate to PCa development. We found that CHD1 loss associated strongly with the presence of an increased luminal cell population compared to wild type controls. Since luminal cells are recognized to be cells of origin in PCa, this observation is consistent with the notion that CHD1 deletion represents a pro-tumorigenic event. Additionally, our data indicates that co-occurrence of CHD1 deletions and SPOP mutations may confer a growth advantage to prostate cells. The overarching goal of this study is to define the coordinated role of CHD1 loss and SPOP mutations in PCa, highlight altered signaling pathways and phenotypes, and nominate potential therapeutic strategies for patients with this PCa subtype.

#3673

BVES dependent regulation of YAP1 in colorectal cancer cells.

Mukul K. Mittal, Anthony Bilotta, Cody E. Keating, Sarah P. Short, Christopher S. Williams. _Vanderbilt University Medical Center, Nashville, TN_.

Colorectal cancer (CRC) has a US incidence rate of almost 150,000 cases and accounts for over 10% of new cancer diagnoses. Dysfunction of epithelial adherens junctions and tight junctions (TJs) have long been implicated in CRC growth and metastasis The Popeye domain containing family of proteins are TJ-associated and appear to nucleate TJ formation. BVES or POPDC1 is one member of this family and is under-expressed in all stages of human colorectal carcinoma (CRC). On the other hand, YAP1 a novel oncogene in the Hippo pathway is up-regulated in many cancers including colon cancer. In the present study, we report an inverse correlation between tumor suppressor protein BVES and oncogene YAP1 in human colorectal carcinoma tumor samples and human colon cancer cell lines. We demonstrate that ectopic expression of BVES in BVES deficient cells decreases YAP1 protein while knocking down BVES in cells expressing BVES increases YAP1 levels. Cycloheximide (CHX) experiments indicate that this is likely occurring at the level of protein stability. CDK6 is a cell cycle regulator and YAP1 transcriptional target, and its levels are increased in the setting of BVES-knockdown in CACO2 cells. Concurrent with this there is increased cell growth. Conversely, when BVES is overexpressed, CDK6 transcription is reduced. Further knocking down of YAP1 in BVES knocked down cells reverses the cell growth advantage suggesting that BVES regulation of cell growth in part is dependent upon YAP1 protein levels. Co-immunoprecipitation (Co-IP) and yeast-2-hybrid (Y2H) assay also shows a direct interaction of BVES and YAP1. In sum, these findings suggest a novel mechanism underlying BVES tumor-suppressive function through regulation of a potential growth modulator, YAP1 in colorectal cancer.

#3674

MIG-6 suppresses epithelial cell proliferation via inhibiting AKT phosphorylation during endometrial tumorigenesis.

Tae Hoon Kim,1 Jung-Yoon Yoo,1 Jae Hee Lee,1 Byung Gak Kim,1 Russell R. Broaddus,2 Jae-Wook Jeong1. 1 _Michigan State University, Grand Rapids, MI;_ 2 _University of Texas M.D. Anderson Cancer Center, TX_.

Endometrial cancer is the most frequently diagnosed malignancy of the female genital tract. Hysterectomy is typically the first line therapeutic strategy for endometrial cancer. However, there is an increasing demand for nonsurgical approaches for endometrial cancer, especially for women of reproductive age with endometrial cancer who wish to preserve their fertility. Most endometrial cancers are characterized by actively proliferating epithelial cells, increased AKT signaling and association with prolonged, unopposed E2 exposure.

Mitogen-inducible gene 6 (Mig-6) is an important mediator of progesterone receptor action in the murine uterus. As P4 achieves the inhibition of proliferation by coordinating stromal-epithelial cross-talk, we generated a mouse model in which we specifically ablate epithelial endometrial Mig-6 using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f) to understand the role of epithelial Mig-6 in the uterus. Sprr2fcre+Mig-6f/f mice displayed endometrial hyperplasia upon 10 weeks of age and develop endometrial cancer by E2 treatment for 3 months. The levels of epithelial proliferation by Ki67 staining were significantly increased in epithelial cells of Sprr2fcre+Mig-6f/f mice at 10 weeks of age. The levels of phospho-AKT and phospho-S6, downstream of AKT, were remarkably higher in Sprr2fcre+Mig-6f/f mice. Interestingly, the hyperplasia exhibited by Sprr2fcre+Mig-6f/f mice was prevented by P4 treatment for 1 week in morphological and histological analysis. Aberrant activation of proliferation as well as AKT signaling was decreased in the epithelium of Sprr2fcre+Mig-6f/f mice by P4 treatment. Furthermore, we identified that MIG-6 inhibits AKT phosphorylation in human endometrial cancer cells. These data suggest that stromal P4 signaling including Mig-6 is critical in regulation of epithelial cell proliferation via regulating AKT phosphorylation during endometrial cancer development and progression.

(This work was supported by American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.)

#3675

Loss of Pkhd1 promotes intestinal tumorigenesis in Apc mice.

Bo Hu,1 Wei Li,2 Qingchao Qiu,3 Zhengxi He,4 Xiusheng He,5 Guanqing Wu2. 1 _Univerity of South China, Vanderbilt University, Brentwood, TN;_ 2 _Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China;_ 3 _University of South China, University of Kentucky, Lexington, KY;_ 4 _Xiangya Hospital of Central South University, China;_ 5 _University of South China, China_.

Objectives: PKHD1 (Polycystic Kidney and Hepatic Disease 1), a causal gene for ARPKD (Autosomal Recessive Polycystic Kidney Disease), has been proposed to play a role in the colorectal carcinogenesis. This study is to explore the role of PKHD1 in colorectal carcinogenesis.

Methods: Mouse model with Pkhd1 mutation (Pkhd1-/-) was used to mate with ApcMin/+ mouse in the same genetic background. The resulting ApcMin/+ mice with and without Pkhd1-/- alleles were randomly sorted into two experimental groups (i.e. Pkhd1-/-;ApcMin/+ n=36; ApcMin/+ n=23). Tumorigenic susceptibility of Pkhd1-/-;ApcMin/+ and ApcMin/+ mice was compared. The number, size, pathology of intestinal tumors in the mice were analyzed at the age of 1-, 3-, and 6-month. The survival rates were investigated in the two groups of mice.

Results: There was no statistical difference in tumor number and size between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, the number and size of the intestinal tumors in 3- and 6-month-old Pkhd1-/-;ApcMin/+ mice were significantly increased compared with those of ApcMin/+ mice alone (P=0.017,P=0.022). In addition, the pathology of tumors was also analyzed between the two groups. There was no pathological difference between 1-month-old ApcMin/+ and Pkhd1-/-;ApcMin/+ mice. However, at 3-month-old, Pkhd1-/-;ApcMin/+ mice exhibited severer atypical hyperplasia in the intestinal tumor than those of ApcMin/+ mice; while at 6-month-old, focal necrosis were often seen in intestinal tumors of Pkhd1-/-;ApcMin/+ mice, but none from ApcMin/+ mice. Strikingly, tumor infiltration into the muscular mucosa was also seen in a 6-month-old Pkhd1-/-;ApcMin/+ mice. There was no statistically survival difference between ApcMin/+ and Pkhd1-/-;ApcMin/+ mice.

Conclution: Loss of Pkhd1 promotes the intestinal tumorigenesis and induces tumor malignant transformation in ApcMin/+ mice.

#3676

Prognostic significance of PTEN alteration in Middle Eastern colorectal carcinoma.

Abdul K. Siraj, Shaham Beg, Rong Bu, Maha Al-Rasheed, Nasser Al-Sanea, Fouad Al-Dayel, Khawla Al-Kuraya. _King Faisal Specialist Hospital & Res. Ctr., Riyadh, Saudi Arabia_.

Phosphatase and tensin homolog (PTEN) detected on chromosome 10 is a tumour suppressor commonly reported to be inactivated in colorectal cancers, but the prognostic significance of PTEN remains controversial. Here, we investigated the clinical associations and the significant prognostic value of combined PTEN mutation, genomic deletion and loss of expression in a large cohort of Middle Eastern colorectal cancers. We examined PTEN protein expression using immunohistochemistry in 400 sporadic colorectal cancers with full clinical investigations and follow up data. PTEN mutations were studied using capture sequencing and Sanger sequencing validation. PTEN copy number alteration was carried out using Fluorescence In-Situ Hybridization (FISH). Of the all CRC tumours analysed 83/347 (24%) cases showed absence of PTEN staining, 189/347 (54.5%%) cases showed weak staining and remaining 75/347 (21.5%) cases showed staining similar to normal & internal positive control. 17 mutations in coding region of PTEN were identified in 13 (3.2%) CRC cases. Further, PTEN deletion was identified in 24/338 (7.1%) of CRC cases. No amplification of PTEN gene was detected. Interestingly when combining all PTEN alterations (protein expression loss, PTEN mutation and PTEN genomic deletion), it accounted for 97 (29.7%) CRC cases. Significant association of combined PTEN alterations was seen disease free survival (p=0.0367). On multivariate analysis, this stands true (p=0.0227) when adjusted for Age, Sex, Site, Grade and Stage. Thus, PTEN alteration is an independent marker of poor prognosis in CRC. In conclusion, PTEN gene alteration plays an important role in Middle Eastern colorectal cancer pathogenesis and it can help in identifying a subgroup of CRC with particular poor outcome.

#3677

Loss of IRF5 induces spontaneous murine mammary tumorigenesis.

Dan Li,1 Xiaohui Bi,1 Corey Goyeneche,1 Amy Pitler,1 Betsy Barnes2. 1 _Rutgers University, Newark, NJ;_ 2 _Feinstein Institute, Manhasset, NY_.

Despite significant research efforts, breast cancer is still the most common malignancy in women and the most common cause of cancer death worldwide. A major challenge in breast cancer treatment is disease heterogeneity that is contributed by many factors including intrinsic cell factors, microenvironment, angiogenesis and tumor-specific immune responses. Interferon regulatory factor 5 (IRF5) is a transcription factor that controls inflammatory and immune responses. Analysis of over 3,000 human breast cancer tissues revealed that high expression of IRF5 correlates with increased survival and lower incidence of metastasis, whereas the lower quartile of IRF5 expression is a significant marker of poor prognosis for recurrence-free survival. These data support a tumor suppressor role for IRF5. To examine how loss of IRF5 expression may play a role in spontaneous mammary tumorigenesis, we generated Irf5 knockout (ko) mice in a BALB/C background. In mammary glands harvested from one-year-old female virgin ko mice (n=15), we found an increased incidence (16%) of mammary carcinoma in situ as compared to their wild-type littermates (4%; n=18). Of interest, the number of tumor bearing mice was significantly higher (38%) in retired ko female breeders (n=8); however, there were no abnormalities detected in one-year-old male ko virgins or breeders (n=6). Cellular and molecular analysis of female ko and wild-type littermates revealed that Irf5 deficiency enhanced mammary epithelial cell proliferation as indicated by an increased number of Ki67 expressing cells, and resulted in abnormal hyperbranching, which may contribute to mammary tumorigenesis. Irf5 ko breeders also showed impaired mammary gland involution after weaning, which has been shown to facilitate mammary tumor formation in humans. Taken together, these findings demonstrate that IRF5 is a gender-specific tumor suppressor in breast cancer. In this ongoing study, we are further investigating the intrinsic and extrinsic (microenvironment) role of IRF5 in mammary tumor initiation and progression.

#3678

The tumor suppressive small GTPase DiRas3 (ARHI) inhibits proliferation and activation of NF-κB in glioblastoma.

Amy Rymaszewski, Michael Straza, Anne Frei, Carmen Bergom. _Medical College of Wisconsin, Milwaukee, WI_.

Glioblastoma (GB) is the most aggressive malignancy affecting the central nervous system (CNS) with a median survival of 12 to 15 months even with surgery, radiation and chemotherapy. Previous research demonstrates that increased activation of NF-κB is critical for GB growth, proliferation and the up regulation of genes involved in cytokine production, cell cycle regulation, apoptosis and cell adhesion. Understanding the molecular targets that regulate NF-κB may provide more effective therapeutic targets for GB. DiRas family small GTPases, which are homologous to pro-oncogenic Ras GTPases, are tumor suppressive rather than tumor promoting and include DiRas1, DiRas2 and DiRas3 (ARHI). DiRas1 and DiRas2 have been suggested to be tumor suppressive in CNS malignancies, but the role of DiRas3 in CNS malignancies remains unknown. Here we demonstrate that expression of DiRas3 protein in GB cell lines is absent, although DiRas3 is expressed in non-malignant glial cells. Re-expression of DiRas3 in U-87 cells reduces cell proliferation by 20%. Using a NF-κB transcriptional activity luciferase reporter assay demonstrates that DiRas3 expression reduces NF-κB transcriptional activity by 70% compared to vector control. Further experiments demonstrate that decreased NF- κB activity occurs via reduced phosphorylation of the NF-κB inhibitor IκBα. The reduced phosphorylation of IκBα could be a result of decreased AKT and ERK activity, as increased ERK and AKT activity can stimulate NF-κB pathways. Our lab has previously demonstrated that the most common binding partner for DiRas1 and DiRas2 was the small GTPase binding protein SmgGDS, and DiRas1 and DiRas2 also reduce NF- κB activation. However, DiRas3 does not interact with SmgGDS, suggesting that DiRas3 can reduce NF-κB in a SmgGDS-independent manner. Understanding the role of DiRas3 and its binding partners in mediating NF- κB activation may lead to novel therapeutics for glioblastoma.

#3679

Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response.

Tami Rubinek,1 Tammi Rubinstein,1 Shiri Shahmoon,1 Chen Varol,1 Ehud Zigmond,1 Rina Rosin-Absesfelf,2 Iris Barshack,3 Ido Wolf1. 1 _Tel Aviv Medical Center, Tel Aviv, Israel;_ 2 _Tel Aviv University, Tel Aviv, Israel;_ 3 _Sheba Medical Center, Ramat Gan, Israel_.

Aberrant activation of the canonical Wnt pathway is implicated in colorectal cancer (CC) development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor (TS). Recent data indicate klotho as a TS in an array of malignancies, including CC. Among other activities, klotho inhibits the insulin-like growth factor (IGF)-1 and Wnt pathways. We aimed to decipher the role of klotho as a TS in CC.

We first noted that klotho overexpression inhibited CC cell colony formation. Next, we examined in vivo effects using the azoxymethane mouse model, and found that klotho inhibited colon polyp formation. We then used an orthotopic model by injecting MC38 cells to mice colons, and found that klotho inhibited colonic obstruction and tumor formation.

While we did not see an effect of klotho on the IGF-1 pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. As the inhibitory effect of klotho on CC cell colony formation was only partially rescued by a constitutively active β-catenin, additional mechanisms may be involved. Thus, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR) was observed. Further studies showed that klotho induced elevation in XBP1 RNA, GRP78 protein levels, and eIF2α phosphorylation, all indicative of a role klotho plays in UPR regulation.

Our data indicate klotho as a potent TS in CC, and suggest its role at early stages of tumor formation. The Wnt pathway partially mediates klotho effects in CC, however additional mechanisms, including ER stress response regulation, are involved.

#3680

Loss of ARID1A expression leads to sensitivity to ROS-inducing agents in ovarian cancer cells.

Suet-Yan Kwan, Kwong-Kwok Wong. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

AT-rich interactive domain-containing protein 1A (ARID1A) is frequently inactivated by mutations in a broad spectrum of cancer types, including ovarian and endometrial cancers. However, therapeutic strategies targeting ARID1A-mutant cancers remain limited. In this study, we aimed to identify drugs that selectively target ARID1A-mutant ovarian cancer cells. To this end, we compared the drug sensitivities of ARID1A-mutant and ARID1A-wildtype cancer cell lines using the Genomics of Drug Sensitivity in Cancer database. We found that ARID1A-mutant cancer cell lines were more sensitive to treatment with the reactive oxygen species (ROS)-inducing agent elesclomol compared to ARID1A-wildtype cancer cell lines. We validated this finding in a panel of ovarian and endometrial cancer cell lines, observing that treatment with elesclomol inhibited growth and induced apoptosis more potently in ARID1A-mutant cells than in ARID1A-wildtype cells. Knockdown of ARID1A expression in ARID1A-wildtype cells resulted in increased sensitivity to ROS-inducing agents, whereas re-expression of ARID1A in ARID1A-mutant cells resulted in decreased sensitivity. Finally, we found that knockdown of ARID1A expression resulted in increased intracellular ROS levels and subsequently promoted cell growth. In summary, we identified a novel role for ARID1A in protecting cells from oxidative stress and that ROS-inducing agents may be used to target ARID1A-mutant ovarian cancer cells.

#3681

Nischarin disruption promotes breast tumor development in mouse models.

Shengli Dong, Suresh K. Alahari. _LSUHSC in New Orleans, New Orleans, LA_.

Nischarin is a novel tumor suppressor that was first discovered and characterized in our laboratory. The accumulating evidence suggests that Nischarin is a critical regulator of cell migration and breast tumor growth, metastasis and invasion. However, little is known about the exact function of Nischarin in in vivo physiological conditions, probably due to lack of any transgenic Nischarin animals. We recently generated Nischarin knockout mouse. This Nischarin knockout mouse has a dramatic unusual phenotype of mammary development and overall growth defect. Nischarin knockout mice do not spontaneously develop breast tumors. To determine the importance of Nischarin in regulation of breast cancer development, we bred Nischarin knockout virgin mice with PyMT (polyoma middle T antigen) and Neu background mice and the progeny produced tumors exclusively in the mammary gland. We found Nischarin knockout virgins with PyMT accelerated breast cancer onset and progression, which further suggests Nischarin inhibits tumor growth. Mammary epithelial cells (MECs) are surrounded by basement membrane, which mainly consists of extracellular matrix. We noted that the basement membrane of Nischarin knockout virgins contain much more collagen and fibronectin than those of wild type virgins. In addition, we observed that Nischarin disruption significantly changed extracellular acidification rates (ECAR) in mouse primary breast tumour cells. We suspected that this changed acidic microenvironment might facilitate breast tumor progression. Recent studies indicated that clinical anti-diabetic drug Metformin, an AMPK activator, inhibits breast cancer cell growth. Our data show that Het-PyMT and Null-PyMT primary cells were much more sensitive for metformin treatment. Thus we examined the importance of AMPK signaling in our mouse models. Interestingly, both tumor tissues and primary cells derived from Null-PyMT breast tumors had lower AMPK activity. Together, our data strongly support that Nischarin disruption promotes breast tumor development and AMPK signaling is important for Nischarin-mediated suppression of breast tumor.

#3682

Phosphorylation mediated conformational changes defines nuclear role of phosphatase and tensin homology (PTEN) in tumor suppression.

Prerna Malaney, Jonathan Semidey-Hurtado, Jamaal Hardee, Deepal Patel, Daniel Hennessey, Kate Stanford, Emily Palumbo, Zhi Tian, Diane Allen-Gipson, Vrushank Davé. _University of South Florida, Tampa, FL_.

Loss of function of tumor suppressor PTEN (Phosphatase and tensin homolog) causes cancer in various tissues. PTEN C-terminal phosphorylation (pPTEN) inactivates PTEN, leading to multiple malignancies with increased severity. However, little is known about the molecular mechanisms underlying such inactivation. Therefore, the objective of our work is to ascertain the molecular mechanisms by which PTEN phosphorylation drives lung cancer. PTEN C-terminal phosphorylation at a serine-threonine cluster (Ser380, Thr382, Thr383 and Ser385) conformationally inactivates PTEN, abrogating its tumor suppressor function. Replacement of these serine/threonine residues with alanine generated an artificial phosphorylation-deficient mutant of PTEN (PTEN-4A), which is constitutively active. PTEN-4A suppressed cell proliferation and migration to a greater extent as compared to PTEN-WT. PTEN-4A preferentially localized to the nucleus and suppressed the E2F-mediated transcription of cell cycle genes. The nuclear localization sequence and phosphatase activity of PTEN-4A is critical for this transcriptional suppression. Immuno-precipitation assays show that PTEN physically associates with the transcription factor E2F1, a likely mechanism for its suppressive effect on E2F1 related genes. Further, deletion analysis of PTEN-4A protein revealed that the C2 domain is indispensable for suppression of E2F-related genes. Systematic transcriptional assays identify disease-associated C2 domain mutations that lose their ability to suppress E2F-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Taken together, we reveal nuclear functions of PTEN-4A in tumor suppression that can be therapeutically leveraged for developing adjunctive cancer therapies. Small molecule inhibitors that hinder PTEN phosphorylation maybe utilized to activate PTEN nuclear function in tumors. Such adjunctive therapy has a high likelihood to reduce toxic doses of chemotherapeutic agents and targeted inhibitors, including kinase inhibitors that are being used in clinical settings.

### Tumor Suppressors: TP53 Pathway

#3683

The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination.

Xingyao Li, Chunhong Yan. _Georgia Regents University, Augusta, GA_.

While the tumor suppressor p53 is crucial for the maintenance of genetic integrity upon DNA damage and oncogenic stresses, its level and activity are tightly controlled by a complex network largely mediated by the E3 ubiquitin ligase MDM2. MDM2 catalyzes the addition of ubiqutin chains to the C-terminus of p53 and is the major ubiquitin ligase to promote proteosomal degradation of p53. We previously demonstrated that ATF3, a common stress response mediator, can bind p53 and prevent it from MDM2-mediated ubiquitination. As a consequence, ATF3 can stabilize p53 upon DNA damage and prevent cells from oncogene-induced transformation by promoting p53-mediated senescence, in line with accumulating evidence supporting that ATF3 can act as a tumor suppressor. Interestingly, ATF3 is also a bona fide substrate of MDM2, and thus, the ATF3-MDM2 interaction can fine tune p53 activity in the DNA damage response. However, the mechanism by which ATF3 blocks p53 ubiquitination to activate the latter protein remains largely undefined. Efficient ubiquitination of p53 requires the binding of MDM2 to the N-terminus of p53, and the MDM2 binding to MDMX. Here we show that neither the p53-MDM2 interaction nor the MDM2-MDMX interaction is disrupted by the binding of ATF3 to p53 or MDM2. As both ATF3 and p53 are substrates of MDM2, we explored a possibility that ATF3 competes with p53 for MDM2-mediated ubiquitination. By in-vitro ubiquitination assays, we identified that the 5 lysine residues clustering in aa 100-115 of ATF3 are required for MDM2-mediated ubiquitination. Site-specific mutagenesis indicates that MDM2 mainly ubiquitinates ATF3 at lysine 107 (K107) and lysine 108 (K108) as MDM2 fails to add ubiquitin chains to ATF3 when these two residues were mutated to arginine. Importantly, while mutations at these two residues do not affect the ATF3 binding to MDM2 or p53, the ATF3 mutant devoid of ubiquitination fails to stabilize p53 and prevent p53 from MDM2-mediated ubiquitination. These results thus demonstrate that ATF3 can activate p53 by competing with the tumor suppressor for MDM2-mediated ubiquitination.

#3684

Proteome-wide triple negative breast cancer mutant p53 association index identifies chromatin unwinding for precision therapeutics.

Wei-Gang Qiu,1 Alla Polotskaia,2 Gu Xiao,2 Lia Di,2 John Philip,3 Ronald C. Hendrikson,3 Jill Bargonetti1. 1 _City University of New York at Hunter College and The Graduate Center, New York, NY;_ 2 _City University of New York at Hunter College, New York, NY;_ 3 _Memorial Sloan-Kettering Cancer Center, New York, NY_.

Over 80% of TNBCs express mutant p53 (mtp53) proteins. We coupled cell fractionation with stable isotope labeling with amino acids in cell culture (SILAC) and inducible knockdown of endogenous mtp53 to determine the mtp53 driven proteome in the cytoplasm and chromatin of triple negative breast cancer (TNBC) cells. Using SILAC coupled to tandem mass spectrometry (LC-MS/MS) we identified that R273H mtp53 expression in MDA-MB-468 breast cancer cells both up and down-regulated multiple proteins and metabolic pathways. We sequenced 73,154 peptide pairs that corresponded to 3010 proteins detected under reciprocal labeling conditions in the cytoplasm and 48,825 peptide pairs that corresponded to 5195 proteins in the chromatin fraction. Pathway enrichment analysis ranked the DNA unwinding pathway as the highest chromatin associated pathway regulated by mtp53. Moreover, to summarize and quantify the degree of under- or over-expression of a protein from two reciprocal experiments, a mutant p53 association index (mPAI) was defined as the log (of base 2) ratio of two readings from the two reciprocal experiments. Values of mPAI were normally distributed with a mean close to zero, consistent with the fact that levels of the majority of proteins were not affected by mtp53 knockdown. Standard deviation of mPAI is close to one and mtp53 itself shows mPAI values of greater than 2.0 (z-score > 2.0) in both cytosol and chromatin fractions, consistent with the expectation that its levels are significantly reduced by knockdown experiments. We thereby identified proteins and pathways significantly affected by mtp53 knockdowns as those with mPAI > 1.0 (indicating positive association) or mPAI < -1.0 (indicating negative association). A pathway that was positively associated in both the cytoplasm and the chromatin was the DNA unwinding pathway, which was represented by associated changes for all the minichromosome maintenance protein complex proteins 2 through 7 (MCM2-7). This hexomeric protein ring structure complex is the eukaryotic DNA helicase complex required for DNA replication and elongation of the replication fork. We validated that the MCM4 protein level is positively associated with mtp53 R273H in the TNBC cell line MDA-MB-468 and in the colon adenocarcinoma cell line HT-29. This suggests that targeting inhibition of the MCM hexomeric complex may be a good precision medicine approach for TNBCs. Further studies are in progress to determine the efficacy of targeting inhibition of MCMs 2-7 as a mechanism to block TNBC.

#3685

Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55.

Stephen Chong, Shazib Pervaiz. _National Univ. of Singapore, Singapore, Singapore_.

Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that has critical roles in numerous cellular pathways. The role of PP2A as an essential tumor suppressor in numerous death signaling pathways is well documented. In this regard, recent work from our laboratory has highlighted a novel mechanism of apoptosis inhibition via post-translational redox-modification of PP2A. Increase in intracellular superoxide, either by pharmacological inhibition or siRNA-mediated silencing of superoxide dismutase 1 (SOD1), results in the dissociation of PP2A-AC catalytic core from its B regulatory subunit. Dissociation of the PP2A heterotrimer is a result of the selective nitration [by peroxynitrite derived from the reaction of superoxide with nitric oxide] of a conserved tyrosine residue on B56δ which we identified as Y289 (B56δY289). Importantly, we showed in lymphoma cells that PP2A-AC catalytic core is typically targeted to Bcl-2 by its B56δ regulatory subunit. When O2-¬¬ is elevated, it signals for the sustained phosphorylation of Bcl-2 at S70 by inducing the release of PP2A-AC catalytic core from B56δ-bound Bcl-2. This results in the sustained phospho-activation of anti-apoptotic Bcl-2 and resistance to apoptotic stimuli. Notably, Bcl-2 is not the only substrate of PP2A, nor is B56δ the only PP2A regulatory subunit susceptible to redox modification. Thus, implications from redox-modification of PP2A may extend beyond the phospho-activation of Bcl-2. In the context of carcinogenesis, another notable substrate of PP2A is the tumor suppressor, p53. p53 stability is negatively influenced by its phosphorylation status at threonine-55 (T55). Phosphorylation of T55, on the other hand, is countered by PP2A harboring the B56γ3 subunit. Here we provide evidence that the B56γ3 sub-unit of PP2A complexes with p53 and this complex is sustained upon an increase in intracellular superoxide. Importantly, redox-mediated inhibition of B56γ3-containing PP2A (PP2AB56γ3) prevents the dephosphorylation of p53 at T55, thereby negatively impacting the stability of p53 in tumor cells with elevated superoxide level. This, we hypothesize, may be a novel death inhibitory mechanism that renders tumor cells resistant to conventional DNA-damaging therapeutic agents that rely heavily on the activation of p53 for death induction.

#3686

**Elephant** TP53 **(ep53) retrogene protein expression enhances cellular apoptosis in response to DNA damage.**

Lisa M. Abegglen,1 Rosann Robinson,1 Lauren Donovan,1 Aleah F. Caulin,2 Mor Goldfeder,3 Avi Schroeder,3 Carlo C. Maley,4 Joshua D. Schiffman1. 1 _Huntsman Cancer Institute, University of Utah, Salt Lake City, UT;_ 2 _Driver Group, L.L.C., San Francisco, CA;_ 3 _Technion - Israel Institute of Technology, Haifa, Israel;_ 4 _Arizona State University, Tempe, AZ_.

We previously reported that compared with other mammalian species, elephants have a lower than expected rate of cancer (Abegglen JAMA 2015). While exploring cancer resistance in elephants, we discovered that elephants have 20 copies of TP53 (1 ancestral gene with introns and 19 retrogenes) compared to humans with 1 copy. TP53 amplification was associated with increased p53-mediated apoptosis induced by DNA damage in elephant cells compared to human cells. As a follow up to this study, we further explored the function of these elephant TP53 retrogenes by expressing their protein (ep53) in cells with 2 functional TP53 alleles. NIH/3T3 cells were transfected with a mammalian expression construct encoding TP53 retrogene 9 fused to an epitope tag (myc). Western blot analysis probing for myc confirmed expression of retrogene 9. To measure the functional consequence of expression, we compared apoptosis of cells transfected with empty vector to cells transfected with myc-tagged retrogene 9 after doxorubicin treatment. Apoptosis was measured with a triplex assay that assesses viability, cytotoxicity and apoptosis (Apo Tox-Glo, Promega). We observed a significant increase in apoptosis of NIH/3T3 cells expressing retrogene 9 compared to empty vector transfected control cells (10μM doxorubicin, P<0.001). These results suggest that ep53 is a functional protein contributing to cancer resistance in elephants, and that elephant retrogenes can function in cells from other species to enhance their apoptotic response to DNA damage. These findings may hold significance for human cancer prevention and treatment.

#3687

p53 downregulates CRKL oncogene through miR-200.

Takashi Tokino, Miyuki Tamura, Masashi Idogawa, Yasushi Sasaki. _Sapporo Medical Univ., Sapporo, Japan_.

Tumor suppressive miRNAs targeting oncogenes are frequently downregulated in cancers, which leads to the activation of the oncogene pathways. Thus, tumor suppressive miRNAs as well as their target oncogenes are proposed to be useful for cancer treatment. The downregulation of the miR-200 family has been involved in the progression and metastasis in cancers. The miR-200 family consists of two gene clusters: miR-200b/200a/429 and miR-200c/141 are located on human chromosome 1 and 12, respectively. Here, we identified that p53 response elements are located around both clusters of miR-200s and confirmed miR-200s as transcriptional targets of p53. in silico analysis of miRNA target predicted an oncogene CRKL as a potential target for miR-200b/200c/429. Moreover, miR-200b/200c/429 inhibit the expression of CRKL mRNA and protein by directly targeting its 3'-UTR region. Importantly, endogenous CRKL expression was decreased in cancer cells with introduction of wild-type p53. Moreover, downregulation of CRKL by siRNA in cancer cells inhibits cell growth. Oncomine database shows that CRKL levels are overexpressed in a subset of cancer types. Furthermore, CRKL is significantly overexpressed in primary breast cancer tissues harboring mutant TP53. Our results demonstrate that p53-target miRNAs, miR-200b/200c/429 are negative regulators of the CRKL oncogene.

#3688

The C-terminal domain of tumor-derived mutant p53 is required for its oncogenic gain-of-function activity.

Caleb C. Lee, James Manfredi. _Icahn School of Medicine at Mount Sinai, New York, NY_.

p53 is the most commonly mutated gene in human cancers. Unlike other tumor suppressors in which loss of protein expression is common, the vast majority of changes in p53 arise as point mutations. Tumor associated mutation hotspots are localized to the DNA binding domain, either in residues involved in direct DNA contact or those that regulate its conformation. In either case, such mutations have been shown to confer gain of function activity. This was first shown in soft agar assays in which over-expression of mutant p53 increased transformation of cells on a p53 null background. Furthermore, knock-in mouse models of mutant p53 showed alterations in tumor spectrum with the development of carcinomas not seen in p53 null mice. However, the mechanism by which mutant p53 actively aids in tumorigenesis remains unclear.

Although mutant p53 causes loss of sequence specific DNA binding via the core domain, it retains the ability to interact with multiple proteins including transcriptional cofactors shown to have tumor suppressor activity such as p300 and CBP. These interactions occur through multiple protein binding domains including the C-terminal domain of p53. Our laboratory and others have shown that C-terminal truncation of wildtype p53 results in multiple deficiencies. Binding of mutant p53 to p300/CBP via the C-terminus is therefore an attractive hypothesis to explain mutant p53 gain of function.

To determine the role of the C-terminus in mutant p53 gain of function activity, we have expressed constructs containing a C-terminal truncation in combination with varying hotspot mutations. Transfection of mutant p53 into a p53 null cell line was required for the formation of colonies in a soft agar assay, indicating an increase in tumorigenicity. Furthermore, C-terminal truncation of the mutant p53 abrogated this phenotype suggesting that this domain is required for mutant p53 gain of function. This was independent of expression levels as shown by immunoblot. We have also extended the findings of mutant p53 interaction with p300 by IP analysis of a panel of cell lines with varying p53 hotspot mutations. To confirm the role of the C-terminus in mutant p53 gain of function, we are developing a xenograft model to detect changes in tumorigenicity in vivo. These results will therefore explore the mechanism by which mutant p53 acts as an oncogene.

#3689

Inactivation of p53 in mammary luminal cells leads to their clonal expansion and facilitates development of mammary tumors with loss of luminal identity.

Luwei Tao, Ying Xie, Zhe Li. _Brigham & Women's Hospital, Boston, MA_.

Mammary epithelium is hierarchically organized, with multipotent basal mammary stem cells (MaSCs) sit at the top of the hierarchy, giving rise to both luminal and basal mammary epithelial cells (MECs) during fetal development or upon transplantation to cleared mammary fat pad. Recent studies suggested that most breast cancers, including basal-like breast cancer, might originate from luminal cells, rather than from basal MaSCs, and that oncogenic events, such as ectopic expression of PIK3CA(H1047R) mutation, could induce multipotency in committed luminal cells, leading to acquisition of a MaSC-like phenotype by luminal MECs. TP53 is the most commonly mutated gene in human breast cancer; in particular, its inactivating mutations have been found in most basal-like and claudin-low breast cancer cases, raising a question as to whether TP53 loss-of-function mutations play a key role in dedifferentiation of committed luminal cells to multipotent MaSC-like cells. To study this, we induced disruption of Trp53 conditional knockout alleles in a small number of luminal MECs, either by transient Cre expression from intraductally injected Cre adenovirus under the control of the Keratin 8 (K8) promoter (Ad-K8-Cre virus) or by tamoxifen-induced transient activation of the CreER fusion driven by the same K8 promoter (K8-CreER mice). By in situ lineage-tracing, we found that induced loss of p53 in K8+ luminal cells led to their clonal expansion (by outcompeting their wild-type neighbors), due to increased cell cycle progression and impaired apoptosis control. However, p53-loss in luminal MECs did not directly affect their luminal identity, as determined by flow cytometry, immunostaining, and microarray expression profiling. Intriguingly, all induced female mice eventually developed mammary tumors. The majority of these tumors were large, fast growing claudin-low mammary tumors, based on histological and microarray analyses; a small number of these tumors exhibited features of basal-like mammary tumors. These data demonstrate that although p53 does not dictate the luminal MEC fate directly (i.e., loss of p53 does not directly lead to luminal-to-basal or mesenchymal transition), its inactivation facilitates loss of the luminal identity and predisposes luminal cells to development of claudin-low or basal-like breast tumors, thus in a way similar to the role of p53 in restricting reprogramming of somatic cells to induced pluripotent cells. Lastly, although it has been suggested that claudin-low breast cancer may originate from transformation of basal cells, our data support that upon p53-loss, claudin-low breast cancer can also have a luminal origin.

#3690

Metabolic regulation of mutant p53 stability by the mevalonate pathway.

Tomoo Iwakuma,1 Atul Ranjan,1 Swathi V. Iyer,1 Subhash Padhye,2 Scott Weir,1 Anuradha Roy3. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _Abeda Inamdar Senior College, Pune, India;_ 3 _University of Kansas, Lawrence, KS_.

Introduction: Mutations in the p53 gene are mostly missense mutations and result in accumulation of dysfunctional p53 protein in tumors with oncogenic gain-of-function activities. Increasing evidence indicates that stabilization of mutant p53 (mutp53) in tumors is crucial for its oncogenic activities including tumor progression and drug resistance, while downregulation of mutp53 reduces oncogenicity of cancer cells. These observations suggest that malignant properties of cancer cells are dependent on the presence of mutp53, thus providing a rationale to identify compounds that deplete mutant p53 with little impact on wild-type p53.

Experimental procedures: Toward this goal, we performed high throughput screens of chemical libraries (~9,000 compounds) with Saos2 (p53 null) cells expressing a fusion protein of p53R175H and luciferase, using luciferase as a reporter.

Summary of data: This screening led us to identify "statins", a class of cholesterol-lowering medications, as compounds that induced degradation of p53R175H. We found that other inhibitors of the mevalonate pathway, such as 6-fluoromevalonate and zoledronic acid, failed to induce p53R175H degradation, while statin-mediated inhibition of HMG-CoA reductase and subsequent reduction in mevalonte-5-phosphate triggered p53R175H degradation. These results suggest that statin's effect on p53R175H is specific and independent of protein prenylation/lipidation or cholesterol synthesis. Moreover, nuclear export of p53R175H was required for the statin-mediated degradation, which was mediated through an E3 ubiquitin ligase CHIP, but not MDM2. Interestingly, statins induced degradation of mainly conformational p53 mutants with minimal effects on the levels of wild-type p53 and DNA contact mutants.

Conclusions: This is the first study demonstrating that mutp53 stability is regulated through a specific process of the mevalonate pathway, thereby providing a novel regulatory mechanism of mutp53 degradation. Our findings suggest that p53 mutation status in tumors may have an impact on efficacy of statins in cancer therapy.

#3691

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation.

Donglai Qi,1 David E. Cobrinik2. 1 _Children's Hospital Los Angeles, Los Angeles, CA;_ 2 _Children's Hospital Los Angeles and University of Southern California, Los Angeles, CA_.

Retinoblastomas can arise from cone photoreceptor precursors in response to the loss of pRB function. Some aspects of cone precursor-specific circuitry cooperate with pRB loss to initiate this process and subsequently continue to contribute to the malignancy. Intrinsic high level MDM2 expression is a key component of this circuitry and is thought to inactivate p53-mediated tumor surveillance that could otherwise be induced by aberrant cell cycle entry. However, the MDM2-related MDM4 has also been proposed to abrogate p53-mediated tumor surveillance in the absence of detectable MDM2. Here we report that high-level MDM2 but not MDM4 has a consistent, critical role in retinoblastoma cell proliferation. Reduction of MDM2 and MDM4 only weakly induced p53, yet reduction of MDM2 but not MDM4 severely impaired proliferation and survival through a p53-independent mechanism. Specifically, MDM2 up-regulated the translation of another component of the cone circuitry, MYCN, in retinoblastoma cells. These findings indicate that high-level MDM2 expression is needed in order to perform a critical p53-independent function and may obviate the need for genomic alterations to the p53 pathway in retinoblastoma cells.

#3692

Transcriptome analysis of oral tongue cancer reveals novel insights into wild type and mutant TP53 transcription program.

Raju SR Adduri,1 Padmavathi Kavadipula,1 Viswakalyan Kotapalli,1 Leena Bashyam,2 Anupama Shirke,1 Arun kumar Paripati,1 Swarnalata Gowrishankar,3 Mukta Srinivasulu,4 Mohammed Mujtaba Ali,4 Subramanyeshwar Rao,4 Snehalatha Dhagam,5 Mohana Vamsy Chigurupati,5 Shantveer G. Uppin,6 Vijaya Tourani,7 Murali D. Bashyam1. 1 _Ctr. for DNA Fingerprinting & Diagnostics, Hyderabad, India; _2 _University of Hyderabad, Hyderabad, India;_ 3 _Apollo Hospitals, Hyderabad, India;_ 4 _MNJ Institute of Oncology & Regional Cancer Centre, Hyderabad, India; _5 _Omega Hospitals, Hyderabad, India;_ 6 _Nizam's Institute of Medical Sciences, Hyderabad, India;_ 7 _Care Hospitals, Hyderabad, India_.

The p53 oncoprotein is a tumor suppressor that is stabilized upon various forms of cellular stresses to induce transcription of genes regulating cell cycle arrest or apoptosis. TP53 is the most frequently mutated gene in human cancers and majority of mutations are located in the region encoding the DNA binding domain compromising thereby its transcription activation ability and resulting in loss of function. Recent studies however suggest mutant p53 proteins to exhibit a gain of function property. Specific p53 missense mutations can result in an altered transcription program causing positive regulation of cell proliferation, metastasis and chemoresistance. In our previous studies, we performed comprehensive characterization of squamous cell carcinoma of the oral tongue (SCCOT) with respect to p53, EGFR, Wnt, MSI, LoH of several tumor suppressor loci and HPV status. Mutant p53 was a significant predictor of overall survival and the TP53 codon 72 Proline allele was significantly associated with SCCOT. In order to dissect the role of mutant p53 in tongue cancer, we performed genome wide DNA and RNA profiling of 26 and 40 SCCOT samples, respectively. Both mutant and wild type tumor samples appeared to exhibit comparable levels of DNA copy number alterations. Transcriptome data analyses using a combination of single sample gene set enrichment analysis and comparative marker selection revealed gene sets that could significantly distinguish p53 mutant and wild type tumor samples. Significance analysis of microarrays performed on all genes constituting the differentially enriched gene sets surprisingly identified only two genes to be upregulated in p53 mutant samples at a false discovery rate significantly lower than 10% namely TP53 itself and SMARCD1; the latter a member of the SWI/SNF chromatin remodelling complex. Elevated levels of TP53 transcript in tumors harbouring mutant p53 significantly correlated with levels of ZMAT3, itself induced by p53 and known to stabilize the TP53 transcript. In addition, the analysis revealed several known (ATF3 and others) and novel (GCHFR and others) targets of wild type p53. Differential expression of all targets was validated in additional tongue cancer samples. Ectopic expression of certain (but not all) p53 mutant proteins in p53 null cells induced SMARCD1 (but not canonical wild type p53 targets) while expression of wild type p53 induced GCHFR, ATF3, CDKN1A, etc. (but not SMARCD1). In contrast, p53 stabilization in cells harboring wild type p53 caused elevation of GCHFR, ATF3, CDKN1A, etc., but not of SMARCD1. Validation of novel targets using promoter-luciferase constructs, chromatin immunoprecipitation PCR and a tongue cancer tissue microarray is underway. This is perhaps the first evidence from Head and Neck tumor samples for a gain of function activity of mutant p53. Thus wild type and mutant p53 may support distinct transcription programs in tongue cancer.

#3693

Oligomerization status of p53 serves as an indicator of sensistivity of p53 wildtype tumors to the therapeutic combination of DNA damaging agent and checkpoint inhibitor.

Pawan Puli, Robert Lipski, Aime A. Levesque. _Univ. of Hartford, West Hartford, CT_.

It is well established that cell cycle arrest in response to treatment with DNA damaging agents can be abrogated in p53-defective cells by treatment with the Chk1 inhibitors, but our more recent studies have shown that some p53 wild-type tumors are also sensitive to checkpoint abrogation. Two possible explanations are cytoplasmic sequestration of p53 or a defect in p53 oligomerization. In this study, we investigated the DNA damage response and UCN-01 sensitivity of two p53 wildtype neuroblastoma cell lines: SK-N-SH, and SH-SY5Y. In order to determine the responses of these cells to DNA damage, the cells were treated with concentrations of SN38 ranging from 0-30 ng/ml. Both lines arrested in G2, S, or G1 depending upon SN38 concentration and displayed an increase in p53 levels with increasing SN38. Additionally, p53 was phosphorylated on serine 15 and serine 20 following SN38 treatment in all three cell lines, suggesting that p53 is active. In order to determine whether these cells are susceptible to UCN-01-mediated abrogation of cell cycle arrest, cell were treated with 3 ng/ml SN38 for 24 hours, followed by 25 nM UCN-01 for 6 and 24 hours. The SK-N-SH showed now sensitivity to UCN-01 treatment whereas the SH-SY5Y abrogated S arrest within 6 hours and abrogated G2 arrest within 24 hours. We also analyzed the oligomerization status of p53 using glutaraldehyde crosslinking. The SK-N-SH cells possessed levels of p53 dimers and tetramers similar to what has previously been reported in p53 wildtype MCF10A cells. The SH-SY5Y, however, had extremely low levels of dimers and tetramers. Consistent with this, only SK-N-SH showed activation of p21waf1 and repression of cyclin B in response to SN38 treatment. Previous studies have reported cytoplasmic sequestration as a mechanism of p53 inactivation in p53 wildtype neuroblastomas. In order to determine the sub-cellular distribution of p53, we prepared nuclear and cytoplasmic extracts. Both the SK-N-SH and SH-SY5Y had primarily nuclear p53. The results of this study suggest that oligomerization status may serve as an indicator of sensitivity of p53 wildtype tumors to the therapeutic combination of DNA damage agent and checkpoint inhibitor.

#3694

Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein.

Surendra R. Punganuru, Hanumantha Rao Madala, Debasish Basak, Kalkunte S. Srivenugopal. _Texas Tech University Health Sciences Center, Amarillo, TX_.

Goniothalamin (GTN) is a secondary metabolite styryl lactone isolated from several species of the tropical medicinal tree Goniothalamus. GTN has been shown to be cytotoxic and induce apoptosis in many cancer cell lines. This study sought to define the molecular basis underlying the antiproliferative actions of GTN. We synthesized the R and S enantiomers of GTN and found the R form to be more cytotoxic against a panel of breast cancer and lung cancer cell lines. The IC50 of GTN against breast cancer cell lines was in the range of 10-20 µM and interestingly, the SKBR3 cells, which harbor a R175H mutation in p53 were more sensitive to the drug. In contrast, the normal breast epithelial MCF10A cells were not killed by GTN, and N-acetylcysteine prevented cell-killing, indicating the ROS involvement. Indeed, the flow cytometry and cell staining with DCF-DA showed high levels of ROS generation, and this was accompanied by significant S-glutathionylation of bulk proteins. Other studies showed that GTN forms conjugate with glutathione with ease and deplete GSH levels in cells. Because p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by GTN may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. We also engineered the p53-null H1299 lung cancer cells to stably express the R175H mutant protein by lentiviral technology as an isogenic model. The conformation-specific antibodies for p53, namely Pab1620 that recognize the wt-p53 and pab420 that detects the mutant p53 were used for validating the p53 restoration. Immunoprecipitation and immunostaining using confocal microscopy showed that GTN treatment of R175H p53-containing cells results in a reciprocal loss of mutant protein and increase of wt-like protein. Further, the EMSA revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes. The changes were associated with a G2/M arrest and significant apoptosis. Increased levels of apoptotic markers suggested a priming action of GTN on cell death pathways. GTN also suppressed the SKBR3 cell migration and invasion at 5-10 µM. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay with either GTN alone or in combination with cisplatin. Our results shed light on the multiple mechanisms, including glutathione depletion, generation of redox imbalance, protein glutathionylation and p53 reactivation in the GTN cytotoxicity. We suggest that GTN-induced oxidative milieu facilitates a functional restoration of the mutant p53 through a thiolation of the redox-sensitive cysteines present in the DNA-binding domain. Our study will help to establish redox-perturbation as a paradigm for reactivation of the defective tumor suppressor (supported by a CPRIT grant [RP130266] to KSS).

#3696

Biological significance of the wild-type p53-induced phosphatase 1(Wip1) expression in invasive breast cancer.

Yuka Inoue,1 Nami Yamashita,1 Eriko Tokunaga,2 Hiroyuki Kitao,1 Kimihiro Tanaka,1 Hiroki Ueo,1 Hiroshi Saeki,1 Eiji Oki,1 Yoshihiko Maehara1. 1 _Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;_ 2 _National Hospital Organization Kyushu Cancer Center, Fukuoka, Japan_.

Backgrounds The wild-type p53-induced phosphatase 1(Wip1) is a member of the serine/threonine protein phosphatases, and plays an important role in the nucleus as one of the key components in the DNA damage response (DDR) network. Wip1 is encoded by the protein phosphatase magnesium dependent 1 delta (PPM1D), sited on locus 17q23. PPM1D amplification has been reported in breast cancer.

Aims We evaluated the expression of Wip1 mRNA, Wip1 protein and PPM1D DNA copy number to clarify the relationship between Wip1 expression and the clinicopathological features and prognosis to determine the biological significance of Wip1.

Materials and Methods Breast cancer cells (MCF7, T47D, MDA-MB231, HCC1937, HS578T, BT20 and SKBr3) were used for Wip1 expression analysis and copy number analysis. Primary invasive ductal carcinoma specimens were obtained from Japanese patients who underwent surgery without neoadjuvant chemotherapy or endocrine therapy. Wip1 mRNA expression was evaluated in 140 cases by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Wip1 protein expression was evaluated in 192 cases by immunohistochemistry (IHC). The PPM1D DNA copy number was analyzed by genomic PCR in 33 breast cancer cases and by the single-nucleotide polymorphism-Comparative Genomic Hybridization (SNP-CGH) array in 12 cases. The effects on the cell growth of the Wip1 inhibitor (GSK2830371) was analyzed by the viability assay in MCF7.

Results Wip1 mRNA expression was significantly higher in MCF7, luminal type cell line. Wip1 mRNA expression was divided into four groups, very high, high, low and very low. The very high Wip1 mRNA expression was significantly associated with positive estrogen receptor (ER) expression (p=0.02). There was no significant correlation between Wip1 mRNA expression and the prognosis. Wip1 protein nuclear expression was positive in 21 cases (10.9%). There was no significant association between the Wip1 protein expression levels and the clinicopathological factors and the prognosis. PPM1D DNA copy number significantly correlated with Wip1 protein expression (p=0.0035). Amplification at 17q23 was detected in 6 cases by SNP-CGH array, and all of these six cases showed positive nuclear Wip1 expression. PPM1D amplification was not observed in Wip1 negative cases. In the cell viability assay, the suppression of the MCF7 cell growth was observed by Wip1 inhibitor.

Conclusions Wip1 nuclear protein expression may be regulated by PPM1D amplification, and the Wip1 inhibitor may have the therapeutic effects for the breast cancer with PPM1D amplification or high Wip1 protein expression.

#3697

Gastric adenocarcinoma TP53 mutations in an ethnically admixed population.

Melissa Pool Pizzi, Helano Carioca Freitas, Maria Galli Amorim, Bruna Durães de Figueiredo Barros, Frederico Omar Gleber-Netto, Ana Flávia Mattos Costa, Maria Dirlei Begnami, Adriane Graicer Pelosof, Diana Noronha Nunes, Emmanuel Dias-Neto. _AC Camargo Cancer Center, Sao Paulo, Brazil_.

TP53 protein is one of the most important and studied human tumor suppressors. Whereas TP53 drives the malignant transformation and is the most frequently mutated gene in human cancers, the location and the type of its mutations for specific tumor types in ethnically admixed populations have not been evaluated. In this study we employed an amplicon panel to capture and to deep-sequence all the exons of TP53 in a series of 29 tumor biopsy samples derived from Brazilian individuals diagnosed with gastric adenocarcinomas (GACs), which were compared to Asian and Europeans.

TP53 sequences were obtained from Brazilian samples with the Ion Torrent AmpliSeq TP53-panel and determined in the Ion PGM Torrent and Ion Proton platforms. We generated a mean of 7.25 million reads per patient, resulting in a median coverage >13,000X. All samples were covered above 6,400X. The mutations observed here were compared to those described in patients from different ethnic groups obtained from TCGA composed for 103 Asians patients (Japan, Hong-Kong, South Korea, Vietnam) and 78 cases of Europeans. The Asian and European populations showed higher prevalence of intestinal type GAC (53.7% and 61.6%, respectively) in contrast to our cohort, in which 69% of the patients had diffuse-type GACs (p<0.001). We found 13 distinct TP53 mutations in 14 cases (48.3%). From these 14, 28.5% (4 cases) occurred in intestinal-type GACs and 71.5% (10 cases) in diffuse-type. Whereas Europeans and Asian patients shared 44.3% (n=27) of the mutations, we found 39% of our TP53 mutations to be shared between Brazilian and European tumors and the same percentage between Brazilians and Asian patients. A single missense mutation, 17:g.7577094G>A p.Arg282Trp in the DNA binding domain in exon 8, was found in all three sample groups studied, suggesting a possible role for this mutation in GAC pathogenesis. Although TP53 is the most frequently mutated gene in GACs we found no specific patterns that could explain the differences in the clinical behavior of this tumor between Asian and non-Asian patients. Further analyses are being performed trying to correlate mutations in TP53 and other genes with the different clinical outcomes observed in GACs from Asians and non-Asians, and in a setting of high genetic admixture, as observed in Brazil.

#3698

The effect of epigenetic silencing and p53 mutation on DLL4 expression in human cancer stem cell disorder: The Li-Fraumeni syndrome.

Zhixing Yao, Zaki A. Sherif. _Howard University, Washington, DC_.

Tumorigenesis results from an accumulation of mutational and epigenetic changes that alter normal cell growth and survival pathways. Li-Fraumeni syndrome (LFS) is a clinically and genetically heterogeneous inherited cancer syndrome that can provide powerful insights into our understanding of the somatic mutations present in sporadic cancers. LFS is currently linked to heterozygous mutation in the tumor suppressor gene p53 (TP53), which encodes a transcription factor that responds to diverse cellular stresses through the regulation of target genes that induce apoptosis, cell cycle arrest, DNA repair, and senescence. We previously showed a balanced reciprocal chromosomal translocation t(11;15)(q23;q15) in the non-cancerous skin fibroblasts of a bilateral breast cancer patient in LFS family. This prompted us to investigate the breakpoint region of the translocation, which uncovered a gene that encodes DLL4, (Delta-like ligand 4; locus at 15q15.1), a Notch ligand that is a key target in tumor vasculature. DLL4 regulates T-cell lineage thymopoiesis and further affects cancer immunosurveillance. In the breakpoint region of chromosome 15q15, we examined THBS1, DNAJC17, Rad51, DLL4, CHAC1 and INO80 gene expression levels. We found that specifically DLL4 is abrogated in all the LFS cell lines as well as MDA231 breast cancer cell line, and drastically down-regulated in MCF7 breast and IMR32 brain cancer cell lines and several human organ-specific tumor samples. Furthermore, DNA methylation studies revealed that DLL4 promoter is silenced only in MCF7 and MDA231 but not in LFS cell lines. ChIP and siRNA knockdown assays demonstrated that p53 binds to DLL4 promoter through its association with CTCF, a chromosomal networking protein CCCTC binding factor. These results imply a possible karyotype-phenotype correlation with respect to DLL4 in LFS and breast cancer initiation and progression. The drastic reduction or absence in the expression of DLL4 in normal skin fibroblasts of LFS patients as well as breast and brain cancer cells is significant and supports the concept that this ligand may also play a role in cancer immune-surveillance; and its resuscitation in the tumor microenvironment may stimulate T-cell immunity and suppress tumor growth.

#3699

Aspirin acetylates wild type and mutant p53 in colon cancer cells: Identification of aspirin acetylated sites on recombinant p53.

Guoqiang Ai,1 Rakesh Dachineni,1 Ramesh D. Kumar,2 Srinivasan Marimuthu,3 Lloyd F. Alfonso,4 Jayarama G. Bhat1. 1 _South Dakota State University, Brookings, SD;_ 2 _Department of Aquatic Animal Health Division, CIBA, Chennai, India;_ 3 _Clinical Research Institute for Mother and Child Health Care, Poojapura, India;_ 4 _D'Youville College School of Pharmacy, Buffalo, NY_.

Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anticancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. Aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21CIP1. Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anticancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin.

#3700

Association of the G/G-SNP309 variant in the mdm2 gene with earlier tumor onset in female renal cell carcinoma patients.

Christine G. Stoehr,1 Robert Stoehr,1 Antonia Wenners,2 Arndt Hartmann,1 Simone Bertz,1 Verena Spath,3 Bernhard Walter,4 Kerstin Junker,5 Holger Moch,6 Raoul Hintze,7 Stefan Denzinger,8 Elisabeth E. Bond,9 Gareth L. Bond,9 Karen Bluemke,10 Katrin Weigelt,1 Verena Lieb,1 Elke Nolte,1 Paolo Fornara,10 Bernd Wullich,1 Sven Wach,1 Helge W. Taubert1. 1 _Univ. of Erlangen, Erlangen, Germany;_ 2 _Universitätsklinikum Schleswig-Holstein, Kiel, Germany;_ 3 _Caritas Hospital Bad Mergentheim, Bad Mergentheim, Germany;_ 4 _District Hospitals Altötting Burghausen, Altötting Burghausen, Germany;_ 5 _University of Saarland, Homburg, Germany;_ 6 _UniversitätsSpital Zürich, Zürich, Switzerland;_ 7 _Helios Hospitals Schwerin, Schwerin, Germany;_ 8 _University of Regensburg, Regensburg, Germany;_ 9 _University of Oxford, Oxford, Germany;_ 10 _Martin Luther University Halle-Wittenberg, Halle, Germany_.

Mdm2 (Human mouse double minute 2) is an important opponent of the tumor suppressor p53. The G/G variant of SNP309 in the MDM2 promoter can increase Mdm2 mRNA/protein expression and is associated with an increased risk and earlier onset of different cancers especially in Asian populations. But the frequency and impact of the G/G variant has not been studied in Caucasian renal cell carcinoma (RCC) patients. Therefore, we analyzed an unselected German cohort of 197 consecutive RCC patients and detected the G/G variant in 18 (9.1%) patients, the G/T variant in 116 (58.9%) patients and the T/T variant in 63 (32.0%) patients. Studying the association between age at tumor onset and SNP309 genotypes, no correlation was detected in the entire RCC cohort or among the male RCC patients. However, the female G/G patients (median age 59.5 years)

were diagnosed 13.5 years earlier than the T/T females (median age 73 years). Next, when separating all females into two groups at their median age (68 years), 7 patients and 1 patient with the G/G variant and 9 patients and 13 patients with the T/T variant were noted in these age groups (P=0.024). To study the age dependency of tumor onset further, a second, age-selected cohort of 205 RCC patients was analyzed, which comprised especially young and old patients. Interestingly, the G/G type occurred more often at lower tumor stages and tumor grades compared with higher stages (P=0.039 and P=0.004, respectively). In females, the percentage of the G/G variant was only slightly higher in the younger age group, whereas in males, the percentage of the G/G variant was remarkably higher in the younger age group (19.4% vs. 8.0%). In summary, female Caucasian RCC patients with the MDM2 SNP 309 G/G variant showed a significantly earlier tumor onset than patients with the wildtype T/T genotype.

#3701

Transcriptomic consequences of MDMX (MDM4) oncoprotein expression knockdown in MDMX-amplified breast cancer cells.

Yi-hsuan Ho, Claire Hutton, Herbie Newell, John Lunec. _Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom_.

The tumour suppressor p53 is activated by cellular stress to induce cell cycle arrest and/or apoptosis. Although the TP53 gene is frequently mutated in cancer, approximately half of human cancers express wild type and functional p53. However, p53 activity is often suppressed by its negative regulators, MDM2 and MDMX. Small molecule antagonists have been developed to inhibit p53-MDM2 binding to release the growth inhibitory and/or pro-apoptotic functions of p53. Previous studies have indicated that MDMX amplification and/or increased protein expression may be associated with resistance to MDM2-p53 binding antagonists. However, specific inhibitors for MDMX have yet to be developed and an MDM2/MDMX-p53 co-inhibitor (RO-5963) does not show better efficacy against high MDMX expressing cells than single target MDM2 inhibitors. To better understand the cellular function and therapeutic potential of targeting MDMX, we have investigated the downstream transcriptomic consequences of knocking down MDMX expression.

Knockdown of MDMX protein expression, demonstrated by Western blot analysis, was achieved in MRK-nu-1 MDMX amplified and TP53 wild-type breast carcinoma cells using both lentiviral shRNA and siRNA. MRK-nu-1 cell growth suppression was observed after knockdown of MDMX. Affymetrix Human Transcriptome Array 2.0 was used to detect differences in the expression of full genes and alternatively spliced forms between negative control and MDMX knockdown groups. Transcriptome Analysis Console 3.0 software was used to analyse the microarray data to identify significant changes in gene expression, alternative splicing and associated functional pathways. Alterations in mRNA expression for a selected panel of genes was confirmed by qRT-PCR.

A greater degree of MDMX expression knockdown was achieved with the siRNA (>95%) than the lentiviral shRNA (>50%) This was reflected by increased suppression of growth and larger changes in gene expression patterns with the siRNA knockdown. Although a similar set of genes was affected by both the siRNA and shRNA knockdown, the magnitude of changes was greater following the more pronounced knockdown with siRNA. The expression of a number of p53 transcriptional target genes was found to be altered, including CDKN1A, MDM2, CCNG2, RRM2B, BTG2, ZMAT3 and FAS consistent with a role for MDMX in suppression of p53 function in these MDMX amplified cells.

#3702

Src phosphorylated Mdm2 requires MdmX to act as a neddylating ligase.

Paula M. Hauck, Lindsey D. Mayo. _Indiana School of Medicine, Indianapolis, IN_.

Mdm2 is an oncoprotein and, along with its family member MdmX, have been shown to be elevated in human cancers. Like Mdm2, MdmX, is a RING containing protein, yet it lacks intrinsic E3 ligase function. Mdm2 has been shown to facilitate ubiquitination as a monomer, a homodimer, and as a heterodimer with MdmX depending on the cellular stress. Our recent report establishes that under growth conditions that activate Src, Mdm2 functions as a neddylating enzyme. Whether MdmX is necessary for Mdm2 to neddylate p53 has yet to be shown. Here we demonstrate that Src phosphorylation results in increased levels of MdmX, increased binding between Mdm2 and MdmX, and increased neddylation of MdmX and p53. Interestingly, the lack of MdmX (in transient assays or in shRNA cell lines) results in decreased neddylation of p53. These data support a critical role for MdmX as part of the Mdm2 neddylating complex.

#3703

MDM2 regulation of MYC and MYCN in pediatric neural cancers.

Hung N. Tran, Donglai Qi, David Cobrinik. _Children's Hospital Los Angeles, Los Angeles, CA_.

Background

MYC and MYCN have critical roles in a wide range of cancers and can be deregulated through diverse transcriptional and post-transcriptional mechanisms. Recent studies have shown that the high-level MYCN expression needed to drive retinoblastoma and MYCN-amplified neuroblastoma proliferation depends upon efficient MYCN translation mediated by highly expressed MDM2 and acting via a p53-independent mechanism. Here, we assessed MDM2-mediated regulation of MYC as well as MYCN in other pediatric neural tumors, specifically in Group 3 medulloblastoma and in MYC-overexpressing neuroblastoma. We also assessed MDM2-mediated regulation of MYCN in small cell lung cancer (SCLC), which has genetic and morphologic features in common with retinoblastoma.

Methods

MDM2, MYCN, and MYC expression were defined in Group 3 medulloblastoma lines D283, D425, D341; in MYC-overexpressing neuroblastoma lines CHLA 255 and SY-5Y; and in p53-wild type and p53-mutant SCLC lines H69 and H526 by western blotting. Cells were infected with shMDM2 and scrambled control lentivirus and effects on MDM2, MYC, and MYCN protein expression defined using western blot and effects on cell growth defined using Cell Titer-Glo. In neuroblastoma cells, MDM2 knockdown effects were defined with or without p53 co-knockdown.

Results

Group 3 medulloblastoma lines D283, D341, D425 highly expressed MYC but not MYCN. MDM2 knockdown did not decrease MYC expression in these cells. MDM2 knockdown decreased MYC expression In CHLA 255 and SY5Y neuroblastoma cells, along with p53 induction and cell death, and MYC down-regulation and cell death were mitigated by co-knockdown of p53. MDM2 knockdown decreased MYCN expression and impaired viability in p53-wild type H69 SCLC cells and in p53-mutant H526 SCLC cells.

Conclusions

MDM2 is not needed to sustain high-level MYC expression in three Group 3 medulloblastoma cell lines. MDM2 sustains high-level MYC in two MYC-overexpressing neuroblastoma cell lines via a p53-dependent mechanism distinct from the p53-independent MYCN regulation in MYCN-amplified neuroblastomas. In SCLC, MDM2 sustains high-level MYCN expression and viability in a p53-independent manner, similar to MDM2 effects in retinoblastoma and in MYCN-amplified neuroblastoma. These findings suggest that MDM2 selectively sustains MYCN but not MYC expression through a p53-independent mechanism and raise the possibility that cis-acting elements specific to MYCN-RNA mediate MDM2 regulation.

#3704

TRISENOX disrupts MDM2-DAXX-HAUSP interaction and increased promyelocytes formation in murine model of APL.

Sanjay Kumar, Paul Tchounwou. _Jackson State University, Flowood, MS_.

Trisenox (ATO) is widely used in the treatment of all age group of acute promyelocytic leukemia (APL) patients in both induction and consolidation therapy. In vitro studies are shown that it works through oxidative stress, cell cycle regulation and apoptosis to kill APL cells having PML-RAR α oncogene. Some ATO resistant case of APL patients are reported with different oncogenes combination. However, in vivo anti-leukemic mechanism of action of ATO in APL patients poorly known. We have used transgenic murine model to elucidate the anti-cancer properties of ATO through new target. We hypothesized that ATO disrupts MDM2-DAXX-HAUSP interaction and also increased more number of promyelocytes formation inside bone marrow cells of APL mice. To test the hypothesis, we used different groups of younger (4-5weeks old) transgenic APL mice and standardized different dose of ATO (1.25, 2.5, 5.0 and 7.5 mg/kg body wt) after 21days continuous treatment in PBS. We utilized western blotting, immunocytochemistry, confocal imaging and other molecular techniques to identify molecular mechanisms of ATO action in APL mice hepatocytes as well as bone marrow cells. We found that it increases number of promyelocyte formation in bone marrow cells and also degradation of PML-RARαdose dependent fashion. ATO nicely activated p53 expression in both liver tissue as well as bone marrow cells of APL mice through degradation of MDM2. It was also disrupted both expression and association of MDM2-DAXX-HAUSP complex molecules dose dependent manner in mice liver tissues. On the basis of these findings, we conclude that ATO disrupts MDM2-DAXX-HAUSP interaction, increased promyelocytes formation, MDM2 degradation by accumulating P53 in APL mice liver tissues. It is novel target for treatment of APL patients through designing of new anti-leukemic drugs.

Keywords: Trisenox, P53, MDM2-DAXX-HAUSP complex molecules APL Mice hepatocytes and bone marrow cells.

Acknowledgements: This research was financially supported by National Institutes of Health NCRR Grant No. 5G12RR013459 and MIMHD Grant No. 8G12MD007581, through the RCMI-Center for Environmental Health at Jackson State University.

#3705

PLK2 phosphorylates TAp73 and prohibits TAp73 tumor suppressor activity in osteosarcoma cells.

Zhengbo Hu,1 Hai Lu,2 Anmin Jin,1 Xiaohong Liao,3 Zunying Xu,1 Chao Dong1. 1 _Zhujiang Hospital of Southern Medical University, Guangzhou, China;_ 2 _the Third Affiliated Hospital of Southern Medical University, Guangzhou, China;_ 3 _Guangzhou Medical University, Guangzhou, China_.

Background: TAp73, as a member of the p53 tumor suppressor family, is frequently overexpressed in malignant tumors in humans. The abundance of TAp73 expression and phosphorylation modification regulate its transcriptional activity. In previous study, we found that the anti-tumor function of TAp73 was reactivated by protein dephosphorylation. Polo-like kinase 2 (PLK2) was previously found phosphorylated p53 and transcriptionally regulated by p53, and these interactions affected the fate of cells. However whether PLK2 interacts with TAp73, phosphorylates TAp73, and modulate its tumor suppressor function are still unclear. Herein, we investigate how PLK2 phosphorylates TAp73 and affects TAp73 function in human osteosarcoma cells.

Materials and Methods: Osteosarcoma cell lines, Saos2 and MG63 were used as models with differential expression levels of TAp73. Phosphorylation predictor software Scansite 3.0 and the predictor GPS-polo 1.0 were used to theoretically analyze the phosphorylation sites on TAp73. Co-immunoprecipitation (Co-IP), phosphor-tag Western Blot (WB), and indirect immunofluorescence assays were used to determine the interaction between PLK2 and TAp73. TAp73 activity was assessed by testing downstream genes, P21 and PUMA using Western blot and RT-PCR. The physiological effects of PLK2 interacting with TAp73 on cell cycle G1 phase, cell proliferation and cell apoptosis were measured by flow cytometry, cell counting kit-8 and terminal-deoxynucleotidyl transferase mediated nick end labeling assays.

Results: TAp73 expression was low in Saos2 cells when compared with MG63 cells, at both mRNA and protein levels, but both have the similar level of PLK2 expression. DNA damaging drugs, Cisplatin and Adriamycin, up-regulated TAp73 and PLK2 expression on does-dependent way. We found that PLK2 directly bound to and phosphorylated TAp73 when TAp73 induced by DNA damaging. The phosphorylation predictor software identified the potential phosphorylation sites that PLK2 could phosphorylate TAp73. PLK2 phosphorylated TAp73 at residue Ser48, which prohibited TAp73 translocation to the nucleus. Additionally, combination of PLK2 inhibition by siRNA with DNA-damaging drugs up-regulated both p21 and PUMA mRNA expression to a greater extent than DNA-damaging drug treatment alone. Inhibiting PLK2 in TAp73-induced osteosarcoma cells enhanced the effects of the DNA-damaging drugs on G1 phase arrest. Moreover, inhibiting PLK2 in TAp73-induced cells sensitized the effects of DNA-damaging drugs on cell proliferation and apoptosis. However, TAp73-knockdown decreased the antitumor effects of DNA-damaging drugs.

Conclusion: These findings reveal a novel PLK2 function in the phosphorylation of TAp73, which prohibits TAp73 anti-tumor activity in osteosarcoma cells. Further studies could be investigated how to target PLK2 and enhance TAp73 antitumor activity in preclinical and clinical trials.

#3706

p53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression following thymidylate synthase (TS) inhibition.

Prashanth Ravishankar Gokare,1 Niklas Finnberg,1 Jenny Dai,2 Maureen Murphy,3 Wafik El-Deiry1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Pennsylvania State University, Hershey, PA;_ 3 _Wistaria Institute, Philadelphia, PA_.

Nucleotide catabolism by cancer cells can influence malignant behavior and intrinsic resistance to therapy. The rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) contributes to the pharmacokinetics of fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU. Histone H3K9 acetylation marks at the DPYD promoter is diminished concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Notably we find that the P72 allele of p53 suppresses DPYD expression more than the R72 p53 allele following 5-FU treatment in mouse embryo fibroblasts. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition by 5-FU, methotrexate (MTX), raltitrexed and siRNA in cancer cells in vitro as well as in mice in vivo. DPYD repression by p53 is dependent on DNA-PK and ATM-signaling since pharmacologic targeting of these kinases reverses the transcriptional repression of DPYD by p53. Mice lacking p53 in their livers have increased conversion of 5-FU to 5-FUH2 in plasma and elicit a diminished 5-FU therapeutic response in syngeneic colorectal tumor xenografts as compared to littermates with an intact p53 allele consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through its ability to regulate DPYD, particularly following metabolic stress imposed by nucleotide imbalance. The findings have implications for the toxicity and efficacy of the cancer therapeutic 5-fluorouracil.

#3707

Semiconductor-based next-generation sequencing analysis of 409 cancer-related genes for mutations and copy-number variations in oral squamous cell carcinoma.

Takafumi Nakagaki,1 Yasushi Sasaki,1 Masashi Idogawa,1 Ryota Koyama,1 Kenta Kobashi,1 Miyuki Tamura,1 Tomoko Ohashi,1 Kazuhiro Ogi,2 Hiroyoshi Hiratsuka,2 Takashi Tokino1. 1 _Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo, Japan;_ 2 _Department of Oral Surgery Sapporo Medical University School of Medicine, Sapporo, Japan_.

Somatic mutation analysis is standard of practice for human cancers in order to identify therapeutic sensitizing and resistance mutations. To better understand the molecular pathogenesis of oral squamous cell carcinoma (OSCC) patients, we performed comprehensive genomic analyses that use PCR target enrichment and next-generation sequencing on the Ion Torrent semiconductor sequencers (PGM and Proton). DNA (40 ng) was extracted from 45 human OSCC specimens and their corresponding non-cancerous tissues including FFPE samples. Using the Ion Ampliseq Comprehensive Cancer Panel, we sequenced 15992 loci from 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers (covered regions = 95.4% of total, 1.5 megabases of target sequence). We also detected copy number variations (CNVs) in which segments of the genome can be duplicated or deleted from sequencing data.

Each sample underwent on average 5.9 million sequencing reads after quality filtering. The mean read depths were 367.8 x, and >95% of targeted bases were represented .The number of somatic mutations (SNVs and indels) in 45 patients with OSCC ranged from 1 to 36 with a median of 7.33 (6.40/Mb). The most frequent mutations were detected on TP53 (28 of 45; 62.2%). Many of the mutations on TP53 were detected in the DNA-binding domain (23 of 28; 82.1%). NOTCH1 mutations were identified in 10 cases (8 missense, 1 coding frameshift, and 1 essential splice site mutations). CDKN2A mutations were observed in 8 cases; and PIK3CA were mutated 3 cases. Although the most common mutations in OSCC were C/G>T/A transitions, which are consistent with previous reports on head and neck, lung and oesophageal SCCs, the second most frequent mutations were C/G>A/T transversions. We also identified a median of 100 significant CNVs (range of 0-481) per sample. Pathway assessment has shown that somatic aberrations within OSCC genomes are mainly involved in several important pathways, including cell cycle regulation (p53 pathway, 84.4%) and RTK-MAPK-PI3K (62.2%). This targeted next generation sequencing using low amounts of FFPE DNA is a valuable tool for high-throughput genetic testing in research and clinical settings.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Cell Death Pathways and DNA Repair

#3708

Targeting synthetic lethality with PARP inhibition in colorectal cancer.

Titto Augustine,1 Radhashree Maitra,2 Jinghang Zhang,1 Jay Nayak,2 Sanjay Goel2. 1 _Albert Einstein College of Medicine, Bronx, New York, NY;_ 2 _Montefiore Medical Center, Bronx, New York, NY_.

Background:

Small molecule poly ADP-ribose (PAR) polymerase (PARP) inhibitors (PARPi) purportedly result in DNA damage and are promising agents in the treatment of cancer. In a quest to identify best "synthetic lethality" for treatment, we have screened various combination therapies in colorectal cancer (CRC) cell lines. In addition, we have interrogated if the presence of microsatellite instability (MSI) status favor susceptibility of cells to synthetic lethality and thus acts as a prognostic marker of combination therapy.

Methods:

Using the SRB assay, the 12 combinations of the PARPi, veliparib (V), rucaparib (R), olaparib (Ol) and PJ34, and the cytotoxic drugs, irinotecan (I), 5-fluorouracil (5-FU) and oxaliplatin (Ox) were screened in HCT116 and LIM2405 cell lines. We assessed the effect of the combination using the CalcuSyn software for combination index (CI). We next evaluated the effectiveness and sequence dependence of combination in an additional 2 cell lines that had MSI status (RKO and HCT15) and 2 that had microsatellite stable (MSS) status (HT29 and SW837) and in 7 HCT116 isogenic lines (mutants of Bax, p21, p53, PTEN, Kras and DNMT and wild type of PIK). The combination of I and R was confirmed by FACS analysis in HCT116 and LIM2405.

Results:

Ol and R showed synergy in combination with I, whereas, V and PJ34 showed antagonism with Ox, including in Ox-sensitive cells. Single agent cytotoxicity was observed at 200nM for R, whereas V required more than 30uM. Early PARP-1 protein upregulation was observed upon Ox administration; but the changes were not reversed with either R or V. Nevertheless, R at 200nM and V 1uM significantly reduced PAR formation in Ox-sensitive cell lines. FACS analysis showed S phase arrest in SW837 (MSS) (p=0.03) whereas G2-M arrest was observed in LIM2405 (MSI) (non-significant) upon treatment with R 400nM and I 200nM. In addition, studies using isogenic cell lines revealed the importance of p21, p53 and PTEN in exerting synergistic effect while combining R and I. Administration of I prior to R (24h each) was more cytotoxic than vice versa and simultaneous treatment in MSI cell lines.

Conclusion:

Among the various combinations studied, R with I was the most synergistic. The effect appears to be more pronounced when I precedes R, particularly in MSI cell lines. Alteration in cell cycle is MSI status dependent. Further work to identify mechanism of synergy is ongoing.

#3709

PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR).

Ymera Pignochino,1 Federica Capozzi,1 Lorenzo D'ambrosio,1 Carmine Dell'aglio,1 Marco Basiricò,1 Paola Boccone,1 Erica Palesandro,1 Loretta Gammaitoni,1 Dario Sangiolo,1 Maria Serena Benassi,2 Massimo Aglietta,1 Giovanni Grignani1. 1 _IRCCS Candiolo Cancer Institute, Candiolo, Italy;_ 2 _Istituto Ortopedico Rizzoli IRCCS, Bologna, Italy_.

Purpose of study. An attractive strategy to improve antitumor treatments is to inflict cytotoxic DNA damage with chemotherapy, and then impede DNA repair by molecular targeting. TR is a new drug characterized by a peculiar mechanism of action: TR traps DNA repair machinery leading to DNA damage, particularly in BRCA1/2-deficient tumors. We speculated that TR might activate PARP1, a key player in DNA-repair, and that subsequent PARP1 inhibition perpetuates TR-induced DNA damage leading to cell death.

Experimental procedures and results. We developed a preclinical platform of 31 cell lines from different histotypes to explore the potential synergy between TR and the PARP1-Is olaparib (OL) and veliparib. We demonstrated that, regardless of BRCA1/2 status, PARP1-Is significantly increased TR activity, but a 15-fold range of sensitivity to the combination was observed. OL was proven the best PARP-I to combine with TR, probably due to its PARP1 trapping activity. In selected experiments, whole-genome expression profiling and GSEA analysis comparing cells displaying high vs. low synergism of the combination (HS-C vs. LS-C) revealed that DDR, G2/M cell cycle checkpoint, and DNA repair pathways were mechanistically involved in TR+OL synergy. TR induced PARP1 activation in 3/6 cell lines and PARP1-Is completely blocked both basal and TR-induced PARP1 activation. OL enhanced DNA damage response in 6/6 cell lines, but unrepairable DNA fragmentation was obtained in cells with high PARP1expr only. In two independent cell panels TR+OL synergism was directly related to PARP1expr both at mRNA (Pearson score r: 0.70, p=0.00079) and protein level (r: 0.71, p=0.015). Silencing and overexpression experiments validated the functional role of PARP1expr in determining TR+OL synergism: in HS-C the downmodulation of PARP1expr reduced sensitivity to TR+OL while the overexpression of PARP1 in LS-C rose TR+OL activity to levels observed in HS-C. Subcutaneous, intravenous and orthotopic xenografts of one HS-C (DMR) and one LS-C (SJSA-1) in NOD/SCID mice revealed OL significantly increased antitumor and antimetastatic activity of TR in cells with high PARP1expr only. Finally, we demonstrated that basal PARP1expr PARP1 activation by other cytotoxics with a stronger PARP1 activation observed in cells with high vs. low PARP1expr, regardless of the considered drug.

Conclusions. OL enhances and potentially broaden TR cytotoxicity. TR+OL combination is particularly attractive in tumors harboring high PARP1expr and specific DDR-R gene signatures that might become predictive biomarkers of response. Future clinical validation of TR+OL combination may extend the use of PARP1-Is beyond BRCA1/2 defective tumors. Indeed, the crucial role of PARP1expr is confirmed regardless of tumor histotype and BRCA1/2 status. Further studies of combination between PARP1-Is and other cytotoxics should consider basal PARP1expr and activation after drug exposure.

#3710

Synergy between PARP and Wee1 inhibitors suggests homologous recombination repair defect in NSCLC as a mechanistic target for combination therapy.

Daniel X. Yang. _Dana Farber Cancer Institute, Boston, MA_.

Advanced non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality. Despite progress in targeted molecular therapeutics and precision medicine, outcomes in this disease remain poor. Recent evidence suggests that impaired homologous recombination (HR) occurs in a significant subset of NSCLCs and may serve as a predictive biomarker for sensitivity to DNA damaging agents. Poly-ADP ribose polymerase (PARP) and Wee1 inhibition represent two mechanistically distinct approaches to augment the effects of DNA damage. Specifically, the PARP inhibitor olaparib impairs repair of DNA single strand breaks, which during replication lead to the formation of DNA double strand breaks (DSBs), resulting in synthetic lethality in HR deficient tumors. AZD1775 is a Wee1 inhibitor that abrogates the G2 checkpoint and thus removes a safeguard against cell cycle progression with unrepaired DNA damage. Moreover, AZD1775 has been recently reported to exhibit single-agent activity in patients harboring BRCA1/2 mutations. Therefore, we hypothesize that olaparib and AZD1775 would have synergistic effects in a subset of NSCLCs and that HR deficiency could be predictive of tumor response to combination therapy.

Utilizing Rad51 focus formation as a marker of HR deficiency, we prospectively selected representative NSCLC cell lines that either did (e.g. Calu6) or did not (e.g. A549) harbor putative defects in HR repair. We treated Calu6 and A549 and other NSCLC cells with AZD1775 and olaparib with varying drug dosing and sequencing to determine the optimal regimen for synergistic effect. Cytotoxicity was determined by CellTiter-Glo cell viability assays and synergy was quantified by calculating the combination index. Additionally, we investigated mechanistic protein markers by Western blot.

In response to combined olaparib and AZD1775 treatment, Calu6 cancer cells demonstrated markedly more pronounced synergistic sensitivity (median CI = 0.19) compared to A549 cancer cells (median CI = 0.90). Moreover, a similar trend toward a selective synergistic effect was demonstrated in a panel of 10 additional NSCLC lines. On biochemical analysis, we observed inhibition of p-Cdk1, upregulation of p-Chk1, and upregulation of p-KAP1, suggesting abrogation of the G2/M checkpoint and activation of ATM/ATR repair pathways, all consistent with the mechanistic underpinnings of our hypothesis.

Taken together, these results provide early pre-clinical evidence for the rational combination of Wee1 and PARP inhibition in the treatment of advanced NSCLC, and suggest HR deficiency as a predictive marker applicable to NSCLC. Continued mechanistic investigation and further confirmatory studies are warranted to inform the selection of patients who may maximally benefit from such combination treatment.

#3711

Pre-clinical combinations of ATR and PARP inhibitors: Defining target patient populations and dose schedule.

John Pollard,1 Phil Reaper,1 Adele Peek,1 Stuart Hughes,1 Hakim Djeha,1 Steven Cummings,1 Karen Larbi,1 Marina Penney,2 Jim Sullivan,2 Darin Takemoto,2 Chris DeFranco2. 1 _Vertex Pharmaceuticals Inc, Abingdon, United Kingdom;_ 2 _Vertex Pharmaceuticals Inc, Boston, MA_.

Defective DNA damage repair, leading to genomic instability, is a common event during tumorigenesis. Despite enabling the persistence of mutations, any of which can confer a growth advantage to the nascent tumor, these defects place an Achilles Heel reliance on remaining repair pathways for survival from DNA damage. The protein kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) are key mediators of a DNA damage response activated by DNA damage during the S and G2 phases of cell cycle. Together they signal a series of cellular responses including activation of checkpoints and repair by homologous recombination. Loss of ATM pathway function frequently occurs in cancer, commonly from loss of function mutations in the tumor suppressor, p53, a substrate of ATM. This leads to a reliance on ATR that can be exploited for therapeutic benefit. Activation of ATR, by generation of S-phase DNA damage (replication stress, [RS]), can arise from treatment with DNA damaging drugs and certain targeted therapies such as inhibitors of poly ADP ribose polymerase (PARP). PARP is an enzyme involved in several DNA repair pathways, including base excision repair. Some PARP inhibitors have been shown to form an irreversible complex with DNA, potentially generating a direct RS lesion. While initial data indicates that inhibition of ATR and PARP is synergistic in some cancer cells, a comprehensive assessment has not been reported.

Inhibition of ATR was cytotoxic in combination with PARP inhibitors against many cancer, but not non-cancer, cells. This effect was observed with multiple PARP inhibitors irrespective of their potential to form a DNA complex. In large cell panels of over 100 cancer cell lines, greater synergy was observed for the combination of an ATR and PARP inhibitor in cell lines with mutation of the TP53 gene. This was confirmed in isogenic cell lines depleted for ATM or p53, and is consistent with the profile of ATR and DNA damaging drug combinations. Furthermore, a triple combination of a PARP inhibitor, ATR inhibitor and the DNA damaging drug, cisplatin, retained cancer cell specific cytotoxic activity. In vitro dose-scheduling studies with the doublet of a PARP and ATR inhibitor showed optimal activity was achieved with transient concurrent exposure to both agents. This schedule contrasts with that for ATR inhibitors in combination with DNA damaging drugs where sequential dosing was optimal. In a mouse xenograft model concurrent dosing on a twice-weekly schedule was effective and well-tolerated.

These data demonstrate the potential of combining ATR and PARP inhibitors in patients with p53 defective tumors. An optimal dose schedule was defined from cell and mouse studies. Together the data support clinical evaluation of ATR and PARP inhibitor combinations.

#3712

The relationship of Procollagen alpha 1 type 1 (Col1A1) / DDR2 signaling in malignant glioma and sensitivity to STAT 3/5 inhibitor.

Shumei Chen,1 Chunjing Wu,1 Ying-Ying Li,1 Medhi Wangpaichitr,1 Ronald J. Benveniste,1 James Turkson,2 Niramol Savaraj,1 Lynn G. Feun3. 1 _Miami VA Hospital/Univ. of Miami, Miami, FL;_ 2 _Univ. of Hawaii, Honolulu, HI;_ 3 _University of Miami Sylvester Comp. Cancer Center, Miami, FL_.

We have previously shown that Procollagen alpha 1 type 1 (Col1A1) is found in low and intermediate grade glioma and in less aggressive glioblastoma multiforme (GBM) (Cancer Invest. 23:577, 2005). However, these tumor cells are more prone to ER stress -inducing agents such as Befeldine (BFA) which block the transport of Procollagen to the cell surface . We also found that GBM cells which possess Col1A1 also express DDR2. The role of Col1A1/ DDR2 signaling in glioma is not known. We have used three GBM cell lines: Glioma1 established in our laboratory from a patient who progressed from grade 3 to grade 4, U-118, A-172 and U-373 to study this signaling pathway. Both Glioma1 and U-118 express Col1A1 and DDR1 and 2. A-172 and U-373 do not express Col1A1 but express both DDR2. U-373 does not express HSP47 which is an essential protein to fold collagen. In order to define the function of DDR2/ Col1A1 in GBM, we have used knock down COL1A1 and DDR2 . Silencing Col1A1 leads to a significant increase in invasiveness by Matrigel assay which indicated that Col1A1 is important in preventing invasion in GBM and hence is found more in low and intermediate grade glioma. However, it has minimal effect on cell cycle or cell proliferation. Next, we have silenced DDR2 in all 4 cell lines and studied the biochemical changes. We found that silencing DDR2 does not affect cell proliferation, cell death or cell cycle. However, it does affect the sensitivity to BFA. In cell lines which possess Col1A1, the cell viability increased by 20 -30% (p <0.05) . In contrast, cell lines which do not express Col1A1, the cell viability decreased by 25-30% ( p<0.01). However, there is no difference in sensitivity to the DNA damaging agent cisplatin. Thus, DDR2 may function differently depending on the presence of Col1A1. To explore this further, we have performed a limited phoshoprotein kinase array in Glioma 1 with siCol1A1 or siDDR2. We found increase in STAT3, 5 and STAT6 upon silencing either Col1A1 or DDR2 which is further confirmed by immunoblot in Glioma 1 for pSTAT 3 and 5. We then tested the antitumor effect of STAT3/5 inhibitor (SH4-54) a benzoic acid based inhibitor (provided by Dr. James Turkson) which interferes with the SH2 and DNA binding domains as well as tyr705 phosphorylation in Glioma1 and U-118 w/wo SiCol1A1 . At 0.5 uM the cell viability in Glioma 1 is 55.65 + 1.35 and 74.2 + 4.6 in U-118 while SiCol1A1 it decreased to 44.5 + 3.53% in Glioma 1 and 58.2+ 1.9, respectively, with minimal activity in A-172 which does not possess Col1A1. In contrast, there is no effect with another commercially available STAT 3 inhibitor S31-201 which only interferes with DNA binding domain. Overall, our data suggest that Col1A1/DDR2/ STAT signaling may be important in certain GBM cell lines and can be exploited for future treatment in brain tumor (Supported by Wanfang Hospital-Taipei Medical University Fellowship and VA Research Fund).

#3713

Targeting alternative DNA repair pathways for cancer cell killing with heavy ion particles.

Huichen Wang, Premkuma Saganti. _Prairie View A &M, Prairie View, TX_.

Heavy ion therapy is a promising approach for cancer treatment, particularly for locally advanced solid tumors. High linear energy transfer (LET) radiation induces greater lesions density in close proximity (clusters) compared that in low-LET radiation. Such clustered DNA damage is thought to be more difficult to repair than isolated DNA damage produced by equivalent doses of low-LET radiation and persist longer in irradiated cells. It has been known that homologous combination contributes to the repair of clustered DNA damage other than classic non-homologous end-joining (NHEJ). DNA double-strand breaks are also repaired by an alternative NHEJ pathway with PARP1, DNA ligase III and Mre11. This alternative pathway is error prone and contributes to genomic instability, such as chromosomal translocation and telomere fusion. Genetic analysis of tumors in TCGA database revealed that these genes are amplified in human tumors and their overexpression is correlated with poor overall survival of cancer patients. However, influence of alternative NHEJ pathways on the relative biological effectiveness (RBE) of heavy ions remains unclear. Here we investigated the effect of PARP-1 and DNA ligase III on RBE of heavy ion particles on cell survival and chromosomal aberration in breast cancer cells and pancreatic cancer cells. Cells were irradiated with X-ray, Carbon (290 MeV/n, 13 KeV/µM) and Iron (1 GeV/n, 150 KeV/µM). We found that overexpression of PARP-1 and DNA ligase III increased RBE of carbon and iron. PARP inhibitor reduced chromosomal aberration induced by heavy ions. This study may provide predictor of radiation responsive of tumor cells from cancer patients that would lead to patient stratification for heavy ion therapy selection

#3714

Optimisation of quinazolinedione sulphonamides as novel inhibitors of poly(ADP Ribose) glycohydrolase (PARG).

Kate Smith,1 Ben Acton,1 Dominic James,1 Cliff Jones,2 Stuart Jones,1 Allan Jordan,1 Nicola Hamilton,1 Alison McGonagle,1 Daniel Mould,1 Helen Small,1 Alex Stowell,1 Julie Tucker,3 Ian Waddell,1 Bohdan Waszkowycz,1 Donald Ogilvie1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _Astra Zeneca, Manchester, United Kingdom;_ 3 _Newcastle University, Newcastle, United Kingdom_.

The macrodomain protein poly(ADP ribose) glycohydrolase (PARG) has been shown to be a critical component in the repair of single stand DNA breaks and counteracts the function of the ARTD family of poly(ADP ribose) polymerases, commonly known as the PARPs. As PARG exists as a single protein, it presents an attractive target for therapeutic intervention in cancer cells with enhanced dependence upon DNA repair.

Inhibitors of this enzyme have proved difficult to discover and develop. Moreover, intact cell-active tool compounds which have the propensity to be used as robust chemical probes to understand PARG pharmacology, are absent from the literature.

This poster will describe our work in this emerging area, optimising a series of drug-like quinazolinedione derivatives to deliver molecules with the correct physicochemical and biochemical properties to function as in vitro cell probe compounds. These unprecedented agents display potent on-target biochemical (5 nM) and cell (10 nM) activity with a significant window to acute 3-day cytotoxicity. Moreover, these agents are selective against PARP family members and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of the scaffold will be described, alongside the outline pharmacology demonstrating on-target, selective inhibition of PARG in cells. Such tool compounds will be of value in revealing the detailed mechanisms of action of PARG in DNA repair and other PAR chain-mediated cellular processes, with the ultimate goal of delivering novel and clinically relevant therapeutic agents.

#3715

Benzimidazolone sulphonamides - potent, selective and drug-like inhibitors of poly(ADP Ribose) Glycohydrolase (PARG).

Allan Jordan,1 Ben Acton,1 Nicola Hamilton,1 James Hitchin,1 Colin Hutton,1 Dominic James,1 Cliff Jones,2 Stuart Jones,1 Alison McGonagle,1 Helen Small,1 Kate Smith,1 Alex Stowell,1 Julie Tucker,3 Ian Waddell,1 Bohdan Waszkowycz,1 Donald Ogilvie1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _AstraZeneca, Manchester, United Kingdom;_ 3 _Newcastle University, Newcastle, United Kingdom_.

In recent years, many proteins involved in DNA repair, such as ATR, ATM and PARP, have received considerable attention as potential points of therapeutic intervention in cancer. Indeed, these efforts have recently delivered several agents into clinical evaluation or FDA regulatory approval. However, the DNA repair protein poly(ADP ribose) glycohydrolase (PARG), which plays an equally critical role in DNA single stand break repair, to successful drug discovery efforts.

Through our innovative collaboration with AstraZeneca, we have discovered a novel PARG-binding pharmacophore and have employed this information to discover drug-like chemotypes, facilitating the development of potent and selective inhibitors.

This poster will describe our emerging results in this area, where a novel benzimidazolone sulphonamide scaffold has been shown potently to inhibit PARG in both biochemical and cellular assays with potencies of 40 nM and 60 nM respectively. Moreover, these agents display pharmacology consistent with the anticipated mode of action, appropriate drug-like properties and are selective against PARP1 and the close glycohydrolase homologue ARH3. The medicinal chemistry optimisation of this scaffold will be described, alongside the recent biological results obtained. Ultimately, this work has helped deliver tool compounds which may help to elucidate the true pharmacology and roles of PARG in cancer and other disease settings.

#3716

Potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC.

Diane Boucher, Russell Hoover, Yuxin Wang, Yong Gu, David Newsome, Pamella Ford, Cameron Moody, Veronique Damagnez, Reiko Arimoto, Shawn Hillier, Mark Wood, William Markland, Brenda Eustace, Kevin Cottrell, Marina Penney, Brinley Furey, Kirk Tanner, John Maxwell, Paul Charifson. _Vertex Pharmaceuticals Inc, Boston, MA_.

Ionizing radiation (IR), which is widely used for the treatment of cancer, causes double-strand breaks (DSBs) in DNA. If left unrepaired, these DSBs are lethal to the cell. DNA-dependent protein kinase (DNA-PK) is a key enzyme in the non-homologous end joining (NHEJ) pathway that repairs DSBs caused by IR, or chemotherapeutic agents that cause DSBs such as doxorubicin. The goal of these studies was to characterize the radiation enhancing effects of VX-984, a selective and potent ATP-competitive inhibitor of the catalytic subunit of DNA-PK (DNA-PKcs), with a focus on non-small cell lung cancer (NSCLC) cells and tumor xenografts. VX-984 enhances the cytotoxicity of IR in a panel of cancer cell lines including NSCLC cell lines in vitro with dose enhancement factors (DEF) greater than 3. Notably, VX-984 combined with IR in normal human lung fibroblasts minimally enhanced the cytotoxicity compared to IR alone. Additionally, VX-984 decreased DNA-PKcs autophosphorylation on S2056 both in vitro and in vivo in NSCLC cells and attenuated the decay of the DNA damage markers γH2AX and pKAP1 in response to IR. In NSCLC PDX models VX-984, in combination with IR (2 Gy x 3), caused durable complete responses while IR alone only led to a delay in tumor growth, consistent with delayed DNA damage repair. In these models, the combination of VX-984 and IR was well tolerated. These data demonstrate that VX-984 is a potent radiation-enhancing agent and provide a strong rationale for the use of VX-984 in combination with IR for the treatment of NSCLC.

#3717

Defining optimal dose schedules for ATR inhibitors in combination with DNA damaging drugs: Informing clinical studies of VX-970, the first-in-class ATR inhibitor.

John Pollard,1 Phil Reaper,1 Adele Peek,1 Stuart Hughes,1 Scott Gladwell,1 Julie Jones,1 Peter Chiu,1 Mark Wood,2 Crystal Tolman,2 Mac Johnson,2 Peter Littlewood,1 Marina Penney,2 Katherine McDermott,2 Brian Hare,2 Scott Z. Fields,2 Mohammed Asmal,2 Brent O'Carrigan,3 Timothy A. Yap3. 1 _Vertex Pharmaceuticals Inc, Abingdon, United Kingdom;_ 2 _Vertex Pharmaceuticals Inc, Boston, MA;_ 3 _Royal Marsden Hospital, London, United Kingdom_.

Proficient repair of DNA damage is a cause of the poor response many patients experience when treated with commonly used DNA-damaging drugs such as cisplatin, carboplatin and gemcitabine. The protein kinase ataxia telangiectasia mutated and Rad3 related (ATR) is recruited to DNA damage lesions caused by such drugs during the S and G2 phase of cell cycle, where it coordinates a series of responses including checkpoint activation and DNA repair by homologous recombination.

Inhibition of ATR potentiates the cytotoxic activity of DNA damaging drugs in many cancer cells. In stark contrast, non-cancer cells survive inhibition of ATR with just transient growth arrest. Cancer cells carrying common defects in a compensatory repair pathway mediated by the kinase ataxia telangiectasia mutated (ATM) and its principle substrate, p53, are especially sensitive to ATR inhibition. Two ATR inhibitors are in clinical development in combination with DNA damaging drugs, however a comprehensive assessment of dose schedule considerations has not been reported.

In pre-clinical models, the efficacy of an ATR inhibitor in combination with multiple DNA damaging drugs was shown to be dependent on dose schedule. In vitro, transient exposure of cancer cells to an ATR inhibitor (2 hours) was highly effective when added after the DNA damaging drug. Maximum activity was observed when addition of the ATR inhibitor was timed to coincide with peak accumulation of cells in S-phase and concomitant activation of ATR (P-Chk1), following treatment with the DNA damaging drug. In mouse xenograft models, strong synergistic activity was achieved from just a single dose of the ATR inhibitor given once per cycle of the DNA damaging drug.

Optimal efficacy was achieved by administering the ATR inhibitor 12-24 hours after the DNA damaging drug. Dosing the ATR inhibitor prior to, or greater than 48 hours after, the DNA damaging drug provided limited benefit. On this schedule, addition of the ATR inhibitor had minimal impact on the tolerability profile of the DNA damaging drug. VX-970, the first-in-class ATR inhibitor, is being assessed as monotherapy and in combination with gemcitabine, cisplatin and carboplatin in Ph1/2 clinical studies. Based on pre-clinical data, VX-970 is being dosed approximately 24 hours after the DNA damaging drug. Preliminary tumor biomarker data from three patients showed high P-Chk1 24 hours after treatment with carboplatin, which is inhibited by VX-970.

These data suggest the importance of dose scheduling on the efficacy of ATR inhibitors and DNA damaging drug combinations and inform the design of ongoing clinical studies.

#3718

Sensitization of human tumor cells to chemotherapy drugs by antisense downregulation of RAD51: Targeting DNA repair to induce synthetic lethality.

Peter J. Ferguson, Mateusz Rytelewski, Mark D. Vincent, James Koropatnick. _London Regional Cancer Program, London, Ontario, Canada_.

The inherent genomic instability of cancer cells has been exploited as a tumor-selective drug target to treat tumors that are deficient in specific mechanisms of DNA repair. Inhibitors of poly(ADP-ribose) polymerase (PARP) are routinely used clinically against tumors deficient in BRCA1 or BRCA2, due to the poor capacity of these cells to undergo homologous recombination repair (HRR). This exploitation of a tumor cell deficiency to enhance selectivity to a particular drug is "synthetic lethality" (Nature 434: 913, 2005). To make use of this phenomenon in tumors that may not be inherently hypersensitive to a particular treatment, we have sought to induce synthetic lethality by down-regulating essential components of DNA repair, in particular BRCA2, to sensitize cells to chemotherapy drugs [Mol Oncol 8(8): 1429-1440, 2014]. Given that an important function of BRCA2 is to modulate the action of RAD51 in HRR, we determined whether antisense knockdown of RAD51 could enhance tumor cell sensitivity to the PARP inhibitor olaparib and the DNA-crosslinking agent cisplatin. Four different anti-RAD51 siRNA molecules (Dharmacon), targeting coding sequences, were tested against cell lines representative of different tumor types in an in vitro assay of proliferation (non-small cell lung cancer line A549b, colon carcinoma line HT-29, and prostate carcinoma lines DU145 and LNCaP). The siRNAs, as single agents, inhibited proliferation in a concentration-dependent fashion and to varying degrees, and sensitized tumor cells to olaparib. A sequence that targeted region 1169-1187 of the RAD51 cDNA (NM_002875.4) was utilized for further studies. At concentrations of anti-RAD51 siRNA 51a that inhibited proliferation of cell lines by less than 50%, 51a enhanced cytotoxicity of olaparib by over 90% and of cisplatin by 60-90%. In all cell lines except LNCaP (with mutant BRCA2) the combination of siRNAs against RAD51 and BRCA2 acted cooperatively to enhance cytotoxicity of olaparib and cisplatin. Notably, when used together, each siRNA down-regulated expression of its respective target mRNA, as demonstrated by quantitative RT-PCR, without interfering with the activity of the other. RAD51 can be exploited clinically as a target for inherent or induced synthetic lethality to DNA-damaging agents (e.g., cisplatin) or inhibitors of DNA repair (e.g., olaparib). Such treatment can include tumors with BRCA2-deficiency, either inherent or induced, to yield at least an additive anticancer effect. MR is a scholar of the CIHR Strategic Training Program in Cancer Research and Technology Transfer (CaRTT) and a recipient of the CIHR Banting and Best Canada Graduate Scholarship.

#3719

TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation.

Michael A. Milhollen,1 Judy Qiuju Shi,1 Tary Traore,1 Jessica Huck,1 Darshan Sappal,1 Kenichi Iwai,2 Akihiro Ohashi,2 Claudia Rabino,1 Jennifer A. Duffy,1 Eric Lightcap,1 Yuko Ishii,1 Jeffrey Ciavarri,1 Neil Bence,1 Allison J. Berger,1 Marc L. Hyer1. 1 _Takeda Pharmaceutical Company Ltd., Cambridge, MA;_ 2 _Takeda Pharmaceutical Company Ltd., Fujisawa City, Japan_.

Radiation therapy, as a primary therapy or as a combination partner, is used in half of all worldwide cancer treatments. Research is ongoing to identify agents which potentiate the effects of ionizing radiation (IR) in tumor cells. Because IR causes DNA double strand breaks (DSBs), inhibition of DNA damage repair mechanisms could enhance the effects of radiation. DNA repair at DSBs is mediated by the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways, both of which rely on the post-translational modification of proteins by ubiquitin (Ub). A phosphorylation and ubiqutination cascasde at DSBs results in Ub-dependent recruitment of 53BP1 and BRCA1 complexes.

We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), the enzyme responsible for activating > 99% of all cellular Ub. Previously, TAK-243 was shown to inhibit mono-Ub of PCNA and FANCD2, key proteins within the translesion synthesis (TLS) and Fanconi Anemia (FA) DNA repair pathways, and also to inhibit Ub transfer to UBC13, an E2 ubiquitin-conjugating enzyme utilized in DSB repair. We hypothesized that TAK-243 would prevent repair of DSBs and thereby potentiate IR-induced cell death.

Here we show that TAK-243 pre-treatment potentiates the effect of IR on HCT-116 cells in a colony formation assay in vitro. To link this combination benefit to the disruption of DNA damage repair, we demonstrate that TAK-243 pre-treatment blocks the IR-induced recruitment of 53BP1 to sites of DNA damage both in vitro and in vivo. In a patient-derived xenograft (PDX) model of non-small cell lung cancer, formation of IR-induced 53BP1 foci is inhibited when TAK-243 is dosed 1 hour before beam-focused radiation exposure. In contrast, levels of IR-induced pH2Ax are not significantly changed by TAK-243 treatment, suggesting that TAK-243 does not prevent formation or detection of DSBs, but rather acts downstream to prevent DNA damage repair. Additive-to-synergistic effects on tumor growth inhibition were observed in several xenograft models treated with the combination of TAK-243 and beam-focused IR, with persistent tumor regressions noted in some NSCLC and breast cancer models. The results of our experiments provide a mechanistic rationale for combining radiation with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (NCT02045095).

#3720

Tissue-specific blockage of DNA damage responses sensitizes breast cancer chemotherapy.

Rong Xu,1 Haifa Shen2. 1 _Department of pharmacology,Tongji Medical College, Wuhan, China;_ 2 _Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX_.

Systemic chemotherapy is not effective at eradicating metastatic breast cancer, and approaches to improve the outcomes for these patients are urgently needed. The ideal method to enhance chemotherapy would involve targeting a protein that is differentially or exclusively expressed in cancer compared to normal tissue; however, such targets are rare. We sought to target the actively dividing breast cancer cells in the distant metastatic organs with tumor tissue-specific delivery of therapeutic siRNA that suppresses the expression of key DNA damage response (DDR) genes such as CHK1 and RAD51 in order to sensitize chemotherapy. Knockdown of CHK1 and RAD51 expression by the gene-specific siRNA oligos was confirmed in vitro in MDA-MB-231 human breast cancer cells. Treatment of nude mice bearing lung metastatic MDA-MB-231 tumors with tumor-enriched nanoparticle-packaged CHK1 or RAD5 siRNA did not significantly suppress tumor growth. Single agent treatment with biweekly 6 mg/kg doxorubicin treatment did not slow down growth of the metastatic tumors either. In contrast, combination treatment with 6 mg/kg doxorubicin and 15 μg CHK1 or RAD51 siRNA in nanoparticles dramatically inhibited tumor growth and extended animal survival. Our result supports the development of tumor tissue-enriched siRNA therapeutics targeting the DDR pathways to sensitize breast cancer therapy.

#3721

A camptothecin-containing nanoparticle-drug conjugate combination with DDR agents provides a novel approach to increasing therapeutic index.

Lenka Oplustil O'Connor,1 Anderson T. Wang,1 David R. Jones,2 Rajesh Odedra,2 Michael Spreadborough,1 Joanne Wilson,1 Aaron Smith,1 Peter Cotton,2 Jaimini Reens,2 Jen Barnes,1 Victoria Sheridan,2 Scott Eliasof,3 Andres Tellez,3 Alan Lau,1 Claire Sadler,2 Mark J. O'Connor1. 1 _AstraZeneca, Cambridge, United Kingdom;_ 2 _AstraZeneca, Alderley Park, United Kingdom;_ 3 _Cerulean Pharma Inc., Cambridge, MA_.

Topoisomerase I inhibitors are used as standard-of-care chemotherapy in many types of cancer but are associated with significant toxicities. There is potential to improve their efficacy further by combining with inhibitors of the DNA damage response, such as the PARP inhibitor olaparib. However, while preclinical data highlight the improved efficacy of this combination, subsequent clinical trials have struggled due to dose limiting myelotoxicity.

CRLX101 is an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (the most potent topoisomerase I inhibitor known). This agent is preferentially targeted to tumours and demonstrated a favourable toxicity profile in the clinic.

Here, we explored the molecular mechanism and therapeutic potential of combining CRLX101 with either olaparib or the WEE1 inhibitor AZD1775, by testing both efficacy and safety in preclinical models. In vitro studies using NCI-H417a SCLC cells demonstrated that combination with both olaparib and AZD1775 potentiated the efficacy of CRLX101 although by different mechanisms. Cellular analyses revealed that CRLX101 treatment alone predominantly activated ATM-mediated DNA damage response and resulted in late S/G2 cell cycle arrest. Combination with a PARP inhibitor further enhanced the CRLX101-induced DNA damage response and prolonged cell cycle arrest in late S/G2 phase. In contrast, WEE1 inhibition abrogated late S/G2 cell cycle arrest induced by CRLX101, resulting in aberrant mitotic entry and enhanced cell death.

Our in vivo studies using wild type Wistar rat model showed that CRLX101, olaparib and AZD1775, are well tolerated as single agents. However, concurrent combination of CRLX101 with either olaparib or AZD1775 resulted in a dose-dependent decrease in haematological parameters. We investigated sequenced schedules and demonstrated that at a 24h delay between the CRLX101 and olaparib mitigates much of the combined bone marrow toxicity, while improving the efficacy above CRLX101 alone in xenograft tumours from NCI-H417a cells.

Collectively, these preclinical data demonstrate increased anti-tumour efficacy of CRLX101 when combined with DDR inhibitors. The combination schedule for CRLX101 and olaparib identified in our preclinical models as providing an increased therapeutic index has been used to develop protocols to test this combination in a relapsed (2nd line) SCLC human clinical trial (in collaboration with NCI).

#3722

Potent functional inactivation of the MGMT DNA repair protein by dithiocarbamate compounds increases the efficacy of temozolomide in human glioblastoma cells.

Hanumantha Rao Madala. _Texas Tech University Health Sciences Center, Amarillo, TX_.

There is an urgent need for the design and discovery of new and potent inhibitors for the DNA repair protein MGMT (O6-Methylguanine-DNA-methyltransferase) in glioma therapy. MGMT is highly expressed in brain tumors, and plays a primary role in conferring resistance to alkylating agents. The psuedosubstrates for MGMT such as the O6-benzylguanine have not been successful in the clinic due to prolonged inhibition of DNA repair in the bone marrow stem cells. Recently, we showed that the anti-alcoholism drug, disulfiram (DSF) inhibits MGMT activity in the same way as ALDH by conjugating with the active-site cysteine 145 (Carcinogenesis 35, 692, 2014). DSF, a symmetrical molecule, is metabolized and split in half to yield dithiocarbamate residues. Since the dithiocarbamates resulting from DSF decomposition are the ultimate reactive species that inactivate the aldehyde dehydrogenase and other signaling targets, we surmised that dithiocarbamate derivatives by themselves will be active as MGMT inhibitors and exert anticancer activities. Therefore, we tested various dithiocarbamates (pyrrolidine dithiocarbamate (PDTC); diethyldithiocarbamate (EDTC); dimethyldithiocarbamate (MDTC) and dibenzyldithiocarbamate (BDTC) on MGMT activity, protein levels, and other redox-sensitive proteins such as the NF-κB and GSTP1. The cytotoxicity of these dithiocarbamates against the MGMT-proficient SF-188 glioblastoma cells was comparable with that of DSF, with the MDTC being most effective and the benzyl derivative BDTC least potent. Western blot analysis in HT29 and SF-188 cells revealed a concentration-dependent degradation of MGMT by the dithiocarbamates, similar to DSF. All dithiocarbamates except the BDTC were superior to DSF in degrading MGMT protein. MDTC was the most potent followed by PDTC and EDTC in depleting the MGMT protein from tumor cells. Further, MDTC was also effective in reducing the cellular levels of NF-κB protein. We also established that MDTC binds to the active site cysteine145 in MGMT leading to its inactivation. Currently, we have developed the pegylated PLGA nanoparticles loaded with MDTC or zinc-chelated MDTC to target the glioblastoma and other cancers. The efficacy of these formulations in intracranial glioma models developed in nude mice is being evaluated. The lack of cytotoxicity and inability to bind with MGMT by BDTC suggests that the bulky group attached to the dithiocarbamate may hinder interaction with target proteins or interfere with their metabolism. Since MDTC has a good potential to cross the blood brain-barrier, possess reactive thiol groups that can interact with not only MGMT but also numerous signaling proteins, our strategy using this repurposed compound holds promise in glioma treatment (supported by grants from CPRIT [RP130266] and Carson-Leslie Foundation to KSS).

#3723

Regulation of TRAIL-induced apoptotic signaling by the autophagy receptor p62 in acute promyelocytic leukemia cells.

Kelly Airiau,1 Olivier Micheau,2 Anne-Marie Vacher,1 Faten Mehri,1 Pierre Vacher,1 Mojgan Djavaheri-Mergny1. 1 _Inserm U916 - VINCO, University of Bordeaux, France;_ 2 _INSERM U866 - Dijon, UFR des Sciences de Santé - Univ of Bourgogne, France_.

Autophagy is an evolutionary conserved process that degrades and recycles cellular components through the lysosomal pathway to maintain cellular homeostasis. It has long been considered as a pro-survival mechanism to maintain viability under stressful conditions. However, a growing body of evidence demonstrates that autophagy could also promote or act as a pro-death program. Autophagy plays an ambivalent role in cancer: it can operate either as a pro-tumoral mechanism or paradoxically as an anti-tumoral mechanism depending on the context and the stage of the tumours, making it an interesting field of investigation.

p62/SQSTM1 is an adapter protein playing a key role during the autophagy process. It promotes the selective degradation of ubiquitinated cellular substrates (i.e. proteins and organelles). As a multidomain protein adapter, p62 is involved in the regulation of various signaling pathways including those that control cell death pathways. In this regard, p62 can directly interact with the death receptor DR5 and promotes death receptor induced-apoptosis through caspase 8 stabilization.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by an abnormal proliferation of myeloblasts (precursors of myeloid white blood cells) without differentiation. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the translocation t(15;17)(q22;q12) which results to the expression of the chimeric protein PML-RAR alpha. All-trans retinoic acid (ATRA) induces cancer cells differentiation and results in complete clinical remission in APL patients. It is now used as a first-line therapy. Despite the success of this treatment, some patients are refractory to ATRA or relapse. Thus, new therapeutic strategies are needed.

We previously showed that ATRA-induced APL cell differentiation was associated with autophagy induction and p62/SQSMT1 accumulation, a response that confers a survival advantage to mature APL cells. As recent findings showed several molecular links between p62 and extrinsic apoptosis pathway, we decided to investigate the link between p62/SQSMT1 and cell death in AML cells upon treatment by TRAIL, an inducer of extrinsic apoptosis. We found that p62/SQSTM1 is required for apoptotic responses in APL cells. Our results also reveal the synergic cooperation between TRAIL and ATRA to increase apoptosis. We are currently investigating the molecular mechanisms underlying the role of autophagy as well as the autophagy receptor p62/SQSM1 in TRAIL-induced apoptosis in APL cells.

#3724

Combination of TRAIL and Chal-24 synergistically kills lung cancer cells through autophagy-mediated degradation of IAPs and c-FLIPL.

Jennings Xu,1 Xiuling Xu,1 Shaoqing Shi,1 Qiong Wang,1 Bryanna Saxton,1 Chengguo Xing,2 Yong Lin1. 1 _Lovelace Respiratory Research Institute, Albuquerque, NM;_ 2 _University of Minnesota, Minneapolis, MN_.

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by the Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

#3725

B-Raf inhibition-induced paradoxical activation of MEK/ERK signaling enhances DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells.

You-Take Oh, Jiusheng Deng, Ping Yue, Shi-Yong Sun. _Emory University Winship Cancer Institute, Atlanta, GA_.

B-Raf inhibitors have been used for the treatment of some B-Raf-mutated cancers such as melanoma and thyroid cancer. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but lead to a paradoxical activation of MEK/ERK signaling in Ras-mutant cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. Given the critical role of MEK/ERK signaling in the positive regulation of DR5 expression as we have previously demonstrated, this study focuses on determining the impact of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-treatment of Ras-mutated cancer cells with PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells. Our results may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition. (This study was supported by Emory Winship Cancer Institute Robbins Scholar awards to YTO and to JD and Halpern Research Scholar award to SYS. SYS is a Georgia Research Alliance Distinguished Cancer Scientist and Halpern Research Scholar)

#3726

Predicting cellular response to small molecule Mcl-1 antagonists.

Leah Hogdal, John Sensintaffar, Allison Arnold, Craig Goodwin, Zhiguo Bian, Subrata Shaw, Chris Tarr, Taekyu Lee, Edward Olejniczak, Stephen Fesik. _Vanderbilt University, Nashville, TN_.

Myeloid cell leukemia -1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which many cancers overexpress and depend on for survival. Mcl-1 inhibits apoptosis by binding to Bax and Bak, inhibiting the effectors of apoptosis from oligomerizing at the outer membrane of the mitochondria to promote cell death. Due to its role in cancer and resistance development to conventional chemotherapies, efforts to potently and specifically target MCL-1 have been ongoing. We have discovered novel small molecule inhibitors that selectively disrupt Mcl-1-Bim binding in the sub-nanomolar range. Anti-proliferative activities of our leading Mcl-1 inhibitors were assessed in a panel of hematological and solid cancer cell lines. We found that our compound promoted apoptosis in a subset of cell lines tested. As there was a variable response, we used BH3 profiling, which is a functional assay that rapidly measures dependence on any of the anti-apoptotic Bcl-2 family proteins for an individual sample to predict in vitro sensitivity to our Mcl-1 antagonists. Using the MS1 peptide, a known potent and selective Mcl-1 binder as a positive control in the BH3 profiling assay, we found a correlation between the BH3 profiling measurement of anti-apoptotic dependence to Mcl-1 and increased cytotoxicity caused by our compounds. These studies demonstrate that our compounds promote apoptosis through a selective inhibition of Mcl-1 mechanism of action and supports further investigation of our compounds in in vivo models of cancer.

#3727

**Small molecule Mcl-1 inhibitors induce apoptosis and death in multiple cancer subtypes** in vitro **.**

John L. Sensintaffar, Allison Arnold, Craig Goodwin, Leah Hogdal, Subrata Shaw, James C. Tarr, Taekyu Lee, Edward Olejniczak, Stephen W. Fesik. _Vanderbilt School of Medicine, Nashville, TN_.

Mcl-1 is a member of the Bcl-2 family of proteins that play a major role in conferring resistance to apoptosis in cancer cells. Inhibiting Mcl-1 using peptides or RNAi has been shown to induce apoptosis in a broad array of cancer cell lines in numerous studies, making Mcl-1 a compelling target for anticancer therapy. We have discovered potent and selective small molecule Mcl-1 inhibitors that bind to the BH3 binding site of Mcl-1 with sub nanomolar affinities. These agents rapidly induced apoptosis in the Mcl-1 dependent NCI-H929 myeloma cell line, as demonstrated by mitochondrial membrane depolarization, caspase activation, and decreased viability. We measured the anti-proliferative activity of our compounds in cell lines from several cancer subtypes and found a broad spectrum of sensitivity to Mcl-1 inhibition. In cell lines that were resistant to Mcl-1 antagonists, combination with the dual Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) greatly enhanced the activity of both compounds. These findings demonstrate that pharmacologic inhibition of Mcl-1 as a single agent or in combination with other cancer therapeutic agents is an effective way to modulate the intrinsic apoptotic pathway and promote cell death in cancer cells.

#3728

Targeting MCL-1 expression, through the inhibition of CDK9 and super enhancer driven transcription, offers multiple opportunities for rational drug combinations.

Wontak Kim, Katherine K. Soh, Ye Sol Lee, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, Steven Weitman, David J. Bearss, Steven L. Warner. _Tolero Pharmaceuticals, Inc., Lehi, UT_.

Downregulating the expression and function of MCL-1 through the inhibition of cyclin-dependent kinase-9 (CDK9) has proven to be a valuable strategy to target this important pro-survival signal in malignant cells of numerous cancer types. This is exemplified by the ability of alvocidib, a potent CDK9 inhibitor, to inhibit the expression of MCL-1 at both the transcript and protein levels in multiple cell lines from both hematological and solid tumor origins. The timing and duration of MCL-1 knockdown varies between cell type; however, the knockdown is consistent and in some cell lines persistent after the removal of drug. Although alvocidib has demonstrated single agent activity in both the clinic and in nonclinical models, strategies that exploit MCL-1-dependent drug resistance, are allowing for the more rational use of alvocidib in combination with standard-of-care and investigational agents. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone (regimen called FLAM), is synergistic in nonclinical models of acute myeloid leukemia (AML). The FLAM regimen results in a significant increase in apoptosis in comparison to any of the single agents alone. This synergy correlates with the downregulation of MCL-1 expression by alvocidib treatment, which places the cancer cells into a heightened state to undergo apoptosis when induced by cytarabine and mitoxantrone treatments. Additionally, the FLAM regimen has demonstrated robust clinical activity in both front-line and relapsed/refractory AML patients. The knockdown of MCL-1 by alvocidib can also be exploited when used in combination with 5-azacytidine (5-aza). BCL-2 family members, including MCL-1 have been described as mechanisms of resistance to 5-aza. Treatment of cells with alvocidib, to repress MCL-1 expression prior to 5-aza treatment, reduced the 5-aza cell viability EC50 more than 2.5-fold, from 1.8 µM to 0.6 µM in MV4-11 cells. The alvocidib/5-aza combination also resulted in synergistic increases in caspase activity relative to either single agent within the combination, at multiple dose levels. MCL-1 dependence is a known mechanism of resistance to BCL-2-targeting agents, such as venetoclax (ABT-199). Alvocidib is an effective approach to targeting MCL-1 leading to the sensitization of cancer cells to venetoclax. Finally, the rational drug combinations described here are further supported by the finding that MCL-1-dependence, measured by NOXA priming, correlates with clinical benefit from treatment with an alvocidib-containing regimen (eg. FLAM) in AML patients. In conclusion, MCL-1 is a key downstream target of inhibiting CDK-9 with alvocidib. Combination strategies using alvocidib have emerged as a powerful solution for overcoming MCL-1 dependent drug resistance.

#3729

Novel radiation mitigators and anticancer drugs.

Robert H. Schiestl,1 Yelena Rivina,2 Michael Davoren1. 1 _UCLA School of Public Health, Los Angeles, CA;_ 2 _Stanford, Palo Alto, CA_.

The possibility of a radiation disaster from a nuclear detonation or accident has existed for over 50 years and spawned much of the basic research in radiobiology in the 1950-60s. The recent Fukushima accident was yet another reminder that there remains a dire need to develop novel therapies against radiation-induced toxicities. Here we report on the development of two novel radiation countermeasure therapies: CJ010 and Yel002. These small, biologically active, drug-like molecules were uncovered in the DEL high throughput assay reducing radiation-induced cyto- and geno-toxicity in yeast. Radiation-modulating activity was further confirmed in yeast plate-based DEL Assay: addition of either CJ010 or Yel002 to irradiated cultures reduced cell death and genomic instability. Further, the compounds increases survival to 75% in vivo following an LD100/30 dose of ionizing radiation (IR) with the first therapeutic injection administered 24 hours post exposure followed by injections at 48,72,96, and 120 hours. Additionally, treatment with Yel001 an analog of CJ010 and Yel002 compounds reduces radiation-induced leukemia from 90% to to 50% and 40% respectively. Of note, treatment with either Yel001 or Yel002 reduced spontaneous leukemia rate from 10% to 0%. Treatment with Yel002 following IR accelerates the recovery of the hematopoietic cells after sub-lethal exposures. In addition, treatment with Yel002 reduces EMS, MMS, UV, radioactive iodine, cigarette smoke extract as well as nitrogen mustard induced toxicity as well as genotoxicity showing a broad application spectrum. It also prolongs live of cells in a senescence assay. In addition Atm deficient mice live 16 weeks longer with weekly injection of Yel002 which is about 12 years in human life expectancy. In addition, Yel002 complements a zebrafish model of Diamond Blackfan Anemia. It works in yeast, CHO cells, different human cells, mice and zebrafish. Toxicity has not been observed in neither in vitro or in vivo administrations. Overall, Yel compounds have much potential as stockpile therapies for radiation-induced lethality and cancer: they are highly effective when administered up to 24hours post exposure, they reduce radiation-induce sequelae such as leukemia, and appear to have an acceptable toxicity profile.

#3730

The PPM1D inhibitor GSK2830371 has p53-dependent anti-lymphoma effects and enhances bortezomib-induced apoptosis in mantle cell lymphoma.

Kensuke Kojima, Mariko Yoshimura, Aya Maeda, Hiroaki Kitamura, Yuki Nishida, Shinya Kimura. _Saga University, Saga, Japan_.

p53 mutations are relatively rare (~15%) in mantle cell lymphoma (MCL). Chemotherapeutic agents induce DNA damage, p53 phosphorylation and pro-apoptotic wild-type (WT) p53 signaling, resulting in lymphoma cell death. However, DNA damaging agents may have severe acute toxicities, accelerate clonal evolution and cause therapy-related neoplasms by adding new mutations to the original clones. PPM1D is a serine/threonine phosphatase that negatively regulates key DNA damage response proteins including p53, CHK2, Histone H2AX, and ATM. PPM1D has been thought to be an oncogenic protein. It has been reported that PPM1D is overexpressed or amplified in breast and ovarian cancers. GSK2830371 (GSK) is a PPM1D inhibitor, which binds to a flap subdomain that regulates enzymatic activity of PPM1D and substrate recognition (Nat Chem Biol 2014). Treatment of tumor cells with the inhibitor GSK has been found to increase phosphorylation of PPM1D substrates and cause growth inhibition in tumor cells harboring wild-type p53. We investigated the clinical significance of PPM1D and anti-lymphoma effects of GSK in MCL. The mRNA expression levels in patient samples were determined using Oncomine. Our gene expression analyses showed an increase in PPM1D mRNA expression in MCL samples versus normal naïve B lymphocytes (P = 0.044) that are the normal counterparts of MCL. PPM1D mRNA levels were positively correlated with CCND1 (encoding Cyclin D1) mRNA levels (r = 0.33, P = 0.0014) and proliferation signature averages (r = 0.54, P < 0.0001). Higher PPM1D expression at diagnosis was associated with a poorer prognosis in MCL patients (i.e., a median overall survival of 3.9 years for cases with PPM1D expression in the lowest third and 1.4 years in the highest third, P = 0.0047). These results indicate that PPM1D overexpression has a negative prognostic impact on MCL overall disease survival and that PPM1D is a potential therapeutic target in MCL. A total of 8 MCL (3 p53 WT and 5 mutant) cell lines were treated with GSK. 10 µM GSK inhibited cell growth of p53 WT Z-138, JVM-2 and Granta-519 cells by 68%, 38% and 39%, respectively. p53 mutant cells were relatively resistant to GSK (14.8 ± 4.7% growth inhibition). p53 knockdown de-sensitized Z-138 and JVM-2 cells to GSK-induced apoptosis. GSK increased total and Ser15-phosphorylated p53 levels and p53 targets p21 and PUMA in p53 WT cells. Basal levels of PPM1D and its substrates were not associated with GSK sensitivity among MCL cell lines. Interestingly, GSK enhanced bortezomib-induced apoptosis in a p53-independent manner, partially through activation of p38 signaling; p38 inhibition attenuated the combination effect. Collectively, PPM1D inhibition may offer a novel therapeutic strategy for MCL.

#3731

TSC deficiency unmasks a novel protective role of RIP1/RIP3 signaling against mitochondrial and oxidative stress-induced cell death.

Piotr T. Filipczak,1 Cindy Thomas,1 Wenshu Chen,1 Andrew Salzman,2 Jacob D. McDonald,1 Yong Lin,1 Steven A. Belinsky1. 1 _Lovelace Respiratory Research Institute, Albuquerque, NM;_ 2 _Radikal Therapeutics, West Tisbury, MA_.

Tuberous sclerosis complex (TSC) is a genetic multi-organ disorder characterized by the development of neoplastic lesions in kidney, lung, brain, heart and skin. It is caused by an inactivating mutation in tumor suppressor genes coding the TSC1/TSC2 complex, resulting in hyperactivation of mTOR- and Raf/MEK/MAPK-dependent signaling that stimulates tumor cell proliferation and metastasis. Despite its oncogenic effect, cells with TSC deficiency were more sensitive to oxidative stress and dependent on mitochondrial metabolism, providing a rationale for a new therapeutic approach. The presented study shows that simultaneous inhibition of two major pathways regulating redox homeostasis using L-Buthionine-sulfoximine (BSO, glutathione synthesis inhibitor) and auranofin (thioredoxin reductase inhibitor) induces oxidative burst, mitochondrial damage and necrotic cell death in TSC2 deficient cells in a highly synergistic and cell context-specific manner. Furthermore, blocking RIP1/RIP3-dependent signaling using chemical inhibitors necrostatin-1 and necrosulfonamide (NSA), reported by others to protect from some cytotoxic stimuli, synergizes with BSO and auranofin in killing TSC2 deficient cells. Expression analysis demonstrated that RIP1 and RIP3 protein levels are elevated in cells with TSC2 deficiency, and their inactivation enhances mitochondrial dysfunction. Finally, supplementation with the mitochondrial metabolite alpha-ketoglutarate, whose synthesis is regulated by RIP1/RIP3, rescues cells from the sensitizing effect of necrostatin-1 and NSA. Together, this study identifies a novel TSC context-dependent role of RIP1/RIP3 signaling in regulating mitochondrial and oxidative homeostasis, and substantiates glutathione, thioredoxin and RIP1/RIP3 pathways targeting as a promising therapeutic approach against TSC-associated tumors.

#3732

Combined targeting of NOXA and GSTpi effectively kills mantle cell lymphoma cells.

Markus Kleih,1 Simon Heine,1 Michael Dengler,2 Lea Schaaf,1 Elisabeth Hoering,1 Heike Horn,1 German Ott,3 Walter E. Aulitzky,3 Heiko van der Kuip1. 1 _Dr. Margarete Fischer-Bosch Institute and University of Tuebingen, Stuttgart, Germany;_ 2 _The Walter & Eliza Hall Institute of Medical Research,, Parkville, Australia; _3 _Robert Bosch Hospital, Stuttgart, Germany_.

Mantle cell lymphoma (MCL) is clinically characterized by an aggressive course and only transient response to chemotherapy. Therefore, new treatment strategies for this malignancy are warranted. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation leading to deregulated expression of cyclin D1. It has been reported recently that cyclin D1 overexpression in MCL is associated with constitutively high levels of GSTP1 and PMAIP1 transcripts, coding for the glutathion S-transferase pi (GSTpi) and for the proapoptotic protein NOXA, respectively. Whereas GSTpi protein is stably expressed, NOXA half-life turned out to be extremely short in this B cell lymphoma leading to constitutively low levels of this BH3-only protein in these cells. We asked if this MCL typical phenotype can be utilized for a selective treatment by stabilizing NOXA protein and concurrent inhibition of GSTpi activity. By screening different compounds known to stabilize NOXA protein and GSTpi inhibitors we found that combination of the fatty acid synthase (FASN) inhibitor Cerulenin together with Ezatiostat or Ethacrynic Acid is not only effective but also selective for MCL cells. Cell death induced by lethal doses of Cerulenin was dependent on Cyclin D1 since siRNA-mediated knockdown of Cyclin D1 partially rescued cells from apoptosis. Combinatory treatment of cells with sub-lethal doses of Cerulenin together with Ezatiostat/Ethacrynic Acid had no effect on viability of fibroblasts from normal donors but effectively killed both MCL cell lines as well as primary cells from MCL patients. Importantly, Cerulenin-mediated NOXA accumulation was significantly further elevated upon combination with GSTpi inhibitors and associated with a concomitant induction of reactive oxygen species (ROS) levels. Cell death was dependent on both NOXA and ROS since either inhibition of ROS by GSH or Catalase or siRNA-mediated knockdown of NOXA was sufficient to almost completely rescue cells from Cerulenin/GSTpi inhibitor induced apoptosis.

In conclusion we demonstrate that combined pharmacological inhibition of GSTpi activity and stabilization of NOXA protein via inhibition of fatty acid synthase might be an effective strategy to selectively kill MCL cells and offer novel treatment options.

#3733

Augmentation of response to therapeutic agents by (-)-gallocatechin gallate through inhibition of RAD51 nuclear translocation.

Ming-Hsi Wu,1 Lie-Chwen Lin,2 Te-Chang Lee1. 1 _Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan;_ 2 _National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, Taiwan_.

Lung cancer is the most common cancer worldwide. Platinum-based combination chemotherapy is one of first line treatments for patients with lung cancer. Most of the cytotoxic drugs are targeting DNA, interfere with DNA synthesis, and triggering DNA damage response (DDR). Since DDR usually activates important defensive pathways to repair damaged DNA, DDR is known as a key factor determining the outcome of chemotherapy. Among them, RAD51, playing a central role in homologous recombination (HR) DNA repair, has been shown to interfere with the prognosis of patients treated with chemoradiotherapy. Since we have demonstrated that inhibition of RAD51 nuclear focus formation synergistically enhances the anticancer activity of cisplatin, we further screened purified natural products in order to find products with activity to inhibit RAD51 nuclear focus formation. In the present study, we found that a green tea catechin derivative, the (-)-Gallocatechin gallate (GCG) has potent inhibitory activity against RAD51 nuclear focus formation in cisplatin-treated H460 cells. The in vitro cytotoxicity assay showed that the combination of cisplatin or irinotecan with GCG significantly increases the sensitivity of H460 cells to these DNA damage agents. We also evaluated the anti-tumor growth activity of cisplatin or irinotecan in combination with GCG using H460 xenografts. Our results showed that cisplatin and irinotecan suppresses tumor growth by 17.6% and 33.8%, while in combination with GCG suppresses tumor growth by 52.2% and 68.9%, respectively. We further demonstrated that GCG is able to inhibit cisplatin-induced ATM and Chk2 phosphorylation. Together, our results suggest that GCG may interfere with ATM-Chk2 pathway and hence suppress RAD51 nuclear focus formation and DNA repair activities.

#3734

Preclinical evaluation of Rad6 inhibition to overcome platinum resistance in ovarian cancer.

Sebastian M. Spencer,1 Ranganatha R. Somasagara,1 Kaushlendra Tripathi,1 David W. Clark,1 Hend Kothayer,2 Andrew D. Westwell,3 Rodney P. Rocconi,1 Komaraiah Palle1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _Zagazig University, Zagazig, Egypt;_ 3 _Cardiff University, Cardiff, United Kingdom_.

Ovarian cancer (OC) is the most lethal gynecological cancer in women in the United States. Advances in surgery and chemotherapy have not significantly changed the overall survival rate of OC for the last few decades, which highlights the need for new therapeutic strategies. Platinum drug resistance and refractory disease pose major challenges in treating this disease and are major factors contributing to the poor survival rate of OC patients. Although most patients initially respond to platinum based chemotherapy, about 80% of cases present with recurrent disease, develop platinum resistance, and die with the advanced disease. Considering the heterogeneity, small fractions of the cells could be inherently resistant to chemotherapy and/or dormant and exhibit stem-like cell properties, contributing to the resistant phenotype and disease recurrence. Although the Cancer stem cell (CSC) theory of therapeutic resistance proposes that the proportion of CSCs correlate to enhanced chemoresistance and early disease recurrence, the specific molecular mechanisms that regulate tumor cell behavior (stemness) and integrate signaling networks with aberrant oncogenic signaling in OC cells are not known. Our analysis of clinical samples revealed upregulation of Rad6, an E2 ubiquitin conjugating enzyme, in more than 80% of ovarian tumors compared to normal ovarian tissues. Upregulation of Rad6 also correlated well with tumor progression. Further analysis of molecular pathways in OC cells revealed a strong correlation between Rad6 upregulation and increased β-catenin and hedgehog signaling, stem cell like characteristics and platinum resistance. Downregulation of Rad6 using siRNAs or inhibition of its catalytic activity by a small molecule inhibitor, attenuated carboplatin induced monoubiquitination of its target proteins such as histone 2B, PCNA and proteins of the Fanconi anemia pathway thereby sensitizing OC cells to carboplatin. Interestingly, inhibition of Rad6 alone in OC cells induced replication stress and reduced cell survival and proliferation by arresting cells in the G2/M phase. Moreover, inhibition of Rad6 in various OC cell lines reduced expression of β-catenin, Gli1 and several OC stem cell markers. Moreover, Rad6 plays an important role in the activation of the trans-lesion synthesis (TLS) pathway by monoubiquitinating PCNA and in the activation of the Fanconi Anemia (FA) DNA repair pathway. These are critical mechanisms for cells to repair DNA crosslinks induced by platinum drugs. Together with these observations, our data suggest that inhibition of Rad6 could be a viable therapeutic target for overcoming platinum resistance and disease recurrence in ovarian cancer.

#3735

A novel ALDH1A selective inhibitor induces necroptosis in ovarian cancer stem-like cells.

Ilana Chefetz-Menaker, Kun Yang, Ron Buckanovich. _University of Michigan, Ann Arbor, MI_.

Background: The high relapse rate in ovarian cancer patients is highly consistent with a cancer stem cell (CSC) model in which rare, inherently chemoresistant CSC are capable of proliferating and differentiating to cause disease. We recently demonstrated ovarian CSC (OvCSC) can be defined by Aldehyde Dehydrogenase (ALDH) enzymatic activity. ALDH expression is increased in chemotherapy resistant OvCSC and ALDH KO can reverse chemotherapy resistance. While ALDH inhibitors exist, they are not isoform specific. We therefore identified and screened numerous putative ALDH inhibitors, based on molecular homology to the known ALDH inhibitor DEAB, and assessed their impact on OvCSC.

Methods and Results: While most ALDH inhibitors, including DEAB, had no impact on CSC, we identified a novel compound, 673A which specifically depletes OvCSC in vitro. Supporting functional depletion of OvCSC, treatment of primary tumor ascites resulted in 4-20 fold decrease tumor sphere formation. ALDH inhibition is highly synergistic with cisplatin both in vitro and in vivo as assessed by cell growth curves and tumor growth. Pre-treatment of tumor cells with 673 significantly reduced tumor in tumor initiation and growth rates. Unlike non-CSC depleting ALDH inhibitors, 673A is a pan-ALDH1A isoform specific inhibitor with IC50<230 nM for ALDH1A1, 1A2, and 1A3. OvCSC Caspase independent cell death was accompanied by negative Annexin V stain and HMB1 translocation to cytoplasm. IF demonstrated nuclear swelling and loss of DNA in a manner consistent with karyolysis and programmed cell necrosis. TEM of treated cells demonstrated clear cellular, nuclear, and organelle swelling and severe vacuolation of cytoplasm consistent with necrosis. Consistent with the induction of programmed cell necrosis, 673A treatment resulted in increased intracellular calcium. Furthermore, the intracellular calcium chelator BAPTA was able to rescue ALDH inhibition-induced necrosis. As RIP1 and RIP3 kinase activity is known to be a major mediator of necroptosis, we next assessed the impact of the RIP1 kinase inhibitor, Nec-1, and down-regulation of RIP1 and RIP3 by siRNA. Inhibition or depletion of RIP Kinase 1 did not prevent 673A mediated necroptosis of OvCSC. 673A initiates necroptotic cell death through translocation of RIPK3 downstream target, MLKL, to membrane and activation of mitochondria fission protein DRP1 and its translocation from cytoplasm to mitochondria.

Conclusions: We have identified a novel ALDH inhibitor, 673A, which selective inhibits three ALDH1A isoforms. 673A mediated ALDH1A isoform inhibition triggers programmed cell necrosis of OvCSC. Our data suggest that pan ALDH1A1 targeted therapy this may be a powerful new therapeutic approach to target OvCSC and bypass their resistance to apoptosis inducing drugs. Future studies will elucidate the importance of mitochondria metabolic pathways (Glycolysis, OXPHOS, and TCA cycle) in ALDH inhibition-induced necroptosis.

#3736

Schlafen 11 (SLFN11) blocks RNA synthesis upon replicative damage, a novel mechanism for killing cancer cells in response to DNA damaging agents.

Junko Murai, Sai-wen Tang, Yves Pommier. _National Institutes of Health, Bethesda, MD_.

The putative DNA/RNA helicase, SLFN11, is a recently discovered determinant of response to DNA damaging agents including topoisomerase inhibitors (camptothecin, irinotecan, topotecan, doxorubicin and etoposide) and cisplatin. SLFN11 is inactivated in approximately 40% of cancer cell lines (NCI-60 and CCLE). Until now, the molecular mechanisms of SLFN11 action in response to DNA damage have been unknown. Using isogenic SLFN11-knockout cells as well as SLFN11-overexpressing cells, we will show that SLFN11 irreversibly arrests cells in S-phase without cell cycle recovery under camptothecin treatment, whereas SLFN11-negative cells continue cell cycle progression. Notably, the action of SLFN11 is independent of ATR-mediated S-phase checkpoint. Co-immunostaining of 5-Ethynyl Uridine (EU) and gamma-H2AX revealed that SLFN11 blocks RNA synthesis upon replicative damage. Comprehensive analysis of cell cycle genes revealed that genes that drive S-phase progression are selectively turned-off in SLFN11-positive cells, whereas p53-dependent genes remain induced by camptothecin. Accordingly, critical proteins for S-phase progression such as cyclin A, cyclin B, CDK1 and CDK2 were suppressed in SLFN11-positive but not in SLFN11-negative cells, which causality links SLFN11-mediated S-phase arrest in camptothecin-treated cells with inactivation of cell cycle-driving pathways. This mechanism is supported by the fact that the CDK inhibitor roscovitine recapitulates the S-phase arrest even in SLFN11-deficient cells. Finally, by contrast to normal SLFN11, overexpression of SLFN11 helicase-dead mutant failed to block RNA synthesis and to kill camptothecin-treated cells, indicating that the helicase activity of SLFN11 is necessary for its RNA synthesis regulatory function. Our study reveals a novel cell cycle regulation mechanism by SLFN11 that links DNA damage and transcriptional responses in cancer cells with replicative DNA damage.

#3737

Inhibition of O-GlcNAc transferase in tamoxifen resistant breast cancer cells.

Anna Barkovskaya,1 Lina Prasmickaite,1 Ian G. Mills,2 Gunhild M. Mælandsmo,1 Siver A. Moestue,3 Harri M. Itkonen2. 1 _Institute for Cancer Research, Oslo, Norway;_ 2 _Center for Molecular Medicine Norway, Oslo, Norway;_ 3 _NTNU, Department of Circulation and Medical Imaging, Trondheim, Norway_.

O-linked N-acetyl-glucosamine transferase (OGT) is an enzyme that catalyzes addition of the O-GlcNAc modification to a wide range of intracellular proteins. The O-GlcNAc modification is a product of the hexosamine biosynthetic pathway, which requires glucose and glutamine as substrates. Uptake of both of these nutrients is often up-regulated in cancer, which in turn leads to an increase in the total protein O-GlcNAcylation. Increased OGT expression has also been reported in most cancer types, including the most frequently diagnosed cancer in women, breast cancer. Many of the breast cancers rely on estrogen receptor alpha (ERα) for proliferation and have shown a strong response to the ERα inhibition, most commonly achieved by treatment with tamoxifen. However, while efficient, prolonged exposure to tamoxifen commonly causes resistance and relapse of the disease. It is therefore vital to uncover mechanisms which contribute to the resistance in order to develop adequate treatment strategy for these patients.

Here, we have investigated the effect of targeting OGT in an isogenic pair of ERα-positive tamoxifen-sensitive MCF7 and tamoxifen-resistant TAMR breast cancer cell lines. OGT inhibition decreased viability and triggered cell death in both cell lines. These responses were associated with over 50% reduction in ERα expression in both MCF7 and TAMR cells. Reduced O-GlcNAcylation has previously been reported to induce endoplasmic reticulum stress and activation of transcription factor C/EBP homologous protein (CHOP), which promotes cell death. Targeting OGT resulted in a strong increase of CHOP expression, which appeared more prominent in the TAMR cells. Finally, targeting OGT induced a very pronounced cell cycle arrest in the G2/M phase in the TAMR cells, while the MCF7 cell lined showed a very modest response.

Taken together, these results indicate that targeting OGT leads to a differential response in the tamoxifen-sensitive and resistant breast cancer cells. Currently, we are using an expanded panel of tamoxifen-resistant cell lines to perform expression microarrays, metabolic flux assays and DNA damage response analysis in order to uncover the cause of the differential response to OGT targeting. This may help us identify potential therapeutic combinations that might be suitable for treatment of tamoxifen-resistant cancers. 

### Gene and Vector-Based Therapy

#3738

IL-24 modulates the high mobility group (HMG) A1/miR222 /AKT signaling in lung cancer cells.

Janani Panneerselvam, Akhil Srivastava, Ranganayaki Muralidharan, Qi Wang, Wei Zheng, Lichao Zhao, Allshine Chen, Yan Zhao, Anupama Munshi, Rajagopal Ramesh. _Stephenson Cancer Center, Oklahoma City, OK_.

Background: High mobility group A1 (HMGA1), a member of the non-histone chromosomal proteins and commonly referred to as architectural transcription factor, regulates transcription of various genes involved in cell growth and survival. Overexpression of HMGA1 has been shown to be associated with tumor progression and metastasis in several cancers, including human lung cancer. A recent study demonstrated that HMGA1 activates AKT function by reducing the activity of the protein phosphatase, phosphatase 2A subunitB (PPPR2A) via the oncogenic micro (mi) RNA-222. We demonstrated that interleukin (IL)-24, a novel tumor suppressor/cytokine, inhibited AKT in lung cancer cells. However, the molecular mechanism of AKT inhibition by IL-24 remains elusive.

Aim: To determine the molecular mechanism of IL-24-mediated AKT inhibition involved the HMGA1/miR-222 axis.

Methods: Human H1299 lung tumor cell line was stably transfected with a tetracycline-inducible plasmid vector carrying the IL-24. After the induced expression of IL-24 protein, expression levels of HMGA1 and its downstream molecular mechanisms were analyzed at the RNA and protein levels in lung cancer cell lines. The inhibitory effect of IL-24 on HMG A1/miR222 /AKT axis in the lung cancer cells is determined by RT-qPCR, western blot, reporter assay, and immunocytochemistry. Mechanistic approaches on overexpression and knockdown of HMGA1 and or miR-222 were utilized and the consequences of its inhibition/overexpression were analyzed on HMGA1/miR222 /AKT signaling axis and in vitro migration and invasion.

Results: Upon induction of IL-24 expression in the H1299 lung tumor cells, we observed a marked reduction in HMGA1protein and mRNA levels. Using a mechanistic approach, we found that IL-24 reduced miR-222-3p and -5p levels, as determined by qRT-PCR. Associated with HMGA1 and miR-222 inhibition was a marked increase in PPP2R2A, with a concomitant decrease in phosphorylated AKTT308/S473 expression. SiRNA-mediated knockdown of HMGA1in combination with IL-24 significantly reduced AKT T308/S473 protein expression and greatly reduced cell migration and invasion compared with individual treatments. Further combination of IL-24 and a miR-222-3p inhibitor significantly increased PPP2R2A expression.

Conclusion: Our results demonstrate for the first time that IL-24 inhibits AKT via regulating the HMGA1/miR-222 signaling node in human lung cancer cells and acts as an effective tumor suppressor. HMGA1 should present a novel target for the effective treatment of lung cancer.

#3739

Efficacy of CD46-mediated Ad5/35 chimeric adenoviral gene therapy in colorectal cancers.

Young Seok Cho,1 Hung Manh Do,1 Sang-Jin Lee,2 Silvio Hemmi,3 Young-Eun Joo,1 Chaeyong Jung1. 1 _Chonnam National Univ. Med. School Gwangju Campus, Gwangju, Republic of Korea;_ 2 _National Cancer Center, Ilsan, Republic of Korea;_ 3 _University of Zurich, Institute of Molecular Life Sciences, Zuerich, Switzerland_.

CD46 is a complement inhibitor membrane cofactor protein and also acts as a receptor for certain pathogenic microbes, including group B denovirus (Ad). Whereas many Ads infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancers preventing effective therapeutics of Ad-based therapies. There have been increasing numbers of studies in cancer gene therapy by modification of Ad fiber knobs to utilize more ubiquitously expressed CD46. However, very limited information is available on expression status of CD46 in many cancers. Our tissue microarray data demonstrated that CD46 was generally overexpressed to cancers from prostate and colon. To seek the evidence whether colorectal cancers (CRC) are good target for Ad-mediated gene therapy, chimeric Ad5 vectors that are capable of targeting CD46 were employed. CD46-ovrexpressed cells showed significantly higher response not only to Ad5/35-GFP but to Ad5/35-TK/GCV suicide therapy. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and cell killing, demonstrating that HCT-116, DLD-1, and Caco-2 cells are highly responsive. Furthermore, injection of Ad5/35-TK/GCV caused significantlyhigher anti-cancer effects in nude mice bearing CD46-overexpressed cancer cells compared to mice with mock cells. Using 80 selected CRC, immunohistochemical studies showed that there is inverted correlation between CD46 expression and clinico-pathological parameters in terms of differentiation, invasion, metastasis, T stage, and survival, suggesting that adenoviral gene therapy may not be as effective to the patients with highly advanced colorectal cancers. Taken

altogether, our study demonstrated that CD46 is generally overexpressed in CRC in which group B-based adenoviral gene therapy seems to be suitable approach but careful consideration needs to be given to select cancer types and cancer status for effective cancer gene therapies.

#3740

Antitumor effect of gene therapy with SOCS-1 in esophageal squamous cell carcinoma.

Takahito Sugase,1 Tsuyoshi Takahashi,1 Serada Satoshi,2 Eri Nakatsuka,3 Minoru Fujimoto,2 Yasuhiro Miyazaki,1 Tomoki Makino,1 Yukinori Kurokawa,1 Makoto Yamasaki,1 Kiyokazu Nakajima,1 Shuji Takiguchi,1 Masaki Mori,1 Yuichiro Doki,1 Tetsuji Naka2. 1 _Osaka University, Graduate School of Medicine, Osaka, Suita, Japan;_ 2 _Department of Dermatology, National Institute of Biomedical Innovation, Osaka, Ibaraki, Japan;_ 3 _Osaka General Medical Center, Osaka, Japan_.

[INTRODUCTION]

Esophageal squamous cell carcinoma (ESCC) is the major histological form of esophageal cancer in East Asian countries. Despite improvements of multimodality treatment, including surgery, radiotherapy and chemotherapy, the prognosis for patients with ESCC remains unsatisfactory compared to other cancer. Therefore, development of novel therapy is urgently required. Constitutive activation of JAK/STAT pathway has been reported in various tumor including ESCC, and associated with progression of tumor. Suppressor of cytokine signaling-1 (SOCS-1) has been cloned as a negative regulator of various cytokine signaling including JAK/STAT pathway. Recently, lines of evidences suggest that overexpression of SOCS-1 has been a promising therapeutic approach for various cancer. This study was aimed to evaluate the therapeutic effect of ESCC using SOCS-1 overexpressed by adenoviral vector (AdSOCS-1).

[METHODS]

For ten ESCC cell line, we evaluated cell growth inhibition via adenotransfection with various dose of AdSOCS-1 by WST-8 assay and Western blotting. Induction of apoptosis was also evaluated. In addition, it was also compared to AdSOCS-1 and JAK inhibitor for antiprolificative effect. To evaluate the therapeutic efficacy of AdSOCS-1, ESCC cell line (TE-14) was subcutaneously implanted into the ICR nu/nu mice. In addition, patient-derived tumor xenografts (PDX) using human ESCC subcutaneously transplanted in NOD-SCID mice were also assessed. Mice were intra-tumorally injection with AdSOCS-1 or control adenovirus vector (AdLacZ) twice a week for 4weeks.

[RESULTS]

AdSOCS-1 markedly suppressed proliferation of all ESCC cell lines in vitro. AdSOCS-1 strongly induced apoptosis via inhibiting not only JAK/STAT pathway but also MAPK and FAK signaling, especially in TE14 which was not significantly suppressed in JAK inhibitor. In TE14 xenograft model, the volume of tumors with AdSOCS-1 therapy was significantly lower than that with AdLacZ. Also, in PDX mice, injection with AdSOCS-1 significantly inhibited the tumor growth compared to AdLacZ.

[CONCLUSIONS]

Our results indicated that overexpression of SOCS-1 inhibited the progression of ESCC both in cell lines and xenograft model mice. Gene therapy by SOCS-1 can be a promising novel approach for intra-lesional therapy in ESCC.

#3741

Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma.

Katja Wosikowski,1 Kathy Hasenbach,1 Jutta Petschenka,2 Diana Barea Roldan,2 Sebastian Kreiter,2 Ugur Sahin,2 Guillaume Serin,3 Julie-Orlane Redon,3 Marc Hillairet de Boisferon,3 Francis Bichat,3 Hanna Kohonen,1 Michel Janicot1. 1 _Isarna Therapeutics, Munich, Germany;_ 2 _TRON, Mainz, Germany;_ 3 _Oncodesign, Dijon, France_.

Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both 'seed and soil' theory and efficacy of combination regimen (immunomodulation) for better clinical outcome.

#3742

Treatment with trabedersen, an anti-TGF-beta 2 antisense, primed tumors to subsequent chemotherapies.

Larn Hwang, Kevin Ng, Wen Wang, Vuong Trieu. _Autotelic Inc, Fountain Valley, CA_.

Background: Overexpression of transforming growth factor-beta 2 (TGF-β2) is associated with poor prognosis and plays a key role in malignant progression of various tumors by inducing proliferation, metastasis, angiogenesis and immunosuppression. Previously reported P-001 trial of Trabedersen - Safety and Tolerability of AP 12009, Administered I.V. in Patients With Advanced Tumors Known to Overproduce TGF-beta 2 was evaluated to determine if treatment with Trabedersen primed the tumors to subsequent chemotherapy.

Methods: A total of 37 patients with advanced pancreatic cancer were treated with Trabedersen by intravenous infusion in escalating doses of 2 treatment schedules (initial schedule: 7-days on, 7-days off; modified schedule: 4-days on, 10-days off; two cycles as core study and up to 10 cycles for expanded study). Blood samples from 18 patients were taken at the screening/baseline (BSL) visit, after completion of the first 2 treatment cycles at Day 29 (D29) and during follow-up (FU) visits, which were conducted every 8 weeks after start of the 1st Trabedersen cycle for the tumor markers of cancer antigen 19.9 (CA19.9) and carcinoembryonic antigen (CEA).

Results: Maximum tolerated dose (MTD) for 7/7 regimen (N=11) was 160 mg/m2/d; whereas MTD was not reached on 4/10 regimen (N=27) even at highest dose of 330 mg/m2/d. There were16 2nd line patients and 11 3rd line and beyond on 4/10 regimen. The progression free survival/overall survival (PFS/OS) of these 2nd line patients in months (mos) were: 1.87/5.60 (N=6), 1.87/9.93 (N=11) and 2.72/11.80 (N=5) at increasing mean dose of 140, 167, and 196 mg/m2/d, respectively. The OS of 9.93 and 11.80 mos are higher than the highest reported OS in the 65 trials reported in the literature during 1997-2015 (range = 2.50-9.20/median = 5.50 mos). The corresponding PFS values were in line with reported literature (range = 0.00-7.65/median = 2.43 mos). The OS of this 4/10 cohort treated with subsequent chemotherapies was 14.7 and 2.93 mos with and without subsequent chemotherapies, p = 0.0023. Chemotherapy on 2nd line followed with subsequent Trabedersen 4/10 regimen as third line was ineffective with OS of 2.80 mos (N=9) versus 9.93 mos (N=11), p =0.046. CA19.9 and CEA biomarker data were consistent with OS data.

Conclusion: Trabedersen treatment was characterized by outstanding OS which was not supported by PFS. The effect was seen primarily when chemotherapies were used as third line after Trabedersen treatment; OS benefit was not observed for the converse- chemotherapies used first followed by Trabedersen. The data supports the enhancement of subsequent chemotherapies following Trabedersen treatment, suggesting a combination trial of Trabedersen and chemotherapies with sequencing.

#3743

New non-integrative MS2-based lentiviral particles for mRNA delivery: A safe and efficient opportunity for gene editing and immunotherapy applications.

Pascale Bouillé,1 Cédric Auffray,1 Jean-Christophe Pagès,2 Christine Duthoit,1 Régis Gayon1. 1 _VECTALYS, Toulouse, France;_ 2 _Université François Rabelais de Tours, INSERM UMR 966, Tours, France_.

Safe and efficient cancer therapies using adoptive transfer of engineered T cells or gene editing are very challenging but very promising approaches nowadays. The scientific and clinical communities have been working for a long time together to encounter substantial clinical advances they have made possible thanks to numerous improvements in cell culture and gene transfer methods. Opportunities to improve gene transfer into primary T cells or hematopoietic stem cells involve a better design of the vectors used. Such improvements must lead to an increase of the efficiency in the percentage of positive cells, as well as a better level and duration of expression, cell phenotype preservation and the number of genes delivered. Lentiviral vectors have seen their use largely increased in clinical protocols over the past few years but safety concerns have been highlighted. First, the permanent genetic modification remains a focus of significant regulatory oversight and even integrase- or reverse transcriptase-deficient lentiviral vectors leads to residual integration events. Moreover it has been shown that some resulting CAR T cells can exhibit toxicity due to high and persistent expression. If mRNA delivery is a versatile, flexible, and safe mean for protein therapies, chemical or electroporation-based transfection protocols are known to induce cell toxicity and phenotype modifications of the target cells.

Here, we describe a new chimeric lentiviral platform that allows mRNA delivery into the target cells without any genomic signature. The respective properties of the MS2 bacteriophage and the lentiviral vectors have been combined to build a non-integrative packaging system in which the wild type HIV packaging sequence is replaced by the MS2 stem-loop repeats and the MS2 Coat sequence is inserted into the NucleoCapsid sequence. The resulting lentiviral particle is able to deliver a non-viral RNA into the target cell, directly available for protein translation.

Transduction of T cells and HSC with these RNA lentiviral particles (RLP) shows an efficient, fast and transient expression of both reporters and functional proteins such as genome editing enzymes. Cell viability of such engineered cells and multiple genes expression analyses will be presented.

This transient mRNA delivery mediated by a lentiviral particle is a powerful tool, useful to induce an efficient CAR delivery and ensure a complete loss of the CAR-driven T cells activity. The possibility to express multiple genes at once in the target cells is an attractive therapeutic perspective. One of the advantages of the MS2RLP system is its ability to utilize lentiviral production platforms already validated in clinical settings. The RNAs transferred by the MS2RLPs are directly expressed into the cytoplasm, which completely removes the risk of integration, an important safety consideration for human use.

#3744

Targeted therapy for breast cancer bone metastases using mRNA engineered mesenchymal stem cells.

Aude Segaliny, Jason Cheng, Weian Zhao. _Department of Pharmaceutical Sciences; Sue and Bill Gross Stem Cell Research Center; Chao Family Comprehensive Cancer Center; Department of Biomedical Engineering, University of California Irvine, Irvine, CA_.

Bone is a primary metastatic site in breast cancer, and bone metastases are associated with poor survival and a dramatic decrease of patient's quality of life. Current treatments lack specificity for bone metastatic niches, leading to poor drug delivery and systemic toxicity. Mesenchymal stem/stromal cells (MSCs) represent ideal trophic vehicles for drug delivery to bone metastases as they are readily available, easy to expand, and display a natural, although inefficient, homing to bone and tumor sites. This work aimed to develop MSCs that are engineered through simple mRNA transfection with both bone vasculature-homing ligands and therapeutics to improve targeted delivery and efficacy. The transfection of protein-coding mRNA is an effective tool to engineer simultaneously and transiently cells with multiple factors to control the fate of transplanted cells, while mitigating risks associated with viral-based engineering.

Human bone marrow-derived MSCs from healthy donors were engineered with synthetic mRNA to express homing ligands P-selectin glycoprotein ligand-1 (PSGL-1)/Sialyl Lewis X (sLeX), and anti-tumor agents cytosine deaminase (CD) and osteoprotegerin (OPG). PSGL-1/sLeX allow MSCs to efficiently target bone tumor vasculatures that overexpress P- and E-selectins. CD is a convertase that locally converts inactive pro-drug 5-Fluorocytosine (5-FC) to active drug 5-Fluorouracil. OPG is a natural decoy receptor for one of the main bone resorptive factors expressed in bone metastases, RANKL (Receptor activator of nuclear factor kappa-B ligand), and thus blocks the vicious cycle existing between tumor growth and bone resorption.

First, we demonstrated that MSCs could be efficiently engineered through mRNA transfection to express simultaneously homing ligands PSGL-1 and sLeX, CD and OPG. We evaluated improved cell homing and anti-tumor functions of engineered stem cells in vitro. Expression of PSGL-1 and sLeX increased MSC rolling and tethering on an activated endothelium under shear flow condition. When co-cultured with human breast cancer cells MDA-MB231 in presence of 5-FC, engineered MSCs induced both breast cancer and MSC death within 5 days. Finally, secreted OPG is able to prevent osteoclastogenesis in vitro using murine osteoclast precursors. We are currently evaluating biodistribution, homing to bone metastatic niches and anti-tumor efficacy of engineered MSCs in vivo using establish models of breast cancer bone metastases (intratibial and intracardiac injection of MDA-MB-231 cells). As bone metastases are osteolytic lesions, bone remodeling will also be measured using histomorphometry.

To conclude, our engineered MSCs can potentially be safer vectors to effectively target and treat bone metastases, thus decreasing off-target systemic toxicity. This next-generation of engineered MSCs has the potential to address an unmet challenge for bone metastases treatment.

#3745

**Tumor fibroblast targeting via uPAR retargeted measles virus:** In vitro **and** in vivo **effects.**

Yuqi Jing,1 Julia Zaias,2 Jaime Merchan1. 1 _University of Miami Sylvester Comprehensive Cancer Center, Miami, FL;_ 2 _University of Miami, Miami, FL_.

Tumor stromal cell components, in particular cancer associated fibroblasts, play an important role in cancer progression. Few studies have focused on stromal fibroblast targeting by oncolytic viruses. The urokinase receptor (uPAR) is a clinically and biologically validated target, which is overexpressed in tumor and stromal cells compared to non-cancer tissues. MV-h-uPA and MV-m-uPA are fully retargeted oncolytic measles viruses directed against human and murine uPAR, respectively, which have shown in vitro and in vivo safety and antitumor effects. Species specific retargeted viral vectors allow to dissect the specific effects of the viruses on murine vs. human tissues in xenograft models. Our aim is to characterize the in vitro and in vivo effects of stromal targeting by oncolytic measles virus via uPAR, with a focus on tumor fibroblasts. In vitro, MV-h-uPA and MV-m-uPA preferentially infected and induced cytotoxicity in human cancer associated fibroblasts (CAF 19, CAF 23) as well as murine fibroblasts (3T3), compared to non-tumorigenic fibroblasts. Murine-murine and human-human fibroblast to cancer cell viral transfer via heterofusion was observed after fibroblast infection by species specific MV-uPA, in breast, colon and renal cancer models, while no viral transfer was observed between cells of different species. In vivo, systemic administration of the murine uPA retargeted virus (MV-m-uPA) significantly decreased tumor progression and prolonged survival in a human breast cancer xenograft model (MDA-MD231), where the host stroma expresses murine uPAR (target of MV-m-uPA). Tumor studies revealed induction of apoptosis (TUNEL assay), while no significant effects on cancer cell proliferation was observed. Dual staining for measles virus nucleocapsid proteins and fibroblasts markers demonstrated viral infection of fibroblasts in treated tumors. Gene expression studies using murine as well as human specific arrays were performed to characterize the effects of stromal targeting by the murine retargeted virus on murine stroma as well as the indirect effects on human cancer cells in vivo.

In conclusion, for the first time our results show feasibility and antitumor effects of stromal fibroblast targeting by oncolytic measles virus via uPAR and demonstrate that stromal fibroblasts are viable targets for oncolytic virotherapy. Studies characterizing other stromal cell components and evaluation of the molecular changes in the tumor stroma as result of viral infection are underway.

#3746

A high dose priming strategy with the novel RNAi therapeutic GS-10 elevates RISC Ago2, Ago4 and GW182 protein levels to enhance RNAi efficacy in a metastatic solid tumor model.

Lucas P. Nacusi, Vicci L. Korman, Alex J. Pretti, Diane K. Tobolt, Alvaro Mendoza, Gretchen M. Unger. _GeneSegues Therapeutics, Chaska, MN_.

A high initial priming dose of the novel RNAi therapeutic GS-10 was investigated as a strategy to elevate important RISC complex proteins that have the potential to increase efficacy of subsequent RNAi treatment regimens in solid tumor cancers. GS-10 is a tumor-targeted 20 nm crystalline capsule bearing single-stranded RNAi oligos against Protein Kinase CK2 (CK2), and a ligand-coated shell derived from Tenascin-C (tenfibgen). Tenascin-C is upregulated in multiple solid tumors, and is a promising targeting ligand for tumors throughout their lifecycle. The ultrasmall capsule size enables efficient raft-mediated delivery of oligos to the perinuclear space of the target cell, and the ability to reach metastases without reliance on EPR. To date, strategies for RNAi-treatment of solid tumor cancers have been sub-optimal due to, at least in part, the varied levels of RNA degradation machinery across patients, tumors and tissues. For example, we have found wide variation in Ago2 protein levels, a key RISC component for RNA cleavage, in human head and neck cancer (HNC) tissues. Here we investigate a strategy for overcoming this limitation for an RNAi therapeutic in solid tumors. We have previously established that SQ doses of GS-10 at 10-100 ug/kg/day have antitumor efficacy, coincident with CK2 protein knockdown, in several mouse models. In an SCC-47 HNC xenograft model, tumor growth inhibition of 86% was observed after GS-10 treatment compared to controls, despite low baseline Ago2 levels. Interestingly, these tumors express relatively high Ago4 levels. FISH analysis of GS-10-treated SCC-47 tumors revealed numerous punctate structures containing mRNA CK2 transcript. Similarly, structures containing Ago4 and GW182 were observed by confocal microscopy, altogether suggesting these RISC proteins mediate transcript sequestration by forming GW-bodies, resulting in tumor inhibition. Now, we are evaluating a strategy using high-dose IV bolus injection as priming to elevate expression of multiple RISC proteins, including Ago2, Ago4 and GW182, in the SCC-47 model. A priming dose of GS-10 at 10 mg/kg showed a 4-fold increase in Ago2 levels in primary tumors vs. treatment controls at 72 hours post-injection. Similarly, a 2-fold increase in Ago4 and GW182 levels was also observed. To establish that GS-10 was reaching tumors in vivo to induce these changes, distribution of an isotopic tenfibgen surrogate of GS-10 to primary tumor and lymph node metastases was confirmed following IV bolus administration. Additional RISC protein and tumor efficacy data from a later timepoint is being analyzed and will be reported. In summary, these data suggest that high-dose IV priming with GS-10 can elevate multiple RISC proteins, setting tumors up for more effective response to subsequent low-dose treatment using this novel therapeutic candidate.

#3747

Oncolytic viruses as a possible therapeutic strategy against malignant mesothelioma.

Carmelina Antonella Iannuzzi,1 Carmela Passaro,2 Silvia Boffo,3 Iris Maria Forte,4 Paola Indovina,3 Marcella Macaluso,3 Giuseppe Portella,2 Antonio Giordano,3 Francesca Pentimalli4. 1 _Department of Medicine, Surgery and Neuroscience, University of Siena, Siena, Italy;_ 2 _Dipartimento di Scienze Mediche Traslazionali Università Federico II, Napoli, Italy;_ 3 _Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA;_ 4 _National Cancer Institute of Naples, Pascale Foundation, Mercogliano, Avellino, Italy_.

Malignant mesothelioma (MM) is a very aggressive tumor associated to asbestos exposure, which is poorly responsive to the current therapeutic strategies, resulting in a dismal prognosis. So, considering also that various studies predict an increase in incidence, new effective therapeutic approaches are urgently needed. Here we investigated at the preclinical level a new possible therapeutic strategy for MM based on the use of oncolytic viruses. Oncolytic viruses show various benefits as anticancer agents: they can replicate selectively in tumor cells reaching 10,000-fold amplification of the input dose; they stimulate the antitumoral immune response; they can be used in combination with other cytotoxic agents; the pleural cavity in which pleural MM arises is easily accessible for such therapeutic approach. In particular, we focused on adenoviruses with a 24 bp deletion in the E1A-conserved region 2, which binds and inactivates the retinoblastoma protein, resulting in a virus (dl 922-947) that cannot trigger S phase entry in normal cells, but can still replicate in cells with an aberrant G1-S checkpoint, a defect observed in over 90% human cancers, including MM. We studied on a panel of mesothelioma cell lines representative of the main different histotypes — the epithelioid NCI-H28 and NCI-H2452, the biphasic MSTO-211H, the sarcomathoid NCI-H2052 — the effects of dl 922-947 treatment used both as a single agent or in combination with other strategies. At first, dl 922-947 cytotoxicity was evaluated through the sulforhodamine B (SRB) assay, which showed that all MM cell lines were susceptible to viral treatment, except NCI-H2052 cells, in which viral entry was not efficient, as shown through infection with a reporter adenovirus transducing GFP. Interestingly and consistently with the cytotoxic effect observed, FACS analysis showed that dl 922-947 treatment induced an increase of the subG1 cell fraction (suggestive of apoptosis induction) and of the hyperdiploid (4N) population (suggestive of mitotic defects). We also investigated by SRB the possible cytotoxic effects of dl922-947 in combination with other therapeutic strategies. In particular, we analyzed the effect of dl922-947 in combination with cisplatin, which is the first-line treatment against MM, and found that, by comparing different schedules of treatment, cisplatin increased the cytotoxic effect of the oncolytic virus. We also tested dl922-947 efficacy in combination with MK-1775, an efficient inhibitor of the WEE1 kinase, which is currently being tested in clinical trials. We found that MK-1775, at doses equal to or above its IC50value, is able to increase the cytotoxic effect of the oncolytic virus treatment.

In conclusion our data indicate that treatment with the dl922-947 oncolytic virus might be a promising new approach against mesothelioma and warrants further investigation both as single agent and in combination strategies.

#3748

Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion.

Takeshi Koujima,1 Hiroshi Tazawa,2 Takeshi Ieda,1 Kazuya Kuwada,1 Terutaka Tanimoto,1 Shinji Kuroda,1 Hiroyuki Kishimoto,1 Masahiko Nishizaki,1 Yasuo Urata,3 Shunsuke Kagawa,1 Toshiyoshi Fujiwara1. 1 _Dept. Gastroenterological Surg., Okayama Univ. Grad. Sch., Okayama, Japan;_ 2 _Ctr. for Innovative Clinical Med., Okayama Univ. Hosp., Okayama, Japan;_ 3 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. More than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis at the time of diagnosis. Moreover, even after curative surgery, PDAC patients still have poor prognosis due to local recurrence and systemic metastasis. Although activation of oncogenic KRAS is implicated in the initiation, promotion and progression of pancreatic cancer, the development of cancer treatment targeting KRAS remains unsuccessful. Moreover, tumor suppressor p53 is also frequently inactivated in PDAC patients. We recently developed two types of tumor-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702, which is modified OBP-301 to express tumor suppressor p53 protein. In this study, we investigated whether oncolytic adenoviruses inhibit cell viability, migration, invasion and KRAS-driven signaling pathway in PDAC cells.

Methods: We used 4 PDAC cells (MIA PaCa-2, Capan-1, Panc-1, BxPC-3). The cell viability was examined by XTT assay. Migration and invasion properties were assessed using transwell chamber assay. The modulation of KRAS-driven signaling pathway was investigated by Western blot analysis.

Results: OBP-301 induced moderate antitumor effect in MIA Paca-2, Capan-1 and BxPC-3 cells and strong antitumor effect in Panc-1 cells in a dose-dependent manner, whereas OBP-702 induced profound antitumor effect in all human PDAC cells, suggesting the broad spectrum of OBP-702's efficacy. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-301 and OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells, and the inhibitory effect of OBP-702 was stronger than that of OBP-301. We also found that oncolytic adenoviruses inhibited the expression of KRAS and KRAS-downstream target MEK/ERK proteins in high-invasive type PDAC cells.

Conclusions: Our results suggest that oncolytic adenoviruses, OBP-301 and OBP-702, inhibit the survival and invasive phenotype of pancreatic cancer by suppressing KRAS-driven signaling pathway and reactivating p53-driven signaling pathway. In vivo experiments are under way to investigate the anti-tumor and anti-invasive effects of oncolytic adenoviruses using BxPC-3 xenograft tumors. The clinical trial of intratumoral administration of oncolytic adenoviruses in patients with invasive PDAC should be also warranted.

#3749

Interaction of 5FU chemotherapy and oncolytic adenovirus in combined cancer treatment.

Jordan Sell, Amanda Oliviera, Eric Jensen, Masato Yamamoto, Julia Davydova. _University of Minnesota-Twin Cities, Minneapolis, MN_.

Previous studies have shown that Interferon(IFN)-based chemoradiation therapy can improve survival after resection of pancreas cancer. However, its clinical utility to this point has been limited due to the severe toxicity related to its use of systemic IFN. Our aim in this study is to evaluate our group's novel oncolytic adenovirus (OAd) which allows targeting IFN treatment to cancer cells while sparing healthy tissue. We have previously shown the ability of IFN to sensitize cancer cells to chemotherapy as well as demonstrated an increased therapeutic effect of the drug in immunocompetent models. This study was conducted to analyze the combination of 5FU chemotherapy and our OAd in vitro in order to assess the interaction of treatments and determine the optimum combined treatment regimen.

Treatments were analyzed in pancreatic cancer and esophageal adenocarcinoma cell lines S2013 and OE19. Recombinant OAds expressing luciferase rather than IFN were used to isolate the combination of 5FU and the virus. Two viral models were evaluated: our therapeutic virus (Cox2) selectively replicative in Cox2 expressing cancer cells and a nearly identical but universally replicative virus (wild type). Cells were treated with 0, 1 or 10 viral particles per cell and 0, 5, 10, or 20 uM 5FU. Three timing regimens were used: simultaneous administration, 5FU 4 hours before virus, and virus 48 hours before 5FU. Crystal Violet and MTS Assays were used to measure cell death. Viral Copy number to assess viral replication was measured using qPCR.

Cell death analysis showed time dependent killing of each virus, with a 2 day delay for the Cox2 virus. 5FU and each virus produced dose dependent cell death independently. There was a significant additive effect seen in cell death from combining treatments when using 5FU before virus in both S2013 and OE19. Simultaneous treatment showed an additive killing effect with the combination in S2013, but a reduced killing by the combination compared to virus alone in OE19. There was also reduced killing when using virus before 5FU in S2013. Viral copy experiments are in progress.

Our Cox2 OAd shows a killing effect similar to wild type in multiple cancer cell lines. When combined with 5FU treatment the expected addition in overall cell death from the independent treatments varies by timing of administrations as well as by type of cancer cell line. The reduced killing of the combination treatment in the simultaneous and virus before 5FU regimens may suggest an inhibition of the virus by 5FU under certain conditions. This also suggests the possibility of a treatment regimen with optimal therapeutic effect, which appears to be 5FU before virus based on the results collected. Further studies investigating different chemotherapeutic drugs and regimens should be conducted to examine these trends.

#3750

CD133-targeted oncolytic adenovirus shows therapeutic effect by attacking colon cancer stem cells.

Mizuho Sato,1 Yoshiaki Miura,2 Masato Yamamoto1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Toyama, Toyama, Japan_.

Colorectal cancer is the third most common cancer in the world and about 50% of patients relapse after treatment. Cancer stem cells (CSCs) have contribution to recurrence, metastasis and and chemotherapy resistant of colorectal cancers. CD133 (Prominin-1), a member of the transmembrane glycoprotein family, is a marker of CSCs in several cancers and is also generally accepted as a colorectal cancer stem cell marker. CD133 expression was correlated with recurrence, metastases and chemotherapy resistance, as well as poor prognosis in colorectal cancer. It is therefore reasonable to develop CSCs-directed therapeutic strategies by employing CD133 as a target molecule. Recently, we have established a method for isolating transductionally-targeted adenovirus by high-throughput screening. In this study, using this adenovirus library screening system, we isolated the CD133-specific Oncolytic Adenovirus (OAd) and tested the oncolytic activity of CD133-targeted Ad in both in vitro and in vivo. CD133-targeted OAd (AdML-TYML) showed strong binding to CD133-positive cells (293-CD133 and LoVo (CD133 (+) human colon carcinoma)), not to CD133-negative cells (parental 293 and LS174T (CD133 (-) human colon carcinoma)) and this virus also showed strong oncolysis selectively in LoVo cells, whereas there was no effect on LS174T cells.

In order to assess the effect of AdML-TYML on stemness, tumor establishment assay was performed. AdML-TYML treatment leads to inhibition of tumor establishment of CD133-positive colorectal cancer cell lines in nude mice. 100% of the mice inoculated with non-infected LoVo cells had developed tumors, while 0% of the mice inoculated with AdML-TYML infected LoVo cells had done so. In addition, when the anti-tumor effect of the CD133-targeted OAd was analyzed in established tumors of CD133(+) colorectal cancer subcutaneous xenografts, intra-tumor (i.t.) administration of AdML-TYML exhibited significantly stronger antitumor effect compared to its equivalent Ad5-WT (wild type Ad5 fiber).

We focused on the CD133 as a target molecule of CSCs in colon cancer and successfully identified potent CD133-targeting OAd using high-throughput screening system. The CD133-targeted OAd exhibited selective infectivity and oncolysis to CD133-positive cells and anti-tumor effect against established colon cancer cells. Our results suggest that CD133-targeted oncolytic adenovirus selectively eliminate CD133 (+) colorectal cancer stem cells from the tumor and also this virus could be a key tool for the prevention of metastases and relapses in a variety of cancers.

#3751

Apoptosis inducer gene delivery by polylysine-calcium complexes attenuates mouse lung carcinoma allograft growth after intravenous injection or intratracheal spray.

Susumu Ishiguro,1 Nabil A. Alhakamy,2 Deepthi Uppalapati,1 Cory J. Berkland,2 Masaaki Tamura1. 1 _Department of Anatomy & Physiology, Kansas State University, Manhattan, KS; _2 _Departments of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS_.

Transfection efficiency and toxicity concerns remain a challenge for gene therapy. Nanoparticle-based gene delivery technique potentially overcomes these concerns and may be applicable to cancer gene therapy. Cell penetrating peptides (CPPs) have been broadly investigated to improve the transfection of genetic material (e.g., pDNA and siRNA). Our previous study demonstrated that an apoptosis inducer, angiotensin II type 2 receptor plasmid DNA (pAT2R) encapsulated in a modified HIV-1 TAT peptide (dTAT-pAT2R), significantly attenuated the growth of Lewis lung carcinoma (LLC) allograft in mouse lungs (Kawabata et al., Cancer Res, 2012). Here, we report a newly synthesized polylysine CPP (K9 peptide)-based gene therapy for lung cancer treatment. The pAT2R and K9 peptide (K9-pAT2R) complexes were condensed using calcium chloride (K9-pAT2R-Ca2+). The resulting complexes were small (~150 nm) and showed high levels of gene expression in vitro. This simple non-viral formulation approach showed negligible cytotoxicity in several different human and mouse cell lines (human cervix, breast, kidney, and human and mouse lung cell lines). Additionally, this K9-pDNA-Ca2+ complex demonstrated cancer targeted gene delivery when administered via intravenous (IV) injection or intratracheal (IT) spray into LLC orthotopic allograft-bearing mice. Average lung weights (mg) of the K9-pAT2R-Ca2+ IT (190.6±48.3) and the K9-pAT2R-Ca2+ IV (201.6±67.0) treated groups were significantly smaller than that of the control PBS group (325.7±69.4, P<0.05). In histological examination of tumors in H&E stained lung, average numbers of tumor nodules in the lungs in the K9-pAT2R-Ca2+ IT (6.0±4.2) and the K9-pAT2R-Ca2+ IV (4.6±3.4) groups were also significantly smaller than that of the control PBS group (17.8±6.0, P<0.01). This tumor attenuating effect was not observed in K9-pLUC-Ca2+ IT and K9-pLUC-Ca2+ IV treatment, thus it is suggested that LLC tumor growth was attenuated by apoptosis inducer gene, pAT2R, delivery. Immunohistochemical analysis confirmed that the complex effectively delivered pAT2R to the cancer cells, where it was expressed mainly in cancer cells along with bronchial epithelial cells. A single administration of these complexes markedly attenuated lung cancer growth offering preclinical proof of concept for a novel non-viral gene delivery method exhibiting effective lung tumor gene therapy via either IV or IT administration. This work is supported in part by Savara Pharmaceuticals (CB and MT), Faculty of Pharmacy of King Abdulaziz University, Jeddah, Saudi Arabia (NAA), University of Kansas Macromolecule and Vaccine Stabilization Center (CB), Kansas State University Johnson Cancer Research Center (MT), NIH grants U43 CA165462 (MT), P20 GM103418 (MT), and Kansas Bioscience Authority Collaborative Cancer Research grant (MT).

#3752

A cell cycle-related kinase (CCRK) self-regulatory circuitry in NAFLD-associated hepatocarcinogenesis.

Hanyong SUN,1 Yuan Tian,1 Weiqin Yang,1 Jingying Zhou,2 Paul B. Lai,3 Joseph J. Sung,1 Grace L. Wong,1 Vincent W. Wong,1 Alfred S. Cheng4. 1 _Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease,The Chinese University of Hong Kong, Hong Kong;_ 2 _School of Biomedical Sciences,The Chinese University of Hong Kong, Hong Kong;_ 3 _Department of Surgery,The Chinese University of Hong Kong, Hong Kong;_ 4 _School of Biomedical Sciences,State Key Laboratory of Digestive Disease, The Chinese University of Hong Kong, Hong Kong_.

Background and aim: Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome including obesity and diabetes which elevate the risk of hepatocellular carcinoma (HCC). Accumulating evidence has unmasked the molecular linkage between androgen receptor (AR) signaling and gender disparity in NAFLD and HCC incidence. Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of AR oncogenic activity in hepatitis B virus-associated HCC through glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling (1-3). Here we investigated whether CCRK plays an oncogenic role in NAFLD-related HCC.

Methods: We investigated the functional role of CCRK in a high-fat high carbohydrate diet-induced obesity model exposed to low-dose diethylnitrosamine using lentiviral-mediated gene knockdown, followed by measurement of glucose/lipid metabolism via intraperitoneal glucose tolerance test and triglyceride/non-esterified fatty acids levels, and HCC incidence. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses.

Results: Lentiviral-mediated Ccrk silencing in male mice significantly reduced obesity-promoted tumor multiplicity and size by >70%. The reduced tumorigenicity was also associated with significant restoration of circulating metabolic profiles and reduction in hepatocellular lipid level. Mechanistic studies demonstrated that the pro-inflammatory cytokine interleukin-6 (IL-6) triggered signal transducer and activator of transcription 3 (STAT3) signaling to up-regulate CCRK expression in an AR-dependent manner. Simultaneously, the phosphorylation of STAT3 by CCRK facilitated the co-occupancy of CCRK promoter by STAT3-AR complex and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. In addition, both in vitro and in vivo data suggested that CCRK phosphorylated GSK-3β to activate not only β-catenin but also mechanistic target of rapamycin complex 1 (mTORC1) signaling pathways that are crucial for cell survival, proliferation and lipogenesis. Furthermore, transgenic over-expression of CCRK in mouse liver led to concordant activation of STAT3, β-catenin and mTORC1 signaling pathways.

Conclusion: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates NAFLD-associated hepatocarcinogenesis and offer novel therapeutic strategies to combat this dreadful disease in an era of global obesity.

Acknowledgement: This project was supported by the Collaborative Research Fund C4017-14G and General Research Fund 14102914.

References:

1. Feng H., et al. (2011). J Clin Invest 121, 3159-3175.

2. Yu Z., et al. (2014). Gut 63, 1793-1804.

3. Feng H., et al. (2015). J Hepatol 62, 1100-1111.

#3753

Improved oncolytic virotherapy by increasing adenovirus spread.

Stephen L. Wechman, Xiao-Mei Rao, Kelly Marc McMasters, Heshan Sam Zhou. _University of Louisville, Louisville, KY_.

Lung cancer is the leading cause of cancer-related death worldwide and kills more patients than the next four cancers combined (Colon, breast, prostate, and pancreas). Currently available lung cancer treatments have not been effective. Therefore the development of novel lung cancer therapeutics is of critical importance. One emerging treatment option is the use of E1b-deleted adenoviruses (Ads) which preferentially lyse cancer cells. Although E1b-deleted Ads have been applied in clinical trials, their efficacy remains low. We have developed a novel oncolytic vector, AdUV, via the bioselection of the cancer selective E1b-deleted Ad Adhz60. We have demonstrated that AdUV is much more potent against multiple cancer cell lines than the parental vector Adhz60 in vitro and suppressed tumor growth in vivo.

We compared AdUV treated cells to cells treated with the wildtype Ad5 and the E1b-deleted Adhz60. AdUV lysed A549 lung cancer cells more efficiently than Adhz60, but neither AdUV nor Adhz60 killed MRC5 and HBEC normal lung cells. Possible mechanisms of action were then investigated to determine how AdUV lysed A549 cells more effectively. AdUV virions were shown to release from infected A549 cells and induce autophagy more effectively than both Adhz60 and Ad5. Autophagy induction is known to support Ad replication and spread through cancer cell monolayers. The plaque assay was used to verify if AdUV could spread more effectively than Ad5 and Adhz60 because viruses which efficiently replicate and release from cells are known to form larger plaques. The average size of plaques formed by AdUV is 340% and 214% larger than those plaques formed by Adhz60 and Ad5, respectively. In vivo, AdUV was non-lethal to athymic nude mice and strongly suppressed A549-xenograft tumor growth, prolonging murine survival. These results indicate that AdUV is a more effective, safe oncolytic Ad vector. Our findings also demonstrate the utility of bioselection of the generation of more effective cancer therapeutic vectors.

### Novel Antitumor DNA-Reactive Agents

#3754

Pre-clinical studies of a highly potent folate receptor targeted DNA crosslinking agent.

Joseph A. Reddy, Melissa Nelson, Theresa Johnson, Christina Dirksen, Marilynn Vetzel, Spencer Hahn, LongWu Qi, Iontcho Vlahov, Christopher Leamon. _Endocyte, Inc., West Lafayette, IN_.

Folate receptor (FR) targeted small molecule drug conjugates (SMDC) have shown promising results in early stage clinical trials with Vintafolide and EC1456. In our effort to develop FR targeted SMDCs with varying mechanisms of action, we have now built a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100 x lower than any other folate SMDC we have created to date. Treatment of nude mice bearing FR positive human xenografts led to cures in 100% of the mice at very low doses (> 500 nmol/kg) and a convenient once a week schedule. The observed activity was not accompanied by any noticeable weight loss (up to 20 weeks post end of dosing) or major organ tissue degeneration. In contrast, no significant anti-tumor activity (0% CR's) was observed in treated animals that were co-dosed with an excess of a benign folate ligand, thus demonstrating its target-specific activity. Complete responses were also observed in other FR positive drug resistant (paclitaxel and cisplatin) models. Taken together, these studies demonstrated that this SMDC with a distinct DNA reacting mechanism has significant anti-tumor growth activity and tolerability, thus lending support to future clinical development of this novel FR-targeted agent.

#3755

Genome-wide transcriptome analysis reveals the distinct gene expression profiles and specific biological effects of fluorescent polyamides, HxIP and azaHxPI.

Luke Pett,1 Vijay Satam,2 Pravin Patil,2 Moses Lee,2 John A. Hartley,1 Konstantinos Kiakos1. 1 _UCL Cancer Institute, London, United Kingdom;_ 2 _Hope College, Holland, MI_.

DNA-binding polyamides target predetermined sequences to inhibit specific transcription factor-DNA interactions and modulate gene expression. Inherently non-genotoxic, these non-covalent binding small molecules exert their biological activity without inflicting DNA damage and provide the basis for potentially less toxic DNA-targeting anticancer therapeutics. However, the genome-wide specificity of polyamide binding remains an outstanding issue in delivering the desired biological response without inducing widespread off-target effects.

In this study, we used RNA-seq transcriptome analysis to assess the global effects of fluorescent polyamides HxIP and azaHxPI, having previously shown that they bind with high affinity and selectivity to their respective target DNA sequences 5'-WWCGWW-3' and 5'-WCGCGW-3' (W=A/T)1,2. Changes to the MDA-MB-231 breast adenocarcinoma transcriptome were measured following 24 h treatment with 2 and 5 μM HxIP or azaHxPI. At 5 μM, HxIP affected 349 genes by two-fold (p<0.05), corresponding to 1.72% of the Ensembl coding gene assembly, of which 171 genes were downregulated and 178 were upregulated. AzaHxPI caused the differential expression of 496 genes (2.44%), with 176 of these genes downregulated and 320 genes upregulated. Both HxIP and azaHxPI affected only a small number of the total gene set without significantly perturbing the rest of the transcriptome, demonstrating the specific nature of polyamide induced biological effects. Only 26 genes were affected by both polyamides (3.1% overlap), suggesting that HxIP and azaHxPI stimulate unique transcriptome profiles as a result of targeting different DNA sequences.

Following a 96 h treatment HxIP had no effect on cell growth (GI50 > 100 μM), whereas azaHxPI causes growth inhibition (GI50 =20.3 μM) and induces apoptosis. Immunoblotting analysis of 𝛾;H2AX showed no evidence of DNA damage, further corroborated by the absence of chk2 phosphorylation. In addition, no ATR-chk1 activation was detected suggesting that azaHxPI does not induce replication stress, despite inhibiting DNA synthesis as shown using the BrdU incorporation assay. Levels of phospho-AKT (Ser472) decreased in response to polyamide treatment, whilst p21 is significantly upregulated and both may be implicated in the mechanism of azaHxPI induced-apoptosis. Taken together these results emphasise the potential application of polyamides as apoptosis-inducing DNA-targeting agents benefiting from enhanced sequence selectivity which in turn confers specific biological activity.

References:

1. Kiakos. et al Chem. Biol. (2015) 22(7):862-75.

2. Satam. et al Bioorg. Med. Chem. Lett. (2015) 25(17):3681-5.

#3756

PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency.

Benjamin H. Lok,1 Ying Feng,2 G. Karen Yu,2 Eric E. Gardner,1 Nadeem Riaz,1 Yuanbin (Kevin) Ru,2 Elisa de Stanchina,1 Simon N. Powell,1 Jerry Shen,2 John T. Poirier,1 Charles M. Rudin1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _BioMarin Pharmaceutical Inc., Novato, CA_.

Poly-(ADP)-ribose polymerase (PARP) was identified as a potential novel therapeutic target for small cell lung cancer (SCLC) in two key proteomic studies. These findings led to the inclusion of a SCLC cohort in a Phase I study of talazoparib (BMN 673) - currently the most potent PARP inhibitor available - with promising efficacy signals. Identifying predictors of response to PARP inhibition would bring a needed therapeutic advance in this recalcitrant disease.

PARP inhibition was first demonstrated to have marked efficacy in BRCA1/2 mutant cancers that are deficient in homologous recombination (HR). Large chromosomal structural alterations characteristic of these tumors can be quantitated by three correlated HR deficiency (HRD) metrics. We sought to determine if HRD would be a relevant predictive biomarker in SCLC.

Thirty-six SCLC cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) were interrogated for sensitivity to talazoparib, olaparib and cisplatin. We found significant correlation between these three pharmacologic agents and sought to determine response predictors. Three different HRD metrics were computed in these 36 SCLC cell lines using publically available CCLE Affymetrix SNP 6.0 array datasets. Surprisingly, none of these three measures of HRD were discriminatory for response to talazoparib in vitro. However, we found that after controlling for HRD and PARP1 target expression, expression of a single gene, SLFN11, was significantly correlated with talazoparib sensitivity as measured by the CCLE Affymetrix U133+2 arrays (q value = 0.008). After deriving SLFN11 knock-out SCLC cell lines by CRISPR/Cas9 gene editing, we found that loss of SLFN11 conferred resistance to talazoparib (IC50 >log difference) as compared to parental SLFN11 proficient cells.

We then treated several SCLC patient-derived xenograft (PDX) models with talazoparib daily and found that SLFN11 expression in vivo was necessary but not sufficient for response. None of the SLFN11 low expressing PDXs responded to therapy, whereas 3/5 (60%) of SLFN11 high expressing PDXs demonstrated significant tumor growth inhibition of 54-75% after 21-28 days of daily treatment with talazoparib 0.3mg/kg.

In conclusion, we demonstrate that SLFN11 expression is correlated with response to PARP inhibition in SCLC in vitro and in vivo. HRD structural aberration scores do not predict for in vitro PARP inhibitor sensitivity, however functional HR interrogation (such as RAD51 foci recruitment) may still be a relevant discriminator in addition to ongoing in vivo analysis. SLFN11 may be a pertinent predictive biomarker in SCLC for therapeutic response to PARP inhibition with considerable clinical implications.

#3757

A novel class of chemicals that react with damaged DNA and specifically kill B-cell cancers.

Shanqiao Wei, Madusha Watuthanthrige, Sophia Shalhout, Ashok S. Bhagwat. _Wayne State University, Detroit, MI_.

Majority of B-cell malignancies arise in germinal center, where the processes of somatic hypermuation (SHM) and class switch recombination (CSR) for antibody maturation are initiated by activation-induced demainase (AID). AID belongs to the AID/APOBEC family and deaminates cytosine in DNA creating uracil. We have previously shown that B-cell lymphoma cell lines and patient tumors express AID at high levels and contain high levels of uracils in their genome. These cells also contain uracil-DNA glycosylase that removes uracils generating high levels of abasic (AP) sites. AP sites exist in equilibrium with the open form of deoxyribose sugar that reacts with alkoxyamines forming a stable oxime. This principle was used to block the repair of excess AP sites in B cell cancers using an alkoxyamine, AA3. We found that AA3 effectively kills B-cell lymphoma cell lines which have high levels of endogenous AP sites. In contrast, AA3 is not toxic to normal human B cells and mouse splenocytes, as well as non-hematological cancers. To elucidate the molecular mechanism of selective cell killing by AA3, we compared the number of AP sites in B cell cancers and non-hematological cancers and confirmed that B cell cancer cells contained many more AP sites in their genome. Additionally, we found that AA3 was covalently linked to B cell cancer genome and AA3-DNA adducts caused cell death by blocking DNA replication and elevating DNA strand breaks. To determine which functionality of AA3 is responsible for its cytotoxicity and to discover better candidates for killing B cell cancers, we designed and synthesized a series of AA3 analogues. Using this approach, we have defined the functional groups required for AA3 toxicity. Overall, we have discovered that AA3 is selectively toxic to B cell cancers. This new family of chemicals could be developed further as novel and unique anti-cancer drugs.

#3758

Quantitative high-throughput screening as a tool to identify novel therapies in bladder cancer: lessons from flavopiridol.

Reema Railkar,1 Achuth Nair,1 Keidren Lewi,1 Spencer Krane,1 Rajarshi Guha,2 Marc Ferrer,2 Craig Thomas,2 Piyush K. Agarwal1. 1 _NCI/NIH, Bethesda, MD;_ 2 _NCATS/NIH, Shady Grove, MD_.

Introduction and Objectives: Bladder cancer (CaB) is the 4th most common cancer among men and 12th most common among women in United States. It is one of the most expensive malignancies to treat from diagnosis to death. No new pharmacological agents have been approved for the treatment of bladder cancer in the last two decades. Therefore, there is an urgent need for development of new treatment therapies. Quantitative high throughput screening (qHTS) of representative cancer cell lines with oncology drugs may identify new treatments and pathways. We utilized this technique to identify new targets and therapies in two primary bladder lines (T24 and UMUC3) and their metastatic derivatives (T24T, SLT3 and FL3 of T24 and LUL-2 for UMUC3).

Methods: We screened 7 bladder cancer cell lines (T24T, SLT3, FL3, LUL-2, RT4, T24, and UMUC3) against 1,912 oncology drugs using a 48 hour cell proliferation assay with an ATP−based readout (CellTiterGlo) to determine activity and potency of compounds in a dose response manner. One of the candidate drugs inhibitory in all cell lines tested is flavopiridol, a pan-CDK inhibitor. We further characterized the mechanism of action of flavopiridol using various cell based assays such as cell proliferation, cell cycle analysis, apoptosis assays, and modified colony forming assays. Finally, mouse xenograft studies were carried out to elucidate the in vivo effects of flavopiridol.

Results: The initial screen identified 95 compounds active in 7 cell lines. The top 50 compounds were further analyzed for molecular size of >200 g/mol and TPSA<90. These parameters are consistent with lipophilicity and may help identify compounds that can be used as intravesical agents. The selection parameters identified mitomycin C and 8 novel compounds. Cell proliferation assays of these 8 novel compounds in cell lines not part of the initial screen revealed that flavopiridol most consistently achieved IC50s of 100-150 nM, consistent with the qHTS data. Flavopiridol induces G2/M arrest; however, it induces little apoptosis as per the Annexin V FITC-PI assay. Growth in Low Attachment (GILA) assay showed inhibition of colony formation in the presence of flavopiridol. Xenograft studies using the UMUC-3 cell line implanted subcutaneously demonstrated cytostatic inhibition of tumor growth in the presence of systemically delivered flavopiridol. However, intravesical administration has not yet been evaluated.

Conclusions: qHTS can identify novel compounds. Flavopiridol is an effective inhibitor both in vitro and in vivo. Although it merely caused cytostatic inhibition in a xenograft model when delivered systemically, its physical properties are most suited for intravesical use which may lead to it being more effective as higher doses can be administered into the bladder with minimal/no systemic toxicities. Studies are now underway to evaluate the use of flavopiridol as an intravesical agent.

#3759

Inhibition of heat shock protein 27 (Hsp27) expression by apatorsen as a therapeutic strategy in breast cancer.

Emilia Komulainen,1 Alice Shia,2 Jasmin Bansal,2 Francessa Cavicchioli,1 Karen O'Leary,1 John Foster,2 Peter Schmid2. 1 _Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom;_ 2 _Barts Cancer Institute, Queen Mary University of London, London, United Kingdom_.

Background: Heat shock protein 27 (Hsp27) is over-expressed in a number of cancers, including breast cancer, and interacts with apoptotic proteins inhibiting the apoptotic process and promoting tumourgenesis. Clinically, Hsp27 expression has been associated with a more aggressive phenotype and resistance to chemotherapy. Apatorsen (OGX-427) is a therapeutic antisense oligonucleotide designed to specifically target Hsp27. Apatorsen has shown single agent clinical activity, and randomised trials in combination with chemotherapy are underway. Here we present pre-clinical validation of inhibition of Hsp27 expression by apatorsen as a novel therapeutic strategy in breast cancer.

Results: Basal expression analysis of Hsp27 by qPCR and western blot in a panel of >20 breast cancer cell lines demonstrated a wide variation with up to 3-log difference in expression between cell lines and good correlation of RNA and protein expression levels. Hsp27 expression increased after treatment with chemotherapy agents. Treatment with apatorsen caused rapid reduction of cellular Hsp27 levels within 24-48 hours; effects on Hsp27 levels were sustained for 5 and 7 days. Knockdown of Hsp27 was associated with significant reduction of cell survival with 38-93% loss of cell viability across the cell line panel (compared to mismatched oligonucleotide). There was no association between response to apatorsen and basal Hsp27 expression or molecular breast cancer subtype. Using whole genome mRNA (Illumina Human HT-12 v4 Expression BeadChip Kit) and methylation analysis (Illumina Human 450K Methylation BeadChip), we were unable to define a predictive signature of response to apatorsen. Co-treatment of breast cancer cell lines with apatorsen and Doxorubicin, Paclitaxel and Vinorelbine chemotherapy showed an additive cytotoxic effect. In vivo validation of the effects of apatorsen is ongoing.

Conclusions: Therapeutic silencing of Hsp27 expression in breast cancer cell lines with apatorsen presents a novel strategy for the treatment of disease. Here we present data to support the use of apatorsen in combination with conventional chemotherapy for the treatment of breast cancer.

#3760

XCE853 is a promising protein disulfide isomerase (PDI) inhibitor exhibiting a strong inhibitory activity in preclinical tumor models.

Gregoire P. Prevost,1 Marine Garrido,2 Maria Serova,3 Jean François Briand,4 Mathieu Gutmann,5 Patrick Ladam,6 Denis Carniato,7 Annemilai Tijeras-Raballand,3 Armand De Grammont,3 Anne Chauchereau,2 Marc-Henry Pitty,1 Paul Foster8. 1 _CIPREVO, Antony, France;_ 2 _Gustave Roussy, Villejuif, France;_ 3 _AAREC Filia Research, Paris, France;_ 4 _CIP, Antony, France;_ 5 _Paris Sud, Antony, France;_ 6 _Université Paris XIII, Bobigny, France;_ 7 _DC, Marcoussis, France;_ 8 _Edgbaston, University of Birmingham, Birmingham, United Kingdom_.

Protein disulfide isomerase (PDI) is a chaperone protein that regulates oxidative protein folding as well as cell viability. Increased PDI levels have been documented in a variety of human cancers, including ovarian, prostate, and lung cancers. Inhibition of PDI activity leads to apoptosis in cancer, suggesting that PDI is a promising druggable target.

XCE853 is a synthetic small molecule displaying an excellent docking with the human PDI. XCE853 inhibits in vitro recombinant PDI activity. In addition, the proliferation of a large panel of human tumor cells is blocked by XCE853 with IC50s in the nanomolar range through an irreversible cytolysis.

XCE853 is also active on a large panel of drug resistant human cancer cells. XCE853 induced an irreversible cytolysis of human tumor cells after a short in vitro exposure independently of efflux pumps leading to a tumor cell death by autophagy and particles release (vesicles or protein aggregates).

In addition, the ex-vivo approach using fresh human tumor explants cultivated in 3 dimensions with low concentrations of XCE853 has shown a strong decrease of the proliferation (KI-67 labeling) in several tumor types.

Finally, XCE853 displayed excellent oral bioavailability in mice and was able to block the growth of several human cancers using in vivo xenograft models leading to a complete tumor growth arrest even after the cessation of the treatment.

Altogether, these data support further efforts on this drug candidate to initiate the preclinical studies and to define the most relevant human tumor types.

#3761

The MDM2 inhibitor AMG 232 causes tumor regression and potentiates the anti-tumor activity of MEK inhibition and DNA-damaging cytotoxic agents in preclinical models of acute myeloid leukemia.

Jude R. Canon,1 Tao Osgood,1 Anne Y. Saiki,1 Jonathan D. Oliner2. 1 _Amgen, Inc., Thousand Oaks, CA;_ 2 _Jon Oliner Consulting, Garrett Park, MD_.

AMG 232 is a potent inhibitor of the MDM2-p53 interaction and is a promising clinical candidate for treating tumors, in particular those harboring wild-type p53. AML represents a compelling indication for AMG 232 given the low rate of p53 mutation, frequent MDM2 overexpression, and unmet medical need. We evaluated the effect of AMG 232 treatment on AML tumors in vitro and in vivo, elucidated the mechanism of anti-tumor efficacy, and tested the effect of combining AMG 232 with targeted agents and chemotherapeutics. Combinations were identified based on evidence of in vitro synergy from cell based screens, or based on biological rationale and clinical opportunity with standard of care agents. Combinations evaluated included MEK inhibitors, and p53-inducing, DNA-damaging cytotoxics cytarabine, doxorubicin, and decitabine. In vitro assays demonstrated that AMG 232 as a single agent was effective at inducing cell death across a panel of p53 wild-type AML cell lines. The anti-tumor efficacy involved activation of the p53 pathway, robust inhibition of the cell cycle and induction of apoptosis. AMG 232 treatment caused AML tumor regression in vivo which was related to dose- and time-dependent induction of the p53 targets p21 and PUMA. The combination of AMG 232 and MEK inhibition resulted in synergistic tumor cell killing in vitro, and enhanced in vivo anti-tumor activity which was significantly better than either single agent. Combinations of AMG 232 with chemotherapies which induce DNA damage resulted in synergistic in vitro cell killing, and superior anti-tumor efficacy in vivo with increased induction of p53 signaling in tumors. These data support a clinical strategy for evaluating AMG 232 as a monotherapy and in combination with targeted and cytotoxic agents to treat AML patients.

#3763

Cucurbitacin B targets EGFR-mutant lung cancer cells via activation of sestrin-3.

Naghma Khan, Farah Jajeh, Mohammad Imran Khan, Eiman Mukhtar, Hasan Mukhtar. _University of Wisconsin-Madison, Madison, WI_.

The standard therapy for advanced non-small cell lung cancer (NSCLC) is based on the presence of epidermal growth factor receptor (EGFR) mutations with a clinical response to the EGFR tyrosine kinase inhibitors. However, despite the initial efficacy of the treatments, almost all patients acquire drug resistance and develop relapse after variable periods of time. One limiting factor for the use of natural and dietary agents is that they exert their effect at high concentrations which are not physiologically attainable. Cucurbitacins are highly oxidized tetracyclic triterpenoids which are arbitrarily divided into twelve categories and are widely distributed in the plant kingdom. Cucurbitacin B is one of the most common and has the highest content in many plants. We found striking effect of cucurbitacin B (0.2-0.6 μM; 24h) on EGFR-mutant lung cancer cells. There was remarkable decrease in the viability of H1975 and H1650 EGFR-mutant lung cancer cells. However, only modest effect of cucurbitacin B occurred on the viability of normal human bronchial epithelial cells. Treatment with cucurbitacin B also resulted in the inhibition of EGFR-mutant H1650 lung cancer cells colonies in a dose-dependent manner with almost complete inhibition at two weeks of treatment. Besides other signaling pathways, over-expression of phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR) is frequently observed in tumors with EGFR mutations. Therefore, inhibition of the PI3K/AKT/mTOR signaling represents a favorable approach for the treatment of lung cancer patients with EGFR mutations. Treatment with cucurbitacin B caused inhibition of PI3K/Akt/mTOR and STAT-3 signaling alongwith simultaneous activation of AMPKα levels in EGFR-mutant lung cancer cells. Sestrins are highly conserved gene family found in all multicellular organisms of the animal kingdom. It is known that there is one sestrin gene in invertebrates and three genes in vertebrates (sestrins 1, 2 and 3). The inhibition of mTOR by sestrins has an impact on many mTOR-reliant processes such as translation, cell growth and proliferation. We found that treatment with cucurbitacin B caused specific increase in the protein and mRNA expression of sestrin-3 in EGFR-mutant lung cancer cells. Mechanistically, cucurbitacin B targeted mTOR via activating cell intrinsic inhibitor sestrin-3. We found that cucurbitacin B specifically activates sestrin-3, which may have resulted in dramatic mTOR inhibition. These results were further confirmed by using sestrin-3 knockdown in cucurbitacin B-treated cells. Sestrin-3 knockdown significantly reduced the effect of cucurbitacin B on cell-viability in EGFR-mutant lung cancer cells. These findings suggest novel mechanism for the action of cucurbitacin B and suggest that it could be explored further as potential therapy for EGFR-mutant lung cancer.

#3764

Trabectedin activity in patient-derived mesothelioma xenografts.

Simonetta Andrea Licandro,1 Roberta Frapolli,1 Ezia Bello,1 Roberta Libener,2 Sara Orecchia,2 Federica Grosso,2 Maurizio D'Incalci1. 1 _IRCCS -Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy;_ 2 _ASO SS Antonio e Biagio e C Arrigo, Alessandria, Italy_.

Malignant pleural mesothelioma (MPM) incidence is increased over the past two decades and it is predicted to increase further in the next 20 years especially in developing countries where asbestos has not yet been banned. Surgery is not an option for the majority of patients due to the diffuse growth of MPM, making chemotherapy the treatment of choice, but prognosis remains very poor with a median survival time of 10-17 months. Trabectedin (ET-743, Yondelis) is a marine alkaloid used for the 2nd line therapy of ovarian cancer (combined with pegylated doxorubicin) and soft tissue sarcomas. It binds the DNA minor groove modulating transcription of cancer cells and has an anti-inflammatory effect modifying tumor microenvironment and reducing tumor associated macrophages.

The aim was to test antitumor activity of trabectedin in newly established patient derived xenografts of MPM.

MPM473, MPM484 and MPM487 xenografts were obtained in athymic nude mice by s.c. injection of 107 cells isolated from the pleural effusion of MPM patients and maintained by serial passages in mice. H&E staining and immunohistochemistry were performed. Mice were randomized when tumor reached about 200 mg to receive trabectedin, cisplatin, pemetrexed, gemcitabine, and imatinib. Antitumor activity was expressed as T/C% were T and C are the mean tumor weight of treated and control mice, respectively.

The three xenografts reproduced the morphology and the histochemical profile of the human tumors. As reported in the table trabectedin was able to induce a 50% reduction in tumor growth in MPM473 and MPM 487 xenografts and only a marginal effect in MPM484 xenograft.  | |  | |

|

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

Treatment | Dose (mg/kg) | Schedule | Best T/C% (Day)

|  | |

MPM473 | MPM484 | MPM487

Cisplatin | 5 | i.v. q7dx3 | 61.9 (69) | 66.1 (69) | 67.3 (39)

Gemcitabine | 100 | i.p. q4dx5 | 83.2 (41) | 67.7 (48) | 49.7 (56)

Imatinib | 200 | p.o. qdx15 | 68.9 (76) | 89.4 (55) | 87.1 (39)

Pemetrexed | 250 | i.p. q4dx4 | 77.4 (52) | 76.8 (49) | 84.3 (39)

Trabectedin | 0.15 | i.v. q7dx3 | 51.0 (73) | 63.6 (69) | 51.3 (56)

In conclusion we report three new MPM xenografts able to mimic the pathological features of human MPM. Although trabectedin showed only a moderate antitumor activity in our experimental models, its efficacy is comparable or better than that of the other drugs currently used for this disease. Studies are in progress in syngenic models that are more appropriate to test also the ability of trabectedin to modulate the immune system.

#3765

Induction of amineoxidases as a possible mechanism of action of tamoxifen metabolites.

T. J. Thomas,1 Tuomo A. Keinanen,2 Mervi T. Hyvonen2. 1 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 2 _University of Eastern Finland, Kuopio, Finland_.

To elucidate the mechanism of action of endoxifen in breast cancer, we determined its effects on estradiol-mediated growth of ERalpha-positive MCF-7 cells. Endoxifen is the active metabolite of tamoxifen; the most widely used selective estrogen receptor modulator for anti-estrogen therapy for women with estrogen receptor alpha-positive breast cancer. Clinical trials recognized that tamoxifen was a prodrug and got converted to active metabolites in the human body. Earlier studies recognized 4-hydroxytamoxifen (4HT) as the active metabolite of tamoxifen, although recent studies showed that the concentration of endoxifen was up to 10-fold higher than that of 4HT in patients treated with tamoxifen. These metabolites are formed by the action of cytochrome P450 2D6 (CYP2D6). We used quantitative PCR analysis to examine the effects of endoxifen on estrogenic stimulation of early response genes, c-myc, c-fos and Tff1. Since the natural polyamines, putrescine, spermidine and spermine, play important roles in estrogenic function, we also measured the activities of enzymes in the polyamine biosynthetic/metabolic pathway: ornithine decarboxylase (ODC), S-adenosyl-L-methionine decarboxylase (AdoMetDC), spermidine/spermine N1-acetyltransferase (SSAT), spermine oxidase (SMO), and acetylpolyamine oxidase (APAO). Polyamine levels were quantified by high performance liquid chromatography. Our results showed that estradiol increased the proliferation of MCF-7 cells by 2- to 3-fold and endoxifen suppressed the effects of estradiol on cell growth. The expression of c-myc, c-fos and Tff1 genes was significantly increased by estradiol, while the addition of endoxifen suppressed estradiol-mediated increase in gene expression. Estradiol increased the activity of ODC and AdoMetDC, whereas endoxifen suppressed the activity of these enzymes. Endoxifen had no effect on SSAT activity, whereas it significantly increased activities of amineoxidases, SMO and APAO. Estradiol increased putrescine and spermidine levels and endoxifen inhibited this increase. There was no significant effect on spermine levels. These data suggest that endoxifen exerts its growth inhibitory effects on MCF-7 cells by suppressing the expression of estradiol-stimulated oncogenes and polyamine biosynthetic pathway. The antiestrogenic effect of endoxifen is also manifested through the stimulation of polyamine oxidase enzymes, SMO and APAO. These results indicate for the first time that the upregulation of SMO and APAO is important in the mechanism of action of endoxifen in breast cancer cells.

#3766

Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs.

Megan Turnidge,1 Justin J. Montoya,2 Apurvi Patel,2 David W. Lee,2 Eiman Aleem,2 Daniel H. Wai,2 Laurence H. Hurley,3 Vijay Gokhale,3 Robert J. Arceci,2 David O. Azorsa2. 1 _Arizona State University, Tempe, AZ;_ 2 _Phoenix Children's Hospital, Phoenix, AZ;_ 3 _University of Arizona, Tucson, AZ_.

Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are 'undruggable' because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 µM for GQC-05 and from 40 nM to 2 µM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures.

#3767

Highly effective inhibitory agent identified by high-throughput screening of genetically characterized anaplastic thyroid cancer cell lines.

Nicole C. Pinto,1 Morgan Black,1 Kara Ruicci,1 John Yoo,2 Danielle MacNeil,2 Kevin Fung,2 Alessandro Datti,3 Stephenie Prokopec,4 Paul Boutros,4 John Barrett,2 Anthony Nichols2. 1 _Western University, London, Ontario, Canada;_ 2 _London Health Sciences Centre, London, Ontario, Canada;_ 3 _The Lunenfeld-Tanenbaum Research Institute, Toronto, Ontario, Canada;_ 4 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada_.

Introduction

While most types of thyroid cancer are treatable, anaplastic thyroid cancer (ATC) is the most lethal human malignancy. Current treatment strategies are largely ineffective with only 10% of patients surviving longer than one year. As such, new treatments are desperately needed to improve patient survival in this aggressive cancer.

Objective

Identify highly effective drugs using high-throughput screening methods with a large panel of kinase inhibitors that can be used in the treatment of ATC.

Methods

High-throughput screening (HTS) of 13 genetically characterized ATC cell lines was carried out over a 6-dose range using 320 kinase inhibitors. Dose-response curves were generated and drugs with high potency were identified. Drugs with the highest activity were selected for further evaluation including investigating mechanism of action and cell death, and assays for migration and invasion.

Results

Based on HTS studies, Lestaurtinib was identified as a highly effective agent with submicromolar mean inhibitory concentrations in all 13 ATC cell lines. Lestaurtinib was found to inhibit STAT5 phosphorylation, as well as cell line migration and invasion. The anti-proliferative effect of Lestaurtinib did not cause apoptosis, autophagy or cell senescence.

Conclusions

With the use of HTS, we have identified many (>20) kinase inhibitors as highly effective agents in the control of ATC. The kinase inhibitor Lestaurtinib was effective at controlling ATC cell proliferation, invasion and migration. Future in vivo analysis of Lestaurtinib with patient-derived xenografts are planned to continue preclinical development of this drug with the ultimate goal of improving outcomes for patients suffering with ATC.

#3768

Targeting of epithelial-to-mesenchyme transition by a novel small molecule inhibitor attenuates prostate and breast cancer invasiveness and bone metastases.

Jan Kroon,1 Onno van Hooij,2 Eugenio Zoni,1 Maaike van der Mark,1 Geertje van der Horst,1 Johan Tijhuis,3 Cindy van Rijt-van de Westerlo,2 Gerald Verhaegh,2 Henk Viëtor,4 Antoine Wellink,4 Peter Maas,3 Jack Schalken,2 Gabri van der Pluijm1. 1 _Leiden University Medical Center, Leiden, Netherlands;_ 2 _Radboud University Medical Center, Nijmegen, Netherlands;_ 3 _SPECS, Zoetermeer, Netherlands;_ 4 _Oncodrone B.V., Nijmegen, Netherlands_.

Transformed epithelial cells can activate embryonic programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype also referred to as epithelial-to-mesenchymal transition (EMT). EMT is associated with poor prognosis in patients with osteotropic cancers. E-cadherin (CDH1) is an essential homotypic cell adhesion molecule that is often down regulated during this process.

EMT-like processes are increasingly linked to therapy resistance and metastasis-initiating cells, thus providing the rationale for the development of novel small-molecule inhibitors that a) block the acquisition of an invasive phenotype in osteotropic cancer cells via EMT or b) revert their invasive, mesenchymal phenotype into epithelial phenotype (MET) by upregulation of CDH1 expression.

High throughput screening of >43,000 LMW compounds, followed by compound design & optimization in vitro led to the identification ten candidate therapeutic compounds. These compounds displayed significant inhibitory effects on cancer cell invasion (>80%) and induced E-cadherin (re)expression, most likely through interference with the binding of transcriptional repressors to the CDH1 E-box elements. We identified a unique compound, OCD155, can effectively and dose-dependently block the acquisition of an invasive phenotype in osteotropic prostate and breast cancer cells (PC-3M-Pro4luc2 and MDA-MB-231/Bluc). When tested in our in vivo models of prostate and breast cancer bone metastasis, treatment of mice with OCD155 strongly and dose-dependently inhibited skeletal metastasis (number of metastases, tumor burden) according to preventive and curative protocols. At the dosages tested, no adverse effects of OCD155 were observed (body weight, liver toxicity parameters). To the best of our knowledge, our studies are the first to demonstrate the efficacy of new small molecule EMT inhibitor in the treatment of experimental skeletal metastasis.

#3769

Continuous, low intensity systemic dosing maximizes the therapeutic margin of Eg5/KSP inhibition in an orthotopic model of urothelial cell carcinoma.

Leigh Williams, Rebecca Ellston, Dawn Trueman, Helen Musgrove, Linette Ruston, Brian Aquila, Elizabeth Pease, Stephanie Klein, Barry R. Davies. _AstraZeneca R &D, Macclesfield, United Kingdom_.

Urothelial Cell Carcinoma (UCC) is the fifth most common cancer, but no new generation molecularly targeted therapies have been registered to treat this disease. Non muscle-invasive bladder cancer can be treated by systemic or intravesical dosing routes. However, the urothelium is one of the most formidable permeability barriers in nature, and may limit the penetration of small molecules into tumour tissue from the intravesical location. Inhibitors of mitosis, including spindle proteins such as Eg5, are attractive targets for cancer therapy, but their efficacy when dosed systemically is severely limited by bone marrow toxicities such as neutropenia and thrombocytopenia. AZ9814, a very potent and selective small molecule inhibitor of Eg5, induces apoptosis and inhibits proliferation of UCC cells in culture with a GI50 of < 1 nM. We have developed an orthotopic model of bladder cancer in the nude rat coupled with a programmable mini-pump system to continuously infuse AZ9814 into the bladder, and compared the therapeutic index of this delivery route with systemic dosing schedules using intraperitoneal injection and continuous subcutaneous infusion using an osmotic mini-pump. Continuous intravesical infusion resulted in severe bladder toxicity including hydropic degeneration and ulceration of urothelium, mucosal erosion and bilateral hydronephrosis, with limited penetration of compound into the tumour, and no significant impact on tumour pharmacodynamics and growth. In contrast, continuous low intensity systemic dosing resulted in significant tumour pharamacodynamic activity and control of tumour growth, albeit with significant but non-lethal effects on bone marrow toxicity. Daily intraperitoneal injection of AZ9814 resulted in very modest anti-tumour effects which were limited by bone marrow toxicity. It is concluded that Eg5 inhibitors have potential to treat UCC, but continuous systemic infusion is the best dosing schedule to maximize therapeutic index.

#3770

HEXIM1 as a pharmacodynamic marker for monitoring target engagement of ABBV-075.

Xiaoli Huang, Xiaoyu Lin, Leiming Li, Rongqi Wang, Lisa Roberts, Paul Hessler, Tamar Uziel, Lloyd Lam, Terry Magoc, Daniel H. Albert, Steven W. Elmore, Guowei Fang, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. _AbbVie Inc, North Chicago, IL_.

Bromodomain and extra-terminal (BET) family are dual bromodomain-containing proteins that play an important role in transcription regulation. ABBV-075 is a small molecule BET bromodomain inhibitor currently in phase 1 clinical trials. Here we report the identification of Hexim1 and other BET-responsive genes as robust pharmacodynamic markers for monitoring ABBV-075 target engagement in tumors and in surrogate tissues such as whole blood and skin. Transcription profiling of cancer cell lines for their responses to BET inhibitors identified a set of 9 BET-regulated genes, including Hexim1, that responded to BET inhibition across cancer cell lines and in xenograft tumors. Further characterization of BET-responsive genes in surrogate tissues such as mouse skin, mouse/human PBMCs and whole blood revealed that the mRNA level of Hexim1 exhibited the best response to BETi treatment across different settings. A dose-dependent increase of Hexim1 expression was detected in the whole blood of ABBV-075 treated tumor bearing mice, and the Hexim1 response was closely correlated with the plasma drug concentration and largely reflected the anti-tumor efficacy at various dose levels of ABBV-075. Hexim1 is part of a protein/RNA complex that sequesters pTEFb and prevents its recruitment by BRD4. It also reportedly mediates the anti-proliferative activity of BRD4 in AML. Taken together, Hexim1 could serve as a functional relevant pharmacodynamic marker for monitoring ABBV-075 target engagement in animal models and in the clinical setting.

#3771

Discovery of a covalent inhibitor of ERK docking-interactions that inhibits A375 melanoma cells proliferation.

Tamer S. Kaoud,1 William H. Johnson,1 Nancy D. Ebelt,1 Andrea Piserchio,2 Mangalika Warthaka,1 Micael Cano,1 Rachel Sammons,1 Qiantao Wang,1 Pengyu Ren,1 Ranajeet Ghose,2 Kevin N. Dalby1. 1 _The University of Texas at Austin, Austin, TX;_ 2 _The City College of New York, New York, NY_.

Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Several ERK inhibitors have already entered clinical trials.

Most of the available ERK inhibitors are reversible inhibitors that either act through an allosteric mechanism, or by targeting the ATP binding site. Taking advantage of our understanding of ERK-docking interactions we tried to discover an irreversible substrate-selective inhibitor that targets the protein-binding site of ERK. Here, we report the discovery of a covalent inhibitor of ERK that targets its protein-docking site. Protein NMR, Mass spectroscopy, mutagenesis and molecular docking studies indicate a covalent interaction of the inhibitor with a conserved cysteine residue, Cys-159. Extensive biochemical studies provide an estimate of its kinetic parameters and its kinase-selectivity profile. The new ERK inhibitor inhibits ERK activation, as well as its ability to phosphorylate downstream substrates (e.g. p90RSK and Elk-1) in HEK293T and A375 melanoma cells. The targeting of ERK in HEK293T cells was confirmed using a chemical-genetic approach where the ERK2 C159A mutant was used to rescue the effects of this compound on ERK2 signaling and cell proliferation. Currently, we are testing the effect of the compound on tumor growth inhibition in an A375 melanoma cancer xenografts model. This covalent inhibitor represents a potentially valuable lead molecule whose development may result in a novel class of pharmacologically useful ERK inhibitors for targeting resistant forms of melanoma.

#3772

SHP-1 determines the radiosensitivity of liver cancer cell and dovitinib acts as a novel radiosensitizer in hepatocellular carcinoma via targeting SHP-1/STAT3 signaling.

Man-Hsin Hung,1 Chao-Yuan Huang,2 Wei-Tien Tai,2 Ming-Hsien Tsai,2 Chih-Ting Shin,2 Szu-Yuan Wu,3 Chung-Wai Shiau,4 Kuen-Feng Chen2. 1 _Taipei Veterans' General Hospital, Taipei, Taiwan;_ 2 _National Taiwan University Hospital, Taipei, Taiwan;_ 3 _Wan Fang Hospital, Taipei, Taiwan;_ 4 _National Yang-Ming University, Taipei, Taiwan_.

Background

Hepatocellular carcinoma (HCC) is one of the most lethal human malignancies and curative therapy is not an option for most patients. There is growing interest in the potential benefit of radiotherapy (RT)-integrated therapy. This study aimed to investigate the biological impacts of a novel tumor suppressor Src homology 2 (SH2) domain-containing phosphatase 1 (SHP-1) and its downstream effecter, STAT3, in regulating the radiosensitivity of HCC cells. Furthermore, we explored the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT.

Material and methods

To understand the impacts of SHP-1/STAT3 signaling affects radiosensitivity, HCC cells with ectopic expression of STAT3, SHP-1 and a catalytic mutant SHP-1 were treated with or without radiotherapy and analyzed by flow cytometry and colony formation assay. Furthermore, five HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T and Huh-7) were treated with dovitnib, RT or both. The in vitro and in vivo effects of above-mentioned treatments were analyzed.

Results

By sub-G1 analysis and colony formation, we found that HCC cell with ectopic expression of SHP-1 was much more sensitive to RT-induced apoptotic effects, while overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Next, we investigated the effects of dovitinib, which showed that dovitinib treatment resulted in SHP-1-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1 diminished the effects of dovitinib on HCC cells. Furthermore, by ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment.

Importantly, we found that dovitinib potentiated the in vitor and in vivo effects of RT in HCC cells through affecting the SHP-1/STAT3 signaling.

Conclusions

SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. A combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects.

#3773

Preclinical evaluation of the CDK4/6 inhibitor LEE011 in nasopharyngeal carcinoma (NPC) cell lines.

Brigette B. Ma, Chi-Hang Wong, Connie Hui, Edwin P. Hui, Anthony T.C. Chan. _Chinese Univ. of Hong Kong, Shatin, Hong Kong_.

LEE011 is a specific CDK4/6 inhibitor that induces G1 cycle arrest by blocking the formation of cyclin D1-CDK4/6 complex and inhibiting Rb phosphorylation. Cyclin D1 is overexpressed in > 90% of NPC and CCND1 gene activation is implicated in pathogenesis. The preclinical activity of LEE011 was evaluated in 4 NPC cell lines (C666-1, HK1, HK1-LMP1, HONE-1) and the immortalized nasopharyngeal epithelial cell line NP69. Under basal condition, phosphorylated (p) Rb was strongly expressed in HK1 and HK1-LMP1, moderately in HONE-1 and NP69, and weakly in C666-1. Cyclin D1, CDK4 and 6 were expressed in all cell lines. The IC50 concentrations for cell growth inhibition after 72 hours of exposure to LEE011 in the respective cell lines were: HK1-LMP1 = 4.10±1.12μM; HK1=5.07±1.37μM, C666-1=12.25±1.63μM, NP69=15.23±1.93μM, HONE-1=19.58±1.54μM. LEE011 could induce over 95% cell growth inhibition in all NPC cell lines. Three representative cell lines (HK1 as the most sensitive to LEE011, and C666-1 and HONE-1 as less sensitive) were chosen to evaluate the effect of LEE011 on kinase signaling, apoptosis and cell cycle. Treatment of these cell lines at or above their respective IC50 concentrations for up to 48 hrs showed a dose-dependent reduction in p- and total Rb expression. A slight increase in the expression of CDK4, CDK6 and cyclinD1 were observed in HK1 and HONE1 cells, but not in C666-1 cells. G0/G1 population was increased by more than 20% in C666-1 and HK-1 cells at up to 48 hrs of exposure to LEE011. The effect of combining LEE011 with the alpha-specific PI3K inhibitor BYL719 on cell growth was studied in C666-1, HK1 and HONE-1 cells. A strong synergistic effect on growth inhibition was seen in C666-1 and HK1, but not HONE-1 at 72hrs, with the respective combination index (CI) of ED50 less than 0.5. Additionally, the combination of LEE011 and cisplatin at different sequences was investigated on their effect on cell growth. The sequential administration of cisplatin followed by LEE011 was the most optimal sequence on cell growth inhibition in C666-1 and HONE-1, but not in HK1 cells. Preliminary result suggests that this schedule was associated with better pRB inhibition than other schedules in C666-1 cells. The IC50 of LEE011 for a cisplatin-resistant HK1-LMP1-cis cell line at 72 hr was similar to its parental HK1-LMP1 cell line. In summary, LEE011 displayed dose- and time-dependent growth inhibitory effect in over 95% of NPC cells examined. A synergistic inhibitory effect on cancer cell growth was observed when LEE011 was combined with BYL719 in vitro and sequential administration of cisplatin followed by LEE011 has shown an optimal inhibitory effect. These results should be confirmed in xenograft models of NPC.

#3774

KD06 is a novel anti-cancer drug that causes cell death in triple-negative breast cancer cell lines and tumor xenografts.

Nancy D. Ebelt,1 Clint D J Taveres,2 Xuemei Xie,3 Youseff W. Naguib,1 Jiney Jose,4 Tinashe B. Ruwona,1 Ashwini K. Devkota,1 Jihyun Park,3 Tamer S. Kaoud,1 Eric V. Anslyn,1 Jeffrey T. Chang,5 Zhengrong Cui,1 Chandra Bartholomeusz,3 Kevin N. Dalby1. 1 _The University of Texas at Austin, Austin, TX;_ 2 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 3 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 4 _Auckland Cancer Society Research Center, New Zealand;_ 5 _The University of Texas Health Science Center at Houston, Houston, TX_.

Development and screening of small molecule compounds for anti-cancer activity has been of prime interest to the scientific community following the success of targeted, large anti-cancer molecules such as therapeutic antibodies. Small molecules pass more easily through cell membranes and may cross the blood-brain barrier. KD06 is a small molecule triptan-like compound whose parent molecule binds and inhibits serotonin receptors. This compound increases apoptosis of the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-157 via caspase activation. Treatment with KD06 also causes increased autophagy as well as activation of ER stress responses. The growth of tumor xenografts of MDA-MB-231 cells in nude mice are significantly inhibited by twice weekly treatment with 30mg/kg KD06. Analysis of signaling changes by KD06 using reverse phase protein array (RPPA) revealed significant decreases in Akt/mTOR signaling leading to decreased activation of the translation initiation factor 4E-BP1. Other notable changes included decreased expression of proteins important for mitosis such as Cyclin B1, Aurora B and PLK1, and increased phosphorylation of EGFR and increased expression of PDGFR. Analysis of PIP3 and ATP levels showed no change after treatment with KD06, indicating that decreased signaling through Akt/mTOR is not likely due to PI3K inhibition or AMPK activation. Immunofluorescence with KD06 treated cells revealed a change in cell shape after 4 hours of treatment that was reminiscent of cells treated with microtubule binding drugs. Akt localization was affected. These results imply that KD06 may have anti-cancer activity through its effect on microtubule dynamics, inhibiting proper localization and signaling of molecules important for survival and protein translation such as Akt and mTOR.

#3775

Mutated cancer cell-specific cell death activity of alkylating Pyrrole-Imidazole polyamide conjugates targeting a variety of oncogenic driver gene mutations.

Hiroki Nagase, Kiriko Hiraoka, Takahiro Inoue, Hiroyuki Yoda, Krishnamurthy Sakthisri, Jason Lin, Takayoshi Watanabe, Nobuko Koshikawa, Atsushi Takatori. _Chiba Cancer Center Research Institute, Chiba, Japan_.

Cancer may be recognized as non-self by antibiotics such as minor groove binders, which show self / non-self recognition partially due to preferential DNA sequence recognition and distinguish from other non-self bacteria. We learned minor groove binders produced from Streptomyces and synthesized Pyrrole-Imidazole polyamide indol-seco CBI conjugate to alkylate specific sites in the cancer genome.

Although a tremendous amount of studies has been made to directly target oncogenic drivers, such as RAS and MYC, yet no drug is clinically available because of difficulties to develop RAS- or Myc-targeted anti-cancer therapeutics due to the smooth 3D surface topology. One of major limitations of targeting the RAS pathway may be intrinsic or acquired resistance as seen in the other molecular target therapy. New approaches that directly target driver genes may provide a more direct route to helps address unmet medical needs for refractory cancer conquest.

We therefore synthesized several Pyrrole-Imidazole polyamide conjugates, each of which specifically alkylated KRAS codon 12 mutant DNA (G12D or G12V), amplified MYCN or mutated DNA of PI3K E542K mutation. All three conjugates reduced expression of the mutated oncogenic-protein by RNA transcription inhibition and induced cancer cell death at low dose (1 to 50 nM). Low dose tail vein injections of conjugates-targeting KRS or MYCN also demonstrated significant anti-tumor effects on xenograft models of human tumors harboring oncogenic mutated driver with minimum host toxicity, but not in xenografts harboring wild type or non-recognized mutations. We also performed a series of biological searches for toxicities by applying Modified SHIRPA (behavioral and functional analysis of mouse phenotype) to test any pathological phenotypes and examinations of blood and urine in ICR mice. Modified SHIRPA screening, blood chemistry, blood cell analysis and urea tests exhibited no toxicologically significant changes. Additionally, we examine pharmacokinetics of PI polyamide conjugates In vivo using LC-mass and fluorescent imaging of tumor-bearing mice. Intriguingly, 48 hours after the administration the highest fluorescence intensity was observed in the tumor-cell nuclear and almost no fluorescent intensity in all other organs, tissues and cells. These data suggest that sequence-dependent alkylating approach using antibiotic mimics of alkylating PI-polyamide conjugates, may open a new strategy not only targeting point mutation of driver oncogene but also targeting key driver gene in the cancer amplicon. This approach should be used for future ' Precision cancer medicine'.

#3776

Mechanistic evaluation of AG311 - an OXPHOS inhibitor - as a potential treatment for breast cancer.

Anja Bastian,1 Satoshi Matsuzaki,2 Kenneth M. Humphries,2 Lora C. Bailey-Downs,1 Aleem Gangjee,3 Michael A. Ihnat1. 1 _Univ. of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _Oklahoma Medical Research Foundation, Oklahoma City, OK;_ 3 _Duquesne University, Pittsburgh, PA_.

About fifty percent of breast cancer patients receiving a combination of surgery, radiation and systemic therapy will not remain cancer-free. Most solid tumors, including breast tumors, have hypoxic regions that can contribute to chemoresistance, radioresistance, and poor differentiation in tumors resulting in a poor clinical outcome in patients with advanced disease. It has been reported that mitochondrial respiration remains active in these hypoxic microenvironments. Complex I of the mitochondrial electron transport chain (ETC) has been shown to switch to a de-active catalytic state under hypoxic conditions. The purpose of this study was to investigate the potential anticancer action of a novel compound with antimitochondrial activity under hypoxic conditions. AG311 (5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine) is a small molecular weight compound shown to inhibit complex I activity in vitro and to drastically reduce mitochondrial oxygen consumption rate by 62.9% at 7.5 µM in breast cancer cells (p<0.001). In two triple negative breast cancer mouse models (MDA-MB-435 and 4T1), AG311 significantly reduced tumor volume by 85% and 81%, respectively (p<0.001 for both). In this current study, the effect of the microenvironment on AG311 mitochondrial inhibition was examined. First, it was shown that AG311 inhibited complex I activity not only in vitro, but also in breast cancer cells (54% inhibition, p<0.001) and tumor homogenate (50% inhibition, p=0.01). AG311 induced greater cytotoxicity in cells (MDA-MB-435) cultured in glucose-depleted media (IC50 15.5 µM vs 20.0 µM, p<0.01), a condition favoring mitochondrial ATP production, as compared to normal glucose concentrations. Further, co-treatment of AG311 with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor and stimulator of oxidative phosphorylation, showed a synergistic effect on cell kill (CI=0.7 at 20 µM). Importantly, hypoxic conditions (1% O2) significantly sensitized cancer cells to AG311-induced cell death (from 43.3% to 30.1%, p=0.019). Further, the effect of AG311 on the deactive form of complex I, which is promoted under hypoxic conditions was assessed by measuring NADH oxidation rate. The switch to the de-active state was thermally-induced in mitochondrial homogenates and once in this state, complex I activity was sensitized to AG311 inhibition (from 45.4% to 65.0% inhibition, p=0.04). Thus, a mitochondrial inhibitor that preferentially inhibits the de-active state of complex I in hypoxic tumor regions could potentially provide a therapeutic benefit.

#3777

TAS4464, a novel and highly potent NEDD8 activating enzyme (NAE) inhibitor, causes apoptosis of sarcomas via cell cycle dysregulation.

Hidenori Fujita, Yayoi Fujioka, Keiji Ishida, Chihoko Yoshimura, Akihiro Hashimoto, Shingo Tsuji, Takashi Mizutani, Shuichi Okubo, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. _Taiho Pharmaceutical Co., Ltd, Ibaraki, Japan_.

Background

Soft-tissue sarcomas (STSs) are heterogeneous tumors that comprise approximately 1% of adult cancers. Patients with advanced STS have a poor prognosis because few chemotherapy options are available.

NAE catalyzes the first step in the NEDD8 conjugation (neddylation) pathway. Because it activates cullin-RING ligase complexes (CRLs) and thus is essential in cancer cell homeostasis, NAE is a promising target for cancer therapy. Here, we investigated the potency of the NAE inhibitor TAS4464 for various STS cell lines.

Material and methods

Cytotoxicity was evaluated through ATP-Based assay. The effects of TAS4464 on NEDD8 conjugation and CRL substrates were evaluated by Western analysis. Cell cycle progression was analyzed by using flow cytometry. Small interfering RNAs (siRNAs) were lipofected into a clear cell sarcoma (CCS) line (SU-CCS-1). The antitumor activity of intravenous TAS4464 was evaluated in xenograft models of the aforementioned CCS and 2 rhadomyosarcoma (RMS) lines (SJCRH30 and RD).

Results

TAS4464 suppressed cell growth and induced cell death in various STS cell lines at lower concentrations than did an investigational NAE inhibitor, MLN4924, and a conventional STS treatment agent, doxorubicin (DXR). Notably, the GI50 values of TAS4464 were less than 10 nM in the tested RMS and CCS cell lines. TAS4464 treatment led to the elimination of cullin neddylation, accumulation of CRL substrate proteins (CDT1, p27, and p21), S phase arrest, and ultimately apoptosis in these STS cell lines. Furthermore, knockdown of CRL substrate proteins by siRNAs markedly attenuated TAS4464-induced cytotoxicity in STS cells. These results suggest that TAS4464-induced cell death was triggered by cell cycle dysregulation based on accumulation of CRL substrate proteins following NAE inhibition.

TAS4464 also led to a decrease in cullin neddylation and accumulation of CRL substrate proteins in RMS and CCS lines subcutaneous xenografts. Weekly administration of TAS4464 (100 mg/kg, IV) completely suppressed tumor growth in mice bearing subcutaneous RD and SJCRH30 xenografts. The effects of TAS4464 significantly exceeded that of DXR and MLN4924. Furthermore, TAS4464 induced tumor regression of ~50% in the pazopanib, a drug approved for STS, insensitive SU-CCS-1 xenograft model.

Conclusion

TAS4464 prominently inhibited cell growth and induced apoptosis in RMS and CSC cells through cell cycle dysregulation and demonstrated impressive antitumor activities in STS xenograft models that respond poorly to DXR and pazopanib. Therefore, TAS4464 may become a valuable therapeutic option for patients with advanced STS.

#3778

In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer.

Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Naoko Hata Sugi, Yukinori Minoshima, Junji Matsui, Masao Iwata. _Eisai Co., Ltd, Tsukuba-shi, Ibaraki, Japan_.

Genetic abnormalities (gene fusion, mutation and amplification) of Fibroblast Growth Factor Receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR2 is mutated in about 10% of endometrial cancer patients and is associated with a higher risk of recurrence. E7090, an oral available FGFR1, 2, and 3 inhibitor that the chemical-structure and basic kinase inhibitory activity of this compound have been presented at AACR2015, is currently under a first-in-human study (NCT02275910) in Japan. In this presentation, the efficacy of E7090 against human endometrial cancer was examined.

E7090 exhibited selective potent antiproliferative activity against 3 human endometrial cancer cell lines harboring FGFR2 mutation with IC50 values of approximately 10 nM among 8 endometrial cancer cell lines. These cell lines harbor S252W mutation (extracellular domain mutation; MFE280) or N549K mutation (kinase domain mutation; AN3CA and MFE296), that are the most common mutations of FGFR2 in endometrial cancer. E7090 also showed antiproliferative activity against Ba/F3 cells expressing mutated type of FGFR2, confirming the inhibitory activity of E7090 against mutated FGFR2. The antiproliferative activity of E7090 against these cell lines was based on inhibition of phosphorylated FRS2α and ERK1/2 as downstream molecules of FGF/FGFR signaling. Especially, the antiproliferative activities of E7090 against AN3CA and MFE296 harboring N550K mutation were more potent compared to these of other FGFR specific inhibitors. In addition, oral administration of E7090 resulted in significant tumor growth inhibition in MFE280, AN3CA and MFE296 xenograft models in mice. Especially, E7090 showed tumor regression in AN3CA xenograft model with a delta T/C value of -66% at a dose of 50 mg/kg.

These results indicated that E7090 showed potent inhibitory activity against mutated type of FGFR2 both in vitro and in vivo, and suggest that E7090 may provide therapeutic opportunities for endometrial cancer harboring FGFR2 mutations.

#3779

Evaluation of efficacy of an RNA aptamer toward non-small cell lung cancer.

Hanlu Wang,1 Haiping Yang,2 Xinxin Ding,3 Wei Dai,4 Yongping Jiang1. 1 _Biopharmaceutical R &D Center, Peking Union Medical College of Tsinghua University, Suzhou, China; _2 _Biopharmagen Corp., Suzhou, China;_ 3 _College of Nanoscale Science, SUNY Polytechnic Institute, Albany, NY;_ 4 _Environmental Medicine, NYU Langone Medical Center, Tuxedo, NY_.

A specific RNA aptamer (RA16) targeting non-small cell lung cancer (HCI-H460 cells) was previously selected by in vivo SELEX. In this study, we report subsequent studies that determined the effect of RA16 on HCI-H460 cell proliferation in vitro and xenogfrat tumor growth in vivo. Firstly, RA16 apatmer, but SCAP control, was capable of inhibiting cell proliferation in a dose-dependent manner. At 300 nM, RA16 apatmer suppressed cell proliferation by 80%. The IC50 for RA16 was determined at 116.7 nM. Intriguingly, RA16 exhibited no inhibitory effect on HeLa cells even at 600 nM. Moreover, inhibition of HCI-H460 cell growth by RA16 was also observed when it was non-covalently conjugated to Epirubicin(EPI)at the ratio of 1:8 (RA16:EPI). Cell inhibition of 93.6±1.48% was observed with RA16:EPI conjugate (0.375μM:3μM), whereas cell inhibition of 89.0±1.67% and 74.8±5.01% was observed with EPI at 3μM and RA16 alone at 0.375μM, respectively. No inhibition of cell proliferation was observed for the control group treated with cell culture medium. Secondly, an in vivo study was performed to investigate the inhibition of RA16 on HCI-H460 xenograft tumors in mice. Twelve mice with tumor sizes ranging from 50 to 100 mm3 were randomly divided into 2 groups (n=6). The mice from each group were administrated via intravenous injections of saline (control) or 2 nM of RA16 on days 0, 3, 5, 7, and 9, respectively. Tumor sizes were measured every other day and tumor volumes were calculated by the formula V (mm3)=1/2×a×b2. The average inhibition rate for mouse tumors of treated group was 54.26±5.87% on day 16 compared with the control group mice. Finally, an in vivo study was performed to evaluate the inhibition of RA16:EPI conjugates on xenograft tumors in mice. Thirty tumor-bearing mice (with tumor sizes about 200~300 mm3) were randomly divided into 6 groups(n=5)and treated with various combinations of RA16 and EPI weekly for 3 times. A strong inhibition (64.38±6.45%) of tumor growth was observed when the mice were treated with RA16:EPI conjugate with PEG (EPI at 1.5 mg/kg/week). A moderate inhibition (47.13±10.21%) was observed in mice treated with EPI alone at 1.5 mg/kg/week. Similarly, a moderate inhibition (39.07±5.65%) of tumor growth was observed in mice treated with RA16-EPI conjugate (EPI at 0.5 mg/kg/week) whereas a weak inhibition (28.76±5.65%) was noticed in mouse group treated with EPI alone at 0.5 mg/kg/week. Furthermore, RA16:EPI conjugate (EPI at 1.5 mg/kg/week) inhibited tumor growth by 32.80±6.99% where no PEG was linked to RA16, suggesting that PEGlyation prolongs the efficiency of RA16 in tumor inhibition in vivo. No any inhibition was observed with saline-treated mice.

In conclusion, our studies demonstrate that RNA aptamer RA16 specifically inhibits proliferation of transformed HCI-H460 cells in vitro and xenograft tumor growth in vivo and that RA16 conjugated with EPI exhibits enhanced activities both in vitro and in vivo.

#3780

Activity of the BET inhibitor INCB054329 in models of lymphoma.

Matthew Stubbs, Robert Collins, Alla Volgina, Mike Liu, Margaret Favata, Mark Rupar, Xiaomng Wen, Richard Sparks, Thomas Maduskuie, Maryanne Covington, Timothy Burn, Bruce Ruggeri, Andrew P. Combs, Wenqing Yao, Reid Huber, Gregory Hollis, Peggy Scherle, Phillip CC Liu. _Incyte Corporation, Wilmington, DE_.

Inhibitors of the BET family of Bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate expression of key survival and cell fate determining genes such as c-myc. Among the various tumor histologies, hematologic malignancies are among the most sensitive cancers to BET inhibition. INCB054329 is a novel, non-benzodiazepine, selective BET inhibitor that is undergoing Phase 1 clinical trials and that has shown encouraging in vitro and in vivo preclinical activity in several models of hematologic malignancy. In the current study, the activity of INCB054329 was evaluated in models of B cell malignancy. INCB54329 effectively inhibited the in vitro growth of a panel of cell lines representing both Hodgkin and non-Hodgkin lymphoma. Treated cells arrested primarily in G1 with sensitive lines also exhibiting dose and time-dependent apoptosis. Within a panel of double-hit lymphoma cell lines, which have activating chromosomal rearrangements in both c-myc and bcl-2, INCB054329 potently inhibited cell growth and was more effective than antagonists of BTK, bcl-2, PIM and PI3Kδ. INCB054329 also showed in vivo efficacy in models of diffuse large B-cell lymphoma (DLBCL). As a single agent, oral administration of INCB054329 inhibited tumor growth in Pfeiffer (GBC) and WILL-2 (GCB, double-hit) subcutaneous xenograft models. The in vivo combination of bendamustine with INCB054329 enhanced anti-tumor efficacy compared with either agent alone in the Pfeiffer model, and the combination was well tolerated. A rational, targeted combination strategy was evaluated involving INCB054329 and a selective, orally active PI3Kδ inhibitor, INCB050465, which is currently in clinical trials in B cell malignancies. Combining INCB054329 with PI3Kδ inhibition markedly enhanced anti-tumor efficacy, increasing the incidence of partial tumor regressions in vivo. In this model, both INCB054329 and INCB050465 treatment led to a reduction in c-Myc protein levels, suggesting a convergence between modulation of BET transcriptional regulation and the PI3Kδ pathway. These data suggest that clinical investigation of INCB054329, both as monotherapy and in combination with standard of care or novel targeted therapies, in several classes of B cell lymphoma, including high risk double hit lymphoma, is warranted.

#3781

Combination therapy with a liver selective acetyl-CoA carboxylase inhibitor ND-654 and sorafenib improves efficacy in the treatment of cirrhotic rats with hepatocellular carcinoma.

Lan Wei,1 Geraldine Harriman,2 Sarani Ghoshal,1 Omeed Moaven,1 Jeremy Greenwood,3 Sathesh Bhat,3 William F. Westlin,2 H. James Harwood,2 Rosana Kapeller,2 Kenneth K. Tanabe,1 Bryan C. Fuchs1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Nimbus Therapeutics, Cambridge, MA;_ 3 _Schrodinger, New York, NY_.

Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Sorafenib is the only FDA-approved drug for the treatment of HCC but its effects are marginal as it only extends survival by a few months. Therefore, new treatment options are urgently needed. Metabolic attenuation is a promising approach to cancer therapy, and rate-limiting steps in key biosynthetic pathways are particularly attractive targets. We recently identified ND-654, a hepatoselective (~3000:1 liver to muscle exposure), allosteric ACC inhibitor that binds to the ACC subunit dimerization site and inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM). Daily oral administration of 10 mg/kg ND-654 reduced tumor incidence by 55% and significantly improved median survival time in a rat model of cirrhosis and HCC. Here, we examine the effects of ND-654 alone and in combination with sorafenib on HCC development in cirrhotic rats. Methods: Male Wistar rats were treated once weekly with 50 mg/kg diethylnitrosamine (DEN) for 18 weeks to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis and when HCCs are first developing (13 weeks), rats were treated daily by oral gavage with either 1) vehicle control, 2) 10 mg/kg ND-654, 3) 10 mg/kg sorafenib, or 4) 10 mg/kg ND-654 and 10 mg/kg sorafenib. After 18 weeks, tumor nodules were counted and liver and tumor tissue was harvested for analysis. Results: Similar to our previous study, simultaneous inhibition of ACC1 and ACC2 significantly reduced HCC incidence by 41% (p < 0.05) which was comparable to results with sorafenib (57% reduction, p < 0.01). Remarkably, the combination of ND-654 and sorafenib significantly reduced HCC incidence by 81% (p < 0.001). We are currently examining the effects of combining ND-654 and sorafenib on markers of tumor proliferation and apoptosis. Conclusions: These results provide further evidence that de novo lipogenesis is an important mediator of hepatic carcinogenesis and that selective inhibition of hepatic ACC is a viable cancer metabolism therapeutic strategy for treating HCC that could be combined with sorafenib.

#3782

Development of a dual inhibitor of pan-RAF and VEGFR2 for the treatment of metastatic colorectal cancer with mutant K-RAS.

Sungpyo Hong,1 Young-Il Choi,1 Jihye Choi,1 Wonyoung Kim,1 Ho-Seok Kwon,2 Yong Bin Park,2 Min-Hyo Ki,2 Hee Jong Shin,2 Michael Lee,1 Soon Kil Ahn1. 1 _Institute for New Drug Development, University of Incheon, Incheon, Republic of Korea;_ 2 _Samjin Pharm. Co. Ltd., Seongnam, Republic of Korea_.

Signal transduction in the RAS-RAF-MEK-ERK (MAPK) pathway plays a key role in cell survival, growth and proliferation. The pathway is controlled by extracellular signals through receptor tyrosine kinases (RTK) and is activated by oncogenic mutations in many types of cancer. K-RAS mutation has been reported in 40% of colorectal cancer patients. These patients have acquired resistance to EGFR tyrosine kinase inhibitors. Therefore, there is a need for the development of new therapeutic approaches for the treatment of these cancers. The B-RAF selective inhibitors have been approved for the treatment of human melanoma with a B-RAF V600E mutation. However, B-RAF specific inhibitor activates C-RAF in RAS mutant cells by inducing RAF dimer formation and paradoxical activation of C-RAF. These observations require the development of pan-RAF inhibitors without inducing paradoxical activation. In addition, as VEGFR2 represents one crucial promoter of tumor angiogenesis in colorectal cancer, the inhibition of VEGFR2 has been expected to be synergistic for the treatment of colorectal cancer.

Therefore, we tried to find dual inhibitors of pan-RAF and VEGFR2 using structure based molecular modeling and identified a selective inhibitor, C1005, which binds to the DFG-out inactive conformation of B-RAF. In addition, C1005 potentially inhibited B-RAF, C-RAF, B-RAF V600E and VEGFR2 with IC50 value of 2.7 nM, 0.7 nM, 5.2 nM and 11nM, respectively. C1005 completely inhibited the phosphorylation of MEK-ERK without paradoxical activation in K-RAS mutant cells compared with Vemurafenib and Regorafenib. We examined the effects of C1005 on cell proliferation of colorectal cancer cells with K-RAS or B-RAF mutation. C1005 displayed potent anti-proliferative activities not only in K-RAS mutant colorectal cancer cells but also in B-RAF mutant colorectal cancer cells. C1005 effectively blocked cell proliferation of HCT-116, LS-513 and WiDr at EC50 of 145 nM, 79 nM and 636 nM, respectively. In addition, growth inhibition of VEGF-stimulated HUVEC indicates that C1005 is a potent inhibitor of the angiogenesis. To study in vivo effect, we tested C1005 in colorectal cancer xenografts with mutant K-RAS. Oral treatment of animals bearing xenograft tumors by 30 and 60 mg/kg, twice daily (BID) of C1005, induced a dose-dependent tumor growth inhibition. We observed 77% and 89% reduction of tumor volume, respectively.

In this study, we discovered an orally active dual inhibitor of pan-RAF and VEGFR2. C1005 has been shown to suppress mutant K-RAS colorectal cancer cells without activating the MAPK pathway. Our findings suggest that C1005 could be an excellent preclinical candidate to treat mutant K-RAS driven colorectal cancer.

#3783

Co-treatment of metastasis-derived colon cancer cells with bromoethyl indole (BEI) enhances camptothecin or TNF alpha-induced cell death.

Rupak Chowdhury,1 Sonni A. Miller,1 Dominique Gales,1 Jason White,1 Upender Manne,2 Temesgen Samuel1. 1 _Tuskegee University College of Vet. Medicine, Tuskegee, AL;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Colorectal cancer is still the third most deadly cancer in the United States. Currently, there are limited choices of targeted anticancer agents that are durably effective against colorectal cancer. Therefore, chemotherapeutic regimens containing the drugs Camptothecin (CPT), 5-FU and Oxaliplatin remain the mainstays for the treatment of advanced colorectal cancer. We recently found that the indole-derivative small compound Bromoethyl Indole (BEI) potently inhibits the proliferation of colon cancer cells and also suppresses NF-kB activation. In this study, we investigated if the combination of BEI with either CPT or TNFα would enhance cell death in vitro. Metastasis-derived parental or engineered NF-kB-reporter colon cancer cells were used to examine 1) the activation of NF-kB signaling by clinically used drugs, 2) the potential for BEI to inhibit drug-induced NF-kB activation, and 3) the potential for combination treatment to enhance cell death in vitro. Cells were treated with either the drugs alone or in combination with BEI at varying concentrations. NF-kB-inducing concentration of CPT (0.6 to 2.5 µM), TNFα (25 to 50 ng/ml), and BEI concentrations of 1 to 50 µM range were tested. Cell cycle profiles and cell death markers were assessed to determine the effects of single or co-treatments. The expressions of Bcl-xL and cIAP2 (BIRC3) proteins were examined to monitor the targets of NF-kB activation.

Our results show that 1) both TNFα and CPT induce NF-kB signaling in metastasis-derived colon cancer cells, 2) combination of BEI and TNFα or CPT inhibits such a drug-induced NF-kB activation and reduces the expression of NF-kB responsive genes, 3) sequential treatment of the cells with CPT and BEI delivers the best outcome, increasing cell death by up to 3-fold compared to either CPT or BEI alone, and 4) co-treatment of the cells with TNFα and BEI increases cell death by up to 2-fold compared to either TNFα or BEI alone, 5) BEI is most effective in inducing cell death at concentrations between 2 and 10 µM.

We anticipate that BEI will improve the therapeutic index of some chemotherapeutic drugs. Therefore, the potential benefit for combination of BEI with NF-kB activating drugs needs to be evaluated in vivo.

### Novel Targets

#3784

The role of the taurine transporter SLC6A6 in promoting prosurvival activity and multidrug resistance of colorectal cancer.

Masahiro Yasunaga, Yasuhiro Matsumura. _National Cancer Center, Kashiwa, Japan_.

The identification of a novel therapeutic target is desired to improve the treatment of colorectal cancer (CRC). In a comprehensive gene expression analysis, we found that the taurine transporter SLC6A6 was highly expressed in CRC. In the functional analysis, SLC6A6-knocked down (KD) CRC cells showed attenuated cell-survival activity accompanying enhanced sensitivity of the drugs (5-FU, DOX and SN-38). Moreover, the number of side population (SP) cells and their cancer stem cell (CSC)-like properties such as tumor-initiation were abrogated by SLC6A6-KD. Conversely, increased the cell-survival activity, the fraction of SP cells and enhancement of multidrug resistance (MDR) were observed in SLC6A6-overexpressed cells. Furthermore, SLC6A6-siRNA treatment enhanced the cytotoxic effect of all 3 drugs as distinct from ABCG2-siRNA treatment, the efficacy of which was limited to its 2 substrate-drug, DOX and SN-38. Finally, we clarified the prosurvival and anti-apoptotic effects of the taurine transporter SLC6A6 in CRC cells. Moreover, we found that SLC6A6 plays an important role in the maintenance of CSC characteristics promoting cell survival signal and MDR. Our findings indicate that the SLC6A6 may be a novel therapeutic target for refractory CRC.

#3785

Targeting PDLIM5 for lung cancer.

Deborah Park, Han Cheng, Tianji Chen, Merve Tor, Nour Khatib, Jason Huang, Qiyuan Zhou, Lijun Rong, Guofei Zhou. _University of Illinois at Chicago, Chicago, IL_.

Rationale: Lung cancer is the most common cause of cancer-related death in the United States and worldwide. Despite decades of efforts on research and smoking cessation, the 5-year survival rate of lung cancer patients remains poor at 15%, and the population of adenocarcinoma cases in nonsmokers is rising. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and 3 LIM domains at its C-terminus. We have previously shown that PDLIM5 regulates TGF-β/Smad family, which are known to participate in the pathogenesis of lung cancer. In this study, we aim to investigate whether PDLIM5 plays a role in lung cancer and establish a high throughput screening (HTS) platform for PDLIM5-targeted drug discovery.

Methods: We searched the Oncomine data base for PDLIM5 gene expression. We suppressed PDLIM5 in A549 cells and determined the expression levels of epithelial-mesenchymal transition markers. We exposed A549 cells to hypoxia or treated them with TGF-β1 and measured the expression levels of PDLIM5. We generated a stable mink lung epithelial cell line (MLEC) containing a TGFβ/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1,200 compounds). We treated MLEC and A549 cells with paclitaxel and determined levels of Smad2/3 and pSmad2/3.

Results: We found that PDLIM5 was overexpressed in non-small-cell lung carcinoma (NSCLC) patients and that the expression levels of PDLIM5 inversely correlated with the survival rate of lung cancer patients. Hypoxia but not TGF-β1 induced PDLIM5 expression levels. Suppression of PDLIM5 induced E-cadherin levels while decreased levels of vimentin and α-SMA. In MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, smad3, and pSmad2. More importantly, we identified and validated that paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells.

Conclusions: We have shown that PDLIM5 is overexpressed in lung cancer cells and its upregulation is hypoxia-dependent but TGF-β independent. Loss of PDLIM5 inhibits EMT. We have identified paclitaxel as a PDLIM5 inhibitor. This system is robust and suitable for PDLIM5 targeted drug discovery. Support: a UIC UICentre Arm1 Inception Grant, a Gilead Sciences Research Scholars Program in Pulmonary Arterial Hypertension award and R01 HL123804 (G. Zhou).

#3786

Rad6 inhibition enhances paclitaxel sensitivity of triple negative breast cancer cells by inducing mitotic spindle defects.

Brittany Haynes, Kristen Cunningham, Malathy Shekhar. _Wayne State University, Detroit, MI_.

Triple negative breast cancers (TNBC) lack estrogen receptors, and progesterone receptors, and HER2/neu receptor amplifications, making targeted therapies unsuitable. The taxane based drug paclitaxel (PTX) is used as a first line chemotherapy. PTX is a microtubule stabilizer that induces G2/M arrest and mitotic catastrophe. Loss of BRCA1 often seen in TNBC patients is associated with PTX resistance. Rad6 is an E2 ubiquitin conjugating enzyme that has two human homologues, Rad6A (UBE2A) and Rad6B (UBE2B), and high Rad6B expression rather than Rad6A is associated with poor survival of TNBC patients. Rad6 is associated with centrosomes at all phases of the cell cycle, and constitutive overexpression of Rad6B in nontransformed MCF10A breast cells leads to centrosome amplification and aneuploidy. Inhibition of Rad6 enzymatic activity, with our novel Rad6-selective small molecule inhibitor (SMI#9) induces G2/M arrest, much like PTX. We hypothesize that the overexpression of Rad6B commonly found in BRCA1 wt and BRCA1 mut TNBCs contributes to taxane resistance by promoting centrosome amplification and enhancing microtubule dynamics. Thus, targeting Rad6 will confer taxane sensitivity by preventing centrosome reduplication and/or microtubule hypernucleation, strengthening G2/M arrest, and ensuring terminal mitotic catastrophe. Immunostaining shows Rad6 is overexpressed in BRCA1 wt and BRCA1 mut breast cancer tissues and TNBC cell lines. Immunostaining of the centrosomal protein pericentrin shows centrosome amplification in both BRCA1 wt and BRCA1 mut TNBC cells. MTT data show SMI#9 synergistically sensitizes both BRCA1 wt (MDA-MB-468) and BRCA1 mut (HCC1937) TNBC cell lines to PTX. Colony forming assays corroborate these data and show that addition of SMI#9 diminishes colony forming efficiency of PTX pretreated MDA-MB-468 cells. SMI#9 treatment of HCC1937 PTX resistant colonies results in increases in cells with enlarged and multiple nuclei (characteristic of mitotic catastrophe). Western blot analysis of Tau, a marker of PTX sensitivity, indicates PTX, SMI#9, and PTX+SMI#9 treatments decrease the steady state levels of the 1N3R and 0N4R, or 1N3R Tau isoforms in MDA-MB-468 and HCC1937 cells, respectively. Analysis of cyclin B1, a marker of G2/M arrest, shows increases in cyclin B1 levels in PTX, SMI#9, and PTX+SMI#9 treated MDA-MB-468 cells, whereas cyclin B1 is degraded in HCC1937 cells treated with these agents (indicative of mitotic catastrophe). Immunofluorescence staining shows that Tau localization to the mitotic spindles is unaffected by PTX, SMI#9, or PTX+SMI#9. However, treatments including SMI#9 resulted in mitotic cells with defective or monopolar mitotic spindles. These data implicate a role for Rad6 in centrosome duplication/separation and provide mechanistic support for inhibiting Rad6 to enhance PTX sensitivity. Supported by NCI R21 CA178117 and T32-CA009531.

#3787

Thyroid hormone induced proliferation of breast cancer cell lines: A novel approach to hormone therapy.

Katarzyna Joanna Jerzak,1 Jessica G. Cockburn,2 John Hassel,2 Kathleen I. Pritchard,1 Sukhbinder K. Dhesy-Thind,2 Bane Anita2. 1 _University of Toronto, Toronto, Ontario, Canada;_ 2 _McMaster University, Hamilton, Ontario, Canada_.

Introduction:

The thyroid hormone (TH) pathway influences cell growth and it may be a novel target for breast cancer (BC) therapy. However, various TH receptor (THR) isoforms exist and some have opposing functions thus complicating the role of THs in BC. Our previous work suggests that THR alpha1 (THRα1) promotes TH-mediated BC proliferation. Here we offer a mechanism that explains these findings. We first evaluated the role of THs on BC cell line proliferation using escalating doses of tri-iodothyronine (T3) and thyroxine (T4). Next, propylthiouracil (PTU) was used to block T4 conversion to T3 demonstrating that observed proliferation was TH dependent. Finally, the anti-proliferative efficacy of a THRα1 inhibitor, dronedarone, was used to demonstrate the necessity of THRα1 downstream of TH signalling. Dronedarone was selected because it is an FDA approved anti-arrhythmic drug, which may also serve as a novel anti-cancer agent.

Methods:

The effect of increasing concentrations of T3 and T4 on the proliferation of 3 BC cell lines (MCF 7, MD-MB-231, BT-474) in 10% charcoal-stripped phenol-free serum were evaluated at 24 and 48 hours following standard MTT-assay protocols. Increasing doses of PTU and dronedarone were added to cells with 200uM T3 or T4 to measure proliferation of MCF-7 and MD-MB-231 cells using MTT assays.

Results:

There was a statistically significant increase in the proliferation of MCF7, MD-MB-231 and BT-474 cells with the addition of T3 and T4 at 24 and 48 hours in a dose dependent manner. Of note, BT-474 cells had a significantly slower proliferation rate and required 9 days of incubation prior to detection of proliferation.

The addition of PTU reduced proliferation by 50% in MCF7 and MD-MB-231 cells in the presence of T4 at roughly 5 mM compared to the presence of T3 which had an IC50 of 12mM. This shows that TH signalling is mediated by T4 conversion to T3 in these cell lines. PTU alone reduced cell proliferation by 50% at concentrations above 12 mM, suggesting that PTU may also act through other mechanisms.

Cell proliferation was not affected by the presence of dronedarone alone, but the combination of 10μM dronedarone and T3 or T4 significantly reduced proliferation of both MCF7 and MD-MB-231 cells. These results suggest that THRα1 is required for TH mediated cell proliferation.

A library of compounds similar in structure to dronedarone is being designed to offer additional candidates for more potent THRα1 inhibition. The properties and structures of these novel compounds will be presented.

Conclusion:

The proliferation of several BC cell lines increases in response to exogenous T3 and T4 when grown in hormone depleted growth media. These results demonstrate the role of specific events contributing to TH-mediated cell proliferation, including T4 conversion to T3 and the necessity of downstream THRα1. Ultimately, in conjunction with our previous work, we propose that THRα1 inhibition is a novel target for cancer therapy.

#3788

Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer.

Radhashree Maitra,1 Titto Augustine,1 Matt Coffey,2 Sanjay Goel1. 1 _Montefiore Medical Center, Bronx, NY;_ 2 _Oncolytics Inc, Calgary, Alberta, Canada_.

Introduction

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for patients whose tumors harbor a mutation in the KRAS oncogene (40-45%). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a dsRNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. We hypothesize that effective expression of host Toll Like Receptors 3 (TLR3) will dampen the infection potential of reovirus propagation by mounting an innate immune response. Development of strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved virus productivity to specifically target the KRAS mutated cancer cells.

Methodology: TLR3 expressing HEK293 cells were treated with reovirus to confirm that TLR 3 is the host pattern recognition motif of the host cellular machinery that is responsible for the detection of dsRNA harboring reovirus. TLR 3 was next down regulated by shRNA technology in KRAS mutated HCT116 CRC cell line and treated with reovirus at dose of 5 MOI (multiplicity of infection) for 48 hours. The cell proliferation profile under this treatment condition was measured by MTT assay and corelated with the proliferation pattern of TLR3 expressing HCT116 cells undergoing similar treatment. Cytokine ELISA was performed for interferon (INF) alpha (α)and beta (β) to determine the downstream consequences of the gene silencing. Finally xenograft tumor models of athymic nude mice (Foxn1nu ) were developed with TLR3 silenced HCT116 cells and treated with reovirus at a daily intra tumoral (IT) dose of 1X107 TCID (tissue culture infective dose) and compared to tumors generated by HCT116 cells receiving identical treatment.

Results

TLR 3 is confirmed to be the host pattern recognition motif for dsRNA containing reovirus

TLR3 which is constitutively expressed by colon epithelium is also a mediator of virus recognition in CRC cell line HCT116.

Down regulation of TLR3 by shRNA technology enhances viral propagation as measured by MTT assay.

Cytokine ELISA assay of INF α β distinctly reveals that downstream activation post reovirus infection is compromised.

In xenograft models, those of TLR3 down regulated HCT116 cells, showed improved control of tumor growth with reovirus treatment as compared to those with TLR 3 expressing HCT 116 cells (p= 0.04)

Conclusion

TLR3 plays an important role in recognition of reovirus and mounting an innate immune response by inducing the secretion of type I INF. Mitigation of the TLR3 receptor mediated pattern recognition by shRNA down regulates host immune response and thus improves virus mediated cell cytotoxicity. The findings can be therapeutically utilized towards improved and beneficial killing of cancer cells by reovirus.

#3789

Targeting cholesterol biosynthesis pathway to inhibit growth of drug resistant ovarian cancer cells.

Yayun Liang,1 Johannes Aebi,2 Kenneth Nephew,3 Salman M. Hyder1. 1 _University of Missouri, Columbia, MO;_ 2 _F. Hoffmann-La Roche Ltd, Basel, Switzerland;_ 3 _Indiana University, Bloomington, IN_.

Despite concerted efforts to develop new strategies for preventing and treating ovarian cancer, almost 25,000 new cases of the disease are reported each year in the United States. Cancers of the ovary have poor prognosis due to drug resistance and metastasis, and have the highest mortality rate of all the known gynecological malignancies. Standard treatments include systemic high-dose toxic chemotherapeutic drugs but drug-resistance almost always occurs. Consequently novel and more effective non-toxic therapies for ovarian cancer are urgently needed. The biosynthetic pathway leading to cholesterol production is an attractive therapeutic target since cholesterol is an essential structural and functional component of cell membranes required for tumor growth. Therapy with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently alternative approaches to inhibit cholesterol biosynthesis are being considered. 2,3-oxidosqualene cyclase (OSC), which converts 2, 3-monoepoxysqualene to lanosterol, has recently emerged as a possible new target for inhibiting this pathway. Since OSC occurs downstream of HMG-CoA reductase during cholesterol biosynthesis, its inhibition is not likely to be associated with the adverse effects caused by statins. Potent small molecule inhibitors of OSC have been identified. While testing anti-cancer properties of one such inhibitor, RO0488071 [4′-[6-(Allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate]) (RO), we found in short-term assays (24 h) that pharmacological doses very effectively reduced the viability of drug-resistant ovarian tumor cells (SK-OV-3 and OVCAR-3), determined by Sulforhodamine B colorimetric assay. In long-term assays (7 days), nM concentrations of the drug were also effective. Importantly, RO (20-40 mg/kg/day; ip) significantly suppressed tumor xenografts obtained with ovarian cancer cells (SK-OV-3; 336 ± 60 mm³ vs 171 ± 20 mm³) with no toxicity to experimental animals (27 treatments once tumors reached 100 mm³). Interestingly, RO elevated levels of the anti-proliferative protein estrogen receptor β (ERβ)in tumor cells in vitro. Initial studies suggest that a combination of RO and an ERβ agonist, enhances the antiproliferative effects of RO in ovarian cancer cells in an additive or synergistic manner. Our study is the first to demonstrate that disrupting cholesterol biosynthesis by inhibiting OSC is a novel and potent means by which to suppress growth of human ovarian cancer cells. Further studies will determine whether combining RO with ERβ agonists is an even more effective therapeutic approach to treat drug-resistant ovarian cancer in vivo.

Support: Peer-reviewed COR faculty grant from the College of Veterinary Medicine, and by generous funds from the donors to the Ellis Fischel Cancer Center, University of Missouri-Columbia.

#3790

Targeting the one-carbon metabolism protein MTHFD2 for cancer therapy: Exploiting the unique redox status of cancer cells.

Andrea Glasauer, Michael Steckel, Andrea Haegebarth, Marcus Bauser, Luisella Toschi. _Bayer Healthcare Pharmaceuticals, Berlin, Germany_.

Two well-established hallmarks of cancer are the need for nucleotides to support continuous cell proliferation and the requirement for cancer cell redox balance. In that respect cancer cells are characterized by an increased rate of reactive oxygen species (ROS) production which can be toxic to cancer cells. In this sense, cancer cells often display an increased ROS scavenging capacity, through antioxidants and NADPH that prevents ROS levels from reaching cytotoxic levels. Mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a critical component of one-carbon metabolism and promotes the reaction of 5, 10-methylene tetrahydrofolate to 10-formyl tetrahydrofolate which is coupled to purine synthesis and the production of NAD(P)H. In its role, MTHFD2 is required for maintaining nucleotide biosynthesis and cancer cell redox balance. Recent studies have shown that MTHFD2 expression is elevated in many cancers compared to normal tissue and expression is correlated with poor survival in breast cancer. In order to validate MTHFD2 as a potential cancer target we show that genetic knockdown of MTHFD2 led to impaired cancer cell survival and proliferation. In breast cancer cells MTHFD2 inhibition caused a decrease in NADPH, an increase in the levels of oxidized glutathione and also promoted the expression of DNA damage and death markers specifically in cancer cells with high MTHFD2 protein expression. Interestingly, ROS-scavenging antioxidants reversed these phenotypes in the presence of MTHFD2 knockdown. Normal cells and low MTHFD2 expressing breast cancer cells had a higher tolerance for the inhibition of the protein. Based on our data, MTHFD2 inhibition leads to the impairment of cancer cells' antioxidant capacity and ROS-mediated cell death. Therefore, MTHFD2 inhibition may have clinical potential for the treatment of patients with breast cancer, and potentially various other cancers.

#3791

TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents.

Alli Murugesan,1 Philippe Tremblay,2 Ming Han,1 Bithika Ray,1 Tyler Lutes,3 Tony Reiman4. 1 _University of New Brunswick, Saint John, New Brunswick, Canada;_ 2 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 3 _Soricimed Biopharma Inc, Sackville, New Brunswick, Canada;_ 4 _Saint John Regional Hospital, Horizon Health Network, Saint John, New Brunswick, Canada_.

Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts.

#3792

**PAPSS1 (3'-phosphoadenosine 5'-phosphosulfate synthase 1) inhibition sensitizes non-small cell lung cancer to cisplatin treatment** in vivo **.**

Ada W.Y. Leung,1 Chansey J. Veinotte,2 Nicole Melong,2 Ian Backstrom,1 Corinna Warburton,1 Edie Dullaghan,3 Jason N. Berman,2 Marcel B. Bally1. 1 _BC Cancer Research Centre, Vancouver, British Columbia, Canada;_ 2 _IWK Health Centre, Halifax, Nova Scotia, Canada;_ 3 _Centre for Drug Research and Development, Vancouver, British Columbia, Canada_.

We previously reported that 3'-phosphoadenosine-5'-phosphosulfate (PAPS) synthase 1 (PAPSS1), an enzyme that synthesizes the biologically active form of sulfate (PAPS) for all sulfation reactions, is a novel therapeutic target that when suppressed enhances the activity of multiple DNA damaging agents in NSCLC cells. PAPSS1 was the lead hit in a synthetic lethal screen completed using chemotherapy-naive NSCLC cells exposed to the IC10 of cisplatin (CDDP). PAPSS1 silencing was more effective in potentiating CDDP activity than our positive control (BRACA2). Here, we evaluated PAPSS1 as a CDDP-sensitizing target in three different model systems: 3D spheroids, zebrafish xenografts, and a mouse xenograft model. siRNA-transfected A549 cells were seeded in round bottom ultra-low attachment plates for spheroid formation. Spheroids were formed over a period of three days and then treated with CDDP. The spheroids were imaged using the IncuCyte ZOOM® Live Cell Imaging system every 3 hours for 8 days to monitor changes in spheroid size. To evaluate PAPSS1 in zebrafish, transfected A549 cells were microinjected into the yolk sack of zebrafish embryos and then maintained in CDDP-containing media for 48 hours. The human cells were harvested from 20 fish per treatment group and counted to determine the change in cell number as a measure of tumor growth in vivo. For mouse studies, RAG2M mice were inoculated subcutaneously with 5x106 parental, non-targeting shRNA, or shPAPSS1-expressing A549 cells. The mice were treated 7 days later with 3 mg/kg CDDP (IV, Q4Dx3). Tumor size was measured using an electronic caliper and tumor volumes were calculated using the equation (lxw2)/2. PAPSS1-silenced cells formed spheroids of comparable size as the scramble control. CDDP (12.5µM) was effective against both control and PAPSS1-silenced spheroids with a reduction of 31% and 46% in spheroid size, respectively. PAPSS1-knockdown spheroids were significantly more sensitive to CDDP even when added at an 8-fold lower dose (1.56 µM). At this concentration, the control spheroids grew about 37% in size while the size of the PAPSS1-silenced spheroids was reduced by 21% (p<0.0001). In zebrafish, the number of A549 cells was reduced by approximately 50% with the combination of PAPSS1 knockdown and CDDP treatment relative to non-silencing, CDDP-treated controls. In mice, tumor development was significantly delayed in the shPAPSS1 group relative to both parental (p = 0.008) and non-targeting shRNA (p = 0.026) controls following CDDP treatment. Our study demonstrates for the first time that PAPSS1 knockdown enhances CDDP treatment in vivo. To pursue PAPSS1 as a therapeutic target, a small molecule inhibitor screen is warranted. The availability of a small molecule inhibitor will be essential to understand how PAPSS1 inhibition sensitizes cancer cells (but not normal cells) to DNA damaging agents.

#3793

Cell-based, high-throughput screen for small molecule inducers of cell death in HPV-associated head and neck cancers.

Nene N. Kalu, Tuhina Mazumdar, Pan Tong, Li Shen, Jing Wang, Lauren Averett Byers, Faye M. Johnson. _University of Texas MD Anderson Cancer Center, Houston, TX_.

High-risk human papillomavirus (HPV) is an oncogenic virus associated with 90% of cervical cancers, over 60% of oropharyngeal carcinoma cases and over 90% of anogenital cancers. Although HPV-positive cancers are molecularly, clinically and epidemiologically distinct from HPV-negative cancers, there are no specific or targeted therapies for HPV-positive cancers. In order to identify small molecule inhibitors that target HPV-positive cancers, we performed a high throughput drug screen on 24 cervical cancer and head and neck squamous cell carcinoma (HNSCC) cell lines (12 HPV-positive and 12 HPV-negative) to determine if these cell lines display differential sensitivity based on HPV status. HPV-positive cell lines with doubling times of less than 72 hours were selected for the screen. Unsupervised clustering of HNSCC cell lines based on protein expression levels obtained from reverse phase protein array (RPPA) was used to select matched HPV-negative cell lines by Spearman's rank-order correlation. Cytotoxic chemotherapeutics and agents targeting a broad range of processes including cell cycle control and DNA damage response were obtained from commercial vendors and academic collaborators. Drug sensitivity was measured by using nuclear staining to monitor cell death and proliferation after 72h of treatment. The screen of 1062 unique compounds at 6 different concentrations (0-3µM) in 24 cell lines represents 25,448 cell line – drug interactions. To ensure the robustness and reproducibility of the screen, the Z-factor and standard deviation for biological replicates were calculated. A Z-factor greater 0.5 was considered acceptable and the mean standard deviation across all drugs for each cell line was 0.06. Drug sensitivity was determined by calculating IC50 and area under the curve (AUC) values. Overall, HPV-positive HNSCC cell lines showed greater sensitivity to 13 drugs from different classes. Particularly, HPV-positive HNSCC cells were more sensitive to p38 MAPK and B-Raf inhibitors including LY2228820 (p < 0.01), regorafenib (p < 0.01), sorafenib (p < 0.05) and SB590885 (p < 0.05). On the other hand, HPV-negative HNSCC lines showed increased sensitivity to 30 drugs among these, palbociclib (p < 0.01), a CDK 4/6 inhibitor, and ryuvidine (p <0.05) which is reported to inhibit CDK 4. We are currently in the process of performing mechanistic studies and identifying genomic characteristics that influence sensitivity to B-Raf and p38 MAPK inhibitors in HPV-associated cancers using the genomic analyses and drug sensitivity data. Given the difference in clinical outcomes based on HPV status, any validated chemical-genomic interactions we discover will provide strong support for HPV-based treatment plans that may improve efficacy, allow for dose de-escalation and prevent permanent toxicity in patients.

#3794

Novel therapeutic targets in head and neck cancer.

Maria Kondratyev,1 Aleksandra Pesic,2 Stephano Marastoni,2 Troy Ketela,3 Jason Moffat,3 Carl Virtanen,4 Azin Sayad,4 Mikhail Bashkurov,5 Alessandro Dati,5 Laurie Ailles,2 Reidar Grenman,6 Marianne Koritzinsky,2 Brad Wouters2. 1 _UHN, Oakville, Ontario, Canada;_ 2 _UHN, Toronto, Ontario, Canada;_ 3 _Banting & Best Department of Medical Research, Toronto, Ontario, Canada; _4 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 5 _Samuel Lunenfeld Research Institute – Mount Sinai Hospital, Toronto, Ontario, Canada;_ 6 _Dept. of Otorhinolaryngology - Head and Neck Surgery, Turku University and Turku University Hospital, Turku, Finland_.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies.

#3795

Novel and selective MELK kinase inhibitors active in breast cancer cell lines.

Patrizia Carpinelli, Marisa Montemartini, Nadia Amboldi, Dario Ballinari, Sabrina Cribioli, Marina Ciomei, Riccardo Colombo, Stefania Re Depaolini, Nilla Avanzi, Giulia Canevari, Walter Ceccarelli, Helena Posteri, Maria Gabriella Brasca, Daniele Donati, Eduard Rudolf Felder, Antonella Isacchi, Arturo Galvani, Alessia Montagnoli. _Nerviano Medical Sciences, S.r.l., Nerviano, Italy_.

Maternal Embryonic Leucine zipper Kinase (MELK) is a serine-threonine kinase implicated in stem cell renewal, override of cell cycle checkpoints, pre-mRNA splicing and resistance to apoptosis, while MELK gene expression levels correlate inversely with poor prognosis in breast cancer, prostate cancer and glioblastoma patients. Moreover, recent findings underlie the oncogenic role of this kinase in triple negative breast cancer (TNBC), a category of high-grade, invasive tumors which lack expression of estrogen receptor (ER) and progesterone receptor (PR) and HER2 amplification and which is resistant to current cytotoxic and targeted therapies. Furthermore, they are highly heterogeneous with respect to genomic alterations, and common therapeutic targets are lacking, although substantial evidence implicates dysregulated kinase signaling.

Here, we describe the preclinical characterization of novel, potent and selective ATP-competitive MELK kinase inhibitors identified by means of high-throughput screening of the NMS proprietary compound collection. Leading compounds possess biochemical activity against MELK in the nanomolar range with high selectivity against a panel of 60 further kinases representative of the human kinome. Amongst human tumor cell lines tested in 2-dimensional colony outgrowth assays, marked sensitivity was observed in breast cancer cell lines, with sub-micromolar anti-proliferative activity. This effect was accompanied by dose-dependent induction of apoptosis and by modulation of cellular biomarkers, consistent with a MELK-dependent mechanism of action.

Overall, these data provide further evidence that MELK is a promising biological target for the development of novel anticancer therapies.

#3796

Targeting cancer stem cells using ALDH-dependent 5-nitrofuran prodrugs.

Richard Crispin, Nathalie M. Spockeli, Val Brunton, Neil Carragher, Charlie Gourley, Douglas R. Houston, Asier Unciti-Broceta, E. Elizabeth Patton. _University of Edinburgh, Edinburgh, United Kingdom_.

We hypothesise that cancer stem cells with high aldehyde dehydrogenase (ALDHhigh) activity present a new therapeutic target and will be selectively sensitive to 5-nitrofuran pro-drugs.

Cancers are heterogeneous and contain subpopulations of ALDHhigh cells with tumour initiating potential. ALDH enzymes metabolize toxic aldehydes, and are highly expressed in somatic and cancer stem cells (CSCs), although their function in stem cells is not fully understood. In a small molecule screen coupled with target ID, we recently discovered that clinically active 5-nitrofurans (5-NFNs) are substrates of ALDH2 (Zhou et al., 2012). 5-NFNs are a class of pro-drug widely used to treat bacterial and parasitic infections where their relative specificity is driven by nitroreductases, but little is known about the enzymes that bio-activate 5-NFNs in humans. Recent clinical cancer research has found that the 5-NFN nifurtimox has anti-cancer properties and it is currently in Phase 2 clinical trials for neuroblastoma and medulloblastoma (ClinicalTrials.gov Identifier: NCT00601003), however the mechanism underlying this anti-cancer activity is unknown.

In melanoma and other cancers, ALDH1A1 and ALDH1A3 are highly expressed in CSCs. We find that cancer cell lines are highly sensitive to 5-NFNs in cell viability assays, where we use a logarithmic drug dose range and assess cell viability by PrestoBlue™ (e.g. A375 melanoma cells EC50=86nM). To test if ALDH1 isoforms are substrates of 5-NFNs, we preformed in vitro activity assays by monitoring NADH production (λ=340nm). We find that the clinically active 5-NFNs nifuroxazide and nifurtimox, in addition to our own newly synthesised 5-NFNs, are competitive substrates for human ALDH1A3 activity in vitro (p<0.05). Notably, nifuroxazide was not a substrate for ALDH2, suggesting that nifuroxazide may show selectivity toward ALDH1. Consistent with our enzymatic activity assays, we find that 5-NFNs are competitive substrates for ALDH activity in melanoma cells by Aldefluor™ in vivo, with 5-NFNs displaying a prolonged competitive inhibition compared with the known inhibitor, DEAB. Importantly, no-nitro control compounds show no activity toward ALDH enzymes in vitro or in vivo. Computational docking studies reveal that 5-NFNs have the potential to fit within the interior of the ALDH enzymatic cavity and interact with the catalytic cysteine, thereby offering a potential mechanism for 5-NFN bio-activation. Kinetic living-cell imaging (IncuCyte ZOOM®) reveals that ALDH1A3 siRNA transfected A375 cells are protected from 5-NFN toxicity (p>0.05) and apoptosis (DRAQ7™: p<0.0001), demonstrating a functional role for ALDH1A3 in mediating 5-NFN activity in cancer cells.

Our work demonstrates a novel and biologically relevant 5-NFN-ALDH1 interaction in cancer cells. We propose 5-NFNs have the potential to target ALDHhigh CSCs within a tumour and advance the repurposing of clinical 5-NFN pro-drug antibiotics as anti-cancer therapeutics.

#3797

Validation of a novel antibody against fibulin-3 for targeted therapy of glioblastoma.

Mohan Sobhana Nandhu, Prajna Behera, Vivek Bhaskaran, Ennio Antonio Chiocca, Mariano S. Viapiano. _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Glioblastomas (GBM) are highly invasive brain tumors that resist cytotoxic and antiangiogenic therapies, resulting in a high rate of recurrence. Fibulin-3 is an extracellular matrix protein secreted by GBM cells but absent from normal brain. This protein activates Notch signaling to promote tumor invasion and resistance to apoptosis. Conditional knockdown of fibulin-3 reduces GBM growth, invasion and vascularization, thus extending survival. Here we report the initial validation of a function-blocking antibody against this unique GBM target. We first identified the Notch-activating motif of fibulin-3 and developed an immunizing peptide against a key sequence within this motif. A monoclonal antibody generated against this peptide (mAb428.2) showed high affinity for fibulin-3 (Kd 5 nM) and no cross-reactivity against highly homologous fibulin-4 or -5. Antibody mAb428.2 detected fibulin-3 in the stroma and capillaries of human GBM without cross-reactivity against normal brain. The antibody (50-250 μg/ml) blocked the activation of Notch induced by fibulin-3 in GBM cells and caused GBM cell cytotoxicity but had no effects on astrocytes or HEK293 cells. Treatment of mice carrying subcutaneous GBM stem-cell (GSC) derived tumors with mAb428.2 (30 mg/kg IV, q24h x 8 days) resulted in tumor slowdown and extended median survival (47% and 64% in two different GSC models). mAb428.2-treated tumors showed decreased BrdU uptake and increased inflammatory reaction in the tumor stroma (macrophage infiltration and cytokine levels). Mice bearing intracranial tumors did not respond to mAb428.2 when delivered by IV or IP routes, likely due to the inability of the antibody to cross the blood brain barrier. This deficiency was overcome by direct intraparenchymal delivery of the mAb428.2 using chronic infusion (0.3 mg of mAb in 200 μl delivered at 1 μl/h over 7d), which resulted in a 26% increase in median survival. These encouraging results suggest that targeted reagents against fibulin-3 may be of clinical importance for novel combination therapies against GBM.

#3798

A glycan-binding malaria protein provides therapeutic access to cisplatin-resistant bladder cancer.

HTOO ZARNI OO,1 Roland Seiler,1 Sherry S. Lee,1 Davide Tortora,1 Gunjan Kumar,1 Chris Wang,1 Thomas M. Clausen,2 Mette Ø. Agerbæk,2 Jamie R. Rich,3 John S. Babcook,3 Peter C. Black,1 Ali Salanti,2 Mads Daugaard1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _University of Copenhagen, Copenhagen, Denmark;_ 3 _Centre for Drug Research and Development, Vancouver, British Columbia, Canada_.

Bladder cancer is a disease of compelling morbidity and mortality mainly due to its high recurrence rate. Cisplatin-based chemotherapy is an integral part of muscle invasive bladder cancer (MIBC) treatment. Although responses are common, a significant number of patients develop resistance to cisplatin and second line treatment options are not well established. No improvement of the MIBC treatment strategy has been achieved in the past two decades and therefore; new approaches to systemic therapy are urgently needed.

The Plasmodium falciparum host-cell anchor protein VAR2CSA has been evolutionarily optimized to bind distinctly modified chondroitin sulfate (CS) glycosaminoglycan (GAG) chains expressed exclusively in the mammalian placenta. This is the underlying key event behind pregnancy-associated malaria outbreaks in endemic regions of the world. We have recently discovered that placental-type CS chains are re-expressed in the malignant compartment as a secondary oncofetal CS (ofCS) modification (Salanti et al. 2015, Cancer Cell). In the present study, we have analyzed the expression and role of ofCS in cisplatin-resistant bladder cancer and evaluated the potential of a VAR2-drug conjugate (VDC) to target cisplatin-resistant tumors.

Using recombinant VAR2CSA protein (rVAR2) as an ofCS detection reagent, we analyzed a tissue microarray of 52 chemotherapy-naïve MIBC samples, with 36 matched post-chemotherapy cystectomy specimens from patients receiving neoadjuvant gemcitabine/cisplatin. In chemoresistant tumor specimens, of-CS expression was significantly upregulated in residual patient tumors after neoadjuvant chemotherapy (p=0.001) and it was associated with advanced tumor stage (ypT3/4, p=0.005) and poor overall survival (p=0.04). Microarray analysis of primary human bladder tumors and subsequent in situ proximity ligation assay validation identified S100A9 and CD44 as the major ofCS-modified proteoglycans in chemoresistant bladder cancer. Binding of rVAR2 to ofCS chains on bladder cancer cells facilitated rapid internalization of the protein. Moreover, a rVAR2-drug conjugate (VDC) efficiently killed all bladder cancer cells in the low nanomolar IC50 concentration range in vitro and retarded growth of chemoresistant orthotopic MIBC xenografts in vivo.

In summary, we demonstrate how a glycan-binding malaria protein can be utilized to gain therapeutic access to cisplatin-resistant bladder cancer. Thus, we provide a method to target cancer-specific glycan modifications for therapeutic intervention as a second line treatment in MIBC, not responding to cisplatin.

#3799

The PAK4 allosteric modulator (KPT-9274) attenuates the growth of renal cell carcinoma.

Robert H. Weiss,1 Omran Abu Aboud,1 Erkan Baloglu,2 William Senapedis,2 Sharon Shacham2. 1 _UC Davis, Davis, CA;_ 2 _Karyopharm, Newton, MA_.

Renal cell carcinoma (RCC) is an increasingly prevalent cancer type that is frequently asymptomatic on presentation and is associated with poor responses and resistance even to the current targeted therapies. Thus, novel therapeutic approaches to treat this disease are urgently needed. P21-activated kinase 4 (PAK4) is a mediator of filopodia formation and stabilizes β-catenin transcriptional activity, both of which are integral to nephrogenesis and cancer. We hypothesized that inhibitors of the PAK4 signaling pathway would result in salutary effects on RCC. To test this, we evaluated the in vitro response of several human RCC cells and normal kidney proximal epithelial cells (NHKs) to the specific PAK4 Allosteric Modulators (PAMs; KPT-8752 and KPT-9274). 786-O (VHL-mutant RCC) and caki-1 (VHL-wt RCC) cells showed decreases in cell viability (MTT), induction of apoptosis and arrest in G2/M phase when incubated with these inhibitors. These responses were diminished in NHK cells which served as a "normal" control cell line. Target and specific pathway proteins (phospho-PAK4, Phospho-β-catenin, c-Myc and cyclin D1) were reduced after RCC, but not NHK, were incubated with KPT-8752 and KPT-9274. To confirm specificity of the inhibitor to PAK4, all these responses were reproduced in RCC cells using specific PAK4 siRNA. Since ~85% of RCC cases are associated with mutation in vhl we used 786-0 xenograft mouse model to evaluate the clinical candidate KPT-9274. KPT-9274 was orally administered at 100 and 200 mg/kg BIDX5 for 4 weeks, resulting in clear attenuation of tumor growth at both doses. There was no obvious change in the health or weight of any of the animals when compared to vehicle group suggesting manageable tolerability. We are currently evaluating combination therapy and plan to test this inhibitor on a metastatic RCC model. In summary, PAK4 inhibitors show considerable promise as novel treatments of RCC as a single agent and warrants further investigation.

#3800

Novel targets and monoclonal antibodies for cancer therapy.

Matteo Parri,1 Susanna Campagnoli,1 Alberto Grandi,1 Elisa De Camilli,2 Aurelien Lacombe,3 Boquan Jin,4 Serenella Eppenberger-Castori,3 Giuseppe Viale,2 Luigi Terracciano,3 Piero Pileri,1 Renata Maria Grifantini1. 1 _Externautics SpA, Siena, Italy;_ 2 _European Institute of Oncology, Milan, Italy;_ 3 _University Medical Hospital, Basel, Switzerland;_ 4 _The Fourth Military University, X'ian, China_.

The identification of markers targetable by specific mAbs represents a high medical need in cancer therapy. Our objective is to discover novel tumor-associated proteins showing promise as targets for monoclonal antibody (mAb) therapy, and to generate and validate highly specific mAbs for therapeutic applications. The approach we used to discover novel tumor markers is based on a high through-put immune-histochemical (IHC) screening of tumor and normal tissues using collections of murine polyclonal and mAbs raised against recombinant human proteins. In the course of such analysis, we discovered and validated different surface exposed proteins over-expressed in one or more cancers. Here, we report a surface exposed protein mainly over-expressed in ovary and breast cancers. Interestingly, the protein is highly expressed in high grade breast cancers. Approximately 40% of cancers in which the protein is over-expressed belong to the triple negative subtype. In line with IHC data, an expression profile analysis in different cells lines showed that this protein is expressed in ovarian and breast cancer cell lines. In breast cell lines, high expression was found in triple negative cells positive to the androgen receptor. Gene silencing experiments combined to phenotypic analysis, showed that loss of protein expression significantly reduces cell proliferation and invasiveness. Several mAbs able to recognize the target protein on the surface of breast and ovary cancer cell lines have been selected and validated in a number of immunoassays. The specificity of the mAb binding was confirmed by gene silencing and competition assays with peptides encompassing the mAb epitopes. Specific mAbs able to detect the protein in cancer cells in IHC are under validation. These antibodies show limited reactivity on normal human tissues and are negative on PBMC from normal donors. Moreover, these mAbs are efficiently internalized by cancer cells, suggesting that they are amenable to the development of Antibody-Drug-Conjugate. Finally, they efficiently recognize the macaca protein ortholog, thus facilitating future safety studies in non-human primates. Overall, results so far accumulated highlight the potential of this novel tumor-associated protein and available mAbs for the development of targeted therapy against ovarian cancer and triple negative breast cancer. Other promising targets and related monoclonal antibodies will be described during the meeting.

#3801

Pro-chemotherapeutic effects of antibody against extracellular domain of claudin-4 in bladder cancer.

Masaomi Kuwada,1 Yoshitomo Chihara,1 Yi Luo,2 Takamitsu Sasaki,3 Kiyohide Fujimoto,1 Masuo Kondou,4 Hiroki Kuniyasu2. 1 _Urology, Nara Medical University, Kashihara, Japan;_ 2 _Molecular Pathology, Nara Medical University, Kashihara, Japan;_ 3 _Gastroenterological Surgery, Fukuoka University School of Medicine, Fukuoka, Japan;_ 4 _Osaka University, Graduate School of Pharmaceutical Sciences, Osaka, Japan_.

Bladder cancer displays an aggressive phenotype in the muscle-invasive phase, and is associated with a high mortality rate. Therefore, novel molecular therapeutic targets are needed to improve patient survival. A monoclonal antibody against the extracellular domain of the claudin-4 (CLDN4) tight junction protein was established by immunizing rats with a plasmid vector encoding human CLDN4. A hybridoma clone, producing a rat monoclonal antibody recognizing CLDN4 (clone 4D3), was obtained. Immunohistochemistry by using the 4D3 antibody showed that CLDN4 expression was associated with local invasion, nodal metastasis, distant metastasis, and advanced stage in 86 cases of bladder cancer. The 4D3 antibody inhibited growth, invasion, and survival, associated with abrogation of the intratumoral microenvironment; lowered concentrations of epidermal growth factor and vascular endothelial growth factor were found in 3-dimentional cultures of T24 and RT4 cells. In combination with cisplatin therapy, 4D3 enhanced cisplatin cytotoxicity by increasing cellular permeability, leading to increased intracellular cisplatin concentrations. In mouse models of subcutaneous tumors and lung metastasis, 4D3 enhanced tumor growth inhibition, alone and with concurrent cisplatin treatment. The anti-tumor activity of the newly established 4D3 antibody suggests that it may be a powerful tool in CLDN4-targeting therapy, and in combination with chemotherapy.

#3802

Evaluation of microfilament-directed cytochalasins as novel antineoplastic agents.

Matthew Trendowski, Timothy D. Christen, Christopher Acquafondata, Thomas P. Fondy. _Syracuse University, Syracuse, NY_.

Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. Cytochalasins are mycotoxins known to potently inhibit the polymerization of filamentous (F)-actin, and have long been used in vitro to examine the importance of microfilaments in fundamental cellular processes. It has previously been demonstrated that cytochalasins preferentially multinucleate malignant cells, suggesting that the congeners may exert novel antineoplastic activity. We have taken these findings, and further expanded upon their potential importance. We have shown in vitro efficacy with multiple congeners, including cytochalasin B, which potentiates substantial sensitivity to clinically approved antineoplastic agents, X-radiation, and low frequency ultrasound. Further, we have shown that cytochalasin B is more cytotoxic against neoplastic cells selected for high metastatic propensity than are their less invasive counterparts. Interestingly, we have also demonstrated that nine cytochalasin congeners may be substantially less affected by drug efflux due to overexpression of ATP-binding cassette (ABC) transporters than are doxorubicin, paclitaxel, or vinblastine, as demonstrated with SK human ovarian carcinoma cell lines of varying levels of drug resistance. Finally, we have shown that cytochalasins B and D elicit substantial antitumor and antimetastatic activity in numerous preclinical mammalian models of malignancy, suggesting that the novel mechanisms by which these congeners exert antineoplastic activity is worth further examination.

#3803

Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis.

Sierrah Grigsby,1 Jennifer Chase,2 James Ropa,1 Justin Serio,1 Chenxi Shen,1 Martha Larsen,3 Preston Donover,4 Melvin Reichman,4 Andrew Muntean,1 Ivan Maillard,2 Zaneta Nikolovska-Coleska1. 1 _Department of Pathology, University of Michigan, Ann Arbor, MI;_ 2 _Department of Internal Medicine, University of Michigan, Ann Arbor, MI;_ 3 _Center for Chemical Genomics, University of Michigan, Ann Arbor, MI;_ 4 _Chemical Genomics Center, Lankenau Institute for Medical Research, Wynnewood, PA_.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression.

#3804

Structural changes to SN79 result in ligands that stimulate a metabolic function of sigma-2 receptors.

Hilary Elaine Nicholson,1 Walid Alsharif,2 Christopher R. McCurdy,2 Wayne D. Bowen1. 1 _Brown University, Providence, RI;_ 2 _University of Mississippi, University, MS_.

Sigma-2 receptors have been of interest as targets for imaging, diagnosis, and treatment of cancer nearly since their discovery. These membrane-bound proteins are highly upregulated in cancer cells compared to healthy tissue, with further upregulation in rapidly proliferating cancer cells compared to quiescent cells. Activation of sigma-2 receptors by traditional agonists induces programmed cell death. However, this function of sigma-2 receptors is inconsistent with upregulation in cancer cells, which is typically associated with survival benefit. We have recently shown the ability of sigma-2 receptors to induce an independent, non-toxic, metabolically stimulative function that is consistent with survival benefit to both cancer cells and rapidly proliferating normal cells, indicated initially by an increase in MTT reduction. CM764, a novel analog of the sigma-2 antagonist SN79, increased total NADH/NAD+ and ATP levels, decreased basal ROS, and increased VEGF levels via induction of HIF1a expression in SK-N-SH neuroblastoma cells (Nicholson et al., JPET, 2015). We also previously described single-element structural changes to the core structure of SN79 and the effects of these changes on sigma receptor binding affinity and ability to induce cell death (Nicholson et al., Proc. Amer. Assoc. Cancer Res. 75: #2440, 2015). Here, we describe further characterization of the metabolic stimulatory effect induced by binding of members of this series to sigma-2 receptors in SK-N-SH neuroblastoma cells. Substitution of the oxazolone oxygen or methyl ketone moieties of the heterocyclic ring system of the SN79 core structure resulted in ligands that all retained significant binding affinity for sigma-2 receptors. Drastic changes in cell viability effects were observed in response to changes to the methyl ketone position. As previously reported, -NCS substitutions to the methyl ketone position resulted in cytotoxic ligands. By contrast, substitutions of the SN79 methyl ketone moiety with an -NH2, either alone (CM571) or combined with heterocyclic ring oxygen atom substitution for an -NMe group (WA403), resulted in ligands that stimulated reduction of MTT, consistent with the metabolically stimulative phenotype previously described. This phenotype was also observed with -NMe and -S- substitutions to the heterocyclic ring oxygen atom without combined substitution of the methyl ketone group (NF7 and WA504, respectively), and for the nitro-substituted methyl ketone group alone (CM458). All stimulative ligands demonstrated maximal increase in MTT reduction after 24 h incubation at 30 μM, with WA504 exhibiting the greatest stimulation of 161% of the amount of MTT reduced by cells that were not treated. These data further support a dual role for sigma-2 receptor activation, inducing both pro-survival and apoptotic pathways, and highlight the impact of single-element changes to the binding and action of SN79 analogs at sigma-2 receptors.

#3805

A novel anti-CD24 monoclonal antibody, humanized and affinity maturated for targeting gastrointestinal cancers.

Shiran Shapira, Dina Kazanov, Vered Padler Karavani, Itai Benhar, Nadir Arber. _Tel Aviv Univ., Tel Aviv, Israel_.

Background: CD24 is a cell-surface heavily glycosylated GPI-anchored protein. We had previously shown that CD24 in an important player in the multistep process of GI carcinogenesis (Gastro 2006, Clin Can Res 2007, Can Res 2008). The creation of chimeric, humanized or fully human antibodies was a major breakthrough and led to a wave of US FDA-approved antibodies.

Aim: To further improve the efficacy of the humanized anti-CD24 mAb by increasing its binding strength and thereby generating a novel therapy tool for GI malignancy

Methods: From murine to humanized, unarmed and conjugated, small derivatives and full IgG antibodies were recombinantly engineered. The antibody genes were recovered, amplified and cloned into appropriate vectors. Then the vectors were introduced into a host (mammalian and E.coli) and adequate amounts of functional antibody were achieved. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two-steps selection (CDR walking) and by using phage display technique. The binding of the different derivatives were evaluated on full Glycan array in which more than 70 sugar moieties were printed.

Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using live imaging device (Maestro Cri device). High-affinity antibodies were selected and created from combinatorial phage-displayed antibody libraries that contain varying degrees of diversity at randomized positions. A chosen matured clone was isolated and showed higher binding strength (1.8x10-8), compared to the parental murine and humanized Abs. The matured antibody showed selective recognition and binding to the CD24 antigen which proves that the genetic manipulations carried out did not affect its properties. Its stability was enhanced following the maturation process,as well as its pharmacokinetics parameters which showed a long serum half-life.The matured antibody mediates ADCC (antibody-dependent cell cytotoxicity), 75% of target cell lysis was demonstrated. Combined treatment with standard chemotherapy and natural products, such as monoterpenes (terpinen-4-ol), showed significant reduction in cell viability (90% cell death). Binding of anti-CD24 Ab to glycan microarray could not be detected while high binding intensities were observed where the whole CD24 protein was printed, indicating that the antibodies bind to the core peptide and not to its sugar residues.

Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured humanized anti-CD24 mAb proved to be more effective than the murine parental Ab. The long serum half-life is desirable as it would decrease the need for repetitive injections.

#3806

Phosphaplatins are potent inducers of pigment epithelial- derived factor (PEDF).

Louiza Belkacemi, Armando Rivera, Lu Yang, Rathindra N. Bose, Shaun X. Zhang. _University of Houston, Houston, TX_.

Pigment Epithelial Derived Factor (PEDF) is a secreted glycoprotein that exhibits several biological activities, most notably as a potent anti-angiogenic agent as well as a neurotrophic factor. As such, this molecule has potential application as therapeutics for cancer and a variety of neurological diseases. However, prior art methods directed towards enhancing and stabilizing PEDF expression in vivo have met with little success. Therefore, methods that could lead to the stable expression of PEDF in vivo are still needed. We have recently shown that PEDF protein is overexpressed in tumor cells treated with platinum based drugs; phosphaplatins that are platinum-(II) and platinum-(IV) complexes coordinated to a pyrophosphate moiety. The objective of this study is to extend our previous findings by measuring PEDF expression in both malignant cells and normal neuronal tissues after treating them with these compounds. We also investigated the impact of PEDF upregulation on important biological processes such as apoptosis, proliferation and invasion of tumor cells. Our data showed that there was a significant upregulation (over 4-fold) of PEDF in brain of mice treated with phosphoplatins when compared to the control animals. The compounds were found to stimulate PEDF expression in cultured neuronal cells when assessed in vitro (P<0.05 vs. untreated control groups). In tumor cells, phosphoplatins upregulated PEDF expression in a dose-dependent manner in various tumor cell lines. In one particular mouse breast tumor cell line (4T1), the data showed that phosphaplatin doses in the range of 25 to 100 µM stimulated the expression of PEDF at 12 and 24 hrs, which declined thereafter. Remarkably, a subsequent treatment of the tumor cells with phosphaplatins at 24 hrs prevented the decline of PEDF expression, suggesting a direct correlation between phosphaplatins and PEDF overexpression. The upregulation of PEDF in the tumor cells was paralleled by increased apoptosis and decreased cell proliferation and invasion. In summary, phosphaplatins mediated overexpression of PEDF in vivo suggests a potential neuroprotective effect of these compounds. While in tumors, phosphaplatins may exhibit antitumor activity via multiple effects, including anti-angiogenesis, enhanced apoptosis, and decreased proliferation and invasion, predominately through stimulating overexpression of PEDF.

#3807

Histone deacetylase inhibitors repress hypoxia signaling through affecting Hsp90.

Chunzhang Yang, Zhengping Zhuang. _NINDS, Bethesda, MD_.

Histone deacetylase inhibitors (HDACis) are among the most promising recently developed anti-cancer agents. Recent work suggests that HDACis suppress both malignant cell metabolism and progression by interfering with the hypoxia-inducible factor (HIF) signaling, which plays a key role in tumor progression in both actual and falsely perceived cellular hypoxia (pseudohypoxia). However, the precise biochemical mechanism of HDACis repression of HIFs function remains unclear. In this study, we demonstrated that the class-2 histone deacetylase inhibitor SAHA potently inhibits hypoxia signaling via interfering with heat shock protein 90 (Hsp90). HDACis increase in the acetylation of Hsp90, resulting in less HIF-α recognition and nuclear translocation. Accumulated cytoplasmic HIF-α is not transcriptionally active and degradaded through proteosomal pathway. Finally, we demonstrated that SAHA remarkably reduces hypoxia signaling and decreases tumor growth in vivo. These findings provide insight into new possible therapeutic strategies for using HDACis in tumors with known HIF pathway aberrations.

#3808

Novel compound conferring selectivity for cancer cells.

Dilan B. Jaunky, Pat Forgione, Alisa Piekny. _Concordia University, Montreal, Quebec, Canada_.

There is a need to develop novel compounds that can effectively treat a broad range of cancers on their own, or in combination with approved therapies. As personalized medicine is developed, combinatorial approaches will become more common making it crucial to increase the repertoire of available drugs. In the past, drugs often were developed to target a biologically-relevant molecule, but structural limitations, stability and solubility issues, or lack of selectivity have hindered the clinical use of many of these drugs. Our approach was to first find a 'high-quality' compound that is selective for cancer cells, then characterize its mechanism of action and identify its target. High throughput screening (HTS) helped to rapidly identify a subset of compounds with selective toxicity toward MCF-7 (breast cancer) cells. Some of these compounds were further tested for their efficacy in HeLa (cervical cancer) cells, and we found one that selectively causes mitotic arrest at 250 nM in comparison to non-cancerous HFF-1 (foreskin fibroblast) cells. At 200 nM, this compound synergizes with drugs known to affect microtubule dynamics and cause mitotic arrest including Nocodazole and Paclitaxel (currently in use as an anti-cancer drug), causing them to be more effective at lower concentrations. Excitingly, this compound also provides a shielding effect for HFF-1 cells treated with Paclitaxel. To learn the mechanism of action for this compound, we performed immunofluorescence microscopy on HeLa and HFF-1 cells treated with a range of concentrations. We found that the mitotic spindle is improperly organized in HeLa cells at 250 nM, but not in HFF-1 cells. Interestingly, microtubules are completely gone in mitotic HeLa cells and are reduced in mitotic HFF-1 cells treated with >500 nM. Given that this compound synergizes with drugs that directly bind to tubulin subunits to modify their dynamics of assembly and disassembly, and differently affects cancerous vs. healthy cells, we hypothesize that it has a unique mechanism of action and may affect microtubule nucleation. We are continuing to characterize the compound, and will identify its molecular target. In addition, we are generating further iterations to explore the Structure-Activity Relationship, and optimize its efficacy. Our in vitro data shows that our approach has the potential to identify novel compounds with the potential for therapeutic use.

#3809

Protein arginine methyltransferase 1 (PRMT1) is a candidate therapeutic target for breast cancers.

David Silvestre,1 Amélie Brisson,1 Bérengère Marty-Prouvost,1 Mengliang Ye,1 Hélène Bonsang,1 Virginie Maire,1 Damarys Loew,1 David Gentien,1 Didier Meseure,1 Fabien Reyal,1 Gordon C. Tucker,2 Sergio Roman-Roman,1 Thierry Dubois1. 1 _Inst. Curie, Paris, France;_ 2 _Institut de Recherches Servier (IdRS), Croissy sur Seine, France_.

Triple-negative breast cancer (TNBC) represents a subgroup of breast cancers associated with the most aggressive clinical behavior. No targeted therapy is currently available for the treatment of patients with TNBC. In the present study, we found that Protein Arginine Methyltransferase 1 (PRMT1) is overexpressed in TNBC at the mRNA level. At the protein level, PRMT1 is overexpressed in all breast cancer subtypes compared to normal breast tissues. The depletion of PRMT1 using siRNA in breast cancer cell lines triggered apoptosis, reduced cell viability and the ability to form colonies in an anchorage-independent manner. Treatment with a PRMT1 inhibitor blocked proliferation specifically in breast cancer cells, with no effect in normal breast cells. Importantly, the expression of PRMT1 is an indicator of prognosis and response to treatment specifically in TNBC patients. To address the cellular pathways regulated by PRMT1, we identified its protein partners by mass spectrometry and the transcriptomic changes following its depletion in TNBC cell lines. Interestingly, we found that PRMT1 directly activates key oncogenic pathways. Furthermore, we found a synergistic interaction between PRMT1 inhibitors and inhibitors for some of those pathways. We show that PRMT1 activity is necessary for breast cancer cell survival and oncogenic pathway activation. Altogether, our results point out PRMT1 as an emerging target for the treatment of breast cancers.

#3810

ARID1A is a novel HuR target: Implications in breast cancer radiotherapy.

Daniel Andrade,1 James Griffith,1 Meghna Mehta,1 Ranganayaki Muralidharan,1 William Berry,1 Myriam Gorospe,2 Rajagopal Ramesh,1 Anupama Munshi1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _National Institute on Aging, National Institutes of Health, Baltimore, MD_.

ARID1A is part of the chromatin remodeling complex SWI/SNF, which can modulate transcription positively or negatively. The role of ARID1A in cancer is controversial. It is described as a tumor suppressor since its expression is found lost in several types of cancer. However, recent investigations suggest that ARID1A could also be an important target to sensitize cancer cells to chemotherapy and radiation. HuR, an important RNA binding protein, post-transcriptionally regulates specific mRNAs by binding to the 3' untranslated region (UTR) of its target mRNAs. Through this action, HuR regulates the expression of its target genes by facilitating either RNA stability or protein translation, thereby affecting several signaling pathways including cell-cycle, DNA damage response and apoptosis.

In order to identify novel mRNAs regulated by HuR that could be targeted for breast cancer therapy, we performed a ribonucleoprotein immunoprecipitation (RNP-IP) microarray using an HuR antibody on irradiated and non-irradiated samples from triple negative breast cancer cell lines. This assay showed ARID1A to be potentially regulated by HuR. Accordingly, analysis of the AU-rich elements (ARE) database of the University of Vienna identifies three potential HuR binding sites on ARID1A 3'UTR, two of which are highly conserved in 100 vertebrate species. To demonstrate that HuR post-transcriptionally regulates ARID1A, we first evaluated the effect of HuR expression on ARID1A mRNA and protein levels in breast cancer cell lines. Genetic or pharmacological inhibition of HuR led to a decrease in ARID1A mRNA and protein levels. Conversely, HuR forced expression led to an increase in ARID1A levels both at the mRNA and protein levels. We also evaluated the effect of HuR on the stability of ARID1A mRNA. In cells where HuR is genetically inhibited, ARID1A mRNA half-life is dramatically reduced compared to the control. These results demonstrate that HuR positively regulates ARID1A by promoting the stability of ARID1A mRNA. To test whether this regulation is exerted by direct interaction of HuR on ARID1A mRNA, we performed an RNP-IP assay, where we pulldown HuR and analyzed the presence of ARID1A mRNA by RT-PCR. Consistent with our previous results, HuR was found to physically interact with ARID1A, thereby promoting its stabilization. Most importantly, through clonogenic assays, we found that genetic inhibition of either HuR or ARID1A results in radiation sensitivity of TNBC cell lines. Conversely, forced expression of ARID1A promotes radiation resistance.

Our results show that (i) ARID1A is a novel HuR target and that (ii) it can dictate radio resistance in HuR overexpressing cancer cells. ARID1A is known to regulate cell cycle and to promote DNA damage repair, which can contribute to resistance to therapy. Altogether, our findings further support an oncogenic role for ARID1A making it an important target for breast cancer treatment.

#3811

The mechanobiome of pancreatic ductal adenocarcinoma: a new, targetable drug space.

Alexandra Surcel, Qingfeng Zhu, Eric Schiffhauer, Robert A. Anders, Douglas N. Robinson. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality, with 37,000 people dying annually in the US. Existing strategies for treating cancer primarily mainly focus on inhibiting cell growth through specific genetic pathways, which typically either fail to completely abolish the disease or which lead to compensatory regulatory changes, and hence, drug resistance. Targeting cell mechanics remains an under-used approach for drug development.

The direct driver of cell shape change events intrinsic to cellular functions, such migration and invasion, is the mechanobiome - a collection of cytoskeletal proteins which are the final determinants of a cell's mechanical attributes and which lie downstream of KRAS and other regulatory molecules. Targeting, and ultimately, inhibiting these processes is less likely to be subject to compensatory regulation by cancer cells. We determined via western blot analysis and immunohistochemistry of patient-derived samples that key players involved in mechanosensation-myosin IIA, IIC, α-actin-4, and filamin B -show increased expression in cancerous ductal epithelial over normal tissue, while non-mechanosensory, or variable mechanosensory, paralogs (myosin IIB, α-actin-1, and filamin A) show decreased expression. This upregulation of highly mechanosensory proteins has initiated an investigation into the necessity and sufficiency of the myosin II paralogs in PDAC metastasis through overexpression and knockdown of expression, coupled with mechanical assays. In addition to resolving the mechanobiome of PDAC, we have previously shown that targeting of myosin IIC by 4-hydroxyacetophenone affects PDAC mechanics. We are testing the in vivo efficacy of 4-HAP by conducting a murine multi-arm study of metastatically human derived pancreatic cancer cells. Preliminary results suggest a protective effect against the metastasis of human pancreatic cancer cells among mice treated with 4-HAP every other day.

#3812

FAK inhibition resensitizes platinum-resistant serous ovarian cancer.

Lisa M. Bean,1 Florian J. Sulzmaier,1 Isabelle Tancioni,1 Sean Uryu,1 Christine Jean,1 Christine Lawson,1 Xiao Lei Chen,1 Elizabeth G. Kleinschmidt,1 Kristen M. Anderson,1 Edward A. Cordasco,1 Joshua Axelrod,1 Vihren N. Kolev,2 Jonathan A. Pachter,2 Dwayne G. Stupack,1 David D. Schlaepfer1. 1 _UCSD Moores Cancer Center, La Jolla, CA;_ 2 _Verastem, Boston, MA_.

Platinum and taxol administration is standard of care chemotherapy for serous ovarian cancer. Tumor recurrence occurs in a high percentage of patients, and this is directly related to poor overall survival. Ovarian cancer stem cell (CSC) resistance to chemotherapy treatment can give rise to tumor recurrence. Focal adhesion kinase (FAK), an intracellular tyrosine kinase, has been linked to mesothelioma and breast CSC survival. Here, we find that FAK activation is elevated in platinum (CP)-resistant ovarian cancer cells and that FAK tyrosine phosphorylation is increased after CP treatment of CP-sensitive ovarian cancer cells. Nanomolar levels of FAK inhibitor (VS-4718) selectively blocked CP-resistant ovarian carcinoma methylcellulose colony growth via cell cycle inhibition but not apoptosis. Oral VS-4718 administration to mice reduces CP-resistant orthotopic tumor burden with a concomitant decrease in tumor-associated aldehyde dehydrogenase (ALDH) activity, a marker of ovarian CSCs. Residual ovarian tumor cells from VS-4718-treated mice exhibit reduced ALDH activity and secondary tumor initiating capacity. CRISPR-mediated FAK knockout or VS-4718 treated ovarian carcinoma cells exhibit diminished Oct-4 transcription factor and ALDH-1A1 CSC-associated protein marker expression. Co-administration of VS-4718 with CP-taxol chemotherapy reduced CP-resistant tumor burden and exhibited additive inhibitory effects on ovarian carcinoma spheroid growth. As we find that CP activates FAK and that FAK activity sustains ovarian carcinoma CSC phenotypes, our results support the testing of FAK inhibitors in combination with CP to prevent recurrent and chemo-resistant ovarian cancer.

#3813

Potent compounds targeting the human MutT homolog 1 (hMTH1) activity and their cytotoxicity evaluation.

Wenjuan Zhou, Liying Ma, Biao Hu, Lingyu Li, Bo Wang, Hong-Min Liu, Wen Zhao. _Zhengzhou University, Zhengzhou, China_.

Recently, it has been reported that MTH1, a sanitizing protein for the oxidized dNTP pool, plays an important role in cancer-associated damage in the dNTP pool and is required for survival in cancer cells. To study the antitumor mechanism of MTH1 and screen effective anti-cancer drugs, the current study was designed to set up an in vitro expressing and activity evaluation system for MTH1, which will be used for MTH1 specific inhibitor screening. Briefly, the acquired recombinant vector pET-28b-MTH1 was transfected into E.Coli BL21(DE3). The MTH1 protein expression was induced by IPTG and purified by Ni2+ affinity chromatography. Through converting dGTP to dGMP and pyrophosphate by MTH1, the latter of which can then be hydrolyzed to inorganic pyrophosphate by inorganic pyrophosphatase, the MTH1 activity is evaluated through measuring the amount of inorganic pyrophosphate after reacting with malachite green and ammonium molybdate at 630nm. Using this method, we have successfully generated a reliable in vitro evaluation system for MTH1 activity. Furthermore, five brand new series of hybrids with total more than one hundred compounds were virtually designed and synthesized. Their potential MTH1 inhibitory effects were screened based on our system. We found that some of them exhibited moderate to excellent inhibitory activity toward MTH1 with nanomole IC50 values, which also showed significant cytotoxicity on several gastric cancer cell lines by MTT assay (with IC50 below 10μM). On the other hand, our studies in human gastric cancer patients also indicated that MTH1 expression levels were significantly enhanced in these patients' cancer tissues, compared to the para-cancer ones. Our findings suggest that MTH1 may be a potential therapeutic target in gastric cancer. Compounds specifically designed to target MTH1 activity may represent a novel and useful strategy for anti-gastric cancer drug discovery.

*Corresponding author: Wen Zhao, Ph.D, Tel.: 01186-15003898187. Email: zhaowen100@139.com

Funding Sources.

This work was supported by National Natural Science Foundation of China (Project No. 81430085, No. 21372206 and No. 81172937 for H.-M.L.; Project No. 81270270 and No. 81470524 for W.Z.); Ph.D Educational Award from Ministry of Education (No. 20134101130001, for H.-M.L. and No. 20134101110013, for W.Z.); The 2013 Scientific Innovation Talent Award from Department of Education of Henan Province (No. 13HASTIT029, for W.Z.). 

### Oncogenes and Tumor Suppressor Genes as Therapeutic Targets

#3814

Synthetic lethal approaches targeting E-cadherin-deficient cancers.

Augustine Chen,1 Bryony J. Telford,1 Andrew Single,1 Henry Beetham,1 Kaylene J. Simpson,2 Parry Guilford1. 1 _University of Otago, Dunedin, New Zealand;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia_.

E-cadherin is a cellular adhesion protein that is frequently mutated in lobular breast cancer and diffuse gastric cancer. The E-cadherin protein which is encoded by the CDH1 gene has key roles in establishing and maintaining cell polarity and differentiation, the organization of cell migration and architecture and the mediation of signaling through various proliferation and survival pathways. E-cadherin also has a tumor suppressor role and its loss in cancer cells would preclude drug targeting by conventional therapy. To circumvent this, we have taken a synthetic lethal approach to exploit any vulnerability created by the loss of E-cadherin. In the therapeutic setting, synthetic lethality refers to a combination of a mutated gene and a drug targeted at a second gene or protein causing cell death (specifically in cancer cells). To identify the vulnerabilities created by E-cadherin loss, we performed a genome-wide siRNA knockdown screen in isogenic MCF10A cell lines with and without E-cadherin expression. From the functional screen, we identified broad classes of G-protein-coupled receptor (GPCR) signaling proteins and families of cytoskeletal proteins which were highly enriched among the synthetic lethal candidates. Indeed, we identified drug classes with linkages to several of the GPCR and cytoskeletal targets that showed evidence of E-cadherin synthetic lethality when we performed a 4,057 known drug screen. These included certain PI3K inhibitors (PI-103), anti-glucocorticoid (mifepristone), tyrosine kinase inhibitor (saracatinib) and multiple histone deacetylase inhibitors (vorinostat and entinostat). Interestingly, the combination of saracatinib and mifepristone gave a synergistic effect (combination index < 1.0) in targeting E-cadherin-deficient MCF10A cells. These results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both of sporadic and familial lobular breast cancer and diffuse gastric cancer.

#3815

Cyclin-dependent kinase 9: a potential therapeutic target for esophageal adenocarcinoma.

Zhimin Tong,1 Devkumar Chatterjee,2 Defeng Deng,3 Omkara Veeranki,1 Alicia Mejia,1 Jaime Rodriguez,4 Jaffer Ajani,5 Wayne Hofstetter,6 Steven Lin,2 Sushovan Guha,7 Scott Kopetz,5 Sunil Krishnan,2 Dipen Maru1. 1 _Departments of Pathology, MD Anderson Cancer Center, Houston, TX;_ 2 _Department of Radiation Oncology, MD Anderson Cancer Center, Houston, TX;_ 3 _Department of System Biology, MD Anderson Cancer Center, Houston, TX;_ 4 _Department of Translational Molecular Pathology, MD Anderson Cancer Center, Houston, TX;_ 5 _Department of Gastrointestinal Medical Oncology, MD Anderson Cancer Center, Houston, TX;_ 6 _Department of Thoracic & Cardiovascular Surgery, MD Anderson Cancer Center, Houston, TX; _7 _UT Health Science Center and Medical School at Houston, Houston, TX_.

Objective: Major limitation of successful implementation of targeted therapy in esophageal adenocarcinoma (EAC) is low frequency of target specific biomarker alteration and intratumoral heterogeneity. Cyclin dependent kinase 9 (CDK9) is a ubiquitous serine threonine kinase whoseinhibition has been used as a therapeutic option in other solid tumors. However, critical in vitro and in vivo information about the efficacy and mechanism of CDK9 inhibition in EAC is lacking and needs to be generated before a clinical trial is proposed.

Design: We assessed the CDK9 protein expression in EAC cell lines and in patient tissue samples of Barrett's esophagus and EAC. Subsequently, we studied effects of genetic downregulation (shCDK9) in SKGT4 and chemical inhibition of CDK9 by CDK9 inhibitor II, a specific, potent competitive inhibitor of CDK9 (Santa Cruz Biotechnology, CA) on proliferation, apoptosis, and cell cycle of SKGT4, FLO-1 and OE33. In vivo effects of shCDK9 and CDK9 inhibitor II was analyzed in xenografts experiments performed on nude mice after injection of FLO-1 or shCDK9-SKGT 4 and control cells in right flank or abdomen. We also assessed the effects of shCDK9 and CDK9 inhibitor II on expression of MCL-1 and phosphorylation of RNA polymerase II in EAC cells.

Results: Higher expression of CDK9 was observed in all EAC cell lines and in tumor samples of patients with EAC as compared to normal squamous epithelium and Barrett`s Esophagus. shCDK9 in SKGT4 cells induced significant reduction in proliferation, increase in apoptosis and G1 arrest as compared to control cells in vitro. In vivo experiment with SKGT4-shCDK9 demonstrated no tumor growth in 4 of 10 mice and tumor volume was significantly smaller in SKGT4-shCDK9 as compared to control SKGT4 cells (72.89 ± 12.88 mm3 v.270 ± 64.07 mm3, p< 0.01) in other mice. Treatment with CDK9 inhibitor II demonstrated dose dependent reduction in proliferation, increased apoptosis and G1 arrest in all three cell lines in vitro and more than 50% reduction in tumor volume in xenografts as compared to control. CDK9 inhibitor II reduced RNA Pol II phosphorylation and expression of MCL-l and up-regulation of MCL-1 expression induced resistance to CDK9 inhibitor II in all three EAC cell lines. .

Conclusions: CDK9 inhibition has strong anti-EAC properties in vitro and in vivo. These properties along with strong diffuse expression of CDK9 in human EAC support further exploration of CDK9 as a potential therapeutic target for EAC.

#3816

Reactive oxygen species and ERK2 phosphorylation are required for NSC59984 to induce mutant p53 protein degradation and restore p53 signaling.

Shengliang Zhang, Lanlan Zhou, David Dicker, Wafik S. El-Deiry. _Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA_.

Tumor suppressor p53 is mutated in over 50% of human cancers. p53 mutation abolishes wild-type p53 function and also endows mutant p53 with a gain-of-function (GOF) that drives tumor growth and drug resistance. Targeting mutant p53 is an attractive strategy for cancer therapy. We recently reported (Zhang et al., Cancer Research, 2015) a small-molecule NSC59984 with dual capabilities to restore p53 signaling and destabilize mutant p53 protein (depleting GOF). We now demonstrate the role of reactive oxygen species (ROS) and ERK2 in the mechanism of action of NSC59984. We observe a sustained-phosphorylation of ERK2 in cancer cells treated with NSC59984. Blockade of ERK2 rescues mutant p53 from NSC59984-mediated degradation, and inhibits restoration of p53 signaling in mutant p53-expressing cells. Thus, sustained ERK2 phosphorylation is required for NSC59984-induced degradation of mutant p53 protein, and depletion of mutant p53 contributes to the restoration of p53 pathway. NSC59984 induces MDM2 phosphorylation that correlates with ERK2 phosphorylation. The effect of NSC59984 on MDM2 phosphorylation is blocked by U0126 and knockdown of ERK2. NSC59984-mediated mutant p53 protein degradation is inhibited by MDM2 knockdown, and enhanced by MDM2 overexpression. Thus, ERK2-dependent MDM2 phosphorylation is a major determinant of NSC59984-mediated mutant p53 degradation. We investigated the role of ROS in the effect of NSC59984 on ERK2 phosphorylation. ROS is induced by NSC59984 and a decrease in ROS by NAC inhibits NSC59984-induced ERK2 phosphorylation, and mutant p53 protein degradation. Increased ROS by BSO treatment enhances the NSC59984 effect on ERK2 phosphorylation, mutant p53 protein degradation and restoration of p53 signaling. We conclude that ROS is required for NSC59984 to sustain ERK2 phosphorylation, which, in turn, is required for NSC59984-induced mutant p53 protein degradation via MDM2. The combination of NSC59984 and BSO synergistically induces cell death in colorectal cancer cells. ROS and ERK2 are two important factors required for NSC59984 to degrade mutant p53 protein, restore p53 signaling and induce cell death. These results provide a rationale for clinical testing of NSC59984 in tumors with high ROS.

#3817

Recombinant stapled proteins for the treatment of chronic myeloid leukemia.

Benjamin J. Bruno, Sean P. Cornillie, Thomas E. Cheatham, Daniel H. Chou, Carol S. Lim. _University of Utah, Salt Lake City, UT_.

The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML). Current therapies target the tyrosine kinase domain of Bcr-Abl, but due to their non-curative nature, resistance to these drugs is common. Therefore, new treatments are needed for those patients refractive to available therapies. Bcr-Abl homo-oligomerization via its N-terminal coiled coil (CC) domain is required for tyrosine kinase activity. Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a peptidomemetic known as CCmut, delivered as a protein using a cell-penetrating peptide (CPP-CCmut). CPP-CCmut selectively enters leukemic cells and inhibits Bcr-Abl, as seen by Western blot, 7AAD, Annexin V, cell proliferation, and colony forming assays.

This leukemia cell-permeable CPP-CCmut protein domain is our lead compound for CML therapy, but to be translatable to the clinic it must be modified to improve proteolytic stability. For this reason, we created stapled versions of this CPP-CCmut. Stapled peptides are resistant to proteolysis, and are emerging as a viable strategy for disrupting protein-protein interactions in diseases including HIV and cancer. Until recently, peptide stapling required incorporation of unnatural amino acids; therefore these peptides were created via solid-state synthesis. However, the synthesis of the 81 amino acid CPP-CCmut is no trivial matter. The ability to staple a longer sequence such as our protein domain is now possible using thiol-ene coupling. This exciting new chemistry allows for the stapling of recombinant proteins at substituted cysteine residues (instead of stapling substituted unnatural amino acids incorporated into synthetically made peptides). Thiol-ene coupling is used here to staple recombinant CPP-CCmut, and provides a path for translation of our lead compound to the clinic.

Molecular modeling has identified optimal locations for staples on CPP-CCmut, which include 29/36, 36/43, 50/57, 57/64, as well as double staples 29/36-50/57 and 36/43-50/57. These staple locations still allow for dimerization of CPP-CCmut with Bcr-Abl, as indicated by free energy calculations and helicity. The staple locations have also been optimized for maximal coverage from proteolytic degradation.

This work acts as a proof-of-concept that proteins (not just peptides) can be stapled, thus greatly expanding the variety and complexity of stapled proteins for use in the exciting field of targeting protein-protein interfaces to treat disease.

#3818

Therapeutic potential of blocking CCR5 by maraviroc in breast cancer bone metastasis.

Asim Pervaiz,1 Michael Zepp,1 Doaa M. Ali,1 Martin R. Berger,1 Hassan Adwan2. 1 _DKFZ, Heidelberg University, Heidelberg, Germany;_ 2 _German University of Cairo, Cairo, Egypt_.

Introduction:

Bone metastasis is a highly unfavorable condition observed in up to 70% of the patients with breast cancer. Treatment options for this condition are not curative, but palliative, only, which highlights the need for exploring new therapeutic options. In this study, we propose that targeting a chemokine receptor (CCR5), often upregulated in primary and secondary breast cancers, can contribute to a more successful treatment of breast cancer bone metastasis.

Methodology:

Following the blockage of CCR5 by maraviroc, cytotoxic effects were measured by MTT assay, while the migratory effects were determined by migration and scratch healing assays. Apoptosis related activities were investigated by nuclear staining and western blot analysis. Maraviroc mediated cytostatic changes were analyzed by a ready-made Human Cell Cycle Regulation Panel (Roche, Germany). In vivo experiments were performed by implanting MDA-MB-231 cells via the saphenous artery to the left hind limb of male nude rats (RNU strain) for inducing bone metastasis. Treatment with maraviroc was started from 2nd and 7th day of transplantation in two groups of rats (n=6/group) designated as A and B, respectively, and was compared with an untreated control group (n=8 rats).

Results:

CCR5 blockage by maraviroc (concentration > 100µM) induced concentration dependent cytotoxicity in MDA-MB-231 and MCF-7 cells. Maraviroc exposure also showed significant inhibition of migration of the cells, while nuclear staining indicated the condensation/fragmentation of nuclear content. Maraviroc exposure induced significant expression of cleaved caspase 7 and PARP, while real time RT-PCR showed pronounced inhibition of multiple genes (≥2fold) including cyclins, CDKs and their down-stream targets. Treatment of tumor bearing animals, with intra-peritoneal injections of maraviroc (25mg/kg, 3-4 weeks daily), reduced the tumor burden significantly (p < 0.05) in group A (50-75%), while the effects were minimal in group B (<25%). Concomitantly, no signs of toxicity were observed in rats of the two groups.

Conclusion:

CCR5 blockage by maraviroc induces significant anti-neoplastic effects in breast cancer cells. At mechanistic level, these effects included induction of apoptosis and alterations in cell cycle. Considerable inhibition in group A, but minimal inhibition of tumor burden in group B indicates that targeting CCR5 by maraviroc during early stages of breast cancer bone metastasis is a promising treatment option which should be further explored.

#3819

PPAR-delta promotes Wnt/B-catenin-driven colorectal tumorigenesis.

Xiangsheng Zuo, Rui Tian, Shen Gao, Micheline J. Moussalli, Weidong Chen Chen, Ling Wu, Imad Shureiqi. _UT MD Anderson Cancer Center, Houston, TX_.

Introduction Aberrant Wnt/B-catenin signaling activation due to mutations in the adenomatous poliposis coli gene, APC, is a critical event in colorectal cancer (CRC). However, to drive CRC tumorigenesis, aberrant Wnt/B-catenin activation requires additional enhancing regulatory mechanisms. The peroxisome proliferator-activated receptor delta gene (PPARD), which is upregulated in CRC, is a downstream target of aberrant Wnt/B-catenin activation in human colon cancer cells. However, PPARD has also been reported to inhibit intestinal tumorigenesis in mice with germline APCmin mutations. Thus, the mechanistic interaction between PPARD and Wnt/β-catenin remains poorly understood. PPARD is a druggable protein for which agonists and antagonists are being developed. Determining PPARD effects on Wnt/B-catenin activation would define the direction of its therapeutic targeting in cancer and other diseases.

Methods We developed a novel mouse model that simulates PPARD overexpression in CRC by inducing PPARD overexpression in intestinal epithelial cells via a villin promoter (villin-PPARD mice). We bred villin-PPARD mice with mice that have Apc580 mutation in intestinal epithelial cells induced by CDX2-Cre recombinase expression (Apc580mu mice) to generate Apc580mu-PPARD-Gut mice. Apc580mu-PPARD-Gut mice and Apc580mu control mice were monitored for CRC tumorigenesis. We also assessed the effects of PPARD expression modulation on Wnt/B-catenin activation in human colon cancer cell lines.

Results PPARD overexpression in colonic epithelial cells increased CRC tumorigenesis in mice with Apc580 mutation. At 10 weeks of age, 100% of Apc580mu-PPARD-Gut mice but only 60% of Apc580mu mice had tumors of any size, and the mean number of tumors per Apc580mu-PPARD-Gut mouse (1.6 ± 2.5) was significantly higher than that per Apc580mu mouse (0.6 ± 0.25; P = 0.02). Tumors >3.5 mm were present in all Apc580mu-PPARD-Gut mice but only 20% of Apc580mu mice, and the mean number of tumors >3.5 mm per Apc580mu-PPARD-Gut mouse (1.4 ± 2.5) was significantly higher than that per Apc580mu mouse (0.2 ± 0.2; p = 0.005). In mice, colonic epithelial PPARD overexpression increased activated B-catenin protein levels and Wnt/B-catenin target gene mRNA levels. In human colon cancer cells, PPARD increased the levels of activated β-catenin, its nuclear localization and transcriptional activity.

Conclusion Our findings indicate that PPARD augments Wnt/B-catenin signaling to promote CRC tumorigenesis and thus support the targeted inhibition of PPARD to suppress CRC tumorigenesis.

#3820

Epigenetic re-expression of fetal IGF2 as therapeutic target in hepatocellular carcinoma.

Iris Martinez-Quetglas,1 Roser Pinyol,2 Daniel Dauch,3 Sara Torrecilla,2 Victoria Tovar,2 Agrin Moeini,2 Clara Alsinet,2 Anna Portela,4 Augusto Villanueva,1 Manel Esteller,4 Lars Zender,3 Josep M Llovet1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _IDIBAPS-Hospital Clínic de Barcelona, Barcelona, Spain;_ 3 _University of Tuebingen, Tuebingen, Germany;_ 4 _Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain_.

Background and aims

Hepatocellular carcinoma (HCC) is a major health problem. Most patients are diagnosed at advanced stages when the only approved therapy is the multi-kinase inhibitor sorafenib. Consequently, there is a great need for the development of new effective treatments. IGF signaling pathway is aberrantly activated in HCC; however, its contribution to HCC pathogenesis is still unclear. Since IGF2 is overexpressed in HCC, we aimed to elucidate the oncogenic potential and mechanism of dis-regulation of this protein and determine the antitumoral efficacy of molecular abrogation of this ligand by targeted therapies.

Methods

Transcriptomic profiling, miRNAs expression, RNA- and whole exome- sequencing and methylation were analyzed in 228 HCCs with a focus on IGF-pathway. Gene Set Enrichment Analysis and Ingenuity Pathway Analysis were carried out in IGF2-overexpressing tumors. Stable HCC cell lines with knock-down and ectopic overexpression of IGF2 were generated. A chemically-induced mouse model of HCC, and two genetically-engineered mosaic mouse models (GEMM) overexpressing IGF2 specifically in the liver were generated to assess IGF2 oncogenicity in hepatocarcinogenesis. The therapeutic potential of a monoclonal-antibody against IGF-ligands (IGF1/2-mAb) alone or in combination with sorafenib was tested in a xenograft model of HCC.

Results

Here, IGF2-overexpression occurred in 15% of HCC patients as a result of the epigenetic reactivation of IGF2-fetal promoters, mainly through loss of promoters methylation (53% of cases) and deregulation of miR-216b, miR-483-5p and miR-let7-d (35% of cases). Re-expression of IGF2 was associated with a progenitor cell-like, poorly differentiated and aggressive subtype of HCC, and poor prognosis (p<0.0001). In a GEMM model, IGF2-overexpression accelerated HCC progression and reduced survival (p=0.02). Conversely, IGF2-blockage using an IGF1/2-monoclonal antibody (mAb) or specific shRNAs against IGF2 reduced viability and proliferation in vitro (p<0.05), and inhibited IGF-Insulin pathway activation without disturbing insulin metabolism. IGF1/2-mAb delayed tumor growth and increased survival in vivo compared to placebo or sorafenib (p<0.0001), through antiproliferative and antiangiogenic mechanisms.

Conclusions

IGF2 is the first validated epidriver in HCC and has a key role in the hepatocarcinogenic process. These results provide the rationale for testing IGF1/2-mAb in a selected subset of HCC patients.

#3821

Targeted therapy for head and neck squamous cell carcinoma using the novel SMAC-mimetic birinapant.

Adeeb Derakhshan, Danielle Eytan, Grace Snow, Sophie Carlson, Anthony Saleh, Hui Cheng, Stephen Schiltz, Suresh Mohan, Shaleeka Cornelius, Jamie Coupar, Anastasia Sowers, Lydia Hernandez, James Mitchell, Christina Annunziata, Zhong Chen, Carter Van Waes. _National Institutes of Health, Bethesda, MD_.

Head and neck squamous cell carcinoma (HNSCC) is the most prevalent cancer affecting the upper aerodigestive tract, with an annual incidence of 600,000 patients and a five year survival of approximately 60% worldwide. Molecular mechanisms driving the development of HNSCC have recently begun to be discovered, with The Cancer Genome Atlas (TCGA) uncovering the genomic landscape of 279 cases of HNSCC. Alterations in cell death pathways were commonly found in the TCGA analysis, with ~30% of samples harboring 11q13/22 amplifications and overexpression of genes encoding for Fas-associated death domain (FADD) and/or cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2). While overexpression of cIAP1 has been implicated in resistance to cytotoxic therapies, the role of FADD amplification as a target for therapy and in mechanisms of cell death is not well understood. Birinapant is a novel second mitochondria-derived activator of caspases (SMAC)-mimetic that targets and promotes degradation of cIAPs. Its clinical efficacy is currently being investigated in phase II trials of patients with ovarian cancer and leukemia. However, its preclinical and clinical efficacies have not been tested in HNSCC and genomic markers of sensitivity remain to be defined. Here we hypothesized that overexpression of FADD and cIAP1/2 could modulate birinapant sensitivity in HNSCC. To test this hypothesis, we have treated a panel of 11 HPV(-) and 8 HPV(+) HNSCC cell lines with birinapant alone and in combination with death agonists TNFα or TRAIL. UMSCC-46, an HPV(-) cell line which possesses high FADD expression, was the only cell line to reach half maximal inhibitory concentration (IC50) 72 hours post treatment with birinapant alone (IC50 = 10.7 nM); however, 8 of 11 HPV(-) cell lines and all 8 HPV(+) cell lines attained an IC50 (range: 0.1 - 794 nM) when treated with birinapant in combination with either TNFα or TRAIL. We further demonstrated that forced FADD overexpression in a previously resistant cell line (UMSCC-38) led to sensitization when treated with birinapant and TNFα. In vivo, two FADD/cIAP1 overexpressing murine xenograft models of HNSCC, UMSCC-46 and UMSCC-11B, were treated with birinapant at 15 mg/kg or 30 mg/kg every 3 days for a total of 10 treatments. The single modality regimen led to tumor growth inhibition and prolonged host survival. Additionally, combination treatment with birinapant 15 mg/kg and radiation 2Gy/day M-F for 2 weeks synergistically induced TNFα and led to a cure of animals bearing UMSCC-46 xenografts. Mechanistically, birinapant enhanced degradation of cIAP1 and modulated caspase apoptotic or MLKL necroptotic cell death markers in vitro and in vivo. These results suggest that patients harboring genomic alterations in FADD and/or cIAP overexpression may be candidates for treatment with birinapant and radiation.

Supported by NIDCD intramural projects ZIA-DC-000073, and 74.

#3822

Targeting TPX2 arrested cell cycle progression, raised multinuclearity, ploidy, and suppresses tumorigenicity in hepatocellular carcinoma cells.

Hung-Wei Pan,1 Chao-Wen Hsu,2 Yu-Chia Chen,3 Tony Wu3. 1 _Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan;_ 2 _Division of Colorectal Surgery, Department of Surgery, Kaohsiung Veteran General Hospital, Kaohsiung, Taiwan;_ 3 _Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan_.

Background: Hepatocellular carcinoma (HCC) remains one of the most difficult cancers to treat, with chemotherapies being relatively ineffective. Therefore, a better knowledge of molecular hepatocarcinogenesis will provide the opportunity for designed targeted therapy. TPX2 (targeting protein for Xklp2) are overexpressed as a consequence of oncogenic alterations, and they are likely to alter the proper regulation of chromosome segregation in cancer cells. Attacking the machinery that is responsible for mitotic genes involved chromosome instability in cancer cell is one of the most successful strategies of clinical therapy. For this reason, we consider that the targeting TPX2 of investigation in chromosome segregation and its consequential events could provide the novel therapeutic strategies for cancer.

Methods: In human tissue approving, IHC stain addressed the molecular significant of TPX2 in HCC tissue microarray. RNAi was used to knockdown TPX2 protein expression. Clonogenic assay, immunostaining, double thymidine block, image-cytometry analysis, and xenografts mouse model were analyzed its role in tumor cell growth, multinuclearity, ploidy, cell cycle progression and tumorigenicity. RT-PCR, Western blotting were used to analyze anti-cancer mechanism in TPX2 targeting.

Results: In this study, TPX2 protein up-expression was present in 16 (42%) of 38 primary HCCs, and associated with high stage, distant metastatic HCCs and poor prognosis (lower 5-year survival). Knockdown of TPX2 inhibited cancer cells growth and result in down-regulation of cyclin A, cyclin E and CDK2 proteins. Targeting TPX2 caused in tested liver cancer cell lines a rising impaired chromosomal instability resulting in multinuclearity and increased poly-ploidy content fraction cells. An image-cytometry analysis revealed a cell cycle progression arrest after TPX2 inhibition. Correlation between down-regulation protein level of genes related to chromosomal segregation such as securin, seprase, Aurora A, Aurora B, cyclin B1 and cyclin B2 and cell ploidy increasing, indicating mitotic phase progression failure and loss the balance of genomic instability. In vitro tumor spheroids assay and in vivo xenografts mouse model showed a therapeutic opportunity in our findings.

Conclusion: Our findings suggest that targeting TPX2 deregulates chromosome segregation genes, thereby arrested cell cycle progression, raised multinuclearity, ploidy, and suppresses tumorigenicity in liver cancer cells and suggesting TPX2 as a potential therapeutic target for HCC.

Grants support: This work was supported by the grants from Ministry of Science and Technology, Taiwan (MOST103-2320-B-075B-001-MY3 to Hung-Wei Pan ), Kaohsiung Veterans General Hospital Research Program, Taiwan (VGHKS103-G01-4, VGHKS104-G01-1 to Hung-Wei Pan, VGHKS103-040 to Chao-Wen Hsu)

#3823

Staphylococcal nuclease and tudor domain containing 1 (SND1) in development and progression of hepatocellular carcinoma.

Nidhi Jariwala, Devaraja Rajasekaran, Rachel Gredler, Maaged Akiel, Chadia Robertson, Paul Fisher, Arun Sanyal, Devanand Sarkar. _Virginia Commonwealth University, Richmond, VA_.

Staphylococcal nuclease and tudor domain containing 1 (SND1) is identified as an oncogene in hepatocellular carcinoma (HCC) and overexpression of SND1 has been correlated with HCC progression. Here, we present effect of liver specific overexpression of human SND1 in a novel transgenic mouse model. We observe greater tumor load and tumor volume in transgenic mice than wildtype mice, when subjected to chemical carcinogenesis. Approximately 30% of transgenic animals manifest spontaneous tumorigenesis with age. Liver specific expression of cancer stem cell markers such as EpCAM and CD133 as well as inflammatory markers was found to be higher in transgenic mice. SND1 overexpressing hepatocytes show increased activation of insulin and NFκB signaling pathways compared to wildtype hepatocytes. However, no significant differences in liver weight or liver function was noted among transgenic and wildtype animals. Overall, our findings confirm that overexpression of SND1 in vivo plays a vital role in development and progression of HCC. Thus, molecular targeting of SND1 seems to be potential therapeutic intervention for HCC management in patients.

#3824

Targeting MUC1-C oncoprotein inhibits AKT-S6K1-elF4A pathway regulated TIGAR translation in colorectal cancer.

Rehan Ahmad,1 Maroof Alam,2 Omar Al-Obaid,1 Masanori Hasegawa,2 Donald Kufe2. 1 _Colorectal Research Center, College of Medicine, King Saud University, Riyadh, Saudi Arabia;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Colorectal cancer is third most common malignancy and is the second most common cause of cancer-related death. The MUC1 multi-domain oncoprotein is aberrantly overexpressed in colorectal cancer and has been linked to poor outcomes in this disease. However, it is not known if MUC1 is of functional significance to colorectal tumors. Here, we investigate the effects of the MUC1-C inhibitor (GO-203), which disrupts MUC1-C homo-oligomerization, on AKT-mTORC-S6K translation pathway in human colorectal cancer cells. The present study demonstrates that GO-203 treatment was associated with inhibition of AKT-S6K1 signaling in colorectal cancer cells. Targeting MUC1-C with GO-203 downregulates TP53-inducible glycolysis and apoptosis regulator (TIGAR) protein. We show that GO-203-induced suppression of TIGAR is mediated by inhibition of AKT and the downstream mTOR pathway. The results also demonstrate that targeting MUC1-C blocks eIF4A cap-dependent translation of TIGAR. TIGAR promotes the shunting of glycolytic intermediates into the pentose phosphate pathway and thus is of importance for maintaining redox balance. In concert with these results, GO-203-induced suppression of TIGAR was associated with decreases glutathione levels. GO-203 treatment also resulted increases in reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential. Consistent with these results, GO-203 inhibited the growth of colon cancer cells in vitro and xenografts in nude mice. These findings thus indicate that MUC1-C is a potential target for the treatment of colorectal cancer.

#3825

Disrupting p-STAT3-mediated VEGF-A paracrine and autocrine feed-forward loops by targeting SHP-1 suppresses triple negative breast cancer cell metastasis.

Jung-Chen Su,1 Hao-Chieh Chiu,1 Chun-Yu Liu,2 Kuen-Feng Chen,3 Chung-Wai Shiau4. 1 _Department of Clinical Laboratory Sciences and Medical Biotechnology, National Taiwan University, Taipei, Taiwan, Taipei, Taiwan;_ 2 _Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan, Taipei, Taiwan;_ 3 _National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan, Taipei, Taiwan;_ 4 _National Yang-Ming Univ., Taipei, Taiwan_.

Patients with metastatic triple-negative breast cancer (TNBC) relapse quickly, representing an unmet need for effective therapy. Regorafenib is a multi-receptor tyrosine kinase (RTK) inhibitor targeting tumor angiogenesis, oncogenesis and the tumor microenvironment and has been approved for metastatic colorectal cancer and advanced gastrointestinal stromal tumor. Recent studies suggest regorafenib also acts as a SHP-1 phosphatase agonist. Here, we investigated the potential of regorafenib to suppress metastasis of TNBC cells by mitigating autocrine and paracrine feed-forward loops of p-STAT3-mediated VEGF-A signaling through targeting the SHP-1/p-STAT3/VEGF axis. We found a significant correlation between cancer cell migration and SHP-1/p-STAT3/VEGF-A expression in a panel of human TNBC cells. Clinically VEGF-A expression is associated with disease-free and distant metastasis-free survival, independent of tumor stage. Regorafenib induced significant anti-migratory effects, in association with downregulation of p-STAT3 and VEGF-A. Overexpression and knockdown experiments validated the indispensable roles of SHP-1, p-STAT3 and VEGF-A in regorafenib-induced anti-migration. To exclude the role of RTK inhibition in regorafenib-induced inhibition of migration, we synthesized a regorafenib derivative, SC-78, that had minimal effect on VEGFR2 and PDGFR kinase inhibition, while having more potent effects on SHP-1/p-STAT3/VEGF-A signaling. SC-78 demonstrated superior in vitro anti-migration to regorafenib. Furthermore, VEGF-A dependent autocrine/paracrine loops were disrupted by regorafenib and SC-78. SC-78 revealed more potent in vivo efficacy than regorafenib in both xenograft and orthotopic models, as well as in a tail vein metastatic model. This study implies that the more potent SC-78 may be a promising lead for suppressing metastasis of TNBC, and the SHP-1/p-STAT3/VEGF axis is a potential therapeutic target for metastatic TNBC.

#3826

Phenethyl isothiocyanate hampers growth and progression of HER2-positive breast cancers.

Ada Koschorke,1 Lorenzo Castagnoli,1 Gaia C. Ghedini,1 Tiziana Triulzi,1 Claudia Chiodoni,1 Rossella Canese,2 Egidio Iorio,2 Manuela Iezzi,3 Patrizia Nanni,4 Elda Tagliabue,1 Serenella M. Pupa1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _Istituto Superiore di Sanità, Rome, Italy;_ 3 _Aging Research Centre, G. D'Annunzio University, Chieti, Italy;_ 4 _Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy_.

Background: Almost 90% of all breast cancers (BCs) express a splice variant of HER2 lacking exon 16 (d16HER2), which promotes the generation of stable and active d16HER2 homodimers on the tumor cell surface. Comparison of the tumorigenic potential of human d16HER2 and the full-length HER2 (WTHER2) in transgenic mice revealed a significantly shorter latency period and a higher incidence of tumors in the d16HER2 line, suggesting enrichment of this variant in HER2+ BC stem cells. Based on reports that cruciferous vegetable-derived compounds such as isothiocyanates (ITCs) mediate strong anti-tumorigenic effects on numerous oncotypes and target BC stem cells, we tested the effect of the phenethyl ITC (PEITC), alone or together with Trastuzumab (T), which reportedly targets HER2+ BC stem cells, on d16HER2-positive tumor growth and progression in transgenic mice.

Methods: The percentage of tumor cells expressing high levels of CD29 and CD24 and low levels of SCA-1 (CD29high/CD24+/SCA-1low) murine stem cell markers was evaluated by multiparametric flow cytometry in cell models derived from spontaneous d16HER2 and WTHER2 transgenic lesions. Mammosphere forming efficiency (MFE%) was calculated in d16HER2- and WTHER2-positive tumor cells. The therapeutic activity of PEITC, T and their combination was assessed in vitro based on their ability to decrease the MFE% and the expression of stem cell markers in d16HER2- vs. WTHER2-positive tumor cells, and in vivo in d16HER2 transgenic mice. Analyses by western blot, histology/immunohistochemistry and magnetic resonance spectroscopy (MRS) are ongoing to assess apoptosis, necrosis and metabolomic profiles of treated tumors vs. controls.

Results: d16HER2 tumor cells showed a significantly higher MFE% and higher expression of stem cell markers than did WTHER2 tumor cells. PEITC and T significantly reduced MFE% as compared to controls (p=0.0342 and p=0.0083, respectively), and their combination exerted significantly stronger inhibition than T (p=0.001) or PEITC (p=0.0003) alone only in d16HER2 cells. The percentage of tumor cells expressing CD29high/CD24+/SCA-1low in d16HER2 tumor cells after treatment with T, PEITC or both was also reduced. Whereas PEITC monotherapy did not significantly reduce mammary tumor incidence compared to controls, while T effectively suppressed d16HER2-driven tumor growth (p=0.0002), the combination of PEITC and T significantly heightened the impairment of mammary lesion development compared to T alone (p=0.0046). Ex vivo metabolomic analyses by MRS of d16HER2-positive murine and human BC cell lines showed an increase in glycolytic pathway as compared to the respective WTHER2 cells.

Conclusions: Our results provide in vivo evidence that the combination of T and PEITC mediates a significantly greater anti-tumor effect than either alone, most likely targeting BC stem cells.

Supported by the Italian Ministry of Health and AIRC.

#3827

Preclinical characterization of DCR-BCAT as a component of combination therapy.

Shanthi Ganesh, Wendy Cyr, Martin Koser, Girish Chopda, Edmond Chipumuro, Zakir Siddiquee, Kevin Craig, Serena Shui, Dongyu Chen, Cheng Lai, Hank Dudek, Weimin Wang, Bob Brown, Marc Abrams. _Dicerna Pharmaceuticals, Inc, Cambridge, MA_.

Dicer-substrate siRNAs (DsiRNAs) are a potent class of RNA interference (RNAi) triggers capable of silencing any expressed mRNA with high specificity. DCR-MYC, a first-in-class DsiRNA targeting the MYC oncogene is currently in Phase 1b/II clinical trials [ASCO 2015, abstract 11006]. DCR-BCAT is an advanced preclinical development candidate that targets CTNNB1, the gene which encodes β-catenin. The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). Robust preclinical and genetic evidence strongly suggests that inhibiting β-catenin function would yield broad therapeutic benefit in oncology, but efforts to target it using conventional drug modalities have been unsuccessful to-date. We have previously reported extensive preclinical pharmacology for DCR-BCAT in mouse tumor models of diverse origin. Here, we explore DCR-BCAT as a monotherapy and in combination with both standard-of-care and experimental therapeutics. Interestingly, when mice bearing HCC tumors were treated with a combination of CTNNB1 and MYC DsiRNAs, the antitumor efficacy was additive or synergistic relative to either single agent alone. Additionally, since up to 50% of colorectal tumors have both activated Wnt signaling and mutant KRAS, we also explored combination therapy of DCR-BCAT and FDA-approved MEK inhibitors. A major advantage of DCR-BCAT is that the improved nanoparticle formulation is more selective for siRNA delivery to tumors over liver and other normal tissues. These data support continued development of DCR-BCAT as a first-in-class RNAi therapeutic, and highlight the potential for use in combination with other promising agents.

#3828

Genomic and pathway analyses provide insights into the mechanisms underlying malignant peripheral nerve sheath tumors.

Elliot Kahen, Andrew S. Brohl, Diana X. Yu, Justine Clark, Christopher L. Cubitt, Daniel M. Sullivan, Damon R. Reed. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Background: Dysfunction of the NF1 gene, which occurs in patients with neurofibromatosis type 1, is associated with an increased risk of developing Malignant Peripheral Neural Sheath Tumor (MPNST). The neurofibromin protein is a negative regulator of RAS function and 1 in 10 neurofibromatosis type 1 patients develop MPNST in their young adult years. Effective chemotherapy treatments for MPNST are not available and the prognosis for unresectable or metastatic tumors is poor. Genome-wide sequencing of human MPNSTs has identified several key genes in MPNST including NF1, CDKN2A, TP53, SUZ12, and EED. Interestingly, high-throughput drug screening of FDA-approved chemotherapy agents demonstrated differential drug sensitivities amongst a panel of MPNST cell lines. Further elucidation of the molecular mechanisms underlying therapeutic responses in MPNST will allow the development of treatments catered to tumor genetics and may improve understanding of other RAS-associated diseases.

Methods: We used a combination of genomics, high-throughput drug screening, and pathway analysis to better understand how the oncogenic pathways important for MPNST development may dictate treatment outcomes. We performed whole exome sequencing of tumors from 5 MPNST patients as well as 4 established MPNST cell lines (SNF02.2, SNF10.1, SNF94.3, SNF96.2). In the cell lines, we additionally performed RNA sequencing and high-density SNP arrays. With the cell line models, we used our high-throughput screening platform to assess the efficacy of 54 agents of interest alone or in combination at clinically relevant doses, guided by published pharmacokinetics. The cell lines were further subjected to pathway analysis when subject to specific blockades downstream of RAS at the protein level with the use of immunoblotting and antibody arrays.

Results: Our sequencing results indicate alterations in NF1, CDKN2A, TP53, SUZ12, and EED are present in the tumors and cell lines studied. Using cell line models, the predicted genetic alterations resulted in striking and consistent effects on expression at both the RNA and protein level. Within our panel of four cell lines, there was a broad spectrum of NF1 activity, ranging from complete loss to normal expression of full-length transcript. Interestingly, we found that NF1 exhibited a spectrum of loss from unexpressed, to partial expression, and normal expression of the full-length transcript. We found that drug sensitivity to targets downstream of RAS is correlated with the degree of NF1 expression. These findings suggest promising opportunities for developing combination clinical trials that take into account a patients' genetics in order to improve treatment outcomes.

#3829

Preclinical development of a combination inhibitor strategy to block K-Ras effector signaling and autophagy for pancreatic cancer treatment.

Kirsten L. Bryant,1 Tikvah K. Hayes,1 Daniel Zeitouni,1 Samuel D. George,1 Swapnil Kher,1 Ahmed A. Samatar,2 Kris C. Wood,3 Alec C. Kimmelman,4 Channing J. Der1. 1 _Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Theramet Biosciences, Princeton Junction, NJ;_ 3 _Duke University, Durham, NC;_ 4 _Dana Farber Cancer Institute, Harvard Medical School, Boston, MA_.

Pancreatic cancer is the 4th leading cause of cancer deaths in the US and the 5-year survival rate is a dismal 6%. The KRAS oncogene is mutated in ~95% of pancreatic ductal adenocarcinoma (PDAC) and targeting KRAS is one of four key priorities for pancreatic cancer research, as outlined by the NCI. We hypothesize that inhibition of K-Ras effector signaling is the most promising approach to achieve this goal, and that concurrent inhibition of mutant KRAS-mediated metabolic reprogramming, in particular upregulated autophagy, is needed to disrupt resistance mechanisms that render PDAC cells unaffected by inhibition of either pathway alone. Our preliminary data show that PDAC cells increase levels of autophagic signaling upon KRAS silencing as a compensatory mechanism for the cell to maintain anabolic processes, particularly due to the loss of upregulated glycolytic signaling and macropincytotic scavenging. Furthermore, we show that ERK inhibitor treatment, just like KRAS silencing, increases autophagic flux in PDAC cells. Additionally, we have demonstrated in vitro that chloroquine (CQ), an inhibitor of autophagy, synergistically enhances the anti-proliferative capacity of two different ERK inhibitors, one of which is under clinical evaluation. Finally, we show that signaling pathways that promote resistance to ERK inhibitor treatment alone are incapable of promoting resistance to the combination treatment with CQ. Overall, our studies support the further clinical evaluation of duel ERK and autophagy inhibition as a treatment for PDAC.

#3830

Synthesis and anticancer activity of novel prodigiosin analogs.

Xiaobing Tian, Shengliang Zhang, Wafik S. El-Deiry. _Fox Chase Cancer Center, Philadelphia, PA_.

We recently reported that Prodigiosin is a potent p53 pathway restoring small molecule that acts through p73 and by interfering with p73:mutant p53 protein interaction (Hong et al., Cancer Research, 2014). Prodigiosin is the parent member of the tripyrrole alkaloid family of natural products that shows potent anti-cancer activity against tumors with mutated p53. To improve pharmacological and medicinal properties of Prodigiosin including p73 induction and restoration of the p53 pathway in tumors with mutated p53, two new series of analogs were synthesized. We introduced carboxylic group substituents either directly on the C-ring or on the side-chain of the C-ring. Carboxylic group allowed for further derivatization of Prodigiosin. A number of new analogs have been prepared and tested for their anticancer activity. Preliminary biological activity assay showed that new compounds inhibited cancer cell proliferation from 0.1 μM to 10 μM both in p53 mutated SW480 and wild-type HCT116 colorectal cancer cell lines. Some of the newly synthesized compounds can induce p53 transcriptional reporter activity in p53 mutated SW480 colorectal cancer cells. Our ongoing experiments are focused on the signaling mechanism of apoptosis induced by the new analogs, identification of molecular targets of the compounds, and in vivo studies of Prodigiosin analogues as single agents or in combination with chemotherapy or targeted therapy.

#3831

Oncogenic BRAF and concomitant RAS or PTEN/PI3K mutations determine together the response to RAS/RAF/PI3K signaling network inhibition.

Tamas M. Garay,1 Eszter Molnar,2 Dominika Rittler,2 Walter Berger,3 Balazs Döme,4 Jozsef Timar,2 Balazs Hegedus1. 1 _Tumor Progression Research Group, Hungarian Academy of Sciences-Semmelweis University, Budapest, Hungary;_ 2 _Semmelweis University, 2nd Department of Pathology, Budapest, Hungary;_ 3 _Institute of Cancer Research, Medical University of Vienna, Wien, Austria;_ 4 _National Korányi Institute of TB and Pulmonology, Budapest, Hungary_.

Background: BRAF is key component in the growth factor signaling cascade that is often impaired in tumors. Although oncogenic BRAF mutation is considered to be the driver mutation in a variety of malignancies, the response to RAS/RAF/PI3K signaling network inhibition is not uniform among BRAF mutant tumors. Accordingly, we investigated the in vitro effect of RAS/RAF/PI3K signaling network inhibitors (panRAF, mutant BRAF specific, MEK and PI3K/mTOR inhibitors) in relation with BRAF concomitant RAS or PTEN/PI3K mutations in a panel of human cancer cell lines.

Methods: Altogether 12 human cancer cell lines were investigated in vitro to assess the effect of inhibitors acting at the different levels of the RAS/RAF signaling network. The seven melanoma, two colon, two lung and one breast cancer cell lines were divided into 3 groups according to their mutational status: BRAF-mutant with no PTEN/PI3K/RAS mutations (A375, WM35, SK-MEL-28, CRL5885), BRAF + PTEN-PI3K (A2058, WM239, HT29, SW1417) and BRAF + RAS double mutant (WM3629, WM3670, MDA-MB-231, CRL5922). Effect of inhibitors on proliferation, clonogenic potential and migration were investigated SRB and clonogenic assays and videomicroscopy measurements, respectively. Both single agent and combination treatments were applied.

Results: In single agent treatments panRAF and PI3K/mTOR inhibitors did not show mutation dependent differences in proliferation, however, mutant-BRAF and MEK specific inhibitors were more effective in cells without concomitant PTEN/PI3K or RAS mutations. Interestingly, not only the combination of BRAF and MEK inhibitors but the combination of PI3K/mTOR and MEK inhibition showed additive inhibitory effect on proliferation in all subgroups. Importantly, a distinct pattern emerged for the mutational subgroup specific migration inhibitory effects in comparison to the inhibition of proliferation.

Conclusion: BRAF mutant cells carrying various concomitant PTEN/PI3K/RAS alterations display distinct response to RAS/RAF/PI3K network inhibition in terms of proliferation as well as migration. Our data suggests that combination treatments acting concurrently on different targets of the RAS/RAF/PI3K network can be more effective as single therapies.

#3832

Combination use of natural Hsp90 inhibitors to reverse BRCA1-mediated platinum resistance in triple negative breast cancer.

Kelli E. Valdez, Prabhu Ramamoorthy, Shrikant Anant, Roy Jensen. _University of Kansas Medical Center, Kansas City, KS_.

Background: Triple-negative breast cancers (TNBCs) are highly associated with an aggressive clinical course and poor prognosis, largely due to resistance to chemotherapy. Platinum-based therapies act by causing DNA damage, which in the absence of a functional DNA repair system, leads to cancer cell death. BRCA1, a well-studied tumor suppressor, is critical for repairing DNA damage by homologous recombination. This ability makes BRCA1 an attractive therapeutic target for the re-sensitization of cancer cells to platinum chemotherapies. Recently, we have demonstrated that the chaperone protein heat shock protein 90 (HSP90) is required for BRCA1 stability. While targeted elimination of HSP90s results in a loss of BRCA1, current pharmacological inhibitors also exhibit toxicity unrelated to HSP90. We have found that the natural compounds gedunin, triptolide, and celastrol can modulate HSP90 expression without negative side effects. We believe that combinatory use of these non-toxic compounds can further reduce BRCA1 expression beyond their individual effect to overcome BRCA1-mediated resistance to platinum-based chemotherapies.

Study Design: The goal of these experiments is to identify a combination of gedunin, celastrol, and triptolide to maximize BRCA1 degradation (via inhibition of HSP90), impairing the DNA repair system, and allowing carboplatin to effectively eradicate TNBC cells. Using in vitro techniques we will identify the most potent combination of gedunin, celastrol and triptolide to maximize BRCA1 degradation. We will then confirm that this is a HSP90 mediate event that results in the impairment of DNA repair. Using in vivo models of breast cancer we will demonstrate that natural compounds will sensitize BRCA-expressing TNBC cells to carboplatin, inhibiting breast cancer progression and tumor growth.

Results: Using Western analysis, we have found that gedunin, celastrol and triptolide each can attenuate HSP90 activity, leading to BRCA1 degradation in BT-20 and MDA-MB-231 TNBC cells. Additionally, we have observed that gedunin, celastrol, and triptolide can each prevent formation of radiation-induced DNA repair complex, as evident by γ-H2AX staining, in HCC1937BRCA1 TNBC cells. Once we identify the most potent combination of our natural compounds, TNBC xenografts with be created and treated with the combination and carboplatin to demonstrate sensitivity to platinum treatment and inhibition of breast cancer progression.

Significance: In order to increase survival rates in TNBC patients, it is essential that resistance to chemotherapy be overcome. Successful completion of these studies demonstrating that natural compounds can act as HSP90 inhibitors to reverse platinum resistance without added toxicity would facilitate their rapid incorporation into clinical trials and provide a critically needed therapy for women with TNBC.

#3833

Restoration of wildtype structure and function of mutant p53 by thiosemicarbozones using a novel zinc metallochaperone based mechanism.

Xin Yu,1 Adam R. Blanden,2 Ashley T. Tsang,3 Saif Zaman,4 John Gelleran,5 David Augeri,5 S. David Kimball,5 Stewart N. Loh,2 Darren R. Carpizo1. 1 _Rutgers The Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _SUNY Update Medical Umiversity, Syracuse, NY;_ 3 _Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ;_ 4 _Rutgers University, Piscataway, NJ;_ 5 _Rutgers Ernest Mario School of Pharmacy, Piscataway, NJ_.

NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure and function to the most common p53 missense mutant (p53-R175H). We identified that ZMC1 functions as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding of p53 protein, and increasing cellular reactive oxygen species to transactivate the newly conformed p53-R175H (via post-translational modifications). ZMC1 was identified from an in silico screen of the NCI anti-cancer drug screen along with two other thiosemicarbazones (TSCs), NSC319725 and NSC328784. We investigated these TSCs to determine if they could reactivate mutant p53 using a zinc metallochaperone mechanism. We found that indeed these compounds could reactivate mutant p53 by functioning as zinc metallochaperones. In distinction, Triapine the only TSC in clinical development, does not function as a zinc metallochaperone and is not a mutant p53 reactivator.

#3834

scFv antibodies against EphA2 receptor as a therapeutic strategy for AML.

Michael Albert,1 Mayumi Sugita,1 Hongliang Zong,1 Juha Himanen,2 Monica L. Guzman1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

The ephrin (Eph) receptors have been sought as therapeutic targets in liquid and solid tumors. Eph receptors are a subcategory of receptor tyrosine kinases and play vital parts in cell survival and function. Recently, monoclonal antibodies have been designed to bind to the ligand-binding cavity, that block ephrin to bind to ephrin type-A receptor 2 (EphA2). The potential clinical importance has been demonstrated in lymphomas, and the ability of the antibody to reduce proliferation and induce apoptosis (Goldgur et. al 2014).

Acute myelogenous leukemia (AML) is a disease with dismal outcomes and new therapeutic targets have been explored. EphA2 has been described to be critical for the survival in EphA2-positive mouse leukemia models and not essential for normal hematopoiesis. Thus, we examined cell surface expression of EphA2 in peripheral blood, and bone marrow samples from AML patients.

We screened 30 primary AML samples and 5 normal samples and assessed their expression of EphA2 using flow cytometry using the lymphoma cell line (Raji) as a positive control, as reported to express EphA2. The EphA2 expression was evaluated under culture conditions as well as without culture. Prior to culture, 22 (73.3%) of 30 samples were EphA2 positive in more than 90% of blasts. The mean cell surface expression of EphA2 in blasts was 95.4% (range: 64.4-99.7%) with a mean fluorescence intensity (MFI) of 201.5 (range: 18.4-675.0) with a MFI of 4.4 for isotype control. Interestingly, we observed 13.3% increase in EphA2 expression post-culture, bringing the proportion of EphA2-high samples from 73.3% to 86.6% of total samples. In addition to the increase of EphA2-high population, the average MFI of this cohort was also increased by 47.6% post-culture relative to pre-culture. Furthermore, we saw a more significant increase in the CD34+ population and little or no change in the CD34- population. These data shows that many AML express EphA2, and that culturing with cytokines can induce robust EphA2 expression.

After validating EphA2 expression in leukemia cells, we have tested a set of single-chain antibodies (scFv) against EphA2 that have shown activity against Raji cells (Goldgur et. al 2014). We tested two single-chain variable fragments (G2 or D2) on samples with low or high expression of EphA2. The samples were incubated in the serum free IMDM medium supplemented with cytokines. The scFv antibodies were tested at 0.5ug and 1.0ug and evaluated after 24hr- or 72hr-culture. We found that exposure to D2 resulted in a significant decrease in cell number after 72 hr-culture, 52.6% and 99.3% relative to vehicle control treatment for 0.5ug and 1.0ug respectively in EphA2-high AML cells. These data suggests that the scFv antibodies against EphA2 are lethal to AML cells that express high level of EphA2.

Taken together, our data suggests that more than 70% of AML cells highly express EphA2 and can be targeted using scFv antibodies specific to EphA2.

#3835

An anti-MET IgG2 monoclonal antibody degrades both wild-type and exon 14 mutant MET receptor tyrosine kinase through a novel exon 14-independent mechanism and inhibits tumor growth.

Brett S. Robinson, Sreekala Mandiyan, Gerald McMahon, Yan Yang. _Kolltan Pharmaceuticals, Inc., New Haven, CT_.

MET gene amplification and exon 14 mutations have been reported and implicated as oncogenic drivers in several types of cancer representing approximately 5-7% of NSCLC, 4-10% of gastric cancer, and 5-10% of colon cancers. Preclinical research and clinical data derived from patients with tumors containing MET gene amplification and exon 14 mutations showed significant growth inhibition and meaningful clinical benefit after treatment with MET-targeting tyrosine kinase inhibitors (TKIs), supporting the hypothesis that MET gene amplification and exon 14 mutations are actionable biomarkers. We have identified several anti-MET monoclonal antibodies that block MET-dependent signaling and tumor growth through: (1) binding to the Sema/PSI domain, (2) preventing HGF interaction with MET and (3) inducing receptor ubiqutination and degradation. One of the anti-MET antibodies, KTN0073, induces potent degradation of oncogenic exon 14 mutant MET, which has been reported to propagate prolonged MET signaling due to a defect in receptor degradation. Our data suggest that KTN0073 engages a pathway different from HGF-induced receptor degradation and protease-mediated shedding, which is independent of the 47 amino acids that are encoded by exon 14 in the intracellular juxtamembrane region and only partially inhibited by the TKI crizotinib. In summary, we report the use of an anti-MET monoclonal antibody to elucidate an exon 14-independent mechanism of MET degradation that could provide a novel way to design anti-MET therapeutics to inhibit growth of human tumors that are driven by HGF, MET amplification and exon 14 mutations. Based on this work, KTN0073-IgG2 anti-MET antibody has been humanized for further preclinical development to explore its potential as a therapeutic for the treatment of human MET expressing cancers.

#3836

Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile.

Cathy Chang, Gerhard Frey, William J. Boyle, Leslie L. Sharp, Jay M. Short. _BioAtla, San Diego, CA_.

Axl is a TAM family receptor tyrosine kinase that has been implicated in the pathogenesis of many cancer types. The high level of expression on the cancer cell surface has made it an attractive target for antibody therapeutics. However, Axl is expressed on many normal tissues and has been implicated in wide ranging requisite biological processes including response of endothelial cells to vascular injury, hematopoiesis, and regulation of immune responses. This normal tissue expression may limit Axl as a target for antibody-drug-conjugates (ADC). Conditionally Active Biologics (CAB) technology is a proprietary platform that selects antibodies that bind to target antigen in the context of diseased tissues, but not normal tissues, by taking advantage of the unique cancer microenvironment that is produced largely as a result of the Warburg effect. Using our CAB technology, we have identified anti-Axl Abs that reversibly bind to recombinant Axl and Axl expressing cells under conditions that are present in the tumor microenvironment but not in normal tissues.

CAB-Axl antibodies were then conjugated to a model toxin payload to generate CAB-Axl-ADCs. The CAB-Axl-ADCs were active against Axl positive human tumor xenografts with tumor stasis observed at 1mg/kg weekly and tumor regressions observed at 1 mg/kg twice a week dose levels. A non-specific IgG-ADC showed minimal efficacy at the same dose levels. Single dose studies in cynomolgus macaques have demonstrated that CAB-Axl-ADC has reduced liver toxicity and immune system effects compared to Axl-ADCs that bind to Axl under normal conditions.

In conclusion, our data is consistent with our work on CAB-EGFR antibodies, and suggests that ADCs generated using the CAB technology provides biologics with increased therapeutic index. Specifically, the CAB-Axl-ADC is an excellent candidate for evaluation as a treatment for human cancers that are Axl positive.

#3837

Passenger deletion of ENO1 as a collateral lethality target in cancer.

Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Joe Marszalek, Yuting Sun, Marina Protopopova, Maria E. Di Francesco, Barbara Czako, Alan Wang, Ronald A. DePinho, Florian L. Muller. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Large scale genomic characterization efforts such as TCGA have painted an unprecedentedly detailed picture of the genetic alterations that underlie tumorigenesis. Yet, the majority of genetic alterations are passenger rather than driver events and are considered unactionable. We have previously proposed that passenger or collateral deletions could serve as pharmacologically targetable vulnerabilities Collateral Lethality, in case passenger genes are homozygously deleted and member of a paralogous gene family carrying out an essential housekeeping function. We have presented proof-of-principal, whereby passenger deletion of the glycolytic gene Enolase 1 (ENO1) as part of the 1p36-tumor suppressor locus, renders glioma cells harboring such deletions highly sensitive to ablation of its redundant paralogue, ENO2. While our original analysis identified ENO1-homozygous deletions in Glioblastoma (GBM), recent bioinformatics analyzes, backed by immunohistochemistry, show that ENO1-homozygous deletions also occur in Hepatocellular Carcinoma and Cholangiocarcinoma. In GBM, multisector analysis of primary and recurrent tumors, indicate that ENO1 deletion is an early event which is homogenously distributed through the primary tumor and persist during recurrence. To pharmacologically exploit ENO1-deletion, we have pursued two approaches. First, we have synthesized cell-permeable prodrug derivatives of the natural Enolase inhibitor SF2312. The lead compound, POMHEX, shows potent killing of ENO1-deleted glioma cells in the low nM range while ENO1-restored isogenic or normal cells can tolerate μM doses. POMHEX has a short half-life yet can eradicate intracranial xenografted ENO1-deleted tumors, provided extensive breakdown of the blood-brain barrier. Our second approach to targeting ENO1-deletion consisted of chemical biology screening of drug libraries for the ability to kill ENO1-deleted but not isogenic rescued cells. We find that ENO1-deleted cells show a dramatic sensitization to inhibitors of the mitochondrial electron transport chain. These include tool compounds such as rotenone as well as compounds not previously associated with mitochondria, such as Mubritinib and an experimental anti-neoplastic agent previously described as a HIF1-inhibitor, now known to inhibit mitochondrial Complex I. The latter agent shows potent activity against ENO1-deleted intracranial xenografts. The likely cause for this sensitivity is the inability of ENO1-deleted cells to compensatory upregulate glycolysis in response mitochondrial inhibition, the typical response of ENO1-intact glioma cells and normal cells. Together, these data indicate that passenger deletion of ENO1 is an encouraging drug-target and provide support for collateral lethality as a viable therapeutic strategy, which, given the large number of passenger deletions in the cancer genome, may be broadly applicable.

#3838

Upregulation of DLEU1 significantly prolongs survival in a rituximab-treated DLEU1 knockout human Burkitt lymphoma (BL) xenograft NSG mouse model: DLEU1 may act as a tumor suppressor gene in pediatric BL.

Sanghoon Lee, Changhong Yin, Tishi Shah, Janet Ayello, Erin Morris, Carmella van de Ven, Mitchell S. Cairo. _New York Medical College, Valhalla, NY_.

BACKGROUND:

Burkitt lymphoma (BL) is the most common histological subtype of non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood 2007; Miles/Cairo., BJHaem 2012). We identified that pediatric BL patients with chromosome 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al., Leuk 2009; Nelson/Cairo/Sanger et al., BJH 2009). Deleted in lymphocytic leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 region (Dave et al., NEJM 2006) and interacts with C-MYC, TUBB2C and UBR1 (Stelzl et al., Cell 2005). We have previously reported a regulated DLEU1 expression significantly associated with changes in BL proliferation in vitro (Lee/Cairo et al., AACR 2015). We hypothesize that DLEU1 may have function as a tumor suppressor gene; however, functions and mechanisms underlying DLEU1 expression in the BL are poorly understood.

OBJECTIVES:

We hypothesize that DLEU1 may act as a tumor suppressor gene in BL and therefore examined whether up-regulation of DLEU1 results in changes in survival following targeted immunotherapy.

METHODS:

DLEU1 stably overexpressing Raji BL cells (Lee/Cairo et al., ASH 2013) were transfected with a firefly luciferase expression plasmid, kindly provided by Laurence Cooper MD, PhD. Four- to six- week-old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice from The Jackson Lab. Tumor burden and tumor progression were monitored by bioluminescence imaging system. Mice were treated with either rituximab (30 mg/kg) or cyclophosphamide (25 mg/kg) by intraperitoneal injection at 7 day intervals. Survival rates were analyzed by the Kaplan-Meier method using the Prism Version 6.0 software.

RESULTS:

We observed that DLEU1 stably overexpressing Raji BL cells xenografted (GFP-DLEU1) mice had significantly extended survival compared to GFP control mice following treatment rituximab (59 vs 51 days, p<0.05, n=8), cyclophosphamide (50.5 vs 37.5 days, p<0.0001, n=12) and in combination treatment (60.5 vs 53 days, p<0.05, n=12) whereas there were no significant differences in survival between WT and DLEU1-KO mice with PBS treatment. We observed that there were no significant differences of luciferase intensity in between GFP and GFP-DLEU1 mice following rituximab, cyclophosphamide or in combination treatment.

CONCLUSIONS:

We demonstrated that the upregulation of DLEU1 expression significantly improved the survival in DLEU1 overexpressing BL cells xenografted NSG mice following rituximab, cyclophosphamide and in combination treatment. Therefore, the up-regulation of DLEU1 expression in BL may in part result in sensitizing to chemoimmunotherapy induced survival and may result in a consideration of potential therapeutic strategies

### Targeting Protein Kinases, Death Pathways, and the Tumor Microenvironment

#3839

Vitamin E delta-tocotrienol targets human colon cancer stem cells and inhibits colon cancer metastasis and induces apoptosis.

Kazim Husain, Domenico Coppola, Said M. Sebti, Mokenge P. Malafa. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

Background: Colon cancer is the most commonly diagnosed cancer both in men and women and cause of cancer related deaths worldwide. Treatment for the recurrent and metastatic colon cancer remains a center of clinical attention. Cancer stem cells (CSCs) are suggested to be the only cells within tumor with tumorigenic capacity and implicated in metastasis. We have shown that vitamin E delta-tocotrienol (VEDT) is the most bioactive tocotrienols against cancers. This study aimed to evaluate the effect of VEDT on colon cancer growth, metastatic epithelial to mesenchymal transition (EMT), migration/ invasion/angiogenesis and colon CSCs growth and apoptosis.

Methods: Human colon cancer cells HCT-116 and metastatic colon cancer cells SW-620 and colon CSCs were treated with VEDT (10-100 μM) for 72 h and cell viability recorded. VEDT (50 μM) was used for malignant transformation; migration/invasion, CSCs spheroid formation, and pluripotency were determined. Azoxymethane treated Fisher 344 rats were used as colon carcinogenesis model. They were treated with 1) vehicle control and 2) VEDT (200 mg/kg, orally twice a day) for 20 weeks. After 20 weeks the polyps and tumor formation were recorded.

Results: VEDT significantly inhibited the anchorage-dependent and independent cancer cell growth, migration/invasion and induced apoptosis compared to control. VEDT inhibited the CSC spheroid formation EMT (E-cadherin to vimentin), metastasis (MMP9), angiogenesis (VEGF), CSCs transcription factors (Nanog, Oct4, Sox2 and KLF4) compared to control.

Conclusion: Vitamin E delta-tocotrienol inhibited colon cancer growth and metastasis through inhibition of cancer stem cell pluripotency, EMT, migration, invasion, angiogenesis and induction of apoptosis.

#3841

Curcumin enhances the efficacy of docetaxel- induced apoptosis of prostate cancer cell.

Saswati Banerjee, Santosh K. Singh, James W. Lillard, Rajesh Singh. _Morehouse School Of Medicine, Atlanta, GA_.

Despite the different prostate cancer (PCa) treatment therapies it continues to be one of the leading causes of death in men. Docetaxel is a well-established chemotherapeutic agent used to target metastatic PCa, however long-term treatment with docetaxel results in drug resistant and toxicity in PCa cells. Combining agents is common to improve outcomes, but the combination should not significantly increase toxicity. Curcumin is a non-toxic natural compound with multifaceted chemo preventive potential. In current study we evaluated whether curcumin can reinforce the effect of docetaxel on PCa cell lines (RWPE-1, C4-2b, DU145 and PC3). PCa cell lines were treated with curcumin or docetaxel alone or in combination followed by cell viability (MTT assay) and apoptosis assay (TUNEL assay and flow-cytometric analysis of annexin V/propidium iodide-stained cells). The expressions of pro- and anti-apoptotic markers were quantitated with real-time PCR and western blot assay. Our results indicate that curcumin combined with docetaxel led to lower cell viability than treatment with docetaxel or curcumin alone. Annexin V staining followed by flow cytometric analysis demonstrated that curcumin treatment enhanced the docetaxel-induced apoptosis of PCa cells, which was further confirmed by TUNEL assay. The down-regulation of the anti-apoptotic proteins (Bcl2, Mcl-1) and upregulation of pro-apoptotic proteins (Bax, Bid etc.) further confirm that we can considerably reduce docetaxel dose using synergistic combination therapy with curcumin. Thus, our results strongly suggest that we can reduce dose levels of docetaxel and compensate it with non-toxic curcumin and it could be a potential therapeutic contender in enhancing the efficacy of docetaxel in PCa treatment.

#3842

Design and engineering of TRAIL fusion proteins for cancer therapy.

Eric M. Tam,1 Hannah Hudson,2 Tamara Dake,2 Sara Ghassemifar,1 Andreas Raue,1 Yasmin Hashambhoy-Ramsay,2 Stephen L. Sazinsky,3 Anahita Daruwalla,1 Neeraj Kohli,1 Lihui Xu,1 Charlotte F. Mc Donagh,1 Birgit Schoeberl,1 Diana H. Chai1. 1 _Merrimack Pharmaceuticals, Cambridge, MA;_ 2 _University of North Carolina, Chapel Hill, NC;_ 3 _Jounce Therapeutics, Cambridge, MA_.

Protein-based agonists of apoptotic death receptors have shown remarkable preclinical efficacy but limited clinical response. The short circulating half-life of recombinant human TRAIL and the necessity of Fc-mediated clustering for potentiating agonistic antibodies against DR4 and DR5 have been proposed to be major impediments to the clinical success of this class. To address these limitations we have created Fc-scTRAIL, a single fusion polypeptide consisting of an IgG1 Fc region followed by three successive TRAIL monomers connected by two fifteen-amino acid linkers. While Fc-scTRAIL showed potent activity in vitro, we observed a low TM (48 °C) and rapid inactivation in serum indicating protein instability. Subsequently, we applied a directed evolution approach using yeast surface display to identify mutations that would stabilize the TRAIL trimer. When individual mutations were transferred to the Fc-scTRAIL format, we observed a dramatic increase in the TM (66-70 °C) while the combination of three mutations improved serum stability by ten-fold. Stabilized Fc-scTRAIL shows greater pro-apoptotic activity across a panel of cancer cell lines when compared to mapatumumab (anti-DR4) and drozitumab (anti-DR5), or the combination of antibodies even in the presence of anti-Fc cross-linking. Moreover, anti-Fc did not improve Fc-scTRAIL activity suggesting that the hexavalent design of the molecule maximizes death receptor activation. Currently, in vivo evaluation of Fc-scTRAIL for pharmacokinetic properties and activity is underway. We believe this format, when combined with an appropriate patient selection strategy, will result in improved clinical outcomes.

#3843

Design of effective combination therapies for high-grade serous ovarian cancer using patient-derived xenograft models.

Claudia Iavarone,1 Ioannis Zervantonakis,1 Hsing-Yu Chen,1 Sangeetha S. Palakurthi,2 Joyce F. Liu,3 Ursula Matulonis,3 Ronny Drapkin,4 Gordon Mills,5 Joel Leverson,6 Deepak Sampath,7 Joan Brugge1. 1 _Department of Cell Biology, Harvard Medical School, , Ludwig Center at Harvard, Boston, MA;_ 2 _Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, MA;_ 3 _Dana Farber Cancer Institute, Department of Medical Oncology, Center for Molecular Oncologic Pathology, Boston, MA;_ 4 _Obstetrics and Gynecology Department, University of Pennsylvania, Philadelphia, PA;_ 5 _Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX;_ 6 _Oncology Development, AbbVie, Chicago, IL;_ 7 _Translational Oncology, Genentech, South San Francisco, CA_.

Despite advances in understanding the genetics and molecular biology of high grade serous ovarian cancer, there have not been any significant improvements in the outcome of patients treated with approved therapies to date. The overall aim of our studies is to identify synergistic drug combinations for the treatment of high-grade serous ovarian (HGS-Ov) cancer and the biomarkers that predict sensitivity for future translation in clinical trials. In particular, this study focuses on the vulnerabilities of patient-derived ovarian cancer cells to inhibition of anti-apoptotic proteins and the design of novel combination therapies to overcome drug resistance and improve the response of selected patients to a particular set of targeted therapies.

Previous studies from our lab provided evidence that inhibition of BCL-2/XL can significantly enhance the sensitivity of tumor cells to targeted therapies in ovarian and breast cancer cell line models (Muranen T et al., Cancer Cell 2012). In this study, we evaluated the efficacy of this combination treatment in ascites cells derived from 15 high-grade serous patient-derived ovarian cancer xenografts (PDXs). We found that inhibitors of PI3K/mTOR (GNE-493) and BCL-2/XL (ABT-737) act synergistically ex vivo, with an ~15 fold variability among the 15 patient-derived samples. A large scale in vivo experiment is ongoing to evaluate the efficacy of this combined treatment in six PDX models.

To identify biomarkers that predict drug sensitivity we performed proteomic Reverse Phase Protein Array (RPPA) and immunoblot analyses. We found that baseline levels of the pro-apoptotic protein BIM correlate with sensitivity to BCL-2/XL inhibition. This motivated us to examine the basis for low BIM expression. In three of the least sensitive PDX models, we found that low levels of BIM correlated with ERK activation based on increased ERK phosphorylation and that inhibition of MEK by PD-0325901 in these models caused upregulation of BIM, as predicted from previous published reports which demonstrated that phosphorylation of BIM by ERK causes its degradation (Ley R, et al. J Biol Chem. 2003). We then examined the sensitivity of these PDX models to ABT-737 and PD-0325901, MEK162, or CI-1040 ex vivo and observed strong synergy resulting in reduction of cell viability and increased cell death. These results suggest that low BIM/high phosphoERK is a potential biomarker for sensitivity of ovarian tumors to combined MEK and BCL-2/XL inhibition. We now plan to examine the efficacy of drug combinations that target MEK and BCL-2/XL in these PDX models in vivo.

Our studies promise to lead to the identification of new drug combination therapies for HGS-Ov cancer treatment and predictive biomarkers to stratify patients that can benefit from these targeted therapies

#3844

A novel liposomal Clodronate depletes tumor-associated macrophages in primary and metastatic melanoma: anti-angiogenic and anti-tumor effects.

Chiara Brignole,1 Patrizia Perri,1 Francesca Piaggio,1 Fabio Pastorino,1 Daniela Di Paolo,1 Laura Emionite,2 Antonio Daga,3 Vangelis Kondylis,4 Manolis Pasparakis,4 Domenico Ribatti,5 Mirco Ponzoni1. 1 _G. Gaslini Children's Hospital, Genoa, Italy;_ 2 _Animal Facility, IRCCS Azienda Ospedaliera Universitaria San Martino–IST, Genoa, Italy;_ 3 _IRCCS Azienda Ospedaliera Universitaria San Martino–IST, Genoa, Italy;_ 4 _Institute for Genetics, University of Cologne, Cologne, Germany;_ 5 _Department of Basic Medical Sciences, University of Bari, Bari, Italy_.

The depletion of tumor-associated macrophages (TAMs), involved in different stages of cancer development and progression, is an appealing strategy in cancer therapy.

We developed novel Clodronate-containing liposomes (Clo-Lipo-DOTAP) presenting good physicochemical properties (size distribution, polidispersity index and Z-potential).

In vitro, Clo-Lipo-DOTAP inhibited proliferation, reduced viability and induced apoptosis of a macrophage-like cell line in a dose- and time-dependent manner.

In proof of functionality experiments, Clo-Lipo-DOTAP depleted macrophages in a genetic mouse model of chronic hepatitis and hepatocellular carcinoma leading to a significant reduction of F4/80-positive cells in the liver and spleen of treated mice compared to PBS-treated controls. The number of granulocytes, B and T lymphocytes was not affected.

In B16/F10 subcutaneous melanoma-bearing mice, Clo-Lipo-DOTAP significantly reduced the volume of primary tumors (P < 0.001). Within the tumors, the expression F4/80 and α-SMA was significantly lowered. Plasma levels of IL-10, Mo KC, TNF-α, VEGF and PDGF-bb were statistically decreased. In B16/F10 lung metastatic melanoma model, treatment with Clo-Lipo-DOTAP significantly reduced the number of pulmonary nodules (P < 0.05). F4/80-positive cells and microvessel density were statistically decreased.

In conclusion, the depletion of TAMs in primary and metastatic melanoma presents anti-tumor efficacy via inhibition of angiogenesis and modulation of inflammation related cytokines.

#3845

A novel selectivity determinant informs the development of next-generation stapled peptide inhibitors of anti-apoptotic BFL-1/A1.

Annissa J. Huhn, Rachel M. Guerra, Gregory H. Bird, Loren D. Walensky. _Dana-Farber Cancer Institute, Boston, MA_.

The development of selective inhibitors for the individual anti-apoptotic BCL-2 family proteins implicated in oncogenesis and chemoresistance remains a formidable but pressing challenge. Precisely tailored compounds would serve as ideal molecular probes and targeted therapies to respectively study and treat human cancers driven by discrete apoptotic blockades. The remarkable potential of this strategy is exemplified by ABT-199, a potent and selective small molecule BCL-2 inhibitor that is proving effective at reactivating apoptosis in BCL-2-dependent cancers. The BCL-2 protein homologue, BFL-1/A1, has emerged as a resistance factor in a series of human cancers, including melanoma, leukemia, and lymphoma, but remains undrugged. To identify binding and specificity determinants for targeting BFL-1/A1, we screened a library of hydrocarbon-stapled BH3 domain helices and identified a subclass of compounds that engage BFL-1/A1 with exquisite selectivity in vitro and in situ. The mechanistic basis for this specificity revealed a unique topographic feature of the BFL-1/A1 binding groove. Leveraging this natural BFL-1/A1 selectivity factor, we designed next-generation stapled peptide inhibitors that block BFL-1/A1's capacity to both bind BH3 domains and suppress BAX-mediated membrane poration. Our studies inform a new pharmacologic strategy for potent and selective inhibition of anti-apoptotic BFL-1/A1 in human cancer.

#3846

Sensitivity of NOTCH1 mutant T-ALL to ABT-263.

Anahita Dastur,1 Carlotta Costa,1 Anthony Faber,2 Cyril Benes1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Virginia Commonwealth University, Richmond, VA_.

Effective targeted therapies are lacking for refractory and relapsed T-cell Acute Lymphoblastic Leukemia (T-ALL). Analysis of a large cell line screen revealed that NOTCH1 mutant T-ALL cells are sensitive to the Bcl-2/xL inhibitor ABT-263. We show that NOTCH1 inhibits the mTOR complex 1, resulting in low MCL-1 levels and sensitization to ABT-263. Further suppression of mTORC1 using small molecule inhibitors in combination with ABT-263 results in very high levels of apoptosis and tumor regressions in vivo. This suggests new therapeutic opportunities for NOTCH1 mutant T-ALL, including in the setting of gamma-secretase inhibitor (GSI) resistance.

#3847

Creating the tumor microenvironment for effective immunotherapy: Antitumor activity of intratumoral IMO-2125, a TLR9 agonist is further enhanced by inhibition of indoleamine-pyrrole 2,3-dioxygenase (IDO).

Daqing Wang, Wayne Jiang, Bhagat Lakshmi, Jillian DiMuzio, Fugang Zhu, Sudhir Agrawal. _Idera Pharmaceuticals, Inc., Cambridge, MA_.

In recent years, understanding of tumor immunology has led to significant advances in cancer immunotherapy. New approaches include selective blockade of checkpoints, harnessing tumor infiltrating lymphocytes (TILs) and use of intratumoral oncolytic viruses. Evolving data has continued to provide evidence that the tumor microenvironment (TME) is critical for effective immunotherapy. In preclinical studies, intratumoral delivery (i.t.) of the Toll-like receptor (TLR) 9 agonist IMO-2125 resulted in increased IFNα, IL-12, Th1 responses, TIL infiltration and dose-dependent antitumor activity against treated and distant tumors. However, gene expression analysis of the treated tumors showed that levels of IDO1 was increased, thereby hampering the optimal TME. We hypothesized that combination of i.t. IMO-2125 and an IDO1 inhibitor would lead to modulation of the TME and more potent antitumor activity versus either agent alone.

In the present study, we evaluated the antitumor activity of i.t. IMO-2125 in combination with an IDO1 inhibitor in a murine syngeneic colon carcinoma CT26 model. Each BALB/c mouse was implanted with two tumors: subcutaneous solid tumor and lung metastatic tumor by s.c. (1x107) and i.v. (3x106) inoculation with CT26 cells. Treatment was initiated when tumor nodules reached 100-200 mm3 with 2.5 mg/kg intratumoral IMO-2125 and 75 mg/kg oral IDO1 inhibitor. All treatments were well tolerated. In the placebo-treated group, the tumor volume was 1039 ± 262 mm3 (mean ± SD). In groups treated with IMO-2125 and IDO1 inhibitor, the tumor volume was 369 ± 216 mm3 (64% tumor growth inhibition, TGI) and 745 ± 252 mm3 (27% TGI), respectively. In the group treated with the combination of IMO-2125 and IDO1 inhibitor, the tumor volume was 45 ± 75 mm3, showing 95% tumor growth inhibition. Similar treatment-related results were observed in the lung nodules, with the combination group showing a 76% reduction in the number of lung tumor nodules over PBS control. Tumors treated with IMO-2125 alone showed 90-fold increase in IDO1 expression which was inhibited 47% in tumors treated with a combination of IMO-2125 and IDO1 inhibitor. The inhibition of IDO1 in combination with i.t. IMO-2125 also resulted in increased TILs.

Overall, these preclinical data showed that combination of i.t. IMO-2125 and an IDO1 inhibitor stimulated systemic immune responses and primed the TME to enable potent antitumor activity. In previous preclinical studies IMO-2125 has shown potent antitumor activity in multiple tumor models in combination with anti-CTLA4 and anti-PD1 agents.

#3848

Targeting hyaluronidase reduces stromal cell protection of chronic myeloid leukemia to imatinib therapy.

Bryan Hostetler, Olga Uchakina, Robert McKallip. _Mercer University, Macon, GA_.

An estimated 20-30% of CML patients will become resistant to tyrosine kinase inhibitors (TKIs) including imatinib. Recent reports suggest that CML resistance to TKI's is driven by interactions with protective microenvironment niches that influence their survival and self-renewal capacity. Hyaluronic acid (HA) has emerged as a key contributor in this phenomenon of CML resistance to TKI's. HA influences cell viability through molecular weight dependent interactions with cell-surface receptors CD44 and RHAMM. Low molecular weight (LMW) HA, is typically found in association with many cancers including CML and promotes apoptotic resistance, proliferation, and migration of cancer cells. High molecular weight (HMW) HA is primarily produced by fibroblasts and its catabolism to LMW HA is mediated by hyaluronidase (HYAL) activity. HYAL activity may provide a potential therapeutic target in combination with current TKI treatments. Using a co-culture model, we investigated the role of HA produced by bone marrow stromal fibroblasts on CML viability when subject to imatinib (IM) treatment. Furthermore, we investigated the possibility of targeting hyaluronidase as an adjuvant in combination with IM for the treatment of CML. Co-culture with stromal fibroblasts protected CML cells from proliferation inhibition by IM. HA production and its catabolic products including LMW HA were elevated in the supernatant of co-cultures exposed to IM. The expression of genes encoding for HA metabolic proteins including hyaluronic acid synthases and HYALs, were significantly increased in co-cultures subject to IM stress. Additionally, HA treatment of CML mono-culture protected against chemotherapy-induced apoptosis. Treatment of CML co-culture with HYAL inhibitor, glycrrhizic acid (GA), inhibited HYAL activity, greatly reduced proliferation and increased apoptosis of CML cells alone and in combination with IM. This investigation provides additional evidence of the importance of stromal cell support in CML pathology and that resistance to TKI therapy may be mediated in part through upregulation of total HA production and its catabolism. Furthermore, we establish the potent therapeutic effects of GA on CML cells alone and in combination with current IM treatment strategies as a prospective method of circumventing TKI-resistant CML. For the first time we demonstrate GA's effectiveness as a HYAL inhibitor in culture. HYALs are an attractive target not only in CML resistance but in many cancers due to the reported role of its product, LMW HA, in inflammation, proliferation, and apoptotic resistance.

#3849

The antidiabetic drug metformin increases pro-apoptotic effect of TRAIL therapy by reducing X-linked inhibitor of apoptosis protein (XIAP) levels in triple-negative breast cancer cells.

Elena Strekalova, Dmitry Malin, Vincent Cryns. _University of Wisconsin, Madison, WI_.

Most metastatic tumors, such as triple negative breast cancer (TNBC) respond poorly to conventional chemotherapy. We have previously demonstrated that targeting TNF-related apoptosis-inducing ligand receptor-2 (TRAIL-R2) might be an effective pathway against metastatic TNBC. However, many TNBCs are resistant to TRAIL receptor agonists. Here we demonstrate that diabetes drug metformin, previously linked to preventing and treating several types of cancer, enhances sensitivity of TNBC cells to recombinant TRAIL and TRAIL-R2 agonistic humanized monoclonal antibody (lexatumumab)-induced apoptosis. We have demonstrated that combination of metformin with lexatumumab or TRAIL augments response to TRAIL therapy and reduce cancer cell viability via induction of apoptosis and inhibiting long-term survival of three different metastatic TNBC cell lines. Intriguingly, metformin treatment also sensitized transformed MCF10A-RasV12 cells, but not untransformed MCF10A-Vector cells, to TRAIL agonist therapy. This effect was associated with reduction of X-linked Inhibitor of Apoptosis Protein (XIAP) levels after metformin treatment. Similar to metformin treatment, inhibition of XIAP by small interfering RNA sensitized breast cancer cells to TRAIL-induced apoptosis. Moreover, metformin treatment in combination with TRAIL more efficiently inhibited primary tumor growth and lung metastases in orthotopic model of TNBC. Overall, our results demonstrate that metformin treatment in combination with targeting TRAIL receptors may be a good strategy to augment the antitumor effects of TRAIL receptors agonistic therapy and provide the foundation for a clinical trial combining dietary metfomin and TRAIL agonists.

#3850

**First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth** in vivo **.**

Sabrina Deroo,1 Sophie Thiolloy,2 Johan Desmet,3 Franky Baatz,1 Stefan Loverix,3 Karen Vandenbroucke,3 Eric Lorent,3 Paula Henderikx,2 Irma Lemmens,4 Philippe Alard,3 Ignace Lasters,3 Yvonne McGrath3. 1 _Complix Luxembourg S.A., Esch-sur-Alzette, Luxembourg;_ 2 _Complix N.V., Diepenbeek, Belgium;_ 3 _Complix N.V., Zwijnaarde (Ghent), Belgium;_ 4 _Ghent University, Dept. Biochemistry, Cytokine Receptor Laboratory, VIB, Dept. Medical Protein Research, Ghent, Belgium_.

We have developed Cell Penetrating Alphabodies (CPABs), a novel and unique therapeutic class of proteins engineered to efficiently enter cells. In vitro, uptake in a range of tumor and non-tumor cell types occurs rapidly with cytosol levels of up to 1 µM concentration after 2 hours of CPAB exposure. Early forms of these CPABs suffered from rapid serum clearance, thereby limiting their efficacy in vivo and amenability to drug development. The incorporation of an albumin binding region in the body of the protein has allowed extension of serum half-life in mice from a few minutes to more than one hour. These CPABs have been shown to be efficiently delivered to xenograft tumors in mice after IV bolus injection by tissue ELISA and immunohistochemistry. CPABs can be used to target and interfere with intracellular protein-protein interactions involved in tumor survival in a highly specific way.

The anti-apoptotic protein Myeloid Cell Leukaemia-1 (Mcl-1) promotes through its interaction with Bak, the survival of a range of different tumor types including myeloid leukemia, breast cancer and non-small cell lung cancer. Moreover, Mcl-1 overexpression is often associated with chemotherapeutic resistance and disease relapse. Mcl-1, however, has proven difficult to target using the conventional small molecule approach.

Alphabodies which bind to Mcl-1 were engineered by a combination of rational design and phage display library screening. The affinities for Mcl-1 ranged between 18 pM and 750 pM with binding to the closely related proteins Bcl-2 and Bcl-XL being below the limit of detection for the assay. In a Mammalian Two Hybrid assay, these Alphabodies inhibited Bak-Mcl-1 but not Bak-Bcl-XL interactions. Anti-Mcl-1 CPABs were shown to efficiently kill the Mcl-1 dependent multiple myeloma cell line NCI-H929 with IC50s ranging from 0.5 µM to 2 µM as monitored in cell viability assays. The dose responsive cell killing correlated with caspase-3/7 activation in NCI-H929 cells. Other Mcl-1 dependent tumor cell types including non-small cell lung cancer (NCI-H23) and Burkitt's lymphoma (Raji) or tumor cell types with high Mcl-1 expression such as ovarian cancer (A2780) and colorectal adenocarcinoma (COLO-320DM) were also killed efficiently using anti-Mcl-1 CPABs. Despite its short half-life, daily intraperitoneal administration of a prototype Mcl-1 targeting CPAB (without half-life extension) at 30 mg/kg for 14 days resulted in tumor inhibition of 33% as compared to vehicle control. Experiments are underway in mouse models using the more optimal CPABs with extended serum half-life and tumor exposure.

CPABs represent the best-in-class cell penetrating protein therapeutics both in terms of efficiency of uptake and amenability to conversion to viable drugs opening unprecedented opportunities to tackle intracellular protein-protein interactions critical to diseases with unmet medical need.

#3851

Synergy between statins and BCL-2 inhibitors in blood cancers.

Jong-Hoon S. Lee, Thanh-Trang T. Vo, David A. Fruman. _University of California, Irvine, Irvine, CA_.

BCL-2 is a key pro-survival protein that is highly expressed in many leukemias and lymphomas. ABT-199 (venetoclax) is a small molecule inhibitor of BCL-2 that has demonstrated promising clinical potential in chronic lymphocytic leukemia (CLL). However, other hematologic malignancies are less responsive to ABT-199 as a single agent, suggesting that combinations of targeted therapies may be required to elicit more promising responses. We have investigated the potential of combining ABT-199 with HMG-CoA reductase (HMGCR) inhibitors (statins), which have known anti-cancer potential in hematologic malignancies. Using multiple chemically distinct statin compounds, we observed profound synergistic induction of apoptosis when combined with ABT-199 in both human diffuse large B cell lymphoma (DLBCL) as well as acute myeloid leukemia (AML) cell lines. This synergy was also seen in primary murine B lymphoma cells over-expressing MYC and BCL-2. Importantly, addition of exogenous mevalonate completely rescued cells from the combination, confirming on-target efficacy of HMGCR inhibition. Using BH3 profiling, we found that simvastatin significantly primed lymphoma cells for undergoing apoptosis (termed mitochondrial priming). Notably, the degree of priming correlated with its ability to synergize with ABT-199, suggesting that this method may be used to predict patient responses. Strikingly, the combination did not synergize to kill normal human peripheral blood mononuclear cells from healthy donors, suggesting that statins may selectively prime cancer cells for apoptosis. Mechanistic studies support the hypothesis that statins synergize with ABT-199 by suppressing protein prenylation, particularly protein geranylgeranylation. In support, the addition of exogenous geranylgeranyl pyrophosphate (GGPP) completely rescued cells from the effects of simvastatin. Furthermore, selective inhibition of protein geranylgeranyl transferase (GGT) was sufficient to recapitulate the effects of simvastatin in combination with ABT-199. Lastly, we have identified Rap1A de-prenylation as a marker of pharmacodynamic response to statins in vivo. Thus, this project highlights a novel combination for use in aggressive lymphomas, establishes its efficacy and tolerability using preclinical models, and provides proof-of-concept to warrant investigation of its clinical potential.

#3852

U3-1784, a human anti-FGFR4 antibody for the treatment of cancer.

Rene Bartz,1 Keisuke Fukuchi,2 Tanja Lange,1 Katrin Gruner,1 Toshiaki Ohtsuka,2 Ichiro Watanabe,2 Shinko Hayashi,2 Mauricio Redondo-Müller,1 Mizuki Takahashi,2 Toshinori Agatsuma,2 Johannes Bange,1 Reimar Abraham1. 1 _U3 Pharma GmbH, Planegg, Germany;_ 2 _Daiichi-Sankyo, Tokyo, Japan_.

Fibroblast Growth Factor Receptor 4 (FGFR4) is the fourth member of the Fibroblast Growth Factor Receptor (FGFR) family of receptor tyrosine kinases. All of the FGFR's have been implicated in cancer development due to increased activation of their enzymatic activity either by gene mutation, over-expression or inadvertent ligand-mediated stimulation. One important alteration that may lead to FGFR4 activation in cancer is the overexpression of its ligand FGF19 in 20-40% of primary liver cancer.

Here, we report the development of U3-1784, a phage display-derived fully human antibody that specifically binds to FGFR4 but not to isoforms of FGFR1-3. The antibody binds to an epitope in the putative ligand binding domain of the receptor and consequently inhibits ligand binding and downstream signaling. In a panel of 10 tumor models derived from hepatocellular carcinoma, U3-1784 significantly inhibits the growth of FGF19-expressing models up to 90% whereas models without FGF19 expression are insensitive. These results strongly suggest that the FGFR4/FGF19 axis is an oncogenic driver in hepatocellular carcinoma. U3-1784 is currently in phase I clinical trials.

#3853

Potential mechanisms for thrombocytopenia and neutropenia induced by antibody-drug conjugates.

Hui Zhao, Sara Gulesserian, Sathish K. Ganesan, Zhilan Zeng, Jimmy Ou, Veronica Robles, Josh Snyder, David R. Stover, Fernando Donate. _Agensys Inc., Santa Monica, CA_.

Thrombocytopenia and neutropenia are common adverse events in cancer patients treated with antibody-drug conjugates (ADCs). We used human hematopoietic stem cells (HSC) in vitro and various ADCs to investigate the potential mechanisms of thrombocytopenia and neutropenia.

Two ADCs, AGS-16C3F and T-DM1, were selected for the investigation of megakaryocytes (MKs) as they both induced thrombocytopenia in patients. AGS-16C3F is an ENPP3-targeting antibody conjugated to microtubule-disrupting agent MMAF (licensed from Seattle Genetics) via a non-cleavable linker (mcMMAF) tested in a phase 1 trial in kidney cancer patients. T-DM1 (KADCYLA) is an anti-HER2 antibody conjugated to the maytansine derivative DM1, approved for the treatment of advanced Her-2 positive breast cancer. HSCs were differentiated to MKs and percentage of CD41 positivity from FACS analysis was used to measure the effect of ADCs on MK differentiation. Results showed that while MKs do not express ENPP3 or Her-2, both AGS-16C3F (IC50 = 40nM) and T-DM1 inhibited MK differentiation. MKs express FcγRIIA to which T-DM1 (IgG1) and, to lesser degree, AGS-16C3F (IgG2) can bind; however, blockage of FcγRIIA failed to rescue MKs from cytotoxicity induced by either T-DM1 or AGS-16C3F suggesting that binding to FcγRIIA is not critical for MK cytotoxicity. AGS-16C3F is internalized into MKs via macropinocytosis and causes significant disorganization of tubulin structure as observed by confocal microscopy.

HSCs were also differentiated to neutrophils which were characterized by the percentage of CD66b positivity in FACS analysis. Several antibodies to targets absent in neutrophils were conjugated to both MMAE and MMAF and were tested for cytotoxicity during neutrophil differentiation. Our results showed that ADCs containing MMAE via a cleavable linker were more cytotoxic to neutrophil differentiation than the same ADC containing MMAF via a non-cleavable linker, in agreement with clinical results. Different from MKs, results showed that neutrophils have low macropinocytosis activity. Secretion of cathepsins increased during neutrophil differentiation, and a membrane impermeable cathepsin inhibitor blocked ADC cytotoxicity to neutrophils, suggesting that extracellular protease cleavage is critical for neutrophil cytotoxicity in vitro. Neutrophils express high levels of FcγRIIA and saturation of this receptor by control IgG inhibited ADC binding to neutrophils and at least partially rescued neutrophils from ADC cytotoxicity, suggesting that FcγRIIA can play a role in neutropenia induced by ADCs, particularly with IgG1 isotype.

In conclusion, applying these in vitro data to clinical experience suggests that thrombocytopenia may be mediated by macropinocytosis of ADC in MK and neutropenia by secreted proteases and interactions with their FcγRs. Understanding these toxicities may help design new ADCs with improved toxicity profile.

#3854

Successful selective eradication of colorectal cancer cells by adenovirus-based delivery of toxins.

Shiran Shapira,1 Assaf Shapira,2 Dina Kazanov,1 Ilana Nabiochtchikov,1 Sarah R. Kraus,1 Nadir Arber1. 1 _Tel Aviv Sourasky Medical Ctr., Tel-Aviv, Israel;_ 2 _Tel Aviv University, Tel-Aviv, Israel_.

Background: K-Ras gene mutation is an early event in the development of colorectal cancer (CRC) and occurs in ~50% of CRC cases. We propose a strategy that exploits the Ras hyperactive pathway, rather than inhibiting it as was tried and failed many times. We have previously reported that recombinant adenovirus, carrying a pro-apoptotic gene under the regulation of Ras-responsive elements (Ets/AP1), suppressed the growth of cancer cells displaying hyperactive K-Ras (Biomed Pharm, 2005,Cancer Gene Ther,2012, Exp Cell Res,2012). TA systems are evolutionarily successful entities that are prevalent in lower organisms and play important roles in a diverse range of cellular activities.

im: To establish a tight control and improved ras responsive element based on the bacterial MazEF system

Methods: Efficient vectors for cancer-directed gene delivery were constructed and cloned into a "first generation" ΔE1/ΔE3 human type-5 adenoviral-vector. Virus particles were produced, their titer was calculated by the End-Point Dilution Assay (EPDA) and their potency was tested. Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT. FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptosis and dead cells, respectively. In vivo tumor formation was measured in xenograft model. Ad-Py4-SV40-MazEF and Ad-ΔPY4-CMV-MazEF viruses (1×109pfu) or PBS were administrated intraperitoneal twice with a 3-day interval between injections.

Results: Adenovirus therapy induced massive cell death, in a dose-dependent manner; 73% with a titer of 10 MOI in cells with activated K-Ras as compared to 22% in tumor cells having the WT K-Ras. The cytotoxic effect was confirmed qualitatively by colony formation assay. In the absence of K-ras-responsive DNA element increase expression of MazE, the anti-toxin, protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system. FACS analysis confirmed massive cell death, 55% apoptosis and 82% dead cells, following infection with the full toxin-antitoxin encoding viruses.

Control viruses lacking the K-ras responsive element a modest toxicity was seen (18% and 10%, respectively). Impressive tumor shrinkage was demonstrated in vivo following treatment with Ad-Py4-SV40-MazEF-encoding adenovirus (61%) without any toxic or side effects. Ad-ΔPY4-SV40-MazEF treated mice (control group) tumor volume was reduced only by 27% (P<0.05). No growth inhibition was seen following injection of PBS.

Conclusions: A proof-of-concept for a novel cancer gene therapy by exploiting aberrant K-Ras hyperactive pathway was successfully demonstrated. The lack of toxicity holds promise for effective and safe therapy of human cancers carrying K-Ras mutations.

#3855

Refolded albumin inhibits IL-17B-driven pancreatic cancer migration.

Chi-Ming Liang, Ching-Chun Liao, Shu-Mei Liang. _Academia Sinica, Taipe, Taiwan_.

Folding and refolding a protein may change its physical, chemical and biological properties. Here, we used a novel protein refolding protocol to convert globular albumin into another form, namely f-albumin, with melting point 10oC higher than albumin (83oC vs 72oC). f-albumin, unlike albumin, inhibited the migration/invasion of a variety of cancer cells. Recently, anti-IL-17RB antibodies have been shown to block the IL-17B-IL17RB signaling-mediated migration/metastasis of pancreatic cancer cells via silencing multiple chemokines, notably CCL20, CXCL1, IL-8 and TFF1. We thus examined whether f-albumin is effective in blocking the stimulatory effects of IL-17B. We found that f-albumin inhibited the migration of pancreatic cancer cells (BxPC3, MIA-paca2 and Panc1) with IC50 around 0.2 μM. Although IL-17B (50 ng/ml) increased the migration of pancreatic cells, this effect of IL-17B was abolished by f-albumin (0.2 μM). The inhibitory effect of f-albumin, however, was not necessarily correlated with its inhibition of CCL20, CXCL1, IL-8 or TFF1. Additional study revealed that IL-17B increased the phosphorylation of PI3K, Akt and MAPK in pancreatic cancer cells. f-albumin modulated not only the IL-17B-mediated phosphorylation of Akt but also the IL-17B-mediated phosphorylation of MAPK in an integrin-dependent manner. These findings demonstrated that f-albumin downregulates Akt/MAPK signaling via integrin to play a braking role in the IL-17B-driven pancreatic cancer migration.

#3856

The atomic basis for paclitaxel sensitization following loss of CRMP2 phosphorylation in ovarian cancer cells.

Yiyan Zheng,1 Ritika Sethi,2 Frank von Delft,2 Ahmed Ashour Ahmed1. 1 _Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;_ 2 _Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom_.

The chemotherapeutic agent paclitaxel increases cancer cell microtubule stability, induces mitotic arrest and cancer cell death and improves survival of cancer patients. However, in ovarian and other cancers, it only elicits a response in less than half of patients who receive it. We have previously shown that enhancing microtubule stability in cells prior to paclitaxel treatment, further increases paclitaxel-induced microtubule stability and cancer cell death (Ahmed et al., Cancer Cell, 2007;12:514 and Ahmed et al., Cancer Research 2011;71:5806). We now report that the microtubule-associated protein CRMP2, and FER kinase are expressed in almost half of high-grade serous ovarian cancers and that inhibiting the phosphorylation of CRMP2 by FER is sufficient for enhancing paclitaxel-induced microtubule stability and cytotoxicity in multiple ovarian cancer cell lines.

We are also able propose a detailed mechanism underlying these observations. A combination of biochemistry, super-resolution fluorescence microscopy, total internal reflection fluorescence (TIRF) microscopy and electron microscopy reveals that CRMP2 induces microtubule nucleation, elongation, bundling and stability. Moreover, FER phosphorylates CRMP2 at 6 tyrosine residues, two of which (Tyr479 and Tyr499) are critical for modulating CRMP2 function; this agrees with site-directed mutagenesis in cancer cells which points to Tyr479 as a critical site for association between CRMP2 and microtubules. A comparison of the crystal structures of wild-type CRMP2 and Tyr479Glu/Tyr499Glu phosphomimetic mutants indicate that phosphorylation of these sites induces significant conformational changes that prevent CRMP2 from forming tetramers that are known to be critical for its microtubule bundling activity.

This then leads us to propose a molecular model of how CRMP2 associates with microtubules, explaining how targeting CRMP2 phosphorylation can be exploited for enhancing the therapeutic efficacy of paclitaxel.

#3857

Pharmacological reactivation of the tumor suppressor protein phosphatase 2A as a novel approach for the treatment of breast cancer.

Caroline C. Farrington,1 Xiaoyan Wang,2 Mahnaz Janghorban,2 Juan Liang,2 Analisa Difeo,1 Rosalie Sears,2 Goutham Narla1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Oregon Health and Sciences University, Portland, OR_.

PP2A is a phosphatase that is functionally dysregulated and inactivated in over 50% of all breast cancers independent of histological type. While much attention has focused on oncogenic kinases as targets for cancer treatment, therapeutic targeting of phosphatases, the key negative regulators of these same signaling pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects, our laboratory reverse engineered these drugs to generate a novel series of compounds that retain the anti-proliferative effects and favorable pharmacokinetic properties but are devoid of the undesirable central nervous system pharmacology of the parent drugs. It was subsequently demonstrated that these first generation novel derivatives exert anticancer effects in cell culture and xenograft models of cancer (without overt toxicity) by modulation of critical oncogenic signaling pathways. Recently, it was determined that the molecular basis of the actions of these novel derivatives is the activation of PP2A. Comprehensive profiling for activity in a panel of 240 cancer cell lines has shown that these first-in-class small molecule activators of PP2A (SMAPs) have significant anti-proliferative activity in breast cancer cell lines, and studies have been extended to disease relevant in vivo models (patient derived xenograft, GEMM, and traditional xenograft models). PP2A's ability to negatively regulate a diverse set of oncogenic drivers in breast cancer means SMAPs are able to exert their effects through multiple biological mechanisms, swiftly disrupting cellular energetics and metabolism, inducing apoptosis and inhibiting proliferation. This will be exhibited through a variety of biological assays evaluating cell growth, apoptosis and changes in cellular metabolism. Target engagement assessed via western blotting demonstrates inhibition of PP2A targets critical for mediating SMAP activity, notably c-MYC and AKT. As a result, this research demonstrates SMAPs have potent activity in a wide variety of breast cancer contexts where response to current therapies is modest and limited by the development of treatment resistance. Thus, SMAPs may be a viable treatment option for high-risk populations of patients who do not respond to the current standard of care. This research strives to demonstrate that therapeutic reactivation of PP2A may represent a novel approach for breast cancer treatment and that these molecules may favorably impact the lives of women suffering from breast cancer.

#3858

Feasibility and efficacy of a precision treatment approach for triple-negative breast cancer in mouse models.

Hui Liu,1 Charles Murphy,2 Florian Karreth,2 Kangkang Yang,1 Gerburg Wulf,1 Lewis C. Cantley2. 1 _Harvard Medical School, Boston, MA;_ 2 _Weill Cornell Medical College, New York, NY_.

15-20% of human breast tumors are triple negative breast cancer (TNBC), an aggressive and deadly subtype of breast cancer that currently lack targeted therapies, leaving chemotherapy as the only systemic treatment option. There is a pressing need for a better understanding of disease mechanisms of TNBC, and for the development of new treatment options. While activating mutations of PIK3CA are frequently found in ER-positive and Her2-amplified breast cancer, inactivation of lipid phosphatases is more frequent in TNBC. Analysis of large human genomics data in TCGA reveals that heterozygous-loss of INPP4B, a lipid phosphates in the PI3K signaling pathway, is enriched in TNBC subtype, and strongly correlates with loss of ER expression. Guided by human genomics information, we have crossed INPP4B phosphatase deletion mice into TNBC mouse model to determine whether INPP4B-loss cooperates with loss-of-function of p53 and/or Brca1 to promote tumorigenesis in vivo. Our results show dose-dependent increase in tumor development frequency in K14cre; Brca1flox/flox; p53flox/flox mice carrying INPP4B Phosphatase KO allele, HET allele compared to INPP4B WT allele. Importantly, these tumors resemble human TNBC in their pathology, histological patterns and gene expression patterns, providing a valuable platform to test their responsiveness to various therapeutic drugs, including PI3K-inhibitors. Our goal is to generate data that form the basis for a phase I/II clinical study that lead to substantially improved treatment strategies for basal-like breast cancer. Toward this goal, we have banked the endogenous tumors in a way allowing transplantation in nude mice, and have performed randomized drug treatment studies. We found that tumors that are heterozygous or deletion for INPP4B are more sensitive to PI3K-inhibition, suggesting these tumors are more dependent on Pi3K activation to thrive. Although tumors show partial response early on, they inevitably relapsed, begging for an understanding of innate and selected drug-resistant mechanisms. To this end, we have completed large-scale RNAseq and whole exome sequencing (WES), and have performed in-depth analyses on the mouse genomics data to identify genetic alternations including chromosomal number variations (CNVs), mutations and gene fusions. Focusing on gene-fusions, we found that by targeting these fusions, we can achieve better treatment outcomes and in some cases, even complete tumor remission. Significantly TCGA data analyses revealed the presence of similar fusions in human TNBC patients and we are in the process of investigating whether human cells carrying these fusions respond equally well compared to their mouse counterparts. Our approach of precision-medicine guided treatment optimization has lead to substantially improved treatment outcomes.

#3859

Peptidomimetic Src kinase inhibitor KX-01 sensitizes estrogen receptor α-negative breast tumor xenografts to tamoxifen by inducing ERα re-expression.

Murali Anbalagan,1 Mei Sheng,1 Brian Fleischer,1 David Hangauer,2 Brian G. Rowan1. 1 _Tulane Univ. School of Medicine, New Orleans, LA;_ 2 _Athenex Inc, Buffalo, NY_.

Unlike breast cancer that is positive for estrogen receptor α (ERα) or amplified/overexpressed HER2/neu, there are no targeted therapies for triple negative breast cancer (TNBC). ERα is silenced in TNBC through epigenetic changes including DNA methylation and histone acetylation. Restoring ERα expression in TNBC tumors may sensitize patients to endocrine therapy. Expression of c-Src kinase and ERα are inversely correlated in breast cancer suggesting that c-Src inhibition may lead to re-expression of ERα in TNBC. KX-01 is a peptide substrate targeted Src kinase/pretubulin inhibitor in clinical trials for solid tumors. At low dose, KX-01 is an effective Src kinase inhibitor in breast tumor xenografts in mice, but does not affect microtubules. MDA-MB-231 and MDA-MB-468 TNBC xenografts in NUDE mice were treated with low dose (1 mg/kg b.wt. BID) KX-01 for 40 days. KX-01 treatment decreased Src kinase activity in tumors and increased ERα mRNA and protein. Immunohistochemistry and Western blot demonstrated that the epithelial markers progesterone receptor and E-cadherin were increased, whereas the mesenchymal markers vimentin and nuclear β-catenin were decreased, suggesting a mesenchymal to epithelial transition (MET) in the tumors. At the ERα promoter in MDA-MB-231 tumors, KX-01 treatment led to enrichment of transcriptionally active (acetyl-H3, acetyl-H3Lys9) chromatin marks. There was no change in ERα promoter methylation as assessed by methylation PCR analysis and bisulfite sequencing. KX-01 treatment did not alter histone deacetylase 1 (HDAC1) levels or activity in tumors, but did result in HDAC1 dissociation from the ERα promoter, and a concomitant recruitment of RNA Polymerase II as assessed by chromatin immunoprecipitation (ChIP). Tamoxifen-resistant MDA-MB-231 xenografts in NUDE mice were treated with vehicle, tamoxifen alone (5 mg pellet; 60 day release), KX-01 alone (1 mg/kg b.wt. BID), or KX-01 + tamoxifen for 40 days. KX-01 alone and KX-01 + tamoxifen reduced tumor volume by 59% and 70%, respectively, compared to vehicle. Tumor volume for the KX-01 + tamoxifen group was significantly reduced compared to the KX-01 alone group (P= .023), and tumor weight of the KX-01 + tamoxifen group was 32% lower compared to the KX-01 alone group (P< 0.01). Only the KX-01 + tamoxifen group exhibited reduced levels of ERα targets pS2, c-Myc and cyclin D1 indicating that estradiol signaling was attenuated by tamoxifen only in tumors treated with KX-01. In MDA-MB-468 tumors, tamoxifen alone (10 mg pellet; 60 day release) and KX-01 alone (1 mg/kg b.wt. BID) had no effect on tumor volume compared to vehicle, but KX-01 + tamoxifen reduced tumor volume 67% compared to vehicle (P= 0.0025). Collectively, these data demonstrate that the peptidomimetic Src inhibitor KX-01 can restore ERα expression in TNBC through changes in histone acetylation that sensitize TNBC tumors to endocrine therapy (tamoxifen).

#3860

The repurposed anthelmintic mebendazole in combination with trametinib suppresses refractory NRASQ61K melanoma.

Cynthia M. Simbulan-Rosenthal,1 Sivanesan Dakshanamurthy,1 Anirudh Gaur,1 You-Shin Chen,1 Rena Shimizu,1 Maryam AbdusSumad,1 Hengbo Zhou,2 John Zapas,3 Valerie Calvert,4 Emaneul F. Petricoin,4 Michael B. Atkins,1 Stephen W. Byers,1 Dean S. Rosenthal1. 1 _Georgetown University School of Medicine, Washington, DC;_ 2 _University of Colorado, Denver, CO;_ 3 _Surgical Specialists; Monocacy Health Partners, Frederick, MD;_ 4 _George Mason University, Institute for Advanced Biomedical Research, Manassas, VA_.

Structure-based drug repositioning in addition to random chemical screening is now a viable route to rapid drug development. Proteochemometric computational methods coupled with kinase assays showed that MBZ binds and inhibits kinases important in cancer, especially both BRAFWT and BRAFV600E. We find that MBZ synergizes with the MEK inhibitor trametinib to inhibit growth of dabrafenib-resistant BRAFWT-NRASQ61K melanoma cells in culture and in xenografts, and markedly decreased MEK and ERK phosphorylation in vivo. RPPA and immunoblot analyses show that both trametinib and MBZ inhibit the MAPK pathway, and cluster analysis revealed a protein cluster showing strong MBZ+trametinib - inhibited phosphorylation of MEK and ERK within 10 minutes, and its direct and indirect downstream targets related to stress response and translation, including ElK1 and RSKs within 30 minutes. Downstream ERK targets for cell cycle, including cMYC, were downregulated, consistent with S phase suppression by MBZ+trametinib, while apoptosis markers, including cleaved caspase-3, cleaved PARP and a sub-G1 population, were all increased with time. These data suggest that MBZ, an off-patent approved drug with no major side effects, should be considered as a therapeutic option in combination with trametinib, for patients with NRASQ61mut or other non-V600E BRAF mutant melanomas.

#3861

Ruxolitinib inhibits STAT-3 activation in glioblastoma.

Samuel Fehling, Braden McFarland, Etty Benveniste. _University of Alabama at Birmingham, Birmingham, AL_.

Glioblastoma (GBM), a primary Grade IV astrocytoma, is the most aggressive primary brain cancer in humans. Despite combined radiotherapy and chemotherapy, the median survival is 14 months. We and others have validated that levels of phosphorylated JAK1/2 and STAT-3 are increased in GBM tissues. When stimulated with IL-6 family members, JAK1/2 recruits and phosphorylates STAT-3. Phosphorylated STAT-3 dimerizes and translocates to the nucleus where it regulates genes involved in proliferation, angiogenesis, anti-apoptotic activity and immunosuppression. Elevated JAK/STAT activation is generated through an increase in IL-6 family cytokines as well as a decrease in JAK/STAT-3 negative regulators. To therapeutically inhibit the JAK/STAT-3 pathway in GBM we employed ruxolitinib, an FDA approved JAK1/JAK2 inhibitor. Human GBM cell lines U87-MG and U251-MG, as well as patient derived GBM xenograft lines JX6 and JX12, were treated with ruxolitinib in doses ranging from 0.01 to 10 µM. Ruxolitinib was observed to inhibit STAT-3 activation in a dose-dependent manner. Pre-treatment of U87-MG and U251-MG cells with ruxolitinib inhibited stimulus-induced STAT-3 activation and downstream gene expression in a dose-dependent manner. Future studies will examine ruxolitinib effects on migration, invasion, apoptosis and proliferation in vitro. Collectively, these data indicate the potential for ruxolitinib therapy for GBM patients.

#3862

Ormeloxifene, a novel pharmacological activator of PKD1 enhances docetaxel sensitivity in prostate cancer.

Aditya Ganju, Bilal Bin Hafeez, Fathi Halaweish, Wei Li, Man Mohan Singh, Murali Mohan Yallapu, Subhash Chauhan, Meena Jaggi. _University of Tennessee Health Science Center, Memphis, TN_.

Background: Prostate cancer (PrCa) is the second leading cause of cancer-related deaths in American Men. Docetaxel (DTX) is a standard first-line treatment for metastatic castration-resistant PrCa after the failure of hormone therapy. However, most PrCa patients who receive DTX experience only transient benefits and rapidly develop incurable drug resistance. Protein Kinase D1 (PKD1), one of the serine threonine kinases from PKD family is highly expressed in normal prostate tissues and is suppressed during PrCa progression. Accumulative evidence suggest a tumor suppressive role of PKD1 in PrCa, while other isoforms of PKD (PKD2 and PKD3) act as oncogene. In this study, we identified pharmacological agent Ormeloxifene (ORM) which selectively activates PKD1 and inhibits metastasis associated protein 1 (MTA1), thus induces sensitivity to DTX treatment in PrCa cells.

Materials and Methods: We have used androgen-independent human PrCa cells (C4-2) which show low PKD1 expression compared to other PrCa cell lines. Cells were treated with 10 and 15 µM doses of ORM for 24 hrs and various functional assays (cell proliferation, colony formation, motility and invasion) were performed. In a parallel experiment, cells were treated with ORM (10 and 15 µM) for 24 hrs protein and RNA samples isolation. Protein lysates were used to investigate the effect of ORM on PKD1, PKD2, PKD3 and MTA1 protein levels. qRT-PCR was performed to investigate the effect of ORM on PKD1, PKD2 and PKD3 expression at mRNA levels. To investigate if ORM treatment sensitizes the effects of DTX, cells were treated with ORM and DTX alone or in combination. In-silico docking studies were performed to determine the putative molecular interaction of ORM with MTA1.

Results: ORM treatment inhibits proliferation and clonogenic potential of C4-2 cells. We observed that ORM significantly induces PKD1 expression at protein and mRNA level in C4-2 cells. To determine whether this PKD1 inducing effects of ORM in PrCa cells is specific, we examined the effects of ORM on PKD2 and PKD3 at mRNA and protein levels. Interestingly, we observed that ORM treatment inhibits expression of oncogenic PKD3 isoform, however, no effect PKD2 was observed. MTA1 is involved in DTX resistance and ORM treatment effectively inhibited the expression of MTA1. However, there was no effect of DTX treatment on the expression of MTA1. We also observed that ORM treatment significantly potentiates the effect of DTX on cell viability and colony formation of C4-2 cells. In-silico docking studies between ORM and MTA1 showed four potential binding sites with best score at serine 270.

Conclusion: Overall, our study defines ORM as a novel PKD1 activator/modulator which also inhibits a key metastasis associated protein, MTA1 and sensitizes the PrCa cells to DTX. Based on these results, it appears that ORM may be a novel therapeutic modality for advanced stage metastatic PrCa alone or in combination with DTX.

#3863

Cancer immunotherapy: Could TK1 be used as a target for colon cancer.

Michelle H. Townsend, Evita G. Weagel, Wei Meng, Craig Chandler, Richard A. Robison, Kim L. O'Neill. _Brigham Young University, Provo, UT_.

The aim of this study is to determine whether Thymidine Kinase 1 (TK1) would be a suitable target for future immunotherapeutic treatment of colon cancer. The salvage pathway enzyme TK1 has been shown to be upregulated in cancer patients due to active proliferation of cancer cells and the resulting high demand for nucleotides. Present in both the cytosol and the serum of cancer patients, TK1 levels can be used to not only predict cancer occurrence, but also to predict tumor aggressiveness and future remission. While serum levels of TK1 have been established, surface expression of the enzyme have not been fully characterized. In colon cancer cells, TK1 has been shown to strongly associate with the plasma membrane. In this project we explored the presence of TK1 on the surface of colon cancer cells (HT29 and SW620 cell lines) using flow cytometry, confocal microscopy, scanning electron microscopy, membrane separation, and cytoplasmic staining. Using fluorescent antibodies conjugated to FITC, expression levels as high as 19% are observed in HT29's and 12% in SW620's in comparison to sodium potassium ATPase surface levels of 11% and 6% respectively. Confocal microscopy images revealed direct overlap between cells stained with a rhodamine red membrane dye and with a FITC conjugated anti-TK1 antibody which indicates a direct relationship between TK1 and the cellular surface of cancer cells. To further confirm surface expression, TK1 levels were assessed using gold labeling and scanning electron microscopy. Gold levels indicate the physical location of the TK1 on the surface of the colon cancer cells and their relative abundance. TK1 surface levels in colon cancer cells corresponded with the surface expression levels of sodium potassium ATPase and share relatively the same abundance. These results strongly indicate a direct relationship between TK1 and the surface of colon cancer cells, and suggests TK1 as a potential target for future immunotherapeutic treatment.

#3864

The Jak1/2 inhibitor AZD1480 suppresses tumor growth and metastasis in genetically engineered mouse models of PTEN-deficient prostate cancer.

Marco A. De Velasco,1 Yurie Kura,1 Naomi Ando,1 Emiko Fukushima,1 Yuji Hatanaka,1 Takashi Oki,1 Kazuhiro Yoshimura,1 Masahiro Nozawa,1 Barry R. Davies,2 Dennis Huszdar,2 Kazuhiro Yoshikawa,3 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kinki University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _AstraZeneca, Macclesfield, United Kingdom;_ 3 _Aichi Medical University, Nagakute, Japan_.

Activation of signal transducer and activator of transcription (STAT) 3 via the interleukin 6 (IL-6)/IL-6R/Janus kinase (JAK) signaling axis is a feature common in patients with advanced human prostate cancer and has been associated with poor prognosis. STAT3 can promote CRPC growth by activating the androgen receptor signaling pathway. It can also promote survival by modulating tumor cell proliferation, epithelial to mesenchymal transition, angiogenesis and immune response. However, recent preclinical data suggests that STAT3 may also act as a tumor suppressor in PTEN-deficient prostate cancer. In this study, we investigated the effectiveness of AZD1480, a potent JAK1/2 inhibitor, in genetically engineered mouse models of prostate cancer driven by the loss of PTEN and PTEN/P53. In vivo pharmacodynamic studies using conditional PTEN-deficient mice demonstrated that AZD1480 strongly inhibited STAT3 phosphorylation. Moreover, AZD1480 monotherapy in PTEN-deficient mice harboring castration-naïve or castration-resistant prostate tumors resulted in tumor growth reductions of 24.8%, P<0.001 and 15.8%, P<0.001, respectively, compared to vehicle treated control mice. Mice treated with AZD1480 also exhibited reduced levels of tumor cell proliferation and microvessel density, and increased apoptosis compared to controls. Clinically relevant outcomes were evaluated in a late-stage model of prostate cancer driven by the conditional inactivation of PTEN and P53. In this model, PTEN/P53 double knockout mice with established tumors were randomized to vehicle or AZD1480 treatment groups. Mice treated with AZD1480 demonstrated a modest but statistically significant improvement in overall survival compared to control mice, median time 20 days vs. 27 days, P=0.03, respectively. Additionally, the metastatic tumor incidence decreased from 46.6% (7/15) in control mice to 20% (3/15) in AZD1480-treated mice. Our findings provide preclinical evidence supporting the potential use of Jak1/2 inhibitors for the targeted therapy of human prostate cancer.

#3865

Therapeutic activation of protein phosphatase 2A for the treatment of lung cancer.

Jaya Sangodkar,1 Rita Tohme,2 Janna Kiselar,2 Sudeh Izadmehr,1 Divya Hoon,1 Sahar Mazhar,2 Abbey Perl,2 Danica Wiredja,2 Daniela Schlatzer,2 Shen Yao,1 David Kastrinsky,1 Neelesh Sharma,2 David Brautigan,3 Mark Chance,2 Alain Borczuk,4 Michael Ohlmeyer,1 Yiannis Ioannou,1 Goutham Narla2. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Case Western Reserve University, Cleveland, OH;_ 3 _University of Virginia School of Medicine, Charlottesville, VA;_ 4 _Weill Cornell Medical College, New York, NY_.

PP2A is a phosphatase tumor suppressor that is dysregulated and deactivated in lung cancer. It is one of the most abundant cellular proteins and regulates the activity of numerous kinases Where achievable, restoration of PP2A function inhibits cancer progression, and notably, by the inhibition of the downstream effectors of the oncogenic kinases that initiate and drive cancer progression. In this study, we determined PP2A inactivation in human lung cancer with specific molecular genotypes and we ascertained the biological and functional consequences of PP2A reactivation. In assessing a lung cancer TMA, we identified that PP2A inactivation was correlated with poor survival and was significantly higher in patients with Kras mutations. In order to understand the therapeutic potential of restoration of PP2A activity in KRAS mutant lung cancer, our lab developed a series of small molecule activators of PP2A (SMAPs) through reverse engineering of tricyclic neuroleptic drugs. SMAP treatment of lung cancer cell lines resulted in an induction of apoptosis and decreased cell viability. Structural and biophysical studies have identified the site of drug binding and mechanism for PP2A activation by this small molecule series. Additionally, cell lines harboring drug-binding mutations were resistant to SMAP therapy as compared to wild type PP2A and EGFP control. Global phosphoproteomic analysis of SMAP treated KRAS lung cancer cell lines revealed ERK signaling as a commonly perturbed pathway in drug treated cell lines. Given the marked dephosphorylation of ERK upon treatment of cell lines with SMAPs, we overexpressed a constitutively active form of MEK (MEKDD) to blunt SMAP mediated ERK dephosphorylation to determine the relevance of ERK inactivation for the biological effects of SMAPs on cellular apoptosis. Overexpression of MEKDD resulted in a blunted apoptotic response to SMAP treatment. Single agent SMAP treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. Western blotting and immunohistochemical analysis of the tumors demonstrated that SMAP treatment resulted in of ERK, AKT, and PP2A-Y307 dephosphorylation in vivo. Additionally, these compounds demonstrate favorable pharmacokinetics and show no overt toxicity. Furthermore, combination of SMAPs with kinase inhibitors further decreased tumor growth in vivo. Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of KRAS-mutant NSCLC.

#3866

The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes acute myeloid leukemia cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors.

Kshama A. Doshi,1 Patrick R. Baldwin,2 Shivani Kapoor,2 Maria R. Baer2. 1 _University of Maryland, Baltimore, MD;_ 2 _University of Maryland Greenebaum Cancer Center, Baltimore, MD_.

Introduction:

Internal tandem duplication of the fms-like tyrosine kinase-3 receptor (FLT3-ITD) is present in acute myeloid leukemia (AML) cells in 30% of patients and these patients have short disease-free survival following chemotherapy. FLT3 inhibitors are clinically active, but their activity is limited and transient. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, and promotes FLT3 signaling in a positive feedback loop in FLT3-ITD cells. We have previously demonstrated that inhibiting Pim kinase sensitizes AML cell lines and primary AML patient blasts with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitor chemotherapy drugs. Here we studied the effects of the pan-Pim kinase inhibitor PIM447 (formerly LGH447; Novartis Pharmaceuticals), currently in clinical trials, on response of FLT3-ITD-expressing cell lines and AML patient samples to FLT3 inhibitors and to topoisomerase 2 inhibitors, with the ultimate goal of developing clinically applicable combination regimens.

Methods:

Cell lines and AML patient samples with FLT3-ITD were cultured with FLT3 inhibitors or topoisomerase 2 inhibitors at clinically applicable concentrations and PIM447 at a range of concentrations. Apoptosis was measured by Annexin V labeling and PARP cleavage. Mcl-1 expression was measured by immunoblotting. Reactive oxygen species (ROS) were measured with the redox-sensitive dye CM-H2DCFDA and DNA double-strand breaks (DSBs) by immunoblotting for γH2AX.

Results:

Transfected Ba/F3-ITD and 32D/ITD cells, MV4-11 and MOLM-14 human AML cells and primary AML patient blasts, all expressing FLT3-ITD, were treated with FLT3 inhibitors, including 100 nM midostaurin and 1 nM quizartinib, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 produced a concentration-dependent increase in apoptosis induced by each FLT3 inhibitor (p<0.001). FLT3-ITD-expressing cells were also treated with the topoisomerase 2 inhibitors daunorubicin and etoposide at IC50 concentrations, with 0, 10, 100 and 500 nM PIM447. Co-treatment with PIM447 also produced a concentration-dependent increase in apoptosis induced by each topoisomerase 2 inhibitor (p<0.001). Increased apoptosis induced by PIM447 with FLT3 inhibitors was associated with Mcl-1 downregulation, while increased apoptosis induced by PIM447 with topoisomerase 2 inhibitors was associated with increased generation of ROS and increased DNA DSBs.

Conclusions:

The clinically applicable pan-Pim kinase inhibitor PIM447 sensitizes AML cells with FLT3-ITD to induction of apoptosis by FLT3 inhibitors and by topoisomerase 2 inhibitors. Our data support in vivo testing of combination regimens and design of clinical trials aimed at improving outcomes for patients with AML with FLT3-ITD.

#3867

Combined PI3K and AURKA inhibition are efficacious in triple-negative breast cancer models.

Amanda E.D. Van Swearingen, Maria J. Sambade, Shivani Sud, Brian Golitz, Gary L. Johnson, Carey K. Anders. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Introduction: Nearly half of metastatic triple negative breast cancer (TNBC) patients develop brain metastases (BMs) and face a poor prognosis. There are no FDA-approved systemic therapies to treat TNBC BM, due in part to the blood-brain barrier. BCBMs exhibit both activation of the PI3K pathway and AURKA amplification/overexpression relative to primary breast cancers. In this study, we evaluate the efficacy of brain-penetrant, clinically-available inhibitors of PI3K and AURKA in TNBC cell lines that are capable of growing in the mouse brain.

Methods: In vitro characterization of the pan-PI3K inhibitor BKM120 and AURKA inhibitor MLN8237 were conducted in 2 human-derived TNBC cell lines, SUM149 and (MDA-MB-)231Br. A siRNA screen (720 kinase genes) was used to identify synthetic enhancers of lethality with BKM120 treatment. To assess the efficacy of these drugs, the IC50s of BKM120 and MLN8237 and synergy of the combination were determined. To compare the effects of BKM120 and/or MLN8237 treatment on cell cycle progression, FACS analysis was conducted at 24, 48, and 72 hours in parent cells and in cells continuously cultured in MLN8237-treated media for 12 weeks.

Results: The screen confirmed that combined PI3K and AURKA inhibition synthetically enhanced lethality in SUM149 and 231Br cells. SUM149 and 231Br cells and two additional TNBC cell lines (MDA-MB-468 and MDA-MB-436) exhibited similar IC50s (1.3-21 µM) to BKM120. However, there was a >2.5 fold range (26.5-69 µM) in IC50s for MLN8237, with the greatest potency in the 231Br line. Concurrent treatment with BKM120+MLN8237 was synergistic or additive in 231Brs at most doses, whereas the combination was additive to antagonistic in SUM149s. Pretreatment with MLN8237 prior to concurrent BKM120+MLN8237 improved synergy in SUM149s, while BKM120 pretreatment improved synergy in 231Brs.

FACS analysis of BKM120 in the SUM149 and 231Br cells induced a slight G1 arrest from 24 to 72 hours, while MLN8237 initially induced a G2 arrest at 24 hours, polyploidy at 48 hours, and a mixed polypoid/G2 arrested population at 72 hours. These effects were more pronounced in the 231Brs than the SUM149s. Combined BKM120+MLN8237 in both cell lines yielded results similar to MLN8237 alone. Cells continuously exposed to increasing MLN8237 concentrations from 50 nM to 300 nM for 12 weeks were resistant to MLN8237-induced cell cycle changes as compared to passage-matched controls.

Conclusions: Combined PI3K+AURKA inhibition using brain-penetrant compounds is a promising strategy for a patient population with few options. In vivo studies evaluating the efficacy of BKM120+MLN8237 in intracranial TNBC mouse models to provide the translational foundation for future clinical studies are warranted.

#3868

Aggressiveness niche within BRCA1-IRIS/TNBC tumors is supported by reciprocal interactions with the microenvironment.

Abhilasha Sinha, Wael M. ElShamy. _University of Mississippi Medical Center, Jackson, MS, Jackson, MS_.

Introduction: Bi-directional interactions with stromal cells promote metastasis from breast tumors. We aimed at determining the role these bi-directional interactions between BRCA1-IRIS-overexpression (IRISOE) tumor cells and stromal cells in generating aggressive triple negative breast cancers.

Experimental Procedures: We used co-culturing assay, immunohistochemistry, in vitro invasion assay with or without cytokines neutralization, ELISA, western blot analysis, exposure to hypoxic conditions, cytokines receptor inactivation, in vivo orthotopic tumor formation assay, analysis of publically available data sets for the association of selected factors with overall survival, distant metastasis free survival and recurrent free survival to define the role of these bi-directional interactions in enhancing IRISOE tumors aggressiveness.

Results: Here we show formation of an aggressiveness niche within IRIS overexpressing triple negative breast cancers (IRISOE/TNBCs) defined as the necrotic/hypoxic core. To this niche, metabolically stressed IRISOE/TNBC cells attract mesenchymal stem cells (MSCs) to the aggressiveness niche through secreting high levels of IL-1β. IL-1R-negative MSCs are induced to express the receptor upon exposure to medium conditioned by IRISOE/TNBCs. Activated by IL-1β, IL-1R activates AKT, MAPK and NF-κB signaling in MSCs to which they respond by releasing inflammatory cytokines, such as CXCL1. Inhibiting IL-1β signaling utilizing neutralizing antibody attenuates MSCs recruitment and CXCL1 secretion, in vitro and in vivo. CXCL1 receptor; CXCR2 is expressed on IRISOE/TNBCs only and thus CXCL1 acts in a paracrine fashion to entrain IRISOE/TNBCs. Mechanistically, MSCs-entrained IRISOE/TNBCs secrete CCL2 and VEGF that activate and attract tumor associated macrophages (TAMs) and endothelial cells (ECs), respectively to the vicinity of IRISOE/TNBCs in the aggressiveness niche. S100A8 and IL-8 secreted from IRISOE/TNBCs-activated TAMs and ECs, respectively in concert with CXCL1 enhance IRISOE/TNBCs aggressiveness. In pre-clinical model, IRISOE cells co-injected with MSCs (10:1 ratio) developed faster and bigger orthotopic mammary tumors containing more TAMs and ECs compared to IRISOE/TNBCs injected alone. These tumors progressed to develop spontaneous lymph-node or distant metastases (e.g., bone, lung and/or brain).

Conclusion: We propose that in concert with an anti-BRCA1-IRIS drug, inhibitors to the reciprocal interactions between IRISOE/TNBCs and their surrounding stromal cells could be exploited as a viable therapy for IRISOE/TNBCs and their metastases.

## CANCER CHEMISTRY:

### Proteomics and Mass Spectrometry

#3869

Phosphoproteomic-based identification of CDK8/CDK19 substrates in colorectal cancer.

Olajumoke O. Popoola,1 Rahul Samant,1 Maria-Jesus Ortiz-Ruiz,1 Aurelie Mallinger,1 Will Court,1 Steve Hobbs,1 Robert Te-Poele,1 Mark Stubbs,1 Rosemary Burke,1 Christina Esdar,2 Kai Schiemann,2 Dirk Wienke,2 Sue Eccles,1 Julian Blagg,1 Paul Workman,1 Paul Clarke1. 1 _Institute of Cancer Research, London, United Kingdom;_ 2 _Merck KGaA, Darmstadt, Germany_.

Introduction

CDK8 is an oncogenic cyclin-dependent kinase that exists as part of the kinase module within the Mediator complex. This complex interacts with the transcription machinery to regulate transcription; signal transduction pathways, including the WNT pathway; and biological processes, such as cell cycle progression. Recently, we identified a series of 3,4,5-trisubstituted pyridines as inhibitors of CDK8 and, its paralogue, CDK19 in colorectal cancer (CRC). Until now, there have been few validated substrates of CDK8/19. Here, we describe a motif-based phospho-proteomic approach, utilizing our 3,4,5-trisubstituted pyridine inhibitors that we have used to identify substrates of CDK8/19. These substrates could represent useful biomarkers for future drug discovery research.

Experimental Outline

We used a COLO205 cell line (COLO205 C4) carrying a TCF/LEF reporter construct responsive to CDK8/19 inhibition and CCT251545, a compound we have previously shown to be a potent and selective inhibitor of CDK8/19. Cells were treated with 350 nM CCT251545 (10 x EC50) for 2 or 6 hours. Proteins were then extracted from treated and control cells, trypsin-digested and immunoprecipitated for phospho-peptide enrichment using proline-directed motifs: PXS*P, S*PXR/K, PXS*PXR/K + T*PE + ST*P + K/HS*P. Potential substrates were identified by LC-MS/MS and validated by immunoprecipitation and western blotting. Substrates were validated in another CRC cell line, SW620, which harbors CRISPR knockouts for CDK8/19.

Summary of Results

LC-MS/MS analyses of COLO205 C4 cell extracts revealed a number of potential CDK8/19 substrates, including some Mediator complex subunits such as MED13, transcriptional coactivators such as HCFC1, and transcription factors such as STAT1. Phosphorylation of STAT1SER727 was the top ranked hit and follow-up studies, in COLO205 C4 cells and xenografts, confirmed repression of STAT1SER727 phosphorylation in the presence of CCT251545. An inactive analogue, CCT251099, and other kinase inhibitors (flavopiridol, KN-93, PD 0325901 and SB 202190) did not block STAT1SER727 phosphorylation. Repression of STAT1SER727 phosphorylation upon treatment with CCT251545 was also observed in SW620 and LS174T CRC cells and xenografts.

Conclusion

A motif-driven, mass spectroscopy-based phospho-proteomic study identified candidate substrates of CDK8/19. Phosphorylation of STAT1SER727 was validated as a useful marker of target engagement in CRC cell lines both in vitro and in vivo.

#3870

SLFN11 is a biomarker of sensitivity to PARP inhibition and chemotherapy in small cell lung cancer (SCLC).

C. Allison Stewart, Pan Tong, Robert Cardnell, Triparna Sen, Fatemah Mina Masrorpour, Youhong Fan, Jing Wang, Lauren Averett Byers. _UT MD Anderson Cancer Center, Houston, TX_.

Small cell lung cancer (SCLC) is an aggressive disease that accounts for 14% of lung cancers. Very little progress has been made towards the treatment of SCLC in the past four decades and there are no established biomarkers to predict effective therapies for patients. In other cancers, SLFN11 plays an important role in sensitizing cancer cells to topoisomerase inhibitors, DNA synthesis inhibitors and alkylating agents. Previously, our lab identified an increase in poly (ADP-Ribose) polymerase 1 (PARP1), an enzyme involved in DNA damage repair, in SCLC patients and cell lines. PARP inhibitors demonstrate significant anti-tumor activity in cell line and animal models of SCLC and are currently being tested in clinical trials for SCLC patients. In Ewing sarcoma (EWS), SLFN11 has been proposed as a biomarker of PARP inhibitor response. Because both EWS and SCLC have high PARP levels and respond favorably to PARP inhibition, we hypothesized that SLFN11 may also be a biomarker for drug sensitivity in SCLC. Using SCLC patient tumors and a large panel of molecularly profiled SCLC cell lines, we investigated the expression of SLFN11 in SCLC and its association with in vitro sensitivity to PARP inhibition (olaparib) and chemotherapy. SLFN11 mRNA levels are significantly higher in SCLC patient tumors relative to normal lung tissue (P=0.005). In a panel of 51 SCLC cell lines, higher SLFN11 protein expression correlates with both cisplatin (P<0.001) and olaparib (PARP inhibitor; P=0.05) sensitivity. In fact, SLFN11 is the top protein biomarker of cisplatin sensitivity in SCLC cell lines (of 171 proteins measured). Consistent with what has been shown for other cancer types, SLFN11 mRNA expression is also correlated with sensitivity to irinotecan (P=0.005) and topotecan (P=0.05) in SCLC, two drugs frequently used to treat this disease. Expression of other SLFN family members does not correlate with drug sensitivity. SLFN11 protein and mRNA levels are concordant (P<0.001) and both demonstrate a bimodal expression pattern in SCLC cell lines which corresponds to cisplatin (protein, P<0.001; mRNA, P=0.005) and olaparib (mRNA, P=0.05) sensitive (SLFN11 high) versus resistant (SLFN11 low) groups. In EWS, leukemia, colon, breast and prostate cancers, SLFN11 functions as an ETS transcription factor response gene. However, a panel of 26 ETS transcription factors did not correlate well with SLFN11 or PARP1 mRNA expression or cisplatin sensitivity, suggesting that SLFN11 expression may be regulated in a different manner in SCLC. In summary, SLFN11 is a predictor of sensitivity to chemotherapies routinely used in the treatment of SCLC, including alkylating agents (cisplatin) and topoisomerase inhibitors (irinotecan, topotecan). Moreover, SLFN11 predicts in vitro response to PARP inhibition, which may have important clinical implications given the ongoing clinical trials investigating PARP as a novel therapeutic target for SCLC.

#3871

Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics.

Luke A. Wittenburg, Holly Conger, Dominique Ramirez, Daniel Gustafson. _Colorado State Univ., Fort Collins, CO_.

The dog serves as a useful model of spontaneously occurring cancer by virtue of similar biology, therapies and mechansims of chemotherapeutic resistance. The ABC transporters are a superfamily of membrane bound proteins that are widely distributed across mammalian tissues and constitute a substantial component of variability in anticancer drug pharmakokinetics and tumor cell resistance. Thus a quantitative atlas of ATP-binding cassette (ABC) transporters across canine tissues is crucial for improving our ability to extrapolate data from dog to human. We have employed a quantitative proteomic approach based on multiple reaction monitoring in tandem mass spectrometry that relies on detection of novel peptide sequences following trypsin digestion of biological samples and separation of peptides utilizing high-pressure liquid chromatography. The 14 proteins evaluated include P-gp, Bcrp, Bsep, Mrp1-6, Mrp7, Mrp9, and the products of canine Abcg5 and Abcg8. Peptide sequences were identified via an in-silico trypsin digestion of proteins and signature peptides were generated for identification and quantitation using stable isotope-labeled signature peptides as an internal reference. Signature peptides were chosen based on lack of posttranslational modification sites and known polymorphisms. Isolated brain microvessels, liver, kidney, duodenum, jejunum, ileum and lung were collected at necropsy from ten normal dogs and membrane fractions of each tissue were prepared. Additional samples were collected for isolation of mRNA. Membrane protein fractions were digested with trypsin and total protein concentrations determined by BCA assay. Calibration curves for signature peptides were prepared in a bovine serum albumin digestate and ranged from 0.25 fmol to 200 fmol on column. Quantitation of peptides in tissue samples was performed via linear regression analysis of calibration curves with extrapolation using at least two transitions for each peptide. Results were obtained as femtomole of peptide per microgram of total protein in the sample. Differential protein levels across tissues were compared to quantitative real time RT-PCR results for the corresponding genes and differences between tissues with each method were concordant. Our results provide the first quantitative atlas of ABC transporters across normal dog tissues and provide a framework for improved understanding of pharmacokinetic and pharmacodynamic variability among dogs receiving anticancer therapies. In addition, this panel will allow evaluation of the potential role that each of these transporters plays in resistance to chemotherapy in vitro and in vivo in dogs, a relevant preclinical model of naturally occurring cancers.

#3872

Quantification of mutant SPOP proteins in prostate cancer using targeted proteomics.

Hui Wang,1 Christopher Barbieri,2 Jintang He,1 Yuqian Gao,1 Chaochao Wu,1 Athena Schepmoes,1 Thomas Fillmore,1 Tujin Shi,1 Sung-Suk Chae,2 Dennis Huang,2 Juan Miguel Mosquera,2 Wei-Jun Qian,1 Richard Smith,1 Sudhir Srivastava,3 Jacob Kagan,3 David Camp,1 Karin Rodland,1 Mark Rubin,2 Tao Liu1. 1 _Pacific Northwest National Laboratory, Richland, WA;_ 2 _Weill Cornell Medical College, New York, NY;_ 3 _National Cancer Institute, Bethesda, MD_.

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for diagnosis, prognosis or targeted therapy of prostate cancer. To address this issue, selected reaction monitoring (SRM) and PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) -SRM mass spectrometry assays have been developed for quantifying mutant SPOP proteins.

SRM assays for wild-type SPOP protein and 11 prostate cancer-derived mutations were developed. The presence of multiple lysine residues in the mutation regions precludes the use of conventional tryptic digestion. Arg-C was selected instead due to its superior performance in generating mutation site(s) containing SPOP peptides that are more suitable for SRM analysis comparing to other proteases (e.g., Asp-N). Although the resulting Arg-C peptides are longer and more hydrophobic than typical tryptic peptides, all the SRM assays showed a linear dynamic range of more than two orders of magnitude. The limits of quantification for the mutation site(s) containing peptides range from 10 to 100 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze 293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations, which agrees with RT-PCR results. It is unknown if this is related to activity of the SPOP protein. PRISM-SRM assays have shown further improvement in sensitivity.

SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.

#3873

Single cell cisplatin measurements by ICP-MS.

Lauren Amable,1 Stan Smith,2 Chady Stephan2. 1 _National Institute on Minority Health and Health Disparities, Bethesda, MD;_ 2 _Perkin Elmer, Shelton, CT_.

Cisplatin, carboplatin, and oxaliplatin are the most widely used class of cancer chemotherapy drugs in the Western world. Many cancers are initially sensitive to platinum treatment but drug resistance frequently occurs. Cellular uptake of cisplatin is related to tumor burden, thus lower intracellular cisplatin levels are associated with a decreased tumor response. Traditional methods to assess cisplatin uptake in cells involves digesting a cell population and measuring the total platinum content. This approach does not reflect the distribution and individual cellular variation of cisplatin uptake. Here, we present a new method, single cell inductively coupled plasma-mass spectrometry (SC-ICP-MS) to quantitate the platinum concentration within individual cells. Experiments were performed using the A2780 cisplatin-sensitive and the corresponding cisplatin-resistant A2780-CP70 ovarian cancer cell lines. Time course experiments were performed to measure the change of cisplatin uptake over time. Serum starvation experiments were also performed to examine if differences in cisplatin uptake were dependent on the cell cycle. SC-ICP-MS was performed by injecting cell suspensions and analyzing the platinum 195 isotope. Individual cellular cisplatin levels were collected and a histogram representing the cell population was generated using the instrument software (Syngistix Nano Application). Other metals, such as zinc, iron, and copper, were analyzed to measure the variation between cell lines after treatment. We observed a heterogeneous distribution of cisplatin concentrations within the sample reflecting that cisplatin uptake differs from cell to cell. Serum starvation affected the uptake of cisplatin within cells, changing the cisplatin distribution curve. SC-ICP-MS was also effective in measuring other metals within individual cells and differences were observed between the A2780 and A2780-CP70 cell lines. In conclusion, single cell ICP-MS analysis allows for the quantitation of cisplatin within individual cells. The heterogeneous distribution of cisplatin uptake more closely reflects what occurs within tumor cells. SC-ICP-MS allows for the development of strategies to increase cisplatin uptake, translating to better clinical responses.

#3874

Mass spectrometry imaging determines biomarkers of early adaptive precision drug resistance in lung cancer.

Erin H. Seeley,1 Pamela S. Cantrell,1 Callee M. Walsh,1 Sijin Wen,2 Satoshi Komo,2 Xiaoliang Wu,2 Wei Zhang,2 Patrick C. Ma2. 1 _Protea Biosciences, Morgantown, WV;_ 2 _West Virginia University Cancer Institute, Morgantown, WV_.

Drug resistance emergence is a common problem that limits long term outcome benefits in the era of precision cancer therapy. Recently, we identified an early precision drug escape mechanism with adaptive tumor cellular reprogramming emerging within days after drug initiation. Here we present a mass spectrometry imaging (MSI) approach to interrogate the biomolecular changes occurring within residual tumor cells under precision treatment with an ALK-specific kinase inhibitor TAE684 in EML4-ALK fusion (ALK+) lung adenocarcinoma xenograft.

ALK+ H3122 lung adenocarcinoma murine xenograft model was established for in vivo treatment with TAE684, at a daily dose of 25mg/kg by orogastric gavage (n=6). Diluent control was included as comparison (n=6). Tumor measurement revealed expected remarkable tumor response with TAE684. Control tumors and drug-treated residual tumor tissues were harvested for MSI studies, at day 7 and day 14 during tumor response. MSI was carried out on formalin fixed, paraffin embedded tissues to compare peptide profiles between control tumors and 7- and 14-day ALK-TKI treated tumors using a histology guided mass spectrometry approach. Briefly, two sections were collected from each sample, one for mass spectrometry and one for histology. Mass spectrometry sections were deparaffinized, antigen retrieved, and subjected to on-tissue tryptic digestion. Tumoral areas of interest (100 µm diameter, ~20 per sample) were annotated on digital microscopy images of the stained sections. The annotated images were merged with digital images of the unstained sections using Photoshop and this combined image was used to guide data acquisition from the areas of interest. Additionally, frozen control and day 14 TAE684 treated tumors were subjected to full section MSI to determine the ALK inhibitor drug distribution as well as the changing landscape of lipids and metabolites.

Statistical analysis of the peptide data resulted in determination of 580 significant peaks using Wilcoxon rank sum test with a Bonferroni correction. A genetic algorithm classification model consisting of 24 peptide peaks was generated using a leave-20%-out cross validation over 10 iterations that resulted in an overall classification accuracy across the 3 groups of over 98%.

Direct MS/MS fragmentation revealed that TAE684 was detected within the frozen dosed tumors, but was absent from the control tumors. Several lipids (notably, m/z 732.78, 744.67, and 770.72 increased, m/z 769.65 and 820.71 decreased) were found to undergo alterations in expression as a result of TAE684 treatment.

MSI allowed for the direct in situ determination of biomolecules that are changing in expression landscape in ALK+ lung cancer as a result of TAE684 treatment. These results provide a rationale to advance our MSI studies to deepen our insights in mechanisms of adaptive precision drug resistance to improve treatment outcomes.

#3875

Proteome-wide approach to identify novel biomarkers in oral tumorigenesis.

Sivagnanam Ananthi, Naga Padma Lakshmi CH, Anbarasu K, Mahalingam Sundarasamy. _Indian Institute of Technology Madras, Chennai, India_.

Oral cancer is the most common type of intraoral head and neck cancer in humans, with a worldwide prevalence of more than 400,000 cases. In India, oral cancer ranks in top three in comparison with other cancer types. The majority of oral cancer patients are diagnosed at an advanced disease stage. Although, there have been many advances in oral cancer treatment, the overall survival rate of oral cancer patients only improved marginally over the past 30 years. The treatment regimen of oral cancer is mainly based on the tumor, node and metastasis (TNM) classification and histopathological diagnosis. These methods are subjective and often have low sensitivity to detect the disease in early stages. Hence, there is an urgent need for early diagnosis to develop biomarkers; to identify high risk individuals, to improve cancer detection at early stages, to predict disease outcome and response to therapy. Proteomics have been successfully employed in studies of lung, breast, prostate, gastrointestinal cancer, and head and neck squamous cell carcinoma. Reports are not much available to describe well characterized biomarkers which will aid for early detection of oral cancer. Racial differences were reported in many cancers including its morbidity, mortality, treatment response and survival rates. In Indian patient population, there is a significant knowledge gap exists in utilizing proteomics for biomarker discovery.

The aim of this study is to identify novel candidate genes which may be used as prognostic or diagnostic biomarkers for Indian oral squamous cell carcinoma. The tongue tumor and adjacent normal samples were subjected to 2D - Differential in Gel Electrophoresis (2D-DIGE). Differentially expressed proteins in tumor samples were identified using nano-LC-MS/MS and validated by quantitative real time PCR. We found 800 protein spots were differentially expressed and seventy proteins were identified by mass spectrometry. Among them twelve proteins including Myosin isoforms, hemoglobin beta, Annexin A1, Creatinine kinase, Carbonic anhydrase, HSPA8 showed decreased expression while eighteen proteins including Apolipoprotein A1, HSPA5, Alpha 1 antitrypsin, Annexin isoforms, Peptidyl prolyl cis trans isomerase, Serpin isoforms, Tropomyosin, Thioredoxin, Alpha enolase, Calpain, Glutathione S Transferase, Heat shock protein beta 1, S100 isoforms showed increased expression in tongue tumor tissues.

Most of the differentially regulated proteins were known to be involved in calcium homeostasis, cell cycle, cell growth and migration. The major outcome of this study was the identification of novel protein molecules which might be used as predictive, prognostic and diagnostic biomarkers for oral cancer. These proteins can also serve as a therapeutic intervention targets for oral cancer progression. This study would be the first study to map the whole proteome profile of the cancerous as well as normal tongue cheek tissue in Indian scenario.

#3876

EphrinB3 and EphrinB4 receptors: potential therapeutic targets in glioblastoma.

Radhika Sreeraman Kumar, Robert J. B. Macaulay, Hannah C. Rutherford, Natalie Barkey, Jiannong Li, Jongphil Kim, John M. Koomen, David L. Morse. _H.L. Moffitt Cancer Center, Tampa, FL_.

Introduction: Glioblastoma (GBM) is the most common primary malignant neoplasm of the central nervous system in adults with poor median overall survival despite advances in tumor. CD133 is a known putative cancer stem cell marker, and we aimed to identify markers in CD133+ GBM tumor initiating cell lines (CSCs) with an infiltrative phenotype that could serve as therapeutic targets.

Methods: Expression (mRNA) microarray datasets including 22,278 probes for known cell-surface markers in three CD133+ CSCs and 17 normal brain tissue lines were obtained. We identified genes with uniformly high mRNA expression, filtered for known localization at the cell-surface and for non- or low expression in normal brain. Ephrin B3 receptor (EphB3), ephrin B4 receptor (EphB4) and fibroblast growth factor receptor (FGFR1) were highly expressed. Protein expression was established by mass spectrometry using CD133+ cell line extracts and then assessed in 27 patient GBM (IDH-wildtype) tumor samples by immunohistochemistry. Expression was graded based of the percentage of cells positive and intensity of staining the tumor, interface and uninvolved brain. One-way ANOVA was used to test any group difference among any combination of two genes; Tukey honest significant difference method was used to adjust for p value for pairwise comparison in tumor locations. Spearman correlation analysis was applied to determine potential correlation co-expression EphB3 and EphB4. The Gamma statistic was used to identify correlation of intensity of staining with tumor location, and the Fisher's exact test was utilized to elucidate potential significance.

Results: High expression ( > 50% of cells stained) was 7/27 for EphB3, 8/27 for EphB4, and 18/27 for FGFR1. EphB3 and EphB4 are significantly associated with tissue location based on percentage of cells stained in each location (p < 0.05). When compared to the uninvolved brain, EphB3 and EphB4 expression in the tumor and interface tissues were significantly higher (p<0.05). EphB3 expression was significantly correlated with EphB4 expression (p < 0.05) in tumor tissue, though the correlation was weak (r = 0.414) EphB3 and EphB4 intensity were positively associated with tissue location: 0.53 [0.21 - 0.86] and EphB4 0.45 [0.09 - 0.80] respectively. FGFR-1 was not associated with tumor location in terms of both percentage and intensity of staining.

Conclusion: In terms of percentage and intensity of staining at the uninvolved brain, interface and normal tissue, EphB3 and EphB4 were significantly associated with those three tissue locations. EphB3 and EphB4 were highly expressed in tumor and interface tissue relative to the normal tissue. Co-expression of EphB3 and EphB4 in tumor tissue was correlated. Evaluation with clinical parameters may disclose subsets of these tumors with varying infiltrative potential.

#3877

A new frontier for molecular profiling of neoplastic bone tissue.

Claudius Mueller,1 Lance A. Liotta,1 Antonella Chiechi,2 Piero Picci,3 Marco Alberghini,3 Lorenzo Memeo,4 Vincenzo Canzonieri,5 Virginia Espina1. 1 _George Mason University, Manassas, VA;_ 2 _Indiana University, Indianapolis, IN;_ 3 _Istituto Ortopedico Rizzoli, Bologna, Italy;_ 4 _Istituto Oncologico del Mediterraneo, Catania, Italy;_ 5 _Centro di Riferimento Oncologico, Aviano, Italy_.

Background: Tissue is alive and reacting to ex vivo stress during "cold ischemia time". Tumor cells react to the absence of vascular perfusion, hypoxia, acidosis, and accumulation of cellular waste, which can critically mask cancer related signaling network data. Standard formalin fixation of calcified tissue requires decalcification with formic/nitric acid solution, which greatly interferes with downstream molecular analysis. We developed a non-formalin fixative (TheraLin) for preserving biomarker molecules and tissue histomorphology in one step, which greatly facilitates the analysis of phosphoprotein signaling pathways in calcified tumor samples.

Methods: Fifty specimens of neoplastic bone were collected at the Istituto Ortopedico Rizzoli (Bologna, Italy). Matched 0.5 cm samples were fixed in TheraLin preservative; or fixed in 4% buffered formalin and decalcified with formic/nitric acid solution. Specimens were processed into paraffin blocks and 5 µm tissue sections cut onto glass slides. Enriched tumor cell populations were procured using Laser Capture Microdissection. Reverse Phase Protein Microarrays were constructed with the microdissected tumor cells and probed with >40 antibodies to unmodified or phosphorylated proteins. Immunohistochemistry (>200 stains in total) was performed on routine clinical protein targets and a subset of phosphorylated proteins.

Results: TheraLin fixation greatly facilitated molecular profiling of bony tissues by eliminating the need for decalcification (18.5 hour average reduction in processing time) while maintaining or improving histomorphology (>85% of cases) and immunohistology (>75% of cases). In addition, we were able to demonstrate full compatibility with laser capture microdissection, reverse phase protein microarrays and downstream analysis of cell signal protein activation. Protein extractability from microdissected material was significantly increased (>2.5 fold, p<0.01) compared to formalin fixed, decalcified bone tissue.

Conclusion: TheraLin obviates the need for decalcification of bone tissue while simultaneously preserving histomophology and immunohistochemistry. TheraLin enables reliable quantification of phosphorylated signal transduction proteins, the substrates of kinase drug targets, and greatly facilitates tumor cell enrichment compared to standard formalin fixation and decalcification.

#3878

Proteomic approach to identify biomarkers for invasive cervix cancer: a prospective pilot study.

David P. Mysona, Adam Pyrzak, Jennifer Allen, Ashok Sharma, Paul Weinberger, Bunja Rungruang, Jin-Xiong She, Sharad Ghamande. _Georgia Regents University, Augusta, GA_.

Objectives: Cervical cancer is the second most common cancer of women worldwide. In this prospective study, we attempted to identify a set of serum biomarkers, which could identify patients who have invasive cervical cancer and possibly help monitor disease status (recurrence, remission).

Methods: Luminex bead array was used to measure serum protein concentrations of 19 different, promising proteins in cervical cancer patients (n=23) compared to women with precancerous lesions (CIN2-3) (n=20), and patients with normal cervical cytology (n=20) in this IRB approved prospective study. Cervical cancer (adenocarcinoma and squamous carcinoma) patients had blood samples drawn within 30 days of diagnosis and repeat blood samples taken on follow up visits after completing therapy. These results were correlated to clinical data and outcomes. Protein expression was confirmed by immunohistochemistry in patient samples.

Results: CIN and controls have very similar levels of serum proteins. Six proteins (CA153, OPG, CEA, IGFBP7, IGFBP4, and MMP-7) had significantly different levels in cervical cancer patients compared to controls and CIN (p < 0.05) with Area Under the Curve values of 0.74, 0.77, 0.79, 0.83, 0.84, and 0.98 respectively. IGFBP4 had a sensitivity of 75% at 95% specificity for correctly identifying invasive disease. MMP-7 had a sensitivity of 92% at 95% specificity. MMP-7 was confirmed by IHC in patient specific cervical cancer tissue.  | |

|

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

Protein | AUC | P value | Sensitivity at 95% Specificity

MMP7 | 0.98 (0.96-0.99) | 5.29E-13 | 92.11

IGFBP4 | 0.84 (0.8-0.89) | 2.36E-07 | 75

IGFBP7 | 0.83 (0.78-0.87) | 6.23E-07 | 57.89

CEA | 0.79 (0.74-0.84) | 1.30E-05 | 42.11

OPG | 0.77 (0.72-0.82) | 3.80E-05 | 35.14

CA153 | 0.74 (0.69-0.8) | 2.50E-04 | 7.89

Conclusions: Several serum proteins are significantly altered in cervical cancer patients compared to controls and CIN, and are worth studying for further clinical utility. MMP-7 may prove to be an important mediator of cervical cancer.

#3879

Comprehensive delineation of stromal proteins implicated in the multistage carcinogenesis and identification of Tenascin-C as stromal marker for metastasis of colon carcinoma.

Yongheng Chen, Maoyu Li. _Xiangya Hospital, Central South University, Changsha, China_.

Colon carcinoma (CC) is one of the most common malignancies threatening the human health. Early diagnosis or effective biomarker for intervention is still a challenge in clinical application for CC. The stroma in human cancer provides the microenvironment for cancer initiation and development, and furthermore, the stroma has been regarded as a therapeutic target for cancer. However, little is known about the dynamics of proteins within the stroma during the carcinogenic process. To discover the involvement of stroma in carcinogenesis and identify the biomarker candidates, the stromal components at the various stages of colonic carcinogenesis were purified by laser capture microdissection (LCM) and iTRAQ-based quantitative proteomic analysis was used for identifying the differentially expressed stromal proteins at different stages. In total, 222 differentially expressed proteins were identified. The expression of two of the top ranked proteins, extracellular matrix protein TNC and S100 family protein S100A9, was further confirmed by immunohistochemical analysis. Further analysis revealed that TNC was highly expressed in the stroma of metastasis and also abundantly secreted by the high metastatic cells. These results suggested that TNC was potential stromal marker for metastasis of colon carcinoma. Our results will contribute to investigating the role of stroma in the colonic carcinogenesis and might provide clues for pathogenesis, early diagnosis, and clinical therapy of CC.

#3880

Quantitative human secretome: a technology to measure the expression levels of thousand of secreted proteins.

Ruo-Pan Huang, Yingqing Mao, RuoChun Huang, Haw-Han Yen. _RayBiotech, Inc., Norcross, GA_.

The secretome constitutes an increasingly important category of proteins which are involved in a multitude of biological, physiological and pathological processes, and are thus a clinically relevant source for biomarkers and therapeutic targets. The development of cancer often leads to alterations in growth-stimulatory autocrine and paracrine signals which change the expression levels of secreted proteins such as growth factors, cytokines, chemokines, and angiogenic factors. There is thus an urgent need to develop high-content and high-throughput approaches to profile the secretome. Using a sandwich-based ELISA format, we have developed an antibody array technology which can quantitatively measure expression levels of 1000 secreted proteins. Most of the target proteins are detectable at pg/ml levels. We have also developed a multi-layered normalization process (including positive controls, standard curves, and reference samples) which significantly enhances the performance of the platform and may be used for accurate measurement of protein concentrations in a variety of samples, including serum, plasma, tissue lysates, and other fluids. This platform is also high throughput, allowing hundreds of samples to be assayed daily. We have analyzed several hundred serum samples (both normal and cancer patients) using this system. Our results demonstrate that our technologies provide a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to cancer and other human diseases.

#3881

Breast cancer proteogenomics landscape defines subtype specific protein level regulations and reveals proteins coded by pseudogenic loci.

Henrik Johansson,1 Yafeng Zhu,1 Mads Haugland,2 Kristine Sahlberg,2 Erik Fredlund,1 Mikael Huss,3 Nathaniel Vacanti,1 Miriam Range Aure,2 Bengt Sennblad,3 Sanela Kjellqvist,1 Lukas Orre,1 Ola Christian Lingjaerde,4 Anne-Lise Borresen-Dale,2 Janne Lehtio1. 1 _Karolinska Institutet, Solna, Sweden;_ 2 _Institute for Cancer Research, University of Oslo, Oslo, Norway;_ 3 _Science for Life Laboratory, Solna, Sweden;_ 4 _University of Oslo, Oslo, Norway_.

The advancement of mass spectrometry (MS) based proteomics has been tremendous in recent years and is expected to continue due to the rapid developments in instrument technology and bioinformatic analysis. These methods have enabled us to discover predictive protein markers for anti-estrogen treatment of estrogen receptor positive breast cancer 1, 2. Recently proteogenomics has emerged as an exciting area, combining proteomics with genomics and transcriptomics. We have published a proteogenomics workflow which can be used to discover new protein coding loci via an unbiased 6 reading frame translation (6-FT) search even in well-annotated higher eukaryotes such as human and mouse3. Herein we present a quantitative proteomics and proteogenomics analysis, using our high resolution isoelectric focusing (HiRIEF) aided proteogenomics method3,4, on breast tumors obtained from five breast cancer subtypes. Protein products from 12645 genes are identified and roughly 10000 proteins are quantified across the entire cohort of 45 breast cancer tissues. The resulting quantitative proteome landscape recapitulates the PAM50 subtypes. The breast cancer proteome defines clusters indicative of stroma, immune response, basal, adipocyte, proliferation, and steroid response related components. Additionally, a proteogenomics search of the 6-FT of the entire human genome reveals novel pseudogenic protein coding regions (n=150) as well as peptides derived from lncRNA (n=79). Finally, customized databases including variant peptides derived from SNPs, mutations, and splice junction peptides are used to analyze protein level events related to variants. This proteogenomics analysis for both new open reading frames and sequence variants reveals novel proteins expressed in a tumor specific manner in several studied individuals, with some being related to known sub-types. Taken together, this study reveals the proteome profiles are related to transcriptomics, copy number, and metabolic activity in specific tumors, proving our proteogenomics workflow provides novel information on the breast cancer molecular landscape.

1. Johansson, H.J. et al. Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer. Clinical proteomics 12, 8 (2015).

2. Johansson, H.J. et al. Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer. Nat Commun 4, 2175 (2013).

3. Branca, R.M. et al. HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics. Nature methods 11, 59-62 (2014).

4. Boekel J, et.al. Multi-omic data analysis using Galaxy. Nature Biotechnol. 2015 Feb 6;33(2):137-9.

#3882

Patient-specific druggable networks in lethal prostate cancer through proteome-guided multi-omic integration.

Justin M. Drake,1 Evan O. Paull,2 Nicholas A. Graham,3 John K. Lee,4 Bryan A. Smith,4 Tanya Stoyanova,5 Claire M. Faltermeier,4 Daniel E. Carlin,6 Ajay Vashisht,4 Jiaoti Huang,7 James A. Wohlschlegel,4 Thomas G. Graeber,4 Owen N. Witte,4 Joshua M. Stuart2. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _University of California, Santa Cruz, CA;_ 3 _University of Southern California, Los Angeles, CA;_ 4 _University of California, Los Angeles, CA;_ 5 _Stanford University, Stanford, CA;_ 6 _University of California, San Diego, CA;_ 7 _Duke University, Durham, NC_.

Metastatic castration resistant prostate cancer (CRPC) remains incurable due to the lack of effective therapies. The need to identify new actionable targets in this disease is crucial as we begin to examine the resistance mechanisms related to androgen withdrawal. Pathway activation of signaling proteins, such as kinases, are hypothesized to drive the progression of lethal CRPC. We set out to define the global picture of signaling pathways in lethal prostate cancer through dataset integration. We used clinical tissue from lethal metastatic CRPC patients obtained at rapid autopsy to evaluate and integrate previously published genomic and transcriptomic datasets combined with a new complete and extensive dataset of the phosphoproteome in metastatic CRPC for pathway analysis. This included extending our analysis of the phosphoproteome to phosphoserine and phosphothreonine peptides with our published phosphotyrosine peptide data. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues relative to treatment naïve prostate cancer to synthesize a robust signaling network consisting of druggable kinase pathways for therapy. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Further, we were able to gather mRNA and phosphoproteome data from six individual patients where the integration of tissue samples from a single autopsy program allowed us to make inferences on the connections between the mRNA and phosphoproteome datasets. Individual patient profiles developed using these hallmarks revealed clinically relevant pathway information suitable for patient stratification and targeted therapies in lethal prostate cancer. Here we describe these pathways: personalized cancer hallmarks using an integrative phospho-signature (pCHIPs) that sheds light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients.

#3883

Variant peptide identification system for bottom-up proteomics: Finding hidden sequence alterations in MS data.

Marian Hajduch,1 Miroslav Hruska,2 Lakshman Varanasi,3 Jiri Voller,3 Petr Dzubak3. 1 _Institute of Molecular and Translational Medicine, Olomouc, Czech Republic;_ 2 _Institute of Molecular and Translational Medicine, faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic;_ 3 _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic_.

In database search, acquired spectra are matched with theoretical spectra of database peptides. Peptides from large databases are likely to match to more than one spectrum, but only a small proportion of peptide interpretations are correct. Incorrect interpretations are likely to arise for two reasons: random matches and non-random matches to homologous peptides. The former is often eliminated by selection of an appropriate similarity threshold or by utilization of the statistical significance of a spectral match. The match to homologous peptide poses a challenge as there is often large similarity in their fragmentation spectra as well. Non-random, but incorrect matches occur surprisingly often in the identification of peptide sequence variants. For this reason, a high identification score does not imply correctness of interpretation. Increasing spectral score threshold enhances specificity in general, but results in significant loss of sensitivity. Therefore, a single spectral criterion is inadequate for distinguishing between homologous peptides.

In this study, we reconceive the system for elimination of incorrect interpretations. We propose a system that uses multidimensional LC/MS information in combination with a priori knowledge of sample content to filter out unlikely interpretations. The system calculates retention time of candidate peptides, performs isotopic analysis of precursors and constructs preliminary protein assembly to increase separation between correct and incorrect matches. The approach is used for reevaluation of database search results and is generally applicable to the analysis of standard bottom-up proteomic data.

The system was used for identification of alterations in MS2 proteomics data and validated against RNA-Seq data from the same sample. The results demonstrate that non-spectral data can be used to efficiently eliminate peptide interpretations that have no correspondence in the RNA and as such are likely false positives. The approach is sensitive and yields a large proportion of altered peptides that have RNA-Seq support. The method can potentially help overcome the problem of the large database search present, e.g., in proteogenomic studies, particularly important in cancer research and diagnostics. In summary, general prior knowledge of sample content and the use of LC and MS1 data improve on the MS2-based identification of peptides.

#3884

Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry.

Bhavin Patel, Alex Behling, Leigh Foster, Ryan Bomgarden, Carrie Clothier, Kay Opperman, John Rogers. _Thermo Fisher Scientific, Rockford, IL_.

Background: The quantitative measurement of protein expression and modification status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitoring cancer progression and determining treatment response. A major bottleneck in the quantitation of signaling pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from current immunoassay technologies (Western blot, ELISA and Luminex). Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to quantitate proteins of interest and identify protein-protein interactions. The objective of this study was to determine the efficacy of multiplex IP to targeted MS technique for measurement of the total and phosphorylated AKT/mTOR pathway targets and to evaluate whether multiplex IP-MS assays are as effective as the current single-plex immunoassay (WB and ELISA) and multiplex Luminex assays.

Methods: Serum starved HCT116, MCF7 and A549 cells were stimulated with IGF-1. Multiplex IP to targeted MS assays (mIP-tMS) were developed and validated for absolute quantitation of eleven total and ten phosphorylated AKT/mTOR pathway targets (IGF1R, IR, IRS1, PTEN, AKT, mTOR, GSK3ɑ, GSK3β, TSC2, p70S6K and PRAS40). Validated mIP-tMS assays were benchmarked against currently available WB, ELISA and multiplex Luminex immunoassays across three unstimulated and IGF-1 stimulated cell lysates.

Results: In previous work, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads can increase target protein yield with low non-specific background. In this study, we validated multiple antibodies for eleven total and ten phosphorylated AKT/mTOR pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for all eleven total and ten phosphorylated targets in low to sub nanogram concentrations across three unstimulated and IGF-1 stimulated cell lysates. The benchmarking of mIP-tMS assays showed high correlation for quantitation of total target relative abundance compared to WB, ELISA and Luminex assays. However, for some phosphorylated targets, mIP-tMS assays had low concordance to the other immunoassays possibly due to differences in the specificity of anti-phospho antibodies used for each assay.

Conclusion: Overall, the multiplex targeted MS assay can be used for identification and quantification of AKT/mTOR pathway proteins in cancer cell lines or tissue samples. Major advantages of this mIP-tMS assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets and their PTMs.

#3885

Peptide-mediated 'miniprep' isolation of extracellular vesicles for high-throughput proteomics; method evaluation and application in colon cancer.

Connie R. Jimenez,1 Meike de Wit,1 Jaco C. Knol,1 Inge de Reus,1 Tim Schelfhorst,1 Logan Bishop-Currey,1 Valerie Dusseldorp,1 Nicole van Grieken,2 Robin Beekhof,1 Sander R. Piersma,1 Thang V. Pham,1 Egbert F. Smit,3 Remond JA Fijneman,4 Gerrit Meijer,4 Henk MW Verheul1. 1 _OncoProteomics Laboratory, Department of Medical Oncology VU University Medical Center, Amsterdam, Netherlands;_ 2 _Department of Pathology, VU University Medical Center, Amsterdam, Netherlands;_ 3 _Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, Netherlands;_ 4 _Department of Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands., Amsterdam, Netherlands_.

Background: Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since they are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, EVs represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation (UC-EV) are laborious, time-consuming, and require high inputs. Recently a novel isolation method was described, which can be performed at small 'miniprep' scale, utilizes specific Heat Shock Protein (HSP)-binding peptides to aggregate HSP-decorated EVs (Ghosh et al. (2014), PLoS ONE 9:e110443). Using cancer secretome and biofluid samples, the authors showed enrichment of exosome markers for their method (abbreviated HSP-EV here) but a detailed description of the captured EV proteomes in comparison to the gold-standard ultracentrifugation (UC) method and application to small tumor proximal fluid samples is lacking.

Approach: Here we used label-free proteomics of replicate EV isolations from HT-29 cancer cell-conditioned medium to compare EV fractions captured using the new HSP peptide method and UC. Subsequently we applied this novel method to profile EVs released from fresh human colorectal tumors (CRC) (n=17) and colon adenoma (n=4) tissue as well as patient-matched normal colon tissue.

Results: Despite a 30-fold different input scale (UC-EV: 60 ml versus HSP-EV: 2 ml), both methods yielded comparable numbers of identified proteins (3115 versus 3085), with reproducible identifications (72.5% versus 75.5%) and spectral count-based quantification (average CV 31% versus 27%). EVs obtained by either method contained established EV markers and proteins linked to vesicle-related gene ontologies. In the EV fraction of the tissue secretomes 6390 proteins were identified, of which 471 proteins were significantly 5-fold more present in CRC samples than in normal tissue EVs. Gene ontology analysis revealed enrichment of nuclear proteins involved in DNA damage response, chromosome organization and RNA processing in the CRC EVs.

Conclusion: The HSP-EV method provides an advantageous, simple and rapid approach for EV isolation from small amounts of biological samples, enabling high-throughput analysis in a biomarker discovery setting.

#3886

Protein mapping of NSCLC cell lines: Defining mechanisms of acquired erlotinib resistance.

Sarah A. Hayes,1 Christoph Krisp,2 Amanda L. Hudson,1 Rozelle Harvie,1 Csilla Hasovits,1 Stephen Clarke,1 Mark P. Molloy,2 Viive M. Howell1. 1 _Bill Walsh Translational Cancer Research Laboratory, Hormones and Cancer, Kolling Institute of Medical Research, Sydney, Australia;_ 2 _Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia_.

Lung cancer is one of the most common and lethal malignancies globally, with non-small cell lung cancer (NSCLC) accounting for 85% of all lung cancer cases. Patients generally have a poor prognosis without treatment as most patients are diagnosed with advanced metastatic disease, when curative therapeutic options are limited. However, there has been a recent emphasis on identifying driver mutations responsible for patient tumours, which has paved the way for more effective targeted therapies in the treatment of NSCLC.

One such targeted therapy, erlotinib, is used as standard‐of‐care treatment in NSCLC patients with sensitising EGFR mutations. Although use of these tyrosine kinase inhibitors (TKIs) often leads to dramatic and prolonged response, acquired resistance eventually ensues. Understanding and overcoming the molecular basis of resistance to erlotinib remains a challenge for successful long-term treatment.

To identify mechanisms of erlotinib resistance, we used latest-generation mass spectrometry to comprehensively map the proteomes of two NSCLC cell lines: a parental NSCLC cell line sensitive to erlotinib (HCC827, contains a deletion in EGFR exon 19) and its matched erlotinib-resistant subline (HCC827_ER). Cell lines were treated with an IC50 dose of erlotinib or mock treatment. Three days after treatment, each cell line was profiled using the Sequential Windowed data independent Acquisition of the Total High-resolution Mass Spectra (SWATH-MS 2.0) algorithm, conducted on the Sciex 6600 TripleTOF. LC-MS/MS data was extracted for 3416 proteins (peptide confidence >99%) following a Sciex ProteinPilot database search.

Overall, 33 proteins were differentially expressed between HCC827 mock and erlotinib treated cells, while expression levels of 59 proteins were significantly different between HCC827er mock and erlotinib treated cells (Fold Change>2, p<0.05). Ingenuity Pathway Analysis listed "Organismal Injury and Abnormalities" and "Cancer" as the leading Diseases and Disorders in both cell lines, with "Cellular Growth and Proliferation" and "Small Molecule Biochemistry" listed as the top Molecular and Cellular Functions in the sensitive and resistant cell line, respectively. In the parental cell line, identified proteins were associated with the regulation of the actin cytoskeleton, as well as the PI3K-Akt signaling pathway, which is commonly altered in human cancers. In the resistant subline, several differentially expressed proteins mapped to various metabolic pathways (including carbon, glycine, serine and threonine metabolism), with some proteins similarly involved in PI3K-Akt signaling.

This is the first time that lung cancer cell lines have been comprehensively profiled by SWATH-MS. Protein mapping will help to increase the understanding of the mechanisms involved in the acquisition of TKI resistance, which is crucial for the development of rational strategies to overcome resistance in the clinic.

#3887

Proteomic profiling identifies cMyc and TTF1 as biomarkers of response to the aurora kinase inhibitor alisertib in small cell lung cancer (SCLC).

Robert J. Cardnell,1 Lerong Li,1 Fatemeh Masrorpour,1 Huifeng Niu,2 Jeffrey Ecsedy,2 Jing Wang,1 Lauren A. Byers1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Takeda Pharmaceuticals, Deerfield, IL_.

Background: Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, with a 5-year survival rate of only 6%. The treatment of SCLC has not changed in over 25 years, hence the development of new drugs for SCLC represents a major unmet need. A recent phase II trial of the aurora kinase inhibitor alisertib showed single agent activity in a subset (21%) of SCLC (Melichar et al, 2015) and a second study is underway testing alisertib in combination with chemotherapy (NCT02038647). However, there are currently no established biomarkers to identify patients likely to have the greatest benefit from alisertib. We have previously demonstrated the utility of proteomic profiling to identify targets and markers in SCLC. Here we use proteomics to identify markers of response to alisertib in SCLC.

Methods: The sensitivity (IC50) of 51 SCLC cell lines to alisertib was assayed in 5-day proliferation assays. Expression levels of 171 total and/or phosphorylated proteins were measured by reverse phase protein array (RPPA) and correlated with IC50's. Using two approaches – 1) Spearman correlation of IC50 to protein data for all cell lines and 2) t-test comparing protein data between the most and least sensitive cell lines – we generated consensus markers of response.

Results: Proliferation assays showed sensitivity to alisertib at clinically achievable doses in 14/51 (27%) cell lines (based on Cmax=1.8uM, Phase I single agent trial). High cMyc protein was the top marker of sensitivity to alisertib (R=-0.47, p=0.0006 as continuous variables; fold difference =3.52, p=0.008 by t-test comparing extremes). Further analysis revealed a bi-modal distribution of cMyc protein, defined as high and low using a bimodality index. The cMyc protein high group (25%) included cell lines that were both cMyc amplified and non-amplified. While cMyc amplified cell lines (11%) are more sensitive to alisertib than non-amplified (p=0.002), high cMyc protein captures a larger population of SCLC that is sensitive to alisertib.

In contrast, expression of thyroid transcription factor 1 (TTF1, a standard IHC marker used in the diagnosis of lung cancer) was the top marker of alisertib resistance (R=0.38, p=0.006; fold difference =-4.22, p=0.003). TTF1 protein expression was also bi-modal, with 32% of cell lines falling into a distinct TTF1-low (more sensitive to alisertib) group.

Conclusions: High cMyc and low TTF1 protein expression identify a subset of SCLC cell lines (27%) that are sensitive to single agent alisertib. cMyc protein as a marker of response is consistent with other preclinical findings suggesting that cMyc amplified SCLC may be more sensitive to aurora kinase inhibition. The association of low TTF1 expression with alisertib sensitivity may prove to be of particular value in selecting patients for treatment given that immunohistochemical assessment of TTF1 is commonly used in the diagnosis of SCLC.

#3888

Systematic characterization of aberrant signaling induced by oncogenic fusions in non-small cell lung cancer.

Sebastian A. Wagner,1 Petra Beli,2 Hubert Serve1. 1 _Goethe University Frankfurt, Frankfurt, Germany;_ 2 _Insitute of Molecular Biology (IMB), Mainz, Germany_.

Recent studies have greatly expanded the knowledge about genetic alterations in non-small cell lung cancer (NSCLC). Gene fusions involving the tyrosine kinases ALK, ROS and RET are found in 5 - 10 % of NSCLC patients and are considered as oncogenic drivers. However, the cellular signaling activated downstream of oncogenic fusions has not been systematically investigated and it remains unclear if different fusions induce specific targetable signaling patterns.

In this study we have employed quantitative mass spectrometry (MS)-based phosphoproteomics to systematically characterize the signaling induced by oncogenic fusions frequently found in NSCLC. To this end, cDNA sequences of oncogenic fusions containing the tyrosine kinases ALK, ROS and RET and different fusion partners were constructed and ectopically expressed in HEK 293 and lung cancer cell lines. Subsequently, tyrosine phosphorylated peptides were enriched and identified by LC-MS. Stable isotope labeling with amino acids in cell culture was employed to precisely quantify the abundance of phosphorylation sites in cells expressing different oncogenic fusions.

Our data demonstrate that all investigated oncogenic fusions induce massive tyrosine phosphorylation of proteins and lead to increased phosphorylation of the majority of all quantified phospho-tyrosine sites. For instance, we quantified ~1300 unique phosphotyrosine sites from HEK 293 cells expressing EML4-ALK. Of those, 952 increased more than 4-fold in abundance. Comparative analyses of the signaling patterns allowed defining kinase-specific phosphorylation signatures. In addition, comparison of signaling patterns induced by different fusions permitted us to investigate the contribution of the non-kinase fusion partner to the downstream signaling. We were able to identify several phosphorylation events that were associated with specific non-kinase fusion partners. For example, kinase fusions containing the kinesin-1 heavy chain (KIF5B) specifically led to phosphorylation of cytoskeletal proteins, including cytoskeleton-associated protein 5 (CKAP5). We are currently investigating the functional role of these phosphorylation events for the oncogenic transformation.

In summary, we demonstrate that MS-based phosphoproteomics is a powerful tool to investigate aberrant signaling induced by oncogenic fusions in non-small cell lung cancer. Importantly, we find that the kinases involved in the oncogenic fusions largely define the downstream signaling and that the investigated fusion partners only exhibited a minor effect on the downstream signaling.

#3889

An aptamer-based multiplexed proteomic technology reveals differential PKC ligand responses.

Jinqiu Chen, Noemi Kedei, David J. Goldstein, Mariam Q. Malik, Peter M. Blumberg. _National Cancer Institute, Bethesda, MD_.

Bryostatin 1 (Bryo 1) is in clinical trials for cancer and Alzheimer's disease. Whereas Bryo 1, like the phorbol esters, binds to and activates protein kinase C (PKC), it paradoxically antagonizes many but not all phorbol ester responses. The mechanistic basis for this differential response is still unknown.

In the LNCaP human prostate cancer cell line, the typical phorbol ester PMA causes growth inhibition whereas Bryo 1 does not. Previously, we showed that PMA induces dramatic changes in gene transcription in the LNCaP cells as determined by gene expression microarray analysis. The effects of Bryo 1 on gene expression were initially similar at 1 hr but were variably more transient, as seen at 6 hr, depending on the specific gene. We think that this transiency of action is a critical mechanistic feature of Bryo 1.

The current study extends to the comparison of the effects of Bryo 1 and PMA on the level of global protein expression. Using a new generation of protein-capture SOMAmer (Slow Off-rate Modified Aptamer) reagents, the SOMAscan (Somalogic, Boulder, CO) proteomic assay simultaneously measures over one thousand analytes within a minute amount of protein sample. SOMAmer reagents are chemically modified single stranded DNA-based protein affinity reagents that recognize specific conformational epitopes of native 3D proteins with good specificity and dynamic range. The technology is emerging as a highly sensitive, multiplexed and quantitative proteomic tool for biomarker discovery and validation. 1,129 targets (covering major gene families including receptors, kinases, growth factors, and a diverse collection of secreted, intracellular and extracellular proteins or domains) were evaluated in both total cell lysate and nuclear extracts of LNCaP cells treated with DMSO, PMA and Bryo 1 for 60 minute and 6 hours. Compared to the DMSO control, the lysates of cells treated for 60 minute with either PMA and Bryo 1 had over three hundred targets that showed significant changes (p<0.01). While a similar scale of signaling response was observed in the 6 hour PMA treated total cell lysate, less than 50 targets differed from the DMSO control in the 6 hour Bryo 1 treated samples (p<0.01). Similar results were obtained for the nuclear extracts. Good assay reproducibility was observed within replicates. While the protein analysis confirmed the transient nature of Bryo 1 action, comparison of the SOMAscan protein expression data with the mRNA transcript analysis revealed substantial differences, indicating the importance of monitoring signaling response at both the transcriptomic and proteomic levels. It further identified a rich set of protein targets involved in the differential PKC signaling by different ligands. On-going studies seek to characterize the basis for the transient signaling response following Bryo 1 treatment.

#3890

NCI's Clinical Proteomic Tumor Analysis Consortium.

Mehdi Mesri, Emily Boja, Tara Hiltke, Chris Kinsinger, Jerry Lee, Henry Rodriguez. _NIH, Bethesda, MD_.

The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) constitutes a network of Proteome Characterization Centers (PCCs), which coordinate and conduct research and data sharing activities to comprehensively interrogate genomically characterized cancer biospecimens. Such integrated proteogenomics approach brings key insights into genomic abnormalities manifested at the protein network and pathway level. Established in late 2011, CPTAC works closely with genomics initiatives such as The Cancer Genome Atlas (TCGA) to systematically identify proteins that derive from alterations in cancer genomes and related biological processes, and provide this data with accompanying assays and protocols to the public. CPTAC investigators conducted the first in-depth comprehensive proteome (global, phosphoproteome and N-glycoproteome) study on understanding the impact of cold ischemia that has resulted in the development of an ischemic signature database and best practice procedures for biospecimen (tissue) sample collection/processing. The PCCs have deeply characterized three cancer types (colorectal, ovarian and breast tumors) previously analyzed by TCGA and additional tissue collections, all of which are accompanied by genomic datasets. Scientific findings examining 95 TCGA colon and rectal tumors using deep proteomic analysis include: a) five proteomic subtypes were identified, with the TCGA MSI/CIMP transcriptomic subtype separated into distinct proteomic subtypes including one subtype (subtype C) that displayed protein network features characteristic of epithelial-mesenchymal transition (EMT); b) copy number alterations (CNAs) on mRNA abundance analysis showed strong cis- and trans-effects; but few extending to the protein level; and c) chromosome 20q amplicon was shown to contain the largest global changes in both mRNA and protein levels, allowing for prioritizing driver genes. In addition to the publicly available proteomic datasets (https://cptac-data-portal.georgetown.edu/cptacPublic), proteomic targeted assays developed by the consortium are available at CPTAC's Assay Portal (http://assays.cancer.gov). This is a public resource of mass spectrometry-based quantitative targeted proteomic assays for measuring proteins of interest from the program. Lastly, monoclonal antibodies targeting cancer specific proteins and peptides are made available at CPTAC's Antibody Portal (http://antibodies.cancer.gov).

#3891

Development of quantitative methods for assessing metastatic potential of human primary tumors.

Kelly A. Martin, Nicholas Hum, Kurt W. Haack, Bruce A. Buchholz, Gabriela G. Loots. _Lawrence Livermore National Laboratory, CA_.

Background: The inability to effectively treat metastases is the main reason for the limited progress in reducing the rates of cancer morbidity and mortality. One major drawback is the lack of quantitative assays for assessing the size and tissue prevalence of tumors in newly diagnosed individuals. Current methods for quantifying tumor burden are mainly qualitative and include measuring the gross weight of the affected organ, counting tumors on the surface of the organ, or evaluating a small sample of the organ using histologic sections. These methods are crude measures of tumor burden and size distribution, and in the case of histology, they are time consuming, difficult to process an adequate sample size and non-quantitative.

Methods: Animal models of metastasis have been useful in identifying genes that regulate susceptibility to the development and progression of metastasis and helped highlight potential novel targets for drug development. In particular several small animal imaging technologies including magnetic resonance imaging, high frequency ultrasound, and optical imaging have been recently applied to this task. Each of these methods may be useful for specific research projects, based on their unique combination of resolution, image acquisition time, animal throughput, and cost-effectiveness, yet none of these modalities adequately address the need for rapid quantification of tumors across the entire organism, nor do they assess therapeutic effectiveness in eradicating cancer in xenograft models. We have developed an Accelerator Mass Spectrometry (AMS)-based high precision quantitative method for assessing the metastatic potential of primary tumors isolated from newly diagnosed patients.

Results: Our Accelerator Mass Spectrometry-based methodology to study metastasis uses xenograft cancer cells labeled with 14C-labeled thymidine that are delivered intravenously into NSG mice and allowed to develop metastatic cancer over the course of up to 10 weeks. At the end of the experiment, all vital organs are collected; the DNA is isolated and is examined by AMS for the presence of 14C-signal. The labeling was optimized to achieve sufficient signal such that a tumor derived from a single cell could be detected by AMS, in secondary tumors, in vivo, independent of histological data.

Conclusions: Using this novel approach we have evaluated the metastatic potential of several prostate cancer cell lines, characterized stem-cell like sublines derived from prostate cancer cell lines [PC3] and examined tissue tropism of cancer sublines derived from kidney and liver metastatic tumors. Further optimization of these techniques will allow us to explore the metastatic potential of primary tumors, isolated from biopsies and expanded in Avatar mice.

#3892

Unique pleural vs peritoneal mesothelioma exosomal signature: does mesothelial cell susceptibility to asbestos matter.

Arti Shukla,1 Phillip Munson,1 Joyce K. Thompson,1 Julie Dragon,1 Maximilian B. MacPherson,1 Hector Peinado2. 1 _University of Vermont, Burlington, VT;_ 2 _Weill Cornell Medical College, NY, NY_.

Malignant mesothelioma (MM) is a cancer of mesothelial cells of the pleura or peritoneum caused exclusively by asbestos exposure. It is a deadly cancer which is only diagnosed at terminal stages, possibly due to the long latency period of development and lack of biomarkers for early diagnosis. Both pleural and peritoneal mesothelial cells can develop MM in response to asbestos, however, pleural MM (85%) is more common than peritoneal MM (15%). To get an insight of this difference we first isolated exosomes from pleural (Hmeso, H2373) and peritoneal (ORT) MM cells and performed proteomic analysis. We found that 45 proteins are common and specific to MM cells derived from pleural cavity (Hmeso/H2373) while 86 proteins are specific to ORT cells lines derived from peritoneal cavity. On the other hand, the non-transformed mesothelial cell line, LP9, showed a completely different profile with 269 proteins secreted in exosomes not shared with MM models. This led us to hypothesize that mesothelial cells of two different locations may have different susceptibility to asbestos, accounting for differences observed in pleural and peritoneal MM. To test this hypothesis we exposed human primary pleural and peritoneal mesothelial cells to asbestos for 8 h, and performed massively parallel sequencing on the RNA. Gene expression analysis performed on the RNA-Seq data showed a higher magnitude of responses from pleural mesothelial cells as compared to peritoneal mesothelial cells for the same genes. In addition, at a p<0.05 and 2-fold threshold, unique genes expressed in pleural mesothelium were pro-inflammatory genes known to be involved in MM tumorigenesis. Taken together, results from our study suggest that exosomal cargo from pleural and peritoneal MM cells bear unique signatures which may be due to the different responses of pleural and peritoneal mesothelial cells to asbestos. Future directions involve assessing miRNA cargo from exosomes of pleural and peritoneal MM cells and identifying potential biomarker candidates. This work is supported by grants from NIH (RO1 ES021110), DoD (W81XWH-14-1-0199) and the University of Vermont Cancer Center/Lake Champlain Cancer Research Organization.

#3893

Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways.

Aradhana Awasthi Tiwari,1 Delphine C.m. Rolland,2 Mona Elmacken,1 Janet Ayello,1 Lisa Kurien,1 Carmella van de Ven,1 Venkatesha Basrur,3 Kevin Conlon,3 Damine Fermin,3 Matthew J. Barth,4 Christian Klein,5 Kojo S.j. Elenitoba-Johnson,2 Megan Lim,2 Mitchell S. Cairo1. 1 _New York Medical College, Elmsford, NY;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _University of Michigan Medical School, Ann Arbor, MI;_ 4 _Roswell Park Cancer Institute, Buffalo, NY;_ 5 _Roche Pharmaceutical Research & Early Development, Zurich, Switzerland_.

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil.

Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL

Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 µg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions

Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (>1.5-fold) reveals the involvement of the BCR (PLCG2, BTK & GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 & RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1& PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P=0.0002 & 0.000002) but no change in Raji4RH.

Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL.

#3894

A xenograft mouse model coupled with in-depth cell surface proteome analysis facilitates further elucidation of K-Ras driven tumorigenesis in lung carcinoma.

Xiaoying Ye, Gordon Whiteley, Dwight Nissley, Frank McCormick, Josip Blonder. _NCI-Frederick/Leidos Biomed. Research, Inc., Frederick, MD_.

The purpose of this study was to develop a proteomic approach for analysis of cell surface proteins in tissue specimens and applied it on a K-Ras driven mouse model of metastatic lung carcinoma. Metastatic disease is the leading cause of lung cancer-related mortality in the United States. In spite of continuous efforts, effective treatments targeting oncogenic K-Ras in lung and other K-Ras driven malignancies are slow to develop. To expand the treatment options for lung cancer and facilitate better understanding of metastasis new targets need to be identified and characterized at the surface of cancer cells, preferably in their native tissue microenvironment. Towards this goal we developed a mass-spectrometry (MS)-based glyco-proteomic approach targeting specifically cell surface proteins in tissue specimens and applied it on a xenograft lung cancer mouse model. We used A549 cells expressing endogenous K-RasG12S to induce lung tumor xenografts in BALB/c mice via tail vein injection. A comparative surface glyco-proteomics of cultured A549 cells, dissected tumor tissue (TT), adjacent tumor (AT) tissue and normal mouse lung tissue (NT) obtained from saline-injected age-matched littermates yielded high enrichment (i.e., ≥80%) of surface proteins. It resulted in extensive catalogue/map of more than 400 glycoproteins identified on tumor cell surface. More than 50% of proteins identified on the surface of A549 cultured cells were unambiguously identified in mouse TT, featuring proteins and pathways implicated in metastasis, invadopodia formation and cancer cell migration. These markers also provide insight regarding species determination of tissue origin (i.e., human vs. mouse) differentiation of tumor parenchyma from stroma, and regulation of tumor immune response.

#3895

Immunoproteomic profiling in African American men with prostate cancer: Evidence for an autoimmune response to glycolytic enzymes.

Tino Wilson Sanchez,1 Jian-Ying Zhang,2 Liping Dai,2 Susanne Montgomery,1 Colwick Wilson,1 Guangyu Zhang,1 Saied Mirshahidi,1 Nathan Wall,1 Carlos A. Casiano1. 1 _Loma Linda University, Loma Linda, CA;_ 2 _University of Texas El Paso, El Paso, TX_.

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer-related male deaths in the U.S. This cancer is more aggressive in African American (AA) men, who are twice as likely to die from the disease when compared to other racial groups. Despite these racial disparities in PCa mortality, studies on PCa biology and biomarker discovery include mostly patients from European American (EA) backgrounds. There is a critical need to find minimally invasive PCa biomarkers that could aid in diagnosis and prognosis, especially in high risk populations like AA men. Immunoproteomics offers a minimally invasive approach to detect early malignant processes that trigger production of autoantibodies to tumor-associated antigens (TAA). We used serum samples from AA (n=59) and EA (n=50) men with PCa to probe one- and two-dimensional Western blots of protein lysates from aggressive PCa cell lines. AA PCa patient sera showed stronger immunoreactivity to proteins in PC3 cell lysates compared to the EA sera, with a 50 kD protein band frequently targeted by autoantibodies in several AA PCa sera. Serological proteomic analysis with mass spectrometry showed that these AA PCa autoantibodies recognized alpha-enolase (ENO1), a protein important in tumor formation, expansion, and glucose metabolism. In addition, several other glycolytic enzymes important for tumor proliferation were also identified as candidate prostate TAA, including GAPDH, bisphosphate aldolase, phosphoglycerate kinase, and lactate dehydrogenase. The ENO1 autoantibody frequency, measured by ELISA in sera from 400 racially diverse men with and without PCa, was four-fold higher in PCa compared to non-PCa patients. Interestingly, although sera from AA men with PCa had stronger WB reactivity to ENO1 present in PC3 lysates compared to EA men, and the ENO1 autoantibodies were initially discovered in eight AA PCa sera but only in two EA sera, the anti-ENO1 autoantibody frequency measured by ELISA, which used yeast-purified recombinant human ENO1 as a substrate, was higher in the EA PCa cohort. In addition, several anti-ENO1 positive sera from AA men with PCa showed increased immunoreactivity to this protein in the PCa cell lines LNCaP, MDA-PC2b, PC3, and DU145, but not against the purified ENO1 used in ELISA. The opposite was true in the EA PCa cohort, where strong reactivity against the purified ENO1 did not correlate with an equally strong immunoreactivity against the cellular ENO1. The EA PCa sera did not recognize the pattern of ENO1 expression observed with the AA sera in the same panel of prostate cell lines. These differences of immunoreactivity between AA and EA sera against PC3-ENO1 vs recombinant human ENO1 suggest that select AA sera may predominantly recognize ENO1 epitopes not displayed in the purified protein. These discrepancies point to racial differences in the immune response to prostate tumors.

### Therapeutics

#3896

Novel in vivo pan-Hsp90 family protein inhibitor, Panvotinib-401, showed potent anticancer activities without HSF1 activation.

Byoung Heon Kang. _UNIST, Ulsan, Republic of Korea_.

Hsp90 family proteins are implicated in tumorigenesis and many inhibitors have been developed as cancer therapeutics. The inhibitors showed potent inhibitory activities against purified Hsp90 family proteins, Grp94 and TRAP1, as well as Hsp90 in vitro. The mitochondrial TRAP1, however, is not inactivated in vivo due to insufficient drug accumulation in the mitochondria. Here, we showed that simultaneous inactivation of Hsp90 family proteins in different compartments can augment anticancer activity of the Hsp90 inhibitors. When simultaneously inhibited, cytoplasmic calcium elevation and activation of calcineurin inhibited HSF1 triggering heat shock responses such as cytoprotective Hsp70 induction. To improve anticancer efficacy of Hsp90 inhibitors, we developed a small molecule (~380 Da) mitochondrial inner membrane-permeable Hsp90 inhibitor, and named it as Panvotinib-401, briefly Pan-401. Pan-401 showed much improved cancer-selective cytotoxic activity compared with Hsp90 inhibitors or mitochondria-targeted (accumulating) inhibitor, gamitrinib. Collectively, with improved subcellular distribution, cancer-specific cytotoxic activity of Hsp90 inhibitors can be further enhanced.

#3897

A novel c-Met targeting antibody drug conjugate for NSCLC.

Lingna Li,1 Cathrine Fells,1 Julia Guo,1 Pia Muyot,1 Edwige Gros,2 Yanliang Zhang,2 Yingqing Sun,1 Hong Zhang,1 Yanwen Fu,2 Tong Zhu,1 Jian Cao,2 Gunnar Kaufmann,2 Gang Chen,1 Zhenwei Miao1. 1 _Concortis Biosystems, San Diego, CA;_ 2 _Sorrento Therapeutic, San Diego, CA_.

c-Met, the proto-oncogene transmembrane receptor tyrosine kinase, is involved in cell proliferation, survival, motility, and invasion in normal and tumor cells. It is widely expressed and associated with poor prognosis in breast, lung, liver, kidney and brain cancers. Hepatocyte growth factor (HGF) is the only known ligand of c-Met, also found in many tumors and cancers where activity is driven by the c-Met receptor. c-Met is accepted as an attractive anti-cancer target, but the development of cancer therapy targeting c-Met receptor or kinase remain very challenging. Clinical development of c-Met/HGF targeting antibodies has shown initial evidence of clinical efficacy but failed in phase III. Small molecule inhibitors often suffer from selectivity issues. Antibody drug conjugates (ADCs) which combine an antibody with highly potent cytotoxic agents, becomes an attractive approach against c-Met overexpressing cancers such NSCLC. We have identified an anti-c-Met antibody STI-0602 and generated ADCs from a panel of cytotoxic agents such as microtubulin inhibitor and DNA damaging agents. ADCs retained the binding to c-Met receptor and showed potent cell killing in a variety of c-Met-positive cell lines. ADCs also showed in vivo efficacy in a panel of c-Met-positive human NSCLC xenograft models like A549, H292, EBC-1, HCC827 and H1993, which suggest a potential therapy for c-Met overexpressing lung cancer and other cancers.

#3898

Structure-based optimization of small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia (MLL).

Jonathan Pollock, Dmitry Borkin, Katarzyna Kempinska, Trupta Purohit, Xiaoqin Li, Bo Wen, Ting Zhao, Hongzhi Miao, Shirish Shukla, Miao He, Duxin Sun, Tomasz Cierpicki, Jolanta Grembecka. _University of Michigan, Ann Arbor, MI_.

Chromosomal translocations that affect the Mixed Lineage Leukemia (MLL) gene result in acute myeloid and lymphoid leukemias. Fusion of MLL with one of 60 different partner genes generates MLL fusion proteins which lead to enhanced cell proliferation, up-regulation of Hoxa9 and Meis1 genes and block hematopoietic differentiation, ultimately leading to acute leukemia. Importantly, the N-terminal region of MLL is retained in all MLL fusion proteins and represents the interaction site of menin, a critical oncogenic cofactor for MLL fusion proteins. As a result, inhibition of the menin-MLL interaction should abrogate the development and progression of MLL leukemia. Therefore, disruption of the menin-MLL interaction with small molecule inhibitors might represent a therapeutic strategy for patients harboring MLL-rearrangements.

The development of potent small molecule inhibitors of protein-protein interactions with optimized drug-like properties represents a challenging task in the lead optimization process. Here, we employed structure-based design with extensive medicinal chemistry efforts to optimize the thienopyrimidine class of menin-MLL inhibitors represented by the MI-136 compound. Our efforts resulted in development of MI-538, which represents the most potent small molecule inhibitor of the menin-MLL interaction developed to date. MI-538, which binds to menin with a Kd of 6.5 nM demonstrated superior cellular activity in MLL-AF9 transformed leukemic cells with a GI50 = 83 nM. In a mouse xenograft model utilizing MV4:11 human MLL leukemia cells, treatment with MI-538 significantly reduced tumor growth. Overall, systematic exploration of substituents on MI-136 led to the identification of MI-538 with improved activity, selectivity, polarity and pharmacokinetic profile, making it suitable for in vivo studies. Interestingly, we found that simultaneous incorporation of multiple substituents optimized for each site on the cyanoindole ring of MI-136 does not always positively affect the activity or drug-like properties of these compounds. This study demonstrates challenges in optimizing inhibitors of protein-protein interaction for potential therapeutic applications.

#3899

Preclinical development of tetra-branched NT4 peptide theranostics.

Jlenia Brunetti,1 Lorenzo Depau,1 Chiara Falciani,2 Giulia Riolo,1 Elisabetta Mandarini,1 Alessandro Pini,1 Luisa Bracci1. 1 _University of Siena, Siena, Italy;_ 2 _SetLance srl, Siena, Italy_.

The tetra-branched peptide NT4 is a potential cancer theranostic, which very selectively binds to human cancer tissues in different malignancies and can efficiently and selectively deliver drugs or liposomes for cancer cell imaging or therapy, in vitro and in vivo. By using NT4 conjugated to methotrexate or 5FdU we obtained significant reduction of tumor growth in xenografted nude mice. Very recently we reported that conjugation of paclitaxel to NT4 leads to increased therapeutic activity of the drug in an orthotopic model of breast cancer in mice and produces tumor regression which is not achieved with unconjugated paclitaxel in identical experimental conditions. We demonstrated that NT4 specifically binds to sulfated glycosaminoglycans and LRP receptors on cancer cells and tissues.

Considering the role of sulfated glycosaminoglycans in cancer cell interaction with the extracellular matrix, we have analyzed the effect of NT4 in cancer cell adhesion and migration on different supports. NT4 inhibits adhesion and migration of different human cancer cell lines, strongly affecting directionality of cell movement.

We have also constructed and validated a novel theranostics nanodevices, by conjugation of NT4 to quantum dots, for selective diagnosis and imaging of different human carcinomas.

Thanks to their high cancer selectivity and versatile chemical conformation, NT4 peptides can be exploited for constructing cancer theranostics, which may also reduce tumor aggressiveness and metastatic potential by inhibiting cancer cell migration.

References:

Falciani, C. et al. Cancer selectivity of tetrabranched neurotensin peptides is generated by simultaneous binding to sulfated glycosaminoglycans and protein receptors. J Med Chem. 2013, 56, 5009-18.

Brunetti, J. et al. Tumor-selective peptide-carrier delivery of Paclitaxel increases in vivo activity of the drug. Scientific Reports. DOI:10.1038/srep17736 .

#3900

Sustained release of PARP inhibitor Talazoparib and chemotherapeutics from biodegradable implants for treatment of breast and prostate cancer.

Jodi Belz,1 Noelle Castilla Ojo,1 Paige Baldwin,1 Rajiv Kumar,1 Anne van de Ven,1 Karen Liby,2 Robert Cormack,3 Mike Makrigiorgos,3 Srinivas Sridhar1. 1 _Northeastern University, Boston, MA;_ 2 _Michigan State University, East Lansing, MI;_ 3 _Dana Farber Research Institute, BOSTON, MA_.

Sustained localized delivery of cancer therapeutics is a safe and effective unique option for non-metastatic cancers. Here we report a novel biodegradable implant with the capability to encapsulate therapeutics, molecular agents, or nanoparticles for local intratumoral delivery. We have successfully demonstrated in vivo the delivery of PARP inhibitor Talazoparib to treat Brca1-mutated cancers and Docetaxel to treat localized or recurring prostate cancers. This one-time intratumoral injection provides a safe vehicle for the sustained release of PARP inhibitor Talazoparib and chemotherapeutic Docetaxel in contrast to low bioavailability and toxicity associated with oral or systemic delivery.

Methods: Biodegradable implants of 1-2mm length and 0.8mm diameter were loaded with ~50µg Talazoparib (BMN) for BRCA1-mutated breast cancer (BCa) studies and ~500µg Docetaxel (DTX) for prostate cancer (PCa) studies. Implants were characterized using SEM and HPLC, and release studies were carried out in pH 6.0 PBS buffer at 37°C. The IC50's were determined using an MTS assay in cell lines W0069 and W780 (BCa) and PC3 (PCa). In vivo studies were carried out in Brca1 Co/Co;MMTV-Cre; p53+/− spontaneous tumored mice for BCa studies. Subcutaneous PC3 tumors were xenografted in nude mice. PCa studies were done with and without radiation. Implants were injected once intratumorally using an 18G brachytherapy needle.

Results: The release profile of the drug from the implant in buffer showed a highly sustained release for multiple weeks at therapeutically relevant doses for both DTX and BMN loaded implants. BCa cell lines W0069 and W780 were highly sensitive to BMN, most likely due to Brca1 mutation. Following a one-time intratumoral implantation of BMN, tumors reduced in size by an average of 50%, while untreated tumors increased ~5X in size. BMN dosing appeared to be well tolerated by the mice. DTX implants proved to be an effective method for PCa treatment in vivo with no weight loss observed. The local DTX group showed sustained tumor inhibition compared to empty implants and an equivalent DTX dose given systemically. At 40 days 89% survival was observed for mice treated with localized DTX implants compared with 0% in all other treatment groups. Histology samples were taken from sacrificed mice and immunohistochemistry is currently underway.

Conclusions: Sustained local release of therapeutically relevant doses of BMN and DTX were observed in vitro and in vivo. Therapeutics loaded in implants represent a novel delivery modality that is well-tolerated. Sustained release of BMN appears to amplify the therapeutic efficacy of PARP inhibition in BRCA1 mutated breast cancers and sustained release of DTX is an effective chemotherapy option alone or in combination with radiation therapy. These results lay a strong foundation for the use of localized biodegradable implants for the treatment of breast and prostate cancer.

#3901

Polymeric mechanical amplifiers of tumor cell receptor-mediated apoptosis.

Michael J. Mitchell, Robert Langer. _MIT, Cambridge, MA_.

Introduction: It has become evident that tumor cells are responsive to mechanical forces in vivo, and prove critical to tumor cell proliferation and death. Recent work has shown that tumor cells exposed to fluid shear forces increase receptor-mediated signaling. We hypothesized that biocompatible, polymeric particles conjugated to the tumor cell surface act as mechanical amplifiers in presence of fluid shear forces to increase mechanotransduction, and can be exploited to increase the efficacy of therapeutic ligands.

Methods: Polymeric particles (size:100 nm-1 μm) were conjugated to free amines on tumor cells via NHS cross linker chemistry. Particles bound to tumor cells were assessed using flow cytometry, brightfield, and confocal fluorescence microscopy. A cone-and-plate viscometer was used to apply a fluid shear force (2.0 dyn/cm^2) to tumor cell suspensions and to amplify the force exerted by polymeric particles on tumor cells. Tumor cells (COLO 205, PC-3) were treated with 0.1 μg/mL of a TNF-related apoptosis-inducing ligand (TRAIL) to assess amplified mechanotransduction and receptor-mediated apoptosis in the presence of polymeric particles. An annexin-V apoptosis assay was used to characterize the mode of cell death. Caspase colorimetric assays and inhibitors (Z-VAD-FMK) were utilized to assess caspase-dependence in the mechanotransduction response. Tumor cells were injected into C57/BL6 via tail vein to assess amplification of TRAIL apoptosis in vivo.

Results and Discussion: NHS crosslinker chemistry was successfully used to conjugate polymeric particles to the tumor cell surface. In the presence of fluid shear forces, it was found that polymeric particles act to mechanical amplify tumor cell mechanotransduction, as evidenced by increased receptor-mediated apoptosis and decreased tumor cell viability in the presence of the therapeutic ligand TRAIL. Additionally, amplification of TRAIL-mediated apoptosis was increased with particles of increasing size, demonstrating that increasing the force exerted on the cell surface with larger particles amplified the therapeutic response. Annexin-V apoptosis assays showed the addition of conjugated polymeric particles to the cell surfaces nearly doubled tumor cell apoptosis in the presence of TRAIL under shear forces, and inhibition assays revealed the response to be caspase-dependent apoptosis. Polymeric particles amplified

TRAIL-mediated tumor cell killing in mice, and reduced both circulating tumor cells in blood and overall tumor cell burden in vivo by over 90%.

Conclusions: These data demonstrate that polymeric particles, both degradable and non-degradable, act as mechanical amplifiers of tumor mechanotransduction in the presence of shear forces in vitro and in vivo, and are exploited to increase therapeutic efficacy. Clinically, this approach shows that increased mechanical force applied to target tumor cells can increase sensitivity to therapeutic ligands.

#3902

Novel selective estrogen receptor downregulators developed using endocrine-independent breast cancer cell lines.

Rui Xiong, Jiong Zhao, Lauren Gutgesell, Debra Tonetti, Gregory Thatcher. _University of Illinois at Chicago, Chicago, IL_.

Approximately 70% of breast cancer patients are estrogen receptor positive (ER+). Aromatase inhibitors and the selective estrogen receptor modulator (SERM), tamoxifen, are the first line treatments for these patients; however, almost 50% of patients either do not respond or acquire resistance. Multiple mechanisms, including mutations of the ESR1 gene, contribute to resistance via ligand-independent constitutive activation of ER. Selective estrogen down-regulators (SERDs) that block ligand-dependent and independent ER signaling by ablation of ER, offer a therapeutic approach to treatment-resistant, advanced stage and early stage ER+ breast cancer. Therapeutic use of the first generation SERD, fulvestrant (Faslodex), has largely remained 2nd and 3rd line, because of poor physiochemical/pharmacokinetic properties. Novel benzothiophene based SERDs were designed, synthesized, and optimized and assayed in three tamoxifen-resistant (TR), endocrine-independent ER+ MCF-7 and T-47D cell lines. Cell viability, ERE-luciferase response, and ER degradation was measured and compared to parent endocrine-dependent MCF-7 and T-47D cell lines in 2D and/or 3D spheroid cell cultures and compared to SERDs, fulvestrant and GDC-0810. Pharmacokinetic analysis was used to select novel SERDs for xenograft studies.

#3903

Optimizing the antitumor efficacy of AuNR-assisted plasmonic photothermal therapy and its molecular impact.

Mohammad Aminur Rahman,1 Moustafa Ali,2 Zhixiang Zhao,1 Georgia Z. Chen,1 Mostafa A. El-Sayed,2 Dong M. Shin1. 1 _Emory University Winship Cancer Institute, Atlanta, GA;_ 2 _Georgia Institute of Technology, Atlanta, GA_.

Plasmonic gold nanorods (AuNRs) are very promising for biomedical applications because of their strongly enhanced radiation (e.g. absorption and scattering) and non-radioactive photothermal properties due to surface plasmon resonance. In plasmonic photothermal therapy (PPTT), AuNRs absorb near infrared (NIR) laser and induce localized heat (i.e., hyperthermia) which can promote tumor tissue ablation. However, the lack of comprehensive studies to improve the efficiency of AuNRs has hindered their application. The objectives of this study were to perform a systematic analysis to optimize AuNR-PPTT based on different sizes, formulation and concentration along with various laser powers for cancer therapy in vitro and in vivo. We used AuNRs of sizes 26X6 nm and 72X19 nm with concentrations of 2.5, 5 and 10 nM, followed by 2 min of 0.5, 1, 1.78 W/cm2 NIR 808 nm diode laser exposure both in vitro and in vivo. For in vitro studies, we studied several head and neck squamous cell carcinoma (HNSCC) cell lines. We conducted an in vivo antitumor efficacy study in nude mice bearing human HNSCC Tu686 xenograft tumors with three formulations of AuNRs (72X19 nm and 26X6 nm with or without rifampicin (Rf) conjugation).Single dose intratumoral injection of 5 nM and 10 nM AuNRs (26x6 nm), followed by 2 min of 1.78 W/cm2 NIR laser exposure inhibited tumor growth and the 2.5 nM dose led to moderate antitumor efficacy. However, we observed severe skin burning at higher concentrations. In contrast, AuNRs (72x19nm) had no remarkable antitumor efficacy at high laser power. Impressively, small AuNR-conjugated Rf (AuNR-Rf) accumulated AuNRs inside the cell and had very significant antitumor efficacy (p<0.05) without any skin burning at 2.5 nM concentration. We confirmed our observations by immunohistochemistry staining of proliferation marker Ki67 in tumor tissues. To understand the molecular impact, we applied the optimized treatment in vitro. AuNR-Rf was able to induce apoptosis in 24 hours of treatment and decreased cell viability, as supported by immunoblotting of PARP and caspase 3 cleavage. In addition, we found that mutant p53 was completely abolished in AuNR-PPTT treatment. Overall, we have demonstrated that 2.5 nM AuNR with 1.78 W/cm2 NIR laser has no remarkable toxicities and optimal antitumor efficacy was observed by conjugation with Rf. In future studies, we will explore potential novel biomarkers by next generation sequencing (NGS) in mouse tumor tissues to determine the response to AuNR-PPTT. (This study supported by U01CA151802).

#3904

Overcoming resistance to HER2 inhibitors through cell based screening.

Chris J. Novotny,1 Sirkku Pollari,2 Jin H. Park,3 Mark A. Lemmon,3 Peter G. Schultz,2 Weijun Shen,2 Kevan M. Shokat1. 1 _University of California San Francisco and HHMI, San Francisco, CA;_ 2 _California Institute for Biomedical Research (Calibr), CA;_ 3 _University of Pennsylvania, PA_.

Signaling from the human epidermal growth factor receptor (HER) family of proteins is dependent on a well-orchestrated series of interactions between family members to form either homo- or heterodimers. The heterodimeric complex formed by HER2 and HER3 is a particularly potent oncogenic signaling unit that can act as a driver of cancer growth and has also been shown to rescue a large number of cancers from a variety of targeted agents. The currently available therapies targeting HER2, such as Lapatinib or ado-trastuzumab emtansine, have dramatically improved patient outcomes in the clinic but preferentially target the protein in its monomeric state. Because of this, increasing the concentration of the HER2/HER3 heterodimer, either by growth factors or increasing the concentrations of HER2 and HER3 at the membrane, significantly diminishes their activity. In order to find a next generation inhibitor of the active HER2/HER3 oncogenic complex we screened 1 million small molecules against an engineered Ba/F3 cell line dependent on ligand stimulated HER2/HER3 signaling for survival. The removal of non-selective inhibitors using alternative Ba/F3 cell lines resulted in the identification of a single core scaffold hit. A medicinal chemistry campaign enabled by a co-crystal structure of a hit compound complexed to EGFR led to the development of a molecule capable of inhibiting the active state of HER2. As a result, this next generation HER2 inhibitor is capable of inhibiting growth factor stimulated HER2/HER3 heterodimers and mutationally activated forms of HER2, which are resistant to current clinical small molecule HER2 inhibitors.

#3905

Anti-tumor effect of combined irreversible electroporation and liposome-encapsulated NVP-BEZ235.

Tian Li, Lucas Wang, Yang Qiao, Burapol Singhana, Marites P. Melancon. _MD Anderson Cancer Center, Houston, TX_.

Introduction

Irreversible electroporation (IRE) is a technique that uses electrical pulses to cause disruption in the lipid bilayer integrity by creating nanopores. Regions that are close to the probe will have a permanent, irreversible damage to the cells, while farther away regions will subject the cell membranes to re-seal (reversible). This reversible region provides opportunity for enhanced nanoparticle uptake. In this study, we tested the efficacy of combined IRE and nanoparticle-mediated drug delivery in vitro and in vivo in a hepatocellular carcinoma model.

Methods

Liposome-encapsulated NVP-BEZ235 (L-BEZ) was made from hydration-sonication, followed by an optional extrusion step. Particle size was tested via dynamic light scattering. BEZ concentration was quantified by fluorescence. Cytotoxicity of IRE and its drug combinations was tested on Hep3B cells using ECM 830 (BTX Harvard Apparatus) at 1600 V/cm field strength. In vivo efficacy was studied on nude mice bearing Hep3B xenografts with single dose of IRE, or drug alone (L-BEZ or BEZ at 4 mg/kg, intratumoral injection), and its combinations.

Results and Discussion

Liposomes range from 100-500 nm in hydrodynamic volume. Highest loading efficiency achieved was 90% (1.8 mg/mL of BEZ). To test the release of BEZ from the liposome, solution of L-BEZ was treated with IRE and the supernatant was tested against Hep3B cells. The extract from L-BEZ had a significantly higher cytotoxicity (51%) as compared to the empty liposome (20%) with a p-value<0.05. This suggests that IRE can release BEZ from the liposome. The LC50 values of L-BEZ and BEZ was determined to be the same (47 ng/mL). In vitro combination treatment of L-BEZ + IRE showed higher cytotoxicity than the controls (BEZ + IRE, BEZ alone, and IRE alone). In vivo, IRE and its combinations significantly suppressed tumor growth more than non-IRE treated groups. Histology showed a median percent necrosis for L-BEZ + IRE (37%) doubled compared to IRE treatment alone (17%).

Conclusion

The use of minimally invasive IRE in combination with nanoparticle-mediated drug delivery treatment is a promising technique to achieve local disease control in hepatocellular carcinoma.

#3906

A novel cationic liquid crystalline nanoparticle for the delivery of synthetic RNAi-based therapeutics.

Emanuela Gentile,1 Taro Oba,1 Jing Lin,1 Ruping Shao,1 Feng Meng,1 Xiaobo Cao,1 Dong Cai,2 Jack A. Roth,1 Lin Ji1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _University of Houston, Houston, TX_.

Small interference RNA (RNAi)-based therapeutics have been used to silence expression of targeted pathological genes. However, short half-life, poor cellular uptake, and non-specific distribution of small RNAs call for the development of novel delivery systems to facilitate the use of RNAi as a new class of therapeutics. We developed a novel cationic liquid crystalline nanoparticle (CLCN) with microfluidic-facilitated encapsulation of hydrophilic biomolecules for efficiently delivering synthetic RNAi-based therapeutics including siRNAs, PNAs, and micro-RNA mimics. CLCNs were prepared by mixing under high speed homogenization a lipophilic phase with a hydrophilic phase containing an emulsifier/stabilizer such as poloxamer. CLCNs were assembled with synthetic small siRNA molecules in nuclease free water to create CLCN/siRNA complexes. The homogenous and stable CLCNs and CLCN-siRNA complexes dispersed in nuclease free water displayed sizes under 100 nm and positive charges between 25-35 mV on the CLCN surface as demonstrated by the NanoTracking and Zeta potential measurements. The gel retardation assay indicated that the binding between the carrier and the siRNA was strong enough to withstand dissociation during electrophoresis. No cytotoxicity was detected in both lung cancer and normal cells treated with various concentrations of CLCNs (from 0.01 to 100 μM) by in vitro cell proliferation assay. The CLCNs were taken up by human cells though endocytosis after binding with the cell membrane and traveling from early endosomes to the lysosome after 24 h treatment as shown by intracellular trafficking analysis with transmission electron microscopy (TEM). The presence of the fluorescent CLCN/siRNA complexes in the cytoplasm was observed as early as 2 h post treatment by confocal fluorescence imaging analysis. A significant inhibition of gene expression was detected in both EGFP-stable clones and transiently-transfected lung cancer H1299 cells treated with CLCNs/siEGFP complexes 24 hour after transfection compared to the untreated and non-specific CLCN-siRNA controls. Biodistribution analysis showed that the CLCNs were successfully delivered to various organs including liver and lung and into the subcutaneous human lung cancer H1299 tumor xenografts in mice 24 h after systemic administration by tail vein. These results suggest that CLCNs are suitable for the delivery of small synthetic RNAi-based therapeutics in vitro and in vivo. CLCNs are a unique and advanced delivery system capable of protecting siRNA from degradation and efficiently delivering siRNA to the cytoplasm where effective gene silencing is achieved.(This study is partially supported by NIH/NCI grants Lung SPORE 5P50CA070907 and R01CA176568, a CPRIT Grant and a MDACC Moonshot Program Grant.

#3907

Enhanced chemotherapeutic effect with matrix metalloproteinase sensitive liposomes.

Rikke Y. Brogaard,1 Rasmus Eliasen,1 Fredrik Melander,1 Anders E. Hansen,1 Andreas Kjær,2 Thomas Lars Andresen1. 1 _Technical University of Denmark, Lyngby, Denmark;_ 2 _University of Copenhagen, Copenhagen, Denmark_.

Introduction: Drug bioavailability following intra-tumoral accumulation is a major challenge in existing drug delivery systems. A site-specific trigger for obtaining drug release specifically in tumor tissue would overcome this problem and enhance the antitumor effect of the carried drug. Our aim was to develop a new drug delivery platform based on liposomes that are sensitive to matrix metalloproteinases (MMPs).

Methods: In the present work, we have designed a matrix metalloproteinase (MMP)-sensitive liposomal drug delivery system encapsulating oxaliplatin, which by tumor‐specific enzymatic dePEGylation allows for controlling the precision of drug release. The dePEGylation is provided by a cholesterol-anchored PEGylated lipopeptide containing a short peptide sequence cleavable by MMP-2 and MMP-9. These MMP-sensitive liposomes are designed to make a charge-reversal transformation upon encountering MMPs in the tumor environment. The surface charge of the liposome turns from slightly negative to positive, resulting in cationic liposomes. In vitro cellular uptake of the liposomes was assessed using flow cytometry and ICP-MS, and further confirmed by confocal microscopy in CT26 and HT1080 cell lines. In vivo studies was evaluated in a syngeneic murine model of MMP-positive CT26 colon cancer.

Results: We successfully formulated MMP-sensitive, oxaliplatin encapsulated liposomes and characterized their zeta potential, size, stability and encapsulation efficiency. The results showed uniformly dispersed particles, and the charge-reversal properties were confirmed as the surface charge changed from slightly negative to distinct positive upon enzymatic cleavage generating cationic liposomes. These MMP-sensitive liposomes were evaluated in vitro and in vivo with a high correlation. In vivo studies showed a prolonged circulation profile with minimal leakage, and similar accumulation in tumors as conventional Stealth liposomes. Efficacy experiment in the same model demonstrated significantly improved antitumor activity relative to free oxaliplatin, and with a superior effect compared to the conventional Stealth liposomes.

Conclusion: With this study, we establish a promising liposomal drug delivery platform for the carriage and release of numerous anticancer drugs into the microenvironment of an MMP-positive tumor.

#3908

A multifunctional peptide for targeted imaging, localization of transformed cell and chemotherapy for nasopharyngeal carcinoma and other cancers.

Chin-Tarng Lin,1 Jong-Kai Hsiao,1 Hon-Man Liu,1 Hang-Chung Wu,2 Alice Yu3. 1 _National Taiwan Univ. College of Medicine, Taipei, Taiwan;_ 2 _Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan;_ 3 _Linkou Chang Gung Memorial Hospital Stem Cell and Cancer Research Institute, Taoyuan, Taiwan_.

Introduction: In treating cancer with chemotherapy, the ultimate goal is to deliver sufficient amounts of the drug to tumor cells while minimizing the damage of normal tissue. To achieve this purpose, using a phage-displayed random peptide library to screen the nasopharyngeal carcinoma (NPC)-TW01 cell line, we identified a specific peptide (L-peptide), which binds specifically to NPC tumor cells but not normal cells. To investigate whether this L- peptide can also bind to other cancer cell lines, be used for cancer tissue binding, targeted imaging and chemotherapy, we have performed this experiment. Methods: A fluorescein-labelled L-peptide was used to perform FACScan for 7 cancer cell lines. The L-peptide was linked to dextran coated iron oxide (L-P-Fe3O4) nanoparticles and a biotin labeled modified L-peptide (biotin-mL-P) were also used to localize L-peptide targeted protein (X-protein) in NPC and breast cancer surgical specimens. This conjugate was also injected intravenously to SCID mice bearing NPC and breast cancer xenografts for magnetic resonance imaging (MRI) analysis. Furthermore, L-peptide-linked liposomal doxorubicin (L-P-Lipo-Dox) was used to observe the efficacy of peptide-targeted chemotherapy in both cancers. Results and Conclusions: FACscan revealed specific binding of L-peptide to NPC, breast cancer, lung cancer, and neuroblastoma. The X-protein in both cancer specimens localized by biotin-mL-P was seen in many NPC and breast cancer tumor cells with some unstained tumor cells, but not or very weak in normal tissue; the X-protein in formalin-fixed paraffin-embedded section could be localized by L-P-Fe3O4 histochemistry in both cancer specimens. MRI analysis of both cancer xenograft bearing mice after injection of L-P-Fe3O4 revealed significant change in the MR signal intensity. A high efficacy of L-P-Lipo-Dox treatment for both cancers with minimal adverse effect was obtained. This multifunctional L-peptide seems able to be used for localization of its binding protein in each cancer cell type, molecular MRI analysis and targeted chemotherapy with minimal adverse effect in different cancer types.

#3909

Interactions of glioma cells with chlorotoxin: an ATR-FTIR spectroscopy study.

Rana Falahat, Marzenna Wiranowska, Ryan Toomey, Norma Alcantar. _University of South Florida, Tampa, FL_.

Chlorotoxin is a scorpion-derived peptide that preferentially binds to tumor cells of neuroectodermal origin, like glioma, but not to normal non-transformed cells. We have previously reported the development of a targeted nanodelivery system using chlorotoxin. Chlorotoxin has been studied as an imaging and targeting agent for drug and radioisotope delivery, but the mechanism of interaction of chlorotoxin with cancer cells has not been well understood and it needs to be further evaluated in order to optimize its use as a targeting compound for cancer cells.

In this study, we used U87 human glioma cell line to evaluate the binding kinetics of chlorotoxin by Attenuated Total Reflection Fourier Transform Infra-Red (ATR-FTIR) spectroscopy. As a sensitive and label free technique, ATR-FTIR is a powerful diagnostic tool for the comparison of cancer cells with normal cells.

First, we characterized the signature spectra of chlorotoxin and U87 cells by assigning and evaluating the infrared absorption bands and their second derivatives. Next, we studied the spectral differences between U87 cells incubated with and without chlorotoxin for incremental time periods ranging from 15 minutes to 24 hours. We also examined the metabolic changes in U87 cells induced with chlorotoxin at different incubation time points by measuring the ratios of integrated areas corresponding to different chemical conformations of proteins, lipids, carbohydrates, and nucleic acids.

Our results revealed spectral changes in U87 cells at different stages of incubation with chlorotoxin. The most notable change occurred after 30 minutes of incubation, where the band assigned to CH3 bending of lipid membranes shifted from 1456 cm-1 to 1450 cm-1. Consequently, a new shoulder appeared at wavenumber of 1466 cm-1 assigned to CH2 bending of lipid membranes for U87 cells treated with chlorotoxin. These changes indicate a direct interaction due to the presence of a molecular binding site between chlorotoxin and the lipids of the cell membrane.

Another significant alternation occurred after 1 hour of incubation of U87 cells with chlorotoxin in the spectral region assigned for nucleic acids. This interaction included a large upshift of the band at 1036 cm-1 to 1051 cm-1 assigned to C-O stretching vibrations of ribose ring in RNA due to the hydrogen bonding with chlorotoxin.

These findings suggest at least two different interactions of chlorotoxin with U87 human glioma cells that can be further explored for improving chlorotoxin containing drug delivery systems. We are currently using the ATR-FTIR method to conduct control experiments by measuring possible interactions of chlorotoxin with normal human astrocytes.

#3910

Biomimetic proteo-lipid vesicles for the treatment of melanoma.

Roberto Molinaro, Jonathan Otto Martinez, Claudia Corbo, Naama E. Toledano Furman, Enrica De Rosa, Alessandro Parodi, Ennio Tasciotti. _Houston Methodist Research Institute, Houston, TX_.

The systemic administration of pharmaceutics presents several drawbacks, such as: i) reduced drug's stability because of the degradation by lytic enzymes in pathological (secreted by the cancer cells) or physiologic (lysosome acidic environment) conditions, and ii) lack of specific tumor targeting, that requires high doses to achieve therapeutically effective drug concentrations, and increases the risk of off-target effects. Drug encapsulation and delivery through nanocarriers can offer many advantages over free drugs, as prevention of drug degradation, control of drug pharmacokinetic and biodistribution, and improvement of intracellular penetration, as well as drug solubilization. However, regardless of the type of approach, the organs of the mononuclear phagocytic system efficiently clear the particles from circulation while protein opsonization often prevents the proper interaction between targeting ligands and target biomarkers. Most cancers are characterized by inflammation and increased leukocyte infiltration. The surface of the leukocyte, in fact, is enriched with transmembrane proteins that determine self-tolerance, adhesion, and negotiation of the inflamed vascular barrier. Leveraging on leukocyte ability to efficiently recognize and infiltrate the tumor tissues, we developed a liposome-like Biomimetic Vesicle (BioV), formulated with leukocyte membrane proteins able to provide extended biocompatibility, self-tolerance and targeting. We hypothesize that the transferring of leukocyte membrane proteins on the surface on a nanovesicle will enhance BioV selective targeting towards the tumor-associated vasculature, increase payload's accumulation, and improve cancer specific cytotoxicity. C57BL mice were intradermally injected with B16 cells. Intravital microscopy and typical tumor growth inhibition curve associated with histological analysis were used to assess BioV targeting and efficacy, respectively in comparison with conventional liposomes and free drug. Once the tumor reached 70-100 mm3, mice were randomly divided into 6 groups (n=10) and treated with saline (CTRL), free doxorubicin (DOX), empty and DOX-loaded liposomes, empty and DOX-loaded BioVs, at a drug concentration of 7 mg/Kg. Melanoma-bearing mice were treated once per week for one month, and tumor volume and survival rate were investigated. Compared to the other groups, BioVs showed higher tumor targeting and DOX accumulation, increased antitumor efficacy, and prolonged survival rate. Histological analysis on tumor slides showed increased apoptosis and reduced immune cell infiltration in the groups treated with BioVs. We believe that BioV represents a promising drug delivery system possessing advantageous biomimetic properties. The high versatility of this approach and its molecular mechanisms of action make this platform a technology applicable for the treatment of all the inflammation-related cancers.

#3911

Bacteriophage associated silicon particles (BASP) for targeting pancreatic neuroendocrine tumors.

Ziqiang Yuan,1 S Srinivasab,2 Jenolyn Francisca Alexander,2 X Liu,2 Wadih Arap,3 Renata Pasqualini,3 Mauro Ferrari,2 Steven K. Libutti,1 Biana Godin Vilentchouk2. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Houston Methodist Research Institute, Houston, TX;_ 3 _University of New Mexico, Albuquerque, NM_.

The purpose of this project is to design and develop a multifunctional nanosystem for the diagnosis and treatment of pancreatic neuroendocrine tumors (PNETs). PNETs also known as Islet Cell Tumors are rare and slow-growing highly vascularized malignancies with an incidence rate of a thousand new cases per year. Their occurrence has increased five-fold within the past few decades and with surgery being the primary treatment modality, there is an immediate need to devise efficient diagnostic and therapeutic procedures.

For this purpose, the synergistic tissue targeting capabilities of phage, optical and physical properties of gold nanoparticles (AuNP)/phage scaffold and the drug carrying potential of the hydrodynamically engineered silicon-based multistage nanovectors were employed to create the Bacteriophage Associated Silicon Particles (BASP). The BASP were characterized in vitro, optimized for in vivo imaging and are currently being investigated for therapeutic efficiency in vivo when loaded with Abraxane. CRKL is evaluated as a potential target expressed in the PNET microenvironment. CRKL targeting phage is incorporated into the BASP and the targeting capability is explored.

Our data show that bacteriophage, displaying CRKL-recognizing peptide sequences, enhanced cellular association of BASP in human pancreatic cancer and murine microvascular endothelial cell lines. There is a significant increase in association of CRKL targeted BASP with endothelial cells as opposed to non-targeted assemblies mainly under the flow conditions with shear stresses similar these in the tumor vessels. Further, intravital microscopy studies in highly vascularized tumors demonstrated preferential accumulation of BASP in the tumor vasculature, based on their hydrodynamic characteristics and biological targeting. Overexpression of CRKL was confirmed in histological samples from PNET patients and mice with homozygous deletion of the Men1 gene in the pancreas (PNET model). MRI and CT imaging using CRKL targeted BASP loaded with SPION demonstrated that the systems can aid in detecting very small tumors using various imaging modalities. 3D CT images showed increasing density in the pancreatic tumor region in the mice for CRKL targeted BASP loaded with SPION treatment. Furthermore, the mice were imaged and validated the biodistribution and concentration of nanoparticles using a 9.4T MRI scanner. MRI images reveal nanoparticles accumulation in the region of tumor growth in the pancreas of mice at post-injection with CRKL targeted BASP loaded with SPION. This innovative multifunctional nanosystem has the potential to deliver a novel platform for both imaging and therapy for patients with PNETs as well as other cancers.

#3912

MM-310, a novel EphA2-targeted docetaxel nanoliposome.

Dmitri B. Kirpotin,1 Suresh Tipparaju,1 Zhaohua Richard Huang,1 Walid S. Kamoun,1 Christine Pien,1 Tad Kornaga,1 Shinji Oyama,1 Ken Olivier,1 James D. Marks,2 Alexander Koshkaryev,1 Sarah S. Schihl,3 Gerald Fetterly,3 Birgit Schoeberl,1 Charles Noble,1 Mark Hayes,1 Daryl C. Drummond1. 1 _Merrimack, Cambridge, MA;_ 2 _UCSF, San Fransisco, CA;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Taxanes are widely used to treat solid tumors either in the curative or palliative setting, in first or later lines of therapy. Analysis of docetaxel dose-response relationship strongly suggests that a higher dose would lead to high response, however will also lead to higher toxicity. This is likely related to the lack of organ and cellular specificity of docetaxel leading to high exposures in normal tissues and the relatively short circulation half-life which indirectly requires higher doses. With the goal of addressing the pharmacokinetic limitations of free docetaxel and the lack of cellular specificity, we developed a novel docetaxel-based nanoliposome (MM-310), targeted against Ephrin receptor A2 (EphA2) which is overexpressed in a wide range of tumors. MM-310 provides sustained release of docetaxel following accumulation in solid tumors. Preclinical models have demonstrated that MM-310 leverages tumor-specific accumulation through the enhanced permeability and retention effect, and cellular specificity through active targeting of EphA2 with specific scFv antibody fragments conjugated to the surface of the liposomes.

Pharmacokinetic and biodistribution studies were performed in mice and rats to compare MM-310 to free docetaxel. Chronic tolerability studies were performed in rodent and non-rodent models with focus on overall animal health, as well as hematologic toxicities. Several cell-derived models of breast, lung and prostate xenografts were used to evaluate the differences betweenf MM-310 and free docetaxel.

MM-310 had a significantly longer half-life than free docetaxel with prolonged exposure at the tumor site. In chronic tolerability studies, MM-310 was found to be 6-7 times better tolerated than free docetaxel with a maximum tolerated dose of at least 120 mpk, compared to 20 mpk for free docetaxel and no detectable hematological toxicity. At equitoxic dosing, MM-310 50 mpk showed greater activity than docetaxel 10 mpk in several breast, lung and prostate xenograft models.

In conclusion, we developed a novel EphA2 targeted docetaxel nanoliposome with prolonged circulation time and slow and sustained drug release kinetics, to enable organ and cellular targeting. MM-310 was able to overcome hematologic toxicities observed upon treatment with free docetaxel in rodent and non-rodent models. MM-310 was also able to induce tumor regression or control tumor growth in several cell derived xenograft models, and was found to be more active than free docetaxel in most models.

#3913

Conjugate-SELEX, a novel screening method, identifies aptamers that deliver payload to the cytosol of target cells.

Qingshan Mu,1 Akshaya Annapragada,2 Mayank Srivastava,3 Varatharasa Thiviyanathan,4 Xin Li,4 David Gorenstein,4 Annanth Annapragada,3 Nadarajah Vigneswaran1. 1 _School of Dentistry, UT Health Science Center at Houston, Houston, TX;_ 2 _Debakey High School, Houston, TX;_ 3 _The Singleton Department of Pediatric Radiology, Texas Children's Hospital, Houston, TX;_ 4 _Department of Nanomedicine, University of Texas Health Science Center at Houston, Houston, TX_.

Introduction: Delivery of a nanoparticle payload to a specific cellular compartment is a prized goal of cancer nanomedicine. The well-known SELEX (systematic evolution of ligands by exponential enrichment) process is often used to identify aptamers that localize to target cellular compartments, but such aptamers cannot be guaranteed to traffic an attached payload nanoparticle to the target as well. We hypothesized that to find an aptamer capable of trafficking a nanoparticle to a specific cellular compartment, the screened library must consist of aptamer-nanoparticle conjugates. We therefore designed a screening procedure we call "Conjugate-SELEX". In this process, the aptamer library is conjugated to liposomal nanoparticles, creating a nanoparticle-aptamer conjugate library for screening. At each round of Conjugate-SELEX, aptamers recovered from the desired cellular compartment are amplified, and used to create the conjugate library for the next round of Conjugate-SELEX. Negative screens against off-target cells (e.g. hepatocytes, unwanted uptake in which, causes many of the toxicities associated with nanoparticle therapeutics) facilitate the identification of an aptamer that efficiently transports payload into target cells while minimizing the uptake in off-target cells. Using fluorescently tagged particles, imaging the cells at each stage verifies the successful transport of the payload particles into the cytosol.

Methods: We used conjugate-SELEX to screen for aptamers that carried an attached nanoparticle payload to the cytosol of UM-SCC-22A oral cancer cell line, while not being taken up by THLE-3 hepatocytes. IonTorrent sequencing of the remaining aptamers after each round demonstrated convergence to a small family of sequences with only one base difference between members. Mass spectrometric proteomics identified the surface receptor bound by these sequences as the neuroblast differentiation-associated protein AHNAK (desmoyokin) a ubiquitous intracellular protein with surface expression exclusively in certain epithelial cell types. Uptake studies with concertedly synthesized hit aptamer sequences showed enhanced uptake of targeted nanoparticles over controls, and increased cytotoxicity of doxorubicin in the nanoparticles over controls.

Conclusions: Conjugate-SELEX is an excellent means of identifying aptamers with the ability to transport attached nanoparticle payloads to targeted cellular compartments, while minimizing off target effects. The technique also lends itself to the identification of surface receptors mediating the trafficking.[D.G., A.A., and N.V. contributed equally to this work.]

#3914

Repurposing disulfiram for use as an anticancer drug: a story about metabolism and metal binding.

Moe Wehbe,1 Malathi Anantha,1 Ian Backstrom,1 Ada Leung,1 Kent Chen,1 Minghan Shi,2 Armaan Malhotra,1 Katarina Edwards,3 Léon Sanche,1 Marcel B. Bally1. 1 _BC Cancer, Vancouver, British Columbia, Canada;_ 2 _Université de Sherbrooke, Sherbrooke, Quebec, Canada;_ 3 _University of Uppsala- BMC, Uppsala, Sweden_.

Disulfiram (DSF) is an FDA approved agent for the treatment of alcoholism with over 60 years of clinical use. It was identified in several high-throughput screen to be effective against glioma tumor initiating cells; this activity is amplified more than 100-fold in the presence of copper (Cu2+). DSF is metabolized to diethyldithiocarbamate (DDC), an established copper chelating agent. One mechanism attributed to the copper DDC (Cu(DDC)2) complex is proteosome inhibition. Cu(DDC)2 is very poorly soluble in aqueous solution; making it difficult to purse development for any cancer indication. A novel liposomal formulation of Cu(DDC)2 is described here; which represents the first iv injectable Cu(DDC)2. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) liposomes were prepared by extrusion methods in the presence of 300 mM unbuffered CuSO4 to ~100 nm in size. DDC was added to these pre-formed liposomes at room temperature, unentrapped drug was removed using column chromatography. The therapeutic activity of this formulation was determined in vitro against a variety of cancer cell lines. The toxicity and pharmacokinetic (PK) behaviour of this formulation was determined in CD-1 mice. DDC was tested in the presence and absence of Cu2+ in vitro with U251, U87, MDA231-BR and A549 cell lines giving IC50 values greater than >10 μM in the absence and from 300- 900 nM in the presence of Cu2+. The liposomal Cu(DDC)2 formulation was as active as unencapsulated Cu(DDC)2 when tested in vitro. The maximum tolerated dose of liposomal Cu(DDC)2 was determined to be 8mg/kg given iv using a Q2D schedule for two weeks. The PK studies suggested that Cu(DDC)2 was rapidly released from circulating liposomes. Initial efficacy studies were completed using convection enhanced delivery of liposomal Cu(DDC)2 in the F98 rat glioma model. Treatment resulted in a statistically significant delay in tumour growth upon injection of 10 μL of 0.5 mg Cu(DDC)2 /mL solution. Further anti-tumor efficacy studies are being conducted in a subcutaneous leukemia xenograft model (MV-4-11). This work describes a novel formulation of Cu(DDC)2, the active agent generated when administering DSF and Cu to treat cancer.

#3915

PBA2, a dual BCR-ABL and GSK3βkinases inhibitor, represses a wide array of Imatinib resistant cell lines in chronic myeloid leukemia.

Ke Yang, li-Wu Fu. _Sun Yat-Sen Univ. Cancer Ctr., GuangZhou, China_.

Despite the clinical efficacy of the BCR-ABL (Tyrosine Kinase Inhibitors) TKIs for the treatment of Chronic Myeloid Leukemia (CML), BCR-ABL-T315I mutation still confers higher-level resistance to imatinib and the second generation BCR-ABL TKIs. Ponatinib, as the third generation BCR-ABL TKIs, has the potential activity against CML with T315I mutant BCR-ABL whereas high frequency of serious adverse events limits its clinical use. Through a compound library screen, we identified a novel compound PBA2, a dual kinases inhibitor of GSK3β and BCR-ABL, as a promising agent for the treatment of imatinib-resistant CML. Not only can PBA2 inhibit cell growth but also induce terminal cell differentiation in a series of kinase mutant CML cell lines as well as T315I mutation. Remarkably, the mechanism of action of this compound involved the simultaneous inhibition effect of BCR-ABL and GSK3β, which could be a fruitful approach to suppress the β-catenin/CBP signal pathway that promoted the interaction of β-catenin/p300, leading to growth inhibition and subsequent terminal differentiation. More importantly, we found a novel molecular link between CBP and BCR-ABL. We showed that CBP could regulate BCR-ABL expression through activating CREB which binds to the BCR-ABL promoter in CML cells to start transcription, and the BCR-ABL in turns affect the expression level of CBP. This positive feedback pathway between CBP and BCR-ABL represents a novel way for the treatment of CML, and the novel compound PBA2 targeting at this loop deserves further investigation in clinical trial.

#3916

A "moneyball" approach to predicting clinical trial toxicity events.

Kaitlyn Gayvert, Neel Madukhar, Olivier Elemento. _Weill Cornell Medical College, New York, NY_.

Over the past decade, significant strides have been made in the management and treatment of various diseases. Despite this progress, clinical attrition rates have continued to substantially rise. Clinical trials can fail for a variety of reasons, ranging from design issues to drug efficacy and safety problems. Drug-likeness approaches, as first proposed by Lipinski almost two decades ago, have become a key tool for the pre-selection of compounds that are likely to have manageable toxicity in clinical studies. However all these methods consider molecular properties of the drug itself alone. In general, these approaches struggle to simultaneously well-characterize the properties of both FDA approved drugs (which we term the sensitivity) and drugs that fail clinical trials (specificity).

We introduce an approach that integrates chemical properties of a compound, along with that of its targets, to provide a new quantitative measure that helps predict whether drugs in clinical trials will fail for toxicity reasons. When trained on failed clinical trials and FDA approved drugs, this method performs at a high accuracy, specificity and sensitivity (~0.75), as well as high area under the ROC curve (>0.80). In comparison, none of the drug-likeness approaches were able to successfully maintain both high sensitivity and specificity. A feature analysis of the model indicates that it is critical to consider both structural properties and properties of the drug target, with the target's network connectivity and liver toxicity as two important features. The approach was further evaluated by testing the predictions of the trained model on an established independent dataset. We found that our method was able to significantly distinguish a representative set of bioavailable drugs from a representative set of toxic drugs (D=0.2133, p<2.2e-16, Kolmogorov-Smirnov test). Additionally, we found that the measure is strongly correlated with severe toxicity events, such as pleural effusion (ρ=−0.9792) and neutropenia (ρ=−0.9613). Altogether, our method provides a novel, broadly applicable strategy that is able to identify drugs likely to possess manageable toxicity in clinical trials.

## CLINICAL RESEARCH:

### Biomarkers for Breast Cancer

#3917

Breast cancer cells expose thymidine kinase 1 as a new immunotherapy target.

Evita G. Weagel,1 Rachel A. Brog,1 Michelle H. Townsend,1 Edwin J. Velazquez,1 Toshiko A. Becker,1 Camilo A. Mejia,1 Michael R. Downey,2 Juan A. Arroyo,1 Richard A. Robison,1 Kim L. O'Neill1. 1 _Brigham Young University, Provo, UT;_ 2 _Utah Valley Regional Medical Center Laboratory, Provo, UT_.

This project investigates the role of Thymidine Kinase 1 (TK1) as a possible target for cancer therapeutics in breast cancer. Currently, therapeutic cancer treatments frequently damage bystander cells and have a devastating impact on the immune system. Many attempts have been made to engineer therapeutic techniques that are specific to cancer cells, unfortunately, resulting in few breakthroughs. TK1 is a phosphotransferase enzyme that is consistently present at abnormally high levels in cancer patient serum. TK1 assists in DNA repair and is characteristically expressed in the cytosol. We have discovered that TK1 is overexpressed on the surface of breast cancer cells lines MCF-7, MDA-MB-231, and Sk-Br-3. Interestingly, TK1 is not found on the surface of normal cells. We developed a unique monoclonal anti-TK1 antibody specific to human TK1 (A72). A72 was used in conjunction with flow cytometry, confocal microscopy, electron microscopy, and immunohistochemistry to test for TK1 on the cell surface. We stained each breast cancer cell line with A72-FITC conjugate and prepared appropriate controls. The samples were then analyzed using an Attune Flow Cytometer showing a 6% binding of A72 on the normal lymphocytes, while A72 binding was elevated to 20% of the breast cancer cells. Further testing using confocal microscopy was performed. The cells were treated with A72-FITC conjugate and Cell Mask Deep Red (a red membrane dye), and consecutively imaged. Results showed that A72-FITC associate with the cells in the same region of the red membrane stain, demonstrating that A72 binds to the cell membrane. This was further confirmed using electron microscopy by probing the cells with anti-TK1 conjugated to biotin that complexed with gold-streptavidin. The normal cells expectedly showed no significant binding, while gold staining was clearly seen on the surface of the each breast cancer cell line. Immunohistochemistry was completed to confirm the presence of TK1 in actual breast tumors from patients. Normal breast tissues along with breast tumor tissues were obtained from the Utah Valley Regional Medical Center. After treating the slides with anti-TK1 antibodies in conjunction with MACH 4 HRP secondary, DAP peroxidase was used to develop the slides. The tissues were examined using a light microscope. Cancer tissues appeared brown, indicating an overexpression of TK1. As expected, healthy breast tissues showed no significant staining, suggesting little to no presence of TK1. These data strongly suggest that TK1 is located on the surface of breast cancer cells, but is not found on the surface of normal cells. Further testing may open the possibilities of using TK1 as a new breast cancer biomarker. Ultimately, this also indicates the possible use of TK1 monoclonal antibodies in a therapeutic approach.

#3918

Circulating progranulin (GP88/PGRN) level correlates with survival in metastatic breast cancer patients.

Ginette Serrero,1 David Hicks,1 Binbin Yue,1 Douglas M. Hawkins,2 Nancy Tait,3 Katherine H. Tkaczuk3. 1 _A &G Pharmaceutical, Inc., Columbia, MD; _2 _University of Minnesota, Minneapolis, MN;_ 3 _University of Maryland Greenebaum Cancer Center, Baltimore, MD_.

Current monitoring of therapy response in metastatic breast cancer (MBC) patients is dependent on expensive and time consuming imaging methods that have limited sensitivity to detect disease response in a timely manner. Understanding of real-time biological processes through measurement of circulating disease associated biomarkers may provide a clearer understanding of the disease state and thus aid real-time clinical management of MBC patients. Current biomarkers used in MBC include CA15-3, CA27.29 and CEA. While useful, they have limitations in providing clinicians with a reliable insight into real-time monitoring of disease processes. Thus, addition of new circulating biomarkers may improve the management of MBC patients. We characterized a target biomarker, the 88kDa glycoprotein Progranulin (GP88) expressed in tumor tissue and secreted in the circulation of BC patients. Biological studies have established GP88 as one of the critical drivers for tumor cell proliferation, survival, invasiveness and drug resistance. Clinical studies have demonstrated that elevated GP88 tissue levels are prognostic for poor outcome and that breast cancer patients have a statistically elevated GP88 serum level than healthy individuals. Using tissue and serum tests to detect and quantify GP88 could provide an ideal target for monitoring disease progression in BC patients undergoing therapy. In the present study, we examined whether GP88 serum levels were elevated in MBC patients and whether GP88 serum levels were correlated to patient survival.

Under an IRB approved protocol, 92 MBC patients that met the inclusion criteria and were undergoing therapy at the UMGCC Breast Clinic were consented. Clinical and disease characteristics along with serum CA15-3 values were collected as part of the study. Serum samples were collected from each patient during therapy and subsequently the patients were monitored. The serum was stored at -80C until tested for GP88. The validated GP88 assay (A&G Pharmaceutical) is sensitive and linear over 0 to 10ng/ml range.

Statistical analysis using Kaplan-Meier functions examined if there is a correlation between GP88 serum level and overall survival. By analyzing the KM plots at different GP88 cut points we identified two populations with distinct survival characteristics. When examined more thoroughly the difference in survival of patients with <60ng/ml and >60ng/ml was statistically significant (P=0.0002). Correlation analysis of serum GP88 and CA15-3 were performed and will be presented.

We conclude that circulating levels of GP88 in MBC patients are correlated with survival. It would appear that patients that can be managed to have a GP88 below 60ng/ml will survive longer. Thus measuring circulating GP88 levels would provide additional information to that available in today's SOC for monitoring. This valuable insight into real-time disease status will assist clinicians in patient management.

#3919

Phosphorylation of eIF4E is a critical factor in development and progression of breast cancer in women.

Chad A. Dumstorf,1 Larry E. Douglass,1 James A. Deddens,2 Thomas G. Lewis,1 Kyle Darpel,1 Christian Gausvik,1 Jeremy R. Graff,3 Julia H. Carter1. 1 _Wood Hudson Cancer Research Lab., Newport, KY;_ 2 _University of Cincinnati, Cincinnati, OH;_ 3 _Biothera, Egan, MI_.

Breast cancer survival rates vary according to stage, tumor grade, and receptor status. While some cancer patients respond well to targeted therapy many patients respond poorly. Critical for advancing new therapies is developing a better understanding of the molecular drivers of breast cancer tumorigenesis and malignant progression. The synthesis of new proteins (translation) is important for cancer cell survival, growth, and proliferation. eIF4E is a rate limiting step in translation. Targeting translation has been suggested as a novel strategy for therapy of malignancies originating in the epithelium since eIF4E regulates the translation of multiple malignancy associated mRNAs. eIF4E is activated by phosphorylation downstream of the Mnk 1 and 2 kinases. We hypothesized that phosphorylation of eIF4E plays a critical role in driving breast cancer and is elevated in advanced cancers and metastases. Using immunochemistry and semi-quantitative cellular pathology approaches we analyzed expression of eIF4E and phospho-eIF4E in 168 FFPE archived surgical specimens of normal breast, benign breast disease and pre-invasive, invasive, and metastatic breast cancer. Immunohistochemical stain in the cytoplasm and nuclei of breast epithelial cells was evaluated microscopically by the area of epithelium stained and the intensity of stain (Histoscore = Area X Intensity). We found that expression of eIF4E in the cytoplasm and nuclei of breast epithelial cells was highly correlated with tumor progression (p < 0.0001 and p = 0.0025, respectively). Similarly phospho-eIF4E expression increased in concert with tumor progression (p < 0.0001) in both the cytoplasm and nuclei of breast epithelial cells. Interestingly, while expression of both eIF4E and phospho-eIF4E was not significantly increased in benign breast disease, expression of both eIF4E and phospho-eIF4E was significantly increased in atypical duct hyperplasia (p = 0.01) and in carcinoma in situ (p < 0.0001) as well as in invasive ductal carcinoma (p < 0.0001) relative to normal duct epithelium. Expression of eIF4E and phospho-eIF4E was also increased in lobular carcinoma in situ relative to expression in normal lobules. Breast cancers of all receptor types (ER+/-, PR +/-, Her2+/-, and triple negative) had increased expression of eIF4E and phospho-eIF4E relative to normal duct epithelium. These data are consistent with the conclusion that eIF4E and phospho-eIF4E are critical factors in breast cancer development and progression and that targeting either eIF4E or phosphorylation of eIF4E may be an effective strategy for therapy of all subtypes of breast cancer.

#3920

Polymorphisms of DNA-repair pathways and its association with radiotherapy-induced acute skin toxicity among tri-racial breast cancer populations.

Sung Yong Eum,1 Eunkyung Lee,1 Jean L. Wright,2 Christiane Takita,1 Eden R, Martin,1 Susan Slifer,1 Jennifer J. Hu1. 1 _University of Miami Miller School of Medicine, Miami, FL;_ 2 _The Johns Hopkins University School of Medicine, Baltimore, MD_.

Radiotherapy-induced skin toxicity is one of the critical quality of life issues in breast cancer patients receiving adjuvant radiation therapy (RT). Finding a biomarker which can predict the development of acute RT-induced toxicity prior to RT is critical to improve precision medicine in radiation oncology. This study evaluated the association of single nucleotide polymorphisms (SNPs) in DNA-repair genes in 268 breast cancer undergoing adjuvant RT. We analyzed 3,747 polymorphisms in 119 DNA repair-related genes; 15 genes in base excision repair (BER), 11 in mismatch repair (MMR), 26 in nucleotide excision repair (NER), 15 in homologous recombination, 5 in non-homologous end-joining (NHEJ), 15 in DNA polymerases, and 47 in other DNA repair-related pathways. Skin toxicity was assessed at post-RT using the modified NCI's Common Toxicity Criteria for Adverse Events (CTCAE) and multiple logistic regression analyses were conducted to assess the associations between SNPs and grade ≥ 4 skin toxicity n=115). After controlling for covariates, 221 SNPs were significant at p <0.05 and 34 at p < 0.01. Significant associations (p<0.01) were found in 7 SNPs in PAPD5 gene, 1 in POLD1, POLE, and POLI in DNA polymerase; 5 in RAD51L1 and 1 in RAD54L in HR; 4 in MMS19 in NER; 2 in MSH4 and 1 in PMS1 in MMR; 2 in RPA3 in BER; 2 in RAD18, and 1 in UBE2V2 in Rad6 pathway; 2 in CHEK2 in DNA damage response pathway; 1 in MGMT in direct reversal of DNA damage; and 1 in BLM in diseases associated with sensitivity to DNA damaging agents pathways. Among these, most significant association was found in BLM variant (s16944918) which showed that carrying at least one minor T allele was associated with increased risk of RT-induced skin toxicity (odds ratio [OR]=3.36; 95% confidence interval (CI)=1.62-6.97, p=0.001). For RPA3 (rs11978293), carriers with at least one minor G allele were more likely to develop RT-induced skin toxicity (OR=2.17, 95% CI=1.34-3.52, p=0.002). The current study suggests that genetic variations in multiple DNA-repair genes/pathways may contribute to inter-individual variation in RT-induced normal tissue toxicity in breast cancer patients and suggests the possibility of potential predictive values of genetic polymorphisms in RT sensitivity. Further validation studies with larger sample size are warranted.

#3921

A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein.

Lixin Zhou,1 Kehu Yuan,2 Fangfang Ren,3 Lili Ki,2 Min Zhou,1 Wei Fu,2 Xiaozheng Huang,1 Rachel M. Gonzalez,2 Youmin Shu,2 Yi Shen,2 Guangli Wang,2 Donghui Ma,2 Wei-Wu He,2 Jian Chen4. 1 _Department of Pathology, Beijing Cancer Hospital, Beijing, China;_ 2 _OriGene Technologies Inc, Rockville, WA;_ 3 _Department of Human Anatomy and Histoembryology, Medical College of Soochow University, Suzhou, China;_ 4 _Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Soochow, China_.

Increased sensitivity and specificity of immunohistochemical (IHC) detection of HER2 expression is crucial as the role of Trastuzumab have expanded in the treatment of HER2 positive non-breast cancer patients such as gastric cancer patients. Non-specific nuclear and cytoplasmic staining of the HER2 antibody clone 4B5 has been reported by other labs. In this study, we evaluated the specificity of HER2 clone 4B5 and a new HER2 antibody clone UMAB36 using a suite of methods including protein lysate arrays, western blots, FISH and IHC correlation screens on 129 breast and 158 colon cancer cases for HER2 expression. The protein lysate array and western results revealed that clone 4B5 recognizes three proteins: HER2, HER4, and ZSCAN18, a nuclear transcription protein. In comparison, clone UMAB36 recognized only HER2 protein in the same protein lysate array and western screen as clone 4B5. False negative results, based on correlation of IHC with FISH HER2 positives, were generated using clone 4B5 in 1 breast and 5 gastric of the total 287 cancer cases screened. Comparatively, no false negative results were observed using clone UMAB36 in which there was a 100% correlation between IHC and FISH screen. In this study, areas of normal gastric tissue often stained positive by HER2 clone 4B5, so we performed further analysis of 470 normal gastric cases using Ventana's BenchMark instrument. The results show that 278 normal gastric cases had positive stain with clone 4B5 compared to 3 cases with clone UMAB36. The high background by clone 4B5 may be due HER4 being upregulated in adjacent normal gastric tissue. Our results indicate clone UMAB36 has higher specificity and sensitivity than clone 4B5 in screening gastric tumors.

#3922

Prediction of breast cancer progression using nuclear morphometry.

Neil Carleton,1 Guangjing Zhu,2 Linda Resar,2 Lisa Rooper,2 Young Kyung Bae,3 Robert W. Veltri2. 1 _Carnegie Mellon University, Pittsburgh, PA;_ 2 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 3 _Yeungnam University College of Medicine, Republic of Korea_.

Introduction:

The progression of breast cancer (BrCa) involves nuclear morphometric changes, which are used in the pathological diagnosis of breast cancer. Although changes in nuclear morphometry (NM) contribute to histologic alterations observed in breast cancer, an accurate and autonomous quantification of NM changes have remained elusive. Here we created an image analysis macro to quantify changes in nuclear parameters, including size, shape, and DNA content and used these parameters to predict BrCa progression.

Materials & Methods:

Three tissue microarrays (TMAs) with 140 BrCa cases, stratified by TNM stage were used for this study. The TMAs were generated using 6 mm cores from primary tumors of a Korean cohort from Yengnam University Hospital, Daegu, South Korea. Specimens were obtained from surgical resection between January 1995 and January 2004. Each case was represented by 2 cores on the TMAs. Clinical information were provided by the pathology reports and patients' medical records. H&E slides were first used for pathological diagnosis of BrCa after which slides were scanned with the Aperio scanner and the image of each core was separated using Aperio ImageScope software. The H&E stained nuclei from the tumor core were quantified using ImagePro® Premier 9.1 software (MediaCybernetics). For each core, data of all regions of interest were pooled and the covariance of each parameter was generated. Data were first analyzed alone and then in combination using multivariate logistic regression (MLR) to predict the aggressive cases or recurrence. For all analysis, p<0.05 was considered statistically significant.

Results:

Using multivariate logistic regression (MLR) to differentiate aggressive BrCa (TNM T3 & T4) from indolent BrCa (TNM T1 & T2) on the TMAs, our H&E NM model generated an receiver operating characteristic curve-area under the curve (ROC-AUC) of 0.75 with a sensitivity of 50.9% and specificity of 84.3% at the predictive probability cutoff of 0.50. Parameters of nuclei diameter, size, staining intensity, and aspect ratio contributed to the MLR model. In addition, our H&E NM model also showed power in the prediction of recurrence based on the MLR (ROC-AUC = 0.80) with a sensitivity of 25.9% and specificity of 96.3% at the probability cutoff of 0.53. Parameters of nuclei diameter, size, staining intensity, and cluster contributed to the MLR model. The predictive probability (PP) is significantly higher in the aggressive and recurrent cases based on the two MLR models, respectively.

Conclusions:

We discovered that our automated quantification of tissue nuclear morphometry can be used to accurately predict BrCa aggressiveness and recurrence in women who underwent surgical resection of their primary tumors. Although further confirmatory studies our needed, our data suggests that this tool could provide an accurate predictor of tumor behavior and patient prognosis that could be used to inform therapy for BrCa patients.

#3923

High resolution diagnostic platform for breast cancer therapy management.

Alimatou M. Tchafa, Viswanadham Sridhara, Hamid Mirzaei. _UT Southwestern Medical Center, Dallas, TX_.

Breast cancer diagnosis is dependent on the differential expression of key proteins (biomarkers). The standard method of biomarker measurements are antibody-based techniques such as immunohistochemistry (IHC). These techniques however are subjective and developing new antibody assays is time consuming and not always successful. It also requires extensive resources and effort. With advances in genomics and proteomics technology, the number of validated biomarkers is increasing at a pace faster than IHC assays development, preventing quick translation from bench to clinic. Selected Reaction Monitoring (SRM) is a robust accurate mass spectrometry method that can be used to quickly and economically quantify hundreds of biomarkers within complex protein mixture. In this study, we developed SRM-based assays to simultaneously monitor 30 biomarkers in human samples for accurate classification, staging, and identification of best therapies for subclasses of breast cancer.

We chose well-established biomarkers whose expression correlates with breast cancer aggressiveness and therapy response. For increased accuracy and specificity, 2-3 peptides per protein were used. Two types of controls were also used. The first consists of a known concentration of heavy labeled peptide that is spiked into all samples to allow for absolute measurement and the second consists of housekeeping proteins that are used to normalize the loading amount. 110 laser-capture micro-dissected FFPE breast tissues (0.01 mm³ each) were digested and measured. Currently in the clinic, the presence or absence of receptors such as HER2 dictates the therapeutic plan of the patient. We compared SRM results of HER2 receptor status to clinical results obtained by IHC. Although there was a good correlation between the 2 methods, there were still about 30% HER2 negative patients according to IHC with high concentrations of HER2 in their tissue. Since SRM is a quantitative technique that measures ions, it is much less prone to errors. We use the sequence of peptides derived from the biomarker to detect and quantify it while IHC relies on the specificity of the antibody binding the biomarker and presence of posttranslational modifications or degradation can seriously impede antibody binding. In addition, we are using a multi-biomarker profiling algorithm to identify potential therapy-resistant subclasses. Our approach is better able to (1) remove the errors associated with the semi-quantitative characteristic of IHC and (2) distinguish between good and poor responders to treatments.

The SRM high-resolution diagnostic platform is needed to better guide cancer therapy and prevent potential IHC misdiagnosis that can complicate therapy strategies. Future studies will focus on adapting and optimizing our protocols to measure these biomarkers in easily attainable body fluids such as serum, urine, saliva to be able to provide a non-invasive alternative to biopsy-IHC platform.

#3924

AIRE is expressed and associated with good prognosis in breast cancer.

Francesca Bianchi,1 Loris De Cecco,2 Tiziana Triulzi,2 Sandra Romero-Cordoba,3 Michele Sommariva,1 Chiara Storti,1 Piera Aiello,2 Elda Tagliabue,2 Andrea Balsari,1 Lucia Sfondrini1. 1 _Università degli Studi di Milano, Milan, Italy;_ 2 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 3 _National Institute of Genomics Medicine, Mexico City, Mexico_.

The transcription factor Autoimmune Regulator (AIRE) gene plays a fundamental role in tolerance process by promoting the ectopic expression of thousands of genes encoding for tissue-specific antigens (TSAs) in medullary thymic epithelial cells (mTECs). Beside the high expression of AIRE in mTECs, there are few evidences of AIRE expression in other lymphoid organs and very recently, AIRE was described to be expressed in human and mouse keratinocytes and in tumors originating in stratified and pseudostratified epithelia. Therefore, we evaluated the hypothesis that AIRE could be expressed by epithelial tumors and we focused on breast cancer, for which large public datasets are available.

Our analysis on the TCGA, the largest public breast cancer RNA-seq dataset, revealed that AIRE is expressed in 33% of the cases, and Aire protein expression was confirmed in 26% of 115 human primary breast cancer specimens by immunohistochemistry analysis. AIRE expression was revealed associated to a better relapse-free survival in the TCGA patients and also in two public gene expression microarray-based dataset developed on Agilent and Illumina platform (NKI-295 and KM-plotter). In all the datasets analyzed, AIRE expression resulted an independent strong prognostic factor for relapse-free survival, particularly in ER-positive tumors. Ingenuity Pathway Analysis (IPA) showed that AIRE-expressing tumors were enriched in translation-related pathways, accordingly with AIRE's described function in mTECs. Moreover, in breast cancer luminal cell lines MCF-7 and MDA-MB-361 transfected with AIRE-expressing vector, a significant increase of cell in G1 phase and activation of caspases cascade were observed, supporting the hypothesis that genotoxic stress, induced by AIRE-mediated proteins over-translation, leads to cycle arrest and apoptosis.

These data highlight for the first time AIRE expression in breast cancer and an association with a better prognosis was revealed. Imbalance of cellular homeostasis, caused by AIRE transcriptional function, is a new unusual mechanisms to trigger apoptosis in breast cancer cells.

#3925

Crown-like structures and adipocyte size in fat tissue adjacent to breast tumor reflect parameters of obesity, dyslipidemia and serum high-sensitivity C-reactive protein.

Charlotte Vaysse,1 Inger Thune,1 Øystein Garred,2 Catherine Muller,3 Ellen Schlichting,4 Frøydis Fjeldheim,1 Anne McTiernan,5 Hanne Frydenberg,1 Anders Husøy,1 Steinar Lundgren,6 Morten W Fagerland,7 Erik A Wist,1 Jon Lømo2. 1 _The Cancer Center, Oslo, Norway;_ 2 _Department of pathology, Oslo, Norway;_ 3 _Institut de pharmacologie et de biologie structurale, Toulouse, France;_ 4 _Department of breast surgery, Oslo, Norway;_ 5 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 6 _Department of oncology, Trondheim, Norway;_ 7 _Center for biostatistics and epidemiology, Oslo, Norway_.

Background: Adult weight gain and obesity have consistently been associated with breast cancer development but the biological mechanisms operating remain unclear. Adipose tissue may develop low-grade inflammation, observed as apoptotic adipocytes surrounded by macrophages forming characteristic crown-like structures (CLS). We questioned whether CLS and adipocyte size in breast fat tissue are biomarkers of patient's body fat distribution, dyslipidemia and serum high-sensitivity C-reactive protein (hs-CRP), factors associated with breast cancer development.

Material and Methods: Among 55 women, aged 35-75 years, with newly diagnosed invasive breast cancer (stage I/II), measurements of body composition: waist to hips ratio (WHR), body mass index (BMI, kg/m2) and total fat percentage (DEXA, %) were assessed. Concentrations of lipids (cholesterol, triglycerides) and hs-CRP were determined in fasting serum blood samples. Surgical specimens of breast tumours with surrounding fat tissue were examined in Haematoxylin Eosin and CD68 stained slides to assess the size of adipocytes (μm) and CLS density (CLS/cm2). We used linear regression models to study the association between mammary adipose tissue parameters, body composition, serum lipids and inflammatory markers.

Results: The breast cancer patients had the following means: age at diagnosis, 55.2 years, BMI, 25.2 kg/m2, WHR, 0.88, truncal fat, 38.1%, total cholesterol, 5.76 mmol/l, triglycerides, 1.19 mmol/l and hs-CRP 1.75 mg/L. The mean tumour size was 16.3 mm, 93 % of tumors were estrogen receptor positive and 82 % were progesterone receptor positive. Mean adipocyte size was 68.0 μm and mean CLS density was 0.12 CLS/cm2. Adipocyte size and CLS density were positively associated with BMI (padipocytes=0.004, pCLS-density=0.008), WHR (padipocytes=0.003, pCLS-density=0.009) and truncal fat (padipocytes<0.001, pCLS-density=0.005). Overweight/obese patients (BMI ≥25 kg/m2) compared with normal weighted patients, had higher of cholesterol (p=0.016), triglyceride (p<0.001), hs-CRP (p=0.001) and had higher levels of CLS density (p=0.002) and larger adipocytes (p=0.003).

Conclusion: Breast adipose tissue markers such as adipocyte size and CLS, reflecting local low-grade inflammation, were positively associated with excess weight, truncal fat, dyslipidemia and a high level of hs-CRP. In addition to the systemic effect, it is conceivable that fat tissue inflammation in the vicinity of the breast cancer can influence events in a paracrine manner. These findings point to important biomarkers in breast tissue that may co-exist with serum biomarkers associated with breast cancer development.

#3926

Nanomechanical profiling of breast cancer: A novel tool for cancer diagnostics and prognostics.

Marija Plodinec,1 Christian Raez,2 Philipp Oertle,3 Adrian Hodel,4 Maja Fünfschilling,5 Roderick YH Lim,3 Ellen C. Obermann2. 1 _Institute of Pathology, University Hospital Basel & Biozentrum, University of Basel, Basel, Switzerland; _2 _Institute of Pathology University Hospital Basel, Basel, Switzerland;_ 3 _Biozentrum & Swiss Nanoscience Institute University of Basel, Basel, Switzerland; _4 _The London Centre for Nanotechnology, UCL and Imperial College London, London, United Kingdom;_ 5 _Institute of Pathology, Cantonal Hospital Baselland, Liestal, Switzerland_.

A crucial point in making treatment decisions for cancer patients is the assessment of tumor aggressiveness. Currently, for breast cancer, established prognostic markers exist that are routinely assessed by standard pathological examination. However, these parameters are often not sufficient to stratify patients, especially those with early stages of breast cancer and adjuvant therapy is frequently administered to patients who might have been cured by surgery and anti-hormonal treatment alone. The goal to avoid over- and under-treatment has led to an intensive search for prognostic and predictive markers in early breast cancer. Physical interactions between cancer cells and the extracellular matrix (ECM) that occur at the molecular (nanometer) scale are crucial for the metastatic process. Consequently, nanomechanical alterations of cells and ECM due to cancer progression can serves as potentially suitable markers of cancer aggressiveness that may help to optimize treatment strategies.

This motivated us to develop an atomic force microscope (AFM)-based method for measuring nanomechanical (stiffness) profiles of unadulterated tissues in native physiological buffer conditions with an unprecedented stiffness sensitivity resolved at nanometer-scale spatial resolution. An AFM utilizes a ∼10 nm-sharp stylus or tip that makes ∼10'000 miniscule indentations across tissue. In our initial study using transgenic mouse model of human breast cancer we could show that the "softest" nanomechanical phenotype (∼0.4 - 0.8 kPa) present at the primary tumor site closely corresponded to the stiffness of the metastatic lesions obtained from the lungs of the same mouse (Plodinec et. al; Nature Nanotech. 2012).

In this study, we have analyzed human 187 breast cancer samples from breast biopsies and tumor resections including primary breast cancers of various stage and grade, lymph node metastases, and non-neoplastic breast parenchyma. Post-AFM samples were fixed, paraffin embedded in an oriented manner and used for routine histology. Data showed for all human breast cancer samples, distinct stiffness phenotypes in comparison to the surrounding non-neoplastic and morphologically normal breast tissue and revealed specific nanomechanical profiles of phenotypes that lead to metastases. Interestingly, patients presenting nanomechanical changes in the adjacent tissue, which was histologically characterized as tumor free, typically exhibited local and/or distant metastases.

Overall, our findings demonstrate the first application of nanomechanical profiling in a clinical setting that allows for fast, on-site assessment of specimen and does not suffer from inter-observer variability such as other markers (Obermann et al, Pathologe, 2012). The relative size and distribution of nanomechanical profiles can provide an indicator of cancer aggressiveness, and therefore orientate therapy choice, and support patient follow-up.

#3927

The presence of COX-2 and tumor-associated macrophages as a prognostic marker for invasive breast carcinoma patients.

Karla Esbona, Sandeep Saha, Yanyao Yi, Menggang Yu, Kari Wisinski, Lee G. Wilke, Patricia J. Keely. _University of Wisconsin-Madison, Madison, WI_.

Inflammation is an important mediator of tumor progression. The objective of this study was to assess whether the tissue localization of COX-2 and tumor-associated macrophages were associated with clinicopathological features of invasive carcinoma, including collagen deposition and patient survival outcome. A tumor microarray (TMA) of 371 biopsy specimens from patients with invasive breast carcinoma was analyzed for expression of high levels of COX-2, and the macrophage markers CD68 and CD163 in either the tumor nest (TN) or the tumor-associated stroma (TS). The study population for this TMA was female patients, 18 to 80 years of age with a median follow up of 8.4 years. Survival tables were calculated according to the Kaplan-Meier method. We found that elevated collagen deposition was associated with high stromal expression of COX-2 (P < 0.0001); however, the amount of collagen deposition was not a predictor for survival outcome. In order to better analyze patient data, samples were divided into quartiles based on their levels of COX-2 expression and/or macrophage infiltration. Tumor nests with high COX-2 expression had worse patient outcome (P = 0.011). One possible mechanism for this is COX-2 dependent recruitment of macrophages to the tumor microenvironment, supported by our finding of high infiltration of CD68+ macrophages in the TS, and CD163+ macrophages in both TN and TS. This recruitment of macrophages was associated with worse overall survival (OS). Furthermore, patient survival was worsened even more if macrophages expressed COX-2, suggesting positive amplification of COX-2 signaling via macrophage recruitment. This notion is further established by the finding of high presence of CD163+ macrophages in the TN as an independent prognostic factor as revealed by multivariate analysis (P < 0.0001, HR = 4.67). These results suggest that in invasive carcinoma the localization of inflammatory markers within the tumor are biomarkers for patient survival outcome. Therefore, we propose that these patients may benefit from therapy with a selective COX-2 inhibitor such as celecoxib.

#3928

Caveolin-1: Beyond a marker for basal-like breast cancers.

Rebecca A. Feldman,1 Zoran Gatalica,1 Semir Vranic,2 Ryan Bender,1 Sandeep Reddy,1 Anatole Ghazalpour1. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _University Clinical Center Sarajevo, Sarajevo, Bosnia and Herzegovina_.

Introduction: Caveolin-1 (Cav1) is associated with basal-like triple-negative (ER-/PR-/Her2-) breast cancers (TNBC). Its biological contribution to this subtype has not been fully explored and controversy persists regarding the molecular role of Cav1 in carcinogenesis. Experimental Procedures: Thirty-four TNBC (17 Cav1+/17 Cav1-) patients molecularly-profiled with a commercial assay (Caris Life Sciences, AZ) were evaluated retrospectively. Cav1 status was determined by immunohistochemistry (caveolin-1 polyclonal; ≥2+ ≥50%). The majority of specimens (28/34) used for profiling were from primary breast sites and contained ≥50% neoplastic cells. The transcriptomes were profiled using Illumina's HumanHT-12 microarray (v4). Data were normalized using mean normalization procedure. Differential expression analysis was performed using R's Limma package. Pathway analysis was carried out using R's signaling pathway impact analysis (SPIA) package with 69 cancer, immunity, and cell signaling related KEGG pathways. Results: Using a cutoff of two-fold and adjusted p-value of 0.05, we identified 954 genes differentially expressed between Cav1+/- TNBC patients. Included in these were 31 genes which were found to be up-regulated by over five- fold and 3 genes down-regulated by over five fold in Cav1+ TNBC. Genes of notable interest for their role in cell signaling, cell adhesion, tumor invasion and metastasis, included an up-regulation of TGFBR2, SPARC, integrins (ITGA11, ITGB5, ITGBL1), cell adhesion proteins (LAMB3, COL5A3) and molecules which facilitate tumor invasion (LAMB3, MMP1, MMP2, MMP9). In addition, genes found to be down-regulated in Cav1+ patients and notable for their roles in promoting epithelial-mesenchymal-transition (EMT) included Claudin 3(CLD3) and CA125/MUC16 (Mucin 16). We also detected an approximately two-fold down-regulation of CDKN2A in Cav1+ patients. Using SPIA pathway analysis, 12 pathways were found to be differentially activated in Cav1+ vs. Cav1- TNBC. The most differentially activated pathways were the focal adhesion pathway (p=4.51E-18), PI3k-Akt signaling pathway (p=2.01E-6) and TGF-β and MAPK signaling pathways (p=0.005, 0.014, respectively). Conclusions: Differential gene expression patterns and pathway analyses provide evidence for distinct profiles for gene expression between Cav1+/- TNBC. Cav1+ TNBC patients exhibit up-regulation of genes important for cell signaling, extracellular matrix remodeling and tumor invasion, and down-regulation of genes that may facilitate EMT and loss of cell cycle control. The focal adhesion pathway, as well as TGF-β, PI3K and MAPK signaling pathways, were identified as differentially activated among Cav1+/- TNBC. Taken together, these data support the role of Cav1+ in identifying a subtype of TNBC that may have a greater risk for invasion and metastasis. The correlation of this subtype with prognosis and drug response should be investigated in future studies.

#3930

A prospective pilot study of kinetics of high sensitivity troponin T (hs-TnT) and amino terminal (nt-proBNP) in breast cancer patients (pts) treated with doxorubicin (A) or trastuzumab (T).

Pooja Advani, Jonathan Hoyne, Alvaro Moreno-Aspitia, Marcia Dubin, Shelly Brock, Caroline Harlow, Tammy Wollett, Saranya Chumsri, Joseph Blackshear. _Mayo Clinic, Jacksonville, FL_.

Introduction: A and T are associated with the development of cardiac dysfunction. The kinetics of cardiac biomarkers early after A or T treatment (Rx) is undefined.

Methods: We studied hs-TnT (detection limit: 3 ng/ml, myocardial infarction threshold 14 ng/ml) and nt-proBNP (detection limit: 5 pg/ml) levels just prior to (baseline), and on days +1, +2, +3 and +7 during the first and second cycle in breast cancer pts treated with A (n=11) or T (n=11). Baseline and peak values for each cycle, baseline to baseline, peak to peak, time to peak, and baseline corrected area under the curve (AUC) were calculated. Study No. NCT01771549

Results: Group (grp) A pts were younger, 51 ± 18 years, than T grp pts, 60 ± 13 years. Three pts in A grp and 4 pts in T grp used antihypertensives. Baseline ejection fraction (EF) was 52-68% (A grp) and 56-69% (T grp). Hs-TnT was undetectable in 5/11 A grp, and 2/11 T grp pts at baseline. By cycle 2 day +1, all A pts had a detectable level of hs-TnT (p=0.03) compared to 7/11 T pts. Median time to peak level for hs-TnT was 1.5 days for the A grp and 1.2 days for the T grp (NS). In the A grp, significant transient increase in nt-proBNP were also seen (Table). In the T grp, a significant rise in nt-proBNP was not seen in cycle 1 but was seen on cycle 2 day +1 and cycle 2 peak. AUC values were positive for hs-TnT in A pts but not in T pts, and were positive for nt-proBTNP for both A and T. Only 1 pt in A grp had symptomatic EF decline (47%) three months from start of A. None of T grp pts had EF decline.

Conclusion: A, but not T was associated with increase in pre-Rx (cycle 2 only), peak, and AUC hs-TnT levels for cycles 1 and 2 with peak values occurring 1.5 days post-Rx. This study evaluating serial kinetics of cardiac biomarker in pts receiving A or T therapy, suggests that assessment of pre- and day +2 post-Rx values could be a means of quantifying cumulative myocardial injury over the chemotherapy course.

*Roche Diagnostics Corp, Indianapolis, IN provided assay reagent kits.

hs-TnT, ng/ml | A, cycle 1 | A, cycle 2 | P-val | T, cycle 1 | T, cycle 2 | P-val

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

Baseline | 4.6 | 9.3 | <0.002 | 7.7 | 5.0 | NS

Peak | 9.6** | 16.1† | <0.002 | 8.0 | 6.8 | NS

Time to peak, days | 1.6 | 1.5 | NS | 1.1 | 1.3 | NS

AUC above baseline | 7.1±13.4 | 27±19 | <0.001 | -1.4±7.2 | -5±17 | NS

|  | |  | |

|

nt-proBNP, pg/ml | A, cycle 1 | A, cycle 2 | P-val | T, cycle 1 | T, cycle 2 | P-val

Baseline | 94 | 90 | NS | 124 | 80 | NS

Peak | 361* | 359* | NS | 209 | 206† | NS

Time to peak, days | 1.8 | 1.9 | NS | 2.2 | 1.8 | NS

AUC above baseline | 423 | 499 | NS | 78** | -32** | <0.5

*p<0.001, | † p ≤0.01 | ††p=0.002 | NS=statistically non-significant (p>0.05) | **p<0.03 | |

#3931

Tumor COX-2 expression is associated with less aggressive tumors and better five-year prognosis in breast cancer patients.

Maria Simonsson, Sofie Björner, Andrea Markkula, Björn Nodin, Karin Jirström, Carsten Rose, Signe Borgquist, Christian Ingvar, Helena Jernström. _Lund University, Lund, Sweden_.

Introduction: Cyclooxygenase-2 (COX-2) is a mediator of inflammation and regulates aromatase expression, suggesting a potentially important role in breast cancer. The prognostic role of COX-2 expression in breast cancer is still debated. The aim of this study was to analyze tumor COX-2 expression in relation to age and tumor characteristics, and to elucidate whether COX-2 expression was associated with breast cancer prognosis.

Materials and methods: COX-2 expression was evaluated in invasive tumors from 984 primary breast cancer patients from a population-based prospective cohort from Lund, Sweden. The patients were included as of 2002 and were followed for up to 11 years. The median follow-up time for patients still at risk was five years. Disease-free survival was assessed by Kaplan-Meier and LogRank test. Adjusted Hazard Ratios (HR) were obtained by Cox regression and adjusted for invasive tumor size, axillary lymph node involvement, histological grade, estrogen receptor (ER) status, and age at inclusion.

Results: Immunohistochemical COX-2 expression was available for 911 patients and the intensity was classified as negative (n=82; 9.0%) /weak or moderate (n=723; 79.4%) /strong (n=106; 11.6%). Patients 50 years or older at inclusion had higher COX-2 expression compared to younger patients (Ptrend=0.05). Overall, higher COX-2 tumor expression was also observed among patients with ER+ tumors (Ptrend<0.0001) and among patients with tumors of lower histological grade (Ptrend<0.0001) compared to patients with ER- tumors or higher histological grade. COX-2 tumor expression was not associated with tumor size or axillary lymph node status. Higher COX-2 expression was associated with lower risk for any breast cancer event in the univariable (LogRank Ptrend=0.02), but not in the multivariable model, adjusted HR 0.73 (95% CI: 0.49-1.09) per category. However, higher COX-2 expression was associated with lower risk for events during the first five years of follow-up (LogRank Ptrend=0.0003), adjusted HR 0.60 (95% CI: 0.37-0.97) per category.

Conclusion: Higher COX-2 expression was associated with less aggressive tumor characteristics and lower risk for any breast cancer event during the first five years of follow-up but not thereafter, in this population-based cohort of breast cancer patients. This study indicates that COX-2 expression in breast cancer may carry short-term prognostic information independently of established clinical and tumor characteristics.

#3932

Values of single nucleotide polymorphisms identified from genome-wide association studies on risk prediction, risk reclassification, and population-based screening of breast-cancer.

Yubei Huang, Fengju Song, Kexin Chen. _Tianjin Medical University Cancer Institute and Hospital, Tianjin, China_.

Background: With an increasing number of single nucleotide polymorphisms (SNPs) being identified from genome-wide association studies (GWAS), it's important to examine whether these SNPs have values in public health. And it's also unclear how to select the targeted SNPs valuable for population-based screening from numerous GWAS-identified SNPs.

Methods: Based on the fraction of the genetic risk explained by a SNP, the area under the receiver operating characteristic curve (AUC), integrated discrimination improvement, and net reclassification improvement were used to select targeted SNPs and to evaluate the values of genetic risk score (GRS) with targeted SNPs on risk prediction, risk reclassification, and effects of population-based screening among 2 million simulated Chinese women aged 35-69 years.

Results: A total of 11 targeted SNPs from 23 GWAS-identified SNPs would be valuable for population-based screening of breast cancer among Chinese women. After incorporating GRS with targeted SNPs into traditional risk predication based on established risk factors (ERF) of breast cancer, the AUC significantly improved from 65.5% to 67.5% (P<0.001). Additional 25.5% (P<0.001) population would be correctly reclassified into cases or controls. If screening targeted at top 25% high-risk women, screening-detected cancer would significantly increase 4.2% (49.2% vs 45.0%, P<0.001) for screening strategy included GRS and ERF in risk assessment before screening compared with that only included ERF in risk assessment.

Conclusion: GRS based on targeted GWAS-identified SNPs could significantly improve the risk prediction, risk reclassification, and the effects of population-based screening. Further real-world studies are needed to validate this method and these results.

#3933

CETSA as a diagnostic tool to guide personalized breast cancer therapy.

Anette Öberg, Henriette Laursen, Sylvia Packham, Daniel Martinez Molina, Takahiro Seki, Yihai Cao, Johan Hartman, Jonas Bergh, Pär Nordlund, Sara Lööf. _Karolinska Institute, Stockholm, Sweden_.

The increased knowledge of fundamental cancer biology has translated into the development of a large number of targeted drugs. However, the potential for breakthroughs in cancer therapy is hampered by the lack of knowledge regarding which patients would benefit from a specific drug. Therefore, there is a need of diagnostic tools that can predict if the individual patient will respond or develop resistance to a specific treatment plan.

In the present project we aim to meet this challenge by developing stringent clinical diagnostic assays for assessing target engagement (TE; i.e. drug binding to its target protein in situ) of current and emerging drugs in breast cancer treatment. The key to develop these clinical TE assays is our novel technology Cellular Thermal Shift Assay (CETSA). This method allow for the first time direct monitoring of biophysical drug TE in cells, animals and patient samples. The main aim of the project is to correlate the results obtained by CETSA on breast cancer biopsies to the treatment outcome for the biopsied patient, with the goal of creating a diagnostic tool for guiding personalized breast cancer treatment. We also aim to identify new biomarkers of efficacy and resistance for specific drugs, by implementing proteome wide CETSA based on a quantitative mass spectrometry read out.

Our recent data demonstrates that CETSA can assess TE in intact cells of drugs commonly used in breast cancer treatment, such as microtubule inhibitors, anthracyclins and CDK4/6 inhibitors. Further we show that CETSA can be applied in vivo on human breast cancer Xenografts and breast cancer patient samples. These data further strengthens the potential of CETSA to become a valuable clinical tool and complement other diagnostic methods to improve efficacy and minimize adverse effects of cancer drugs.

#3934

Analysis of PKC-ζ protein and mRNA levels in normal and malignant breast tissue.

Christopher Apostolatos, Tracess Smalley, Mildred A. Duncan. _University of South Florida, Tampa, FL_.

It is estimated that in 2015 breast cancer will be the second leading cause of cancer death in women. For this purpose, biochemical markers for breast cancer have been investigated, to assist in early detection and more accurate diagnoses. In breast cancer tissue, the atypical isozyme of protein kinase C zeta, PKC-ζ, has been a topic in research; It has been shown that an overexpression of this protein may be indicative of developing carcinogenesis and contributes to proliferation through the NF kappa B pathway, which is a stress-regulated switch for cell survival. In this investigation, the expression of PKC-ζ was analyzed in normal and malignant female human breast tissue samples by Western blot, immunoprecipitation and PCR. In the preliminary results, the malignant breast tissue samples illustrated a significant expression of PKC ζ when compared to the expression of PKC-ζ in normal breast tissue samples. The same tissues were also processed for total RNA isolation which was followed by cDNA synthesis and Real Time PCR. The level of PKC-ζ mRNA was tested and no overexpression was observed in either malignant or normal breast tissue samples. While protein studies may suggest that PKC-ζ could be considered a biomarker for breast cancer, the same cannot be said about mRNA levels. The overexpression of PKC-ζ protein level and the normal PKC-ζ mRNA level are indicators of possible miRNA activity that may regulate translation in malignant tissues but not the normal.

#3935

Metformin and response to neoadjuvant chemotherapy in patients with breast cancer.

Abdel-Rahman N. Zekri, Abeer A. Bahnassy, Hussam Hussam, Hussin Khaled, A Ahmad, Zeenel Din, Mosaad M. El-Gamal. _National Cancer Inst. Cairo Univ., Cairo, Egypt_.

Introduction: Breast cancer (BC) is the most common cancer in females and the second leading cause of cancer-related death among women, mostly due to distant metastatic relapses after potentially curative multimodality therapy. Metformin, a first line therapy for type 2 diabetes, decreases the incidence of cancer and cancer-related mortality in diabetic patients through activating AMPK by an LKB1-dependent mechanism. We assessed the effect of metformin and the possible genetic pathways involved in metformin-induced therapeutic effect in non diabetic breast cancer patients receiving neoadjuvant chemotherapy

Methods: we assessed the RNA expression levels of 90 genes in the p53, and the PI3K/AKT/m-TOR pathways using the ABSuperarray technology in tumor tissues obtained from 76 invasive breast cancer patients from Egypt who were categorized into responders and non-responders. The results of the array were then confirmed by immunohistochemistry to verify the protein expression of the differentially expressed genes. The array results were then correlation with response to treatment and survival rates.

Results and conclusions: Genetic profiling of the p53/bcl2 and the PI3K/AKT/m-TOR pathways array showed that 41 genes were differentially expressed between the responders and the non-responders in BC patients treated with chemotherapy and metformin. The differentially over-expressed genes are: IGF1R, p73, NFKB1, AKT, PDK1, mTOR, PI3K, ERK, AMPK, LKB1, ATF3, STAT1, CYCLIN D-1, VEGF, CDK1, CDK4, TSC1/2, OCT1, PKA, APAF-1, p53, RPRM, PIDD, FADD, CHEK1/2. The differentially under-expressed genes are: APAF1, TRAF2, p21, FADD, p16, mdm2, TNF, BCL2, BAX, CDKN1A, TRAF2, TNF, PTEN, P53AIP1. A panel of 20 genes: AMPK, LKB1, TSC1/2, OCT1, PKA, APAF-1,p53, PDK1, RPRM, PIDD, FADD, CHEK1/2 (overexpressd) and CYCLIN D-1, VEGF, CDK-4, IGF, NFKB, AKT, mTOR, APAF1 (reduced expression) effectively predicted response to treatment in the two studied groups.

#3936

Identification and validation of the potential biomarker insulin-like growth factor binding protein acid-labile subunit for breast cancer in African American women.

Padma P. Tadi Uppala,1 Carlos Garberoglio,2 Sharon Lum,2 Willie Davis,2 Hon-Chiu Eastwood Leung,3 Michael Liebman,4 Keiji Oda,1 Utkarsh P. Patel1. 1 _Loma Linda University School of Public Health, Loma Linda, CA;_ 2 _Loma Linda University, School of Medicine, Loma Linda, CA;_ 3 _Baylor College of Medicine, Dallas, TX;_ 4 _Windber Research Institute, Windber, PA_.

Breast cancer is the most frequently diagnosed cancer in women, with an estimated 40, 730 breast cancer deaths in the US in 2015. African American (AA) women have a lower breast cancer incidence rate, but a higher breast cancer death rate, than non-Hispanic White women. Research indicates that breast tumor biology in AA women is different from that in Caucasian women. AA women are more likely to be diagnosed with breast cancer at an earlier age and with more aggressive form of the disease, characterized by higher grade and negative estrogen and progesterone receptor status. Because of the aggressive nature of these tumors and current lack of targeted therapies, identification of novel relevant protein markers is of great importance. The purpose of this study was to validate serum proteins that were previously identified by serum proteomic profiling in 22 serum samples by 2D-DIGE/MS analysis and a subset of samples by shotgun LC/MS technology. Methods and new data: The current study included serum samples from 15 African American breast cancer patients and 12 healthy controls. Patients were grouped into triple negative (TN), HER2 and Luminal A and B subtypes. Proteins of biological significance were validated using western blot analysis. For ceruloplasmin, and insulin-like growth factor binding protein acid-labile subunit (IGFBP-ALS), one-way ANOVA was used to compare mean density among the three groups. For Vitamin D Binding protein (VDB), a two-sample t-test was used to compare the density between the groups. Due to the small sample size, we have also conducted nonparametric tests. IGFBP-ALS was significantly lower in triple negative breast cancer patients (p=0.016) and in HER2 (p=0.025) subtypes. There was no significant difference in VDB protein in the luminal A and B subtypes (p=0.98). Future efforts will focus on validating the identified panel of biomarkers to gain insight into their role(s) in the etiology of aggressive breast tumors. Funded by Susan G Komen for the Cure and SEED grant SPH.

#3937

Nestin expression is associated with a basal-like phenotype and poor prognosis in breast cancer.

Kristi Krüger,1 Elisabeth Wik,1 Tor Audun Klingen,2 Ying Chen,3 Turid Aas,4 Lars A. Akslen1. 1 _Centre for Cancer Biomarkers, CCBIO, Department of Clinical Medicine, University of Bergen, Bergen, Norway;_ 2 _Department of Pathology, Vestfold Hospital, Tønsberg, Norway;_ 3 _Department of Pathology, Akershus University Hospital, Lørenskog, Norway;_ 4 _Department of Surgery, Haukeland University Hospital, Bergen, Norway_.

Background:

Nestin, neuroepitheilal stem cell protein, is an intermediate filament protein first described in neural stem or progenitor cells, and its expression has been seen in several tissues, including different types of cancer.

In breast cancer, Nestin expression has been linked to the triple negative and basal-like phenotypes. We here evaluated Nestin and its association with clinico-pathologic markers and survival in two prospective Norwegian breast cancer cohorts.

Materials and methods:

Series I includes 546 women (50-69 years) diagnosed with primary invasive breast cancer (403 screen-detected and 143 interval cancers) as part of the prospective and population-based Norwegian Breast Cancer Screening Program (NBCSP) during 1996-2003 (Hordaland county).

Series II includes 282 women (50-69 years) with invasive cancers (199 screen-detected and 83 interval cancers) diagnosed as part of the NBCSP during 2004-2009 (Vestfold County).

Immunohistochemical staining of Nestin was done on tissue microarrays (TMAs). Expression in tumor cells was graded by a Staining Index (SI) (values 0-9).

Results:

Nestin positivity, observed in 9% and 13% of the breast cancer cases in Series I and II, was associated with higher histological grade (OR, odds ratio 14.3 and 8.1), high tumor cell proliferation by Ki-67 (OR 11.4 and 9.1), larger tumor diameter (OR 1.8 and 2.4), but not with lymph node status or HER2 status. Further, Nestin positivity was associated with negativity for estrogen (OR 14.9 and 11.9) and progesterone receptor (8.5 and 3.4), the triple negative phenotype (OR 29.1 and 17.4) and basal-like differentiation by positivity for Cytokeratin 5 (OR 9.1 and 8.7) or P-cadherin (OR 7.0 and 8.5).

By univariate survival analysis, Nestin positivity was associated with reduced breast cancer specific (p=0.002 and p=0.076) and overall survival (p=0.005 and p=0.039). When including the standard variables tumor size, histologic grade and lymph node status in a multivariate analysis, Nestin was still significantly associated with reduced breast cancer specific survival in the largest cohort (Series I).

Conclusion:

We found that Nestin expression in breast cancer is associated with aggressive features, the triple negative phenotype, basal-like differentiation, and reduced breast cancer specific survival.

#3938

Predicting first line tamoxifen response of recurrent ER+ breast cancer patients based on transcriptional activity of signaling pathways.

Anieta M. Sieuwerts,1 Marcel Smid,1 Márcia A. Inda,2 John A. Foekens,1 Anja van de Stolpe,2 John W.M. Martens,1 Wim F.J. Verhaegh2. 1 _Erasmus MC, Rotterdam, Netherlands;_ 2 _Philips Research, Eindhoven, Netherlands_.

Introduction

Generally, estrogen receptor positive (ER+) breast cancer patients are eligible for hormonal treatment, but roughly half of them respond, and identifying those that do remains a challenge. Predicting hormonal therapy response with higher specificity (without losing sensitivity) is of clinical value, to avoid overtreatment and enable consideration of alternative, more effective (targeted) drugs. We developed Bayesian computational model-based mRNA tests [1] to analyze oncogenic signal transduction pathway activity in cancer tissue samples, for selection of personalized therapy. Here, we show that such a test for the ER pathway can predict hormonal therapy response independent of traditional predictive factors.

Methods

Gene expression levels (Affymetrix HG-U133+PM microarrays) were used to determine transcriptional activity of the ER, AR, PI3K, Wnt, Hedgehog (HH), TGFβ and NFκB pathways, as described earlier [1], in 132 ER+ primary breast tumor samples of patients who did not receive adjuvant hormonal therapy and subsequently relapsed and were treated with first line tamoxifen treatment. Univariate and multivariate Cox proportional hazards regression was used to associate the pathways' activities with progression free survival (PFS) on tamoxifen treatment, together with traditional response prediction factors.

Results

In 102 (77%) of the 132 samples, at least one of the seven pathways was found active. ER pathway activity was observed in 36 samples (27%). Strikingly, 76 samples (58%) had an active NFκB pathway. In 37 samples (28%), two or more pathways were active, predominantly ER with NFκB.

The probability of ER pathway activity was associated with a longer PFS, with a hazard ratio of 0.41 in a univariate analysis (p=0.0039), while activity of the TGFβ pathway was associated with a shorter PFS (HR=2.2, p=0.044). In a multivariate analysis using the seven pathways, ER pathway activity remained significant (HR=0.44, p=0.011), while TGFβ did not (HR=1.67, p=0.24). A Kaplan-Meier analysis as a function of ER activity gave a log-rank p-value of 0.002.

A multivariate analysis for PFS including ER pathway activity and the traditional predictive factors disease free interval (<2yr vs. >2yr), site of relapse, age, menopausal status, ESR1 and PGR mRNA expression and Her2 status, showed that ER activity was independently associated with a favorable PFS (HR=0.43, p=0.047).

Conclusion

In ER+ breast cancer patients who did not receive adjuvant hormonal therapy, transcriptional ER pathway activity as measured by our computational ER pathway mRNA test was associated with a favorable outcome upon first line tamoxifen treatment. The ER pathway test can hence be used as an independent predictor of hormonal therapy response in ER+ breast cancer patients with recurrent disease.

[1] Verhaegh W et al. Cancer Res. 2014;74:2936-45.

#3939

Clinical significance of expression of androgen-receptor splice variant-7 (AR-V7) in neoadjuvant chemotherapy for breast cancer.

Yuka Asano, Shinichiro Kashiwagi, Wataru Goto, Kento Kurata, Tamami Morisaki, Satoru Noda, Tsutomu Takashima, Naoyoshi Onoda, Sayaka Tanaka, Masahiko Ohsawa, Masaichi Ohira, Kosei Hirakawa. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

Background: Triple-negative (TN) breast cancer (BC) patients testing positive for androgen receptor (AR) expression are thought to be chemotherapy resistant, similar to other hormone receptor-positive (HR) BCs. On the other hand, prostate cancer is a hormone-dependent malignant tumor like breast cancer, it has been reported that chemotherapy is more likely to be effective if there is an abnormality in androgen-receptor splice variant-7 (AR-V7). In an immunohistochemical study, as well, AR-V7 expression was reportedly a poor prognostic factor in castration-resistant prostate cancer (CRPC). In this study, we investigated the association between chemotherapy sensitivity and AR-V7 expression in patients treated with neoadjuvant chemotherapy (NAC) using standardized chemotherapy criteria and regimens.

Materials and Methods: A total of 177 patients with resectable early-stage breast cancer were treated with NAC. ER, PgR, HER2, Ki67 and AR-V7 status were assessed immunohistochemically.

Results: In the 43 AR-V7 expression positive group, compared to the 133 negative group, TNBC (p <0.001) and non-HRBC (p=0.001) were significantly more frequent, and the pCR rate were significantly higher (p<0.001). Among the 61 TNBC and the 80 HRBC patients, the pathological complete response (pCR) rate was significantly higher in AR-V7 expression positive group than in the negative group (p=0.008) (p = 0.018). Analysis of all 177 patients receiving NAC revealed that no significant difference in disease-free survival (DFS) was associated with AR-V7 expression (p=0.080, log-rank). However, a significantly extended non-recurrence period was observed in patients with AR-V7-expressing tumors compared to AR-V7 negative tumors, when the analysis was limited to patients with TNBC (p=0.016, log-rank). In univariate analysis, AR-V7 expression made a significant contribution to extending disease-free survival in patients with TNBC (p=0.044, hazard ratio=0.121). However, multivariate analysis indicated that AR-V7 expression was not an independent factor (p=0.083, hazard ratio=0.158).

Conclusion: These findings show that AR-V7 expression is a therapeutic effect predictive marker in BCNAC, and indicates particularly high chemotherapy sensitivity in TNBC.

#3940

Baseline molecular markers and risk of distant relapse in the NeoSphere study.

Giampaolo Bianchini,1 Tadeusz Pienkowski,2 Young-Hyuck Im,3 Giulia Valeria Bianchi,4 Ling-Ming Tseng,5 Mei-Ching Liu,6 Ana Lluch,7 Vladimir Semiglazov,8 Juan de la Haba-Rodríguez,9 Do-Youn Oh,10 Brigitte Poirier,11 Jose Luiz Pedrini,12 Pinuccia Valagussa,13 Luca Gianni1. 1 _IRCCS Ospedale San Raffaele, Milan, Italy;_ 2 _Centrum Onkologii, Warsaw, Poland;_ 3 _Department of Medicine, Samsung Medical Center, Seoul, Republic of Korea;_ 4 _Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy;_ 5 _Taipei-Veterans General Hospital, National Yang-Ming University, Taipei, Taiwan;_ 6 _Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan;_ 7 _Hospital Clínico Universitario, INCLIVA Biomedical Research Institute, Valencia, Spain;_ 8 _NN Petrov Research Institute of Oncology, St Petersburg, Russian Federation;_ 9 _Hospital Reina Sofia, Córdoba, Spain;_ 10 _Seoul National University Hospital Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 11 _Hôpital du Saint-Sacrement, CHU de Québec, Québec, Quebec, Canada;_ 12 _Hospital Ernesto Dornelles, Porto Alegre, Brazil;_ 13 _Fondazione Michelangelo, Milan, Italy_.

Background We investigated the association between gene-expression (GEP) based markers and distant event free survival (DEFS) in HER2+ breast cancer patients (pts) in the NeoSphere study.

Methods In NeoSphere HER2+ pts were randomized to neoadjuvant HD, PHD, PH or PD (H=trastuzumab, P=pertuzumab, D=docetaxel). Affymetrix-based GEPs were generated in 367/417 pts (88%). We evaluated the association with DEFS of 10 biomarkers in all patients with arms pooled and by each arm, and separately by ER status: six immune-related metagenes (CD8, IGG and MHC2, related to T cells, plasma cells and antigen presenting cells, respectively; MHC1, STAT1 and IF.I related to HLA cass I genes, and to genes modulated by interferons), ESR1 and an ER-related score (ERS), a proliferation marker (MKS) and ERBB2 expression. We re-assessed findings in GEPs derived from ER-/HER2+ pts of the NOAH trial treated with neoadjuvant chemotherapy (CT) or CT and trastuzumab (CTH).

Results Median follow-up was 5 years. Overall none of the markers was significant, but an interaction test for biomarkers and ER status was significant for MHC1, MHC2, STAT1, and marginally for MKS.

In ER+/HER2+ tumors, immune markers were not significant, but higher proliferation (MKS; HR 2.12 [1.07-4.19], p=0.03) was linked to higher risk, with a similar trend for low ERS (p=0.097). In ER-/HER2+ tumors higher MHC2 (HR 0.53 [0.36-0.79]; p=0.002), MHC1 (HR 0.41 [0.22-0.77], p=0.005) and STAT1 (HR 0.69 [0.49-0.97], p=0.036) were linked to better DEFS. Outcome for high MHC1 tertile was excellent and similar in all treatment arms. Low/int MHC1 pts treated with PHD had a trend for better DEFS compared to other treatments (HR 0.41 (0.14-1.21), p=0.11). In cases reaching pCR higher MHC1 (p=0.009), MHC2 (p=0.006), IGG (p=0.027) and STAT1 (p=0.008) were linked to better DEFS. For instance, the 5 yrs DEFS for high and low MHC1 tertiles was 100% and 74.6%, respectively.

Similarly, in ER-/HER2+ pts from NOAH, the 5-yrs DEFS in high, int and low MHC1 tertiles was 88.1%, 68.4% and 48.1%, respectively (p=0.015). Prognosis was similar and good in patients with high MHC1 receiving CT or CTH (p=0.674). Instead, in low/int MHC1 groups, CTH compared to CT significantly improved DEFS (HR 0.39 [0.16-0.93], p=0.035). Also in NOAH, the 5 yrs DEFS of pCR cases was influenced by baseline MCH1 (100% and 76.2% with high and low/int MHC1, respectively).

Conclusions In this exploratory analysis of NeoSphere, different biological functions were linked to DEFS in ER+ (proliferation and ER-related genes) and ER- (immune related) cases. In particular outcome of ER-/HER2+ with high MHC1 was good. However, in this group the benefit from adding trastuzumab to CT or pertuzumab in the PHD regimen was relatively small. Instead, the benefit seemed significant and large in cases of low/int MHC1, who had higher relapse risk. Of note, baseline immune markers of ER-/HER2+ tumors were linked to different DEFS also for cases achieving pCR.

### Circulating Biomarkers 3 / Immune Biomarkers

#3941

Novel biomarkers and multiplexed NGS to stratify FFPE NSCLC by tumor infiltrating lymphocytes and histopathology phenotypes.

Ann Mongan,1 Sophie Rozenzhak,1 Geoffrey Bien,1 David Chi,1 Hiroyoshi Nishikawa,2 Fiona Hyland,1 Jim Godsey1. 1 _ThermoFisher, South San Francisco, CA;_ 2 _National Cancer Center East, Kashiwa, Japan_.

There is increasing evidence supporting the association of tumor infiltrating lymphocytes (TIL) and drug sensitivity of solid tumors. In particular, primary and meta-analyses have reported a positive correlation between TIL level and outcome in advanced non-small cell lung cancer (NSCLC) treated with checkpoint inhibitors such as PD-1 and PD-L1. Recent trials with immunotherapies have started including TIL assessment in the study protocol in recognition of this metric as a predictive and prognostic biomarker. TIL levels are typically quantified by visual assessment of H&E-stained tumor sections. While this approach is generally accepted as the standard, there is an increased recognition that visual assessment of H&E-stained slides lacks precision and is relatively subjective (Salgado R, 2015; Schalper KA, 2015). Furthermore, as investigators are often also interested in measurements of additional biomarkers such as IFNg as well as the drug targets, a gene panel approach offers a convenient solution to objectively quantify expression levels of these informative markers. Here we report the discovery and verification of a unique gene expression signature that is capable of stratifying FFPE samples of NSCLC tumors by TIL levels and histopathology phenotypes (adenocarcinoma vs. squamous cell carcinoma). Gene expression was measured by an RNA Ion AmpliSeq Gene Expression research panel* containing 200 assays. Each research sample was measured with replicates at library generation step and sequencing step. Technical replicates were found to have >0.99 correlation among each other. Assays on the panels were also found to be robust with respect to low input amount (1-10 ng RNA). *For Research Use Only. Not for use in diagnostic procedures.

#3942

Increased frequency of proliferating peripheral white blood cells in the blood of hepatocellular carcinoma patients compared with noncancer controls.

Claire Hutton, Suriyon Uitrakul, Laura F. Ogle, Helen L. Reeves, Alastair Greystoke, Gareth J. Veal, David Jamieson. _Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom_.

Introduction

Proliferation of peripheral blood cells, including activated CD8+ T-cells, has been reported in numerous pathologies including haematopoietic cancers and solid tumours. During development of a method to detect and characterise circulating tumour cells by imaging flow cytometry it became apparent that peripheral proliferating lymphocytes, while still rare, are substantially more common than CTCs in all cancer patients and are present in everybody. We investigated the frequency of these cells in patients with hepatocellular carcinoma (HCC) and healthy volunteers, and went on to assess their utility as a surrogate tissue for proof of mechanism studies of anti-proliferative agents.

Methods

Whole blood samples (4ml) from 7 patients with hepatocellular carcinoma and 14 healthy volunteers were collected into EDTA tubes. After RBC lysis and fixation WBCs were stained with fluorochrome conjugated antibodies to Ki67 and CD45 and the nuclei were stained with DAPI to assess relative DNA content. For each sample images of 100,000 cells were collected by imaging flow cytometry. Putative G2 cells were identified as single cells with DNA content equivalent to twice the amount of the majority of cells and the same as images of doublets. The frequency of cells with nuclear localised Ki67 reactivity was assessed independently of DNA content. Whole blood from healthy volunteers was also treated by ex vivo incubation with PMA ± the CDK inhibitor Dinaciclib.

Results

The mean frequency of Ki67 positive G1 peripheral WBCs in blood from patients with HCC was 0.44%, as compared to a frequency of 0.24% in the blood from healthy volunteers (p = 0.007). Incubation of healthy volunteer blood with PMA for 20 minutes at 37°C resulted in a small but reproducible increase in the number of Ki67 positive cells and this increase was abrogated by simultaneous incubation with Dinaciclib (141 ± 41% versus 92 ± 39%, expressed as a percentage of no PMA control, p = 0.0007). Additional staining of the ex vivo samples with an antibody to pMCM2, as a potential proof of mechanism marker for CDC7 inhibitor efficacy, was carried out. Results showed that pMCM2 expression was associated with ki67 and the frequency of pMCM2 positive cells also increased with exposure to PMA

Conclusion

The frequency of proliferating WBCs is increased in patients with HCC. These cells may have utility as a predictive/prognostic biomarker, or possibly in the pharmacodynamic monitoring of anti-proliferative pharmacological agents.

#3943

Baseline LDH serum level as predictive value of activity in patients treated with anti PD-1 and PDL-1 monoclonal antibodies.

Maria Silvia Cona,1 Valter Torri,2 Massimo Di Nicola,1 Marina Garassino,1 Michele Del Vecchio,1 Sara Cresta,1 Diego Signorelli,1 Silvia Damian,1 Matteo Duca,1 Alice Indini,1 Monica Niger,1 Filippo de Braud1. 1 _Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _IRCCS Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy_.

BACKGROUND: Lactate dehydrogenase (LDH) is a key enzyme in the glycolytic metabolism, especially in anaerobic condition. High serum levels at baseline seem to be a negative prognostic marker in many tumor types, as well a negative predictive marker for response to treatment with anti CTL4 antibodies(Ipilimumab)in Malignant Melanoma. Recently, the treatment of several advanced tumors with anti PD-1 or PDL-1 monoclonal antibodies (mAbs) showed better outcome in comparison to Ipilimumab. However, biomarkers for a proper selection of responding patients is not yet available. The aim of this study is to correlate LDH serum level at baseline with clinical outcome in patients (pts) with advanced solid tumors treated with anti PDL-1 and anti PD-1 mAbs.

MATERIAL AND METHODS: We evaluated baseline LDH serum level in 145 pts affected by advanced solid tumors, treated at our Institute with anti PDL-1 and anti PD-1 mAbs. The rate of clinical responses and progression free survival (PFS) were related to normal or elevated baseline LDH serum level. We stratified the pts in 4 groups: normal value (group A),up to 1.5 x ULN (group B); < 2 > 1.5 x ULN (Group C); > 2 x ULN (Group D). We defined as disease control (DC) any complete (CR) and partial response (PR) or stable disease (SD) lasting > 3 months. Correlation between LDH serum levels and DC was assessed by Fisher's exact test; PFS was estimated using the Kaplan-Meier method.

RESULTS: We evaluated a heterogeneous population of 145 patients: 73 pts with lung cancer (50.3%; 67 NSCLC, 6 SCLC); 32 pts with Melanoma (22%) and 40 pts (27.6%) with other solid tumors (8 urothelial, 7 biliary tract, 6 mesothelioma, 4 head and neck, 4 sarcoma, 3 gastric, 3 colon, 2 RCC, 1 thyroid, 1 ovarian, 1 HCC). 79 pts (54.5%) were treated by anti PD-1 and 66 (45.5%) by anti PDL-1 agents. Overall as "best response", 79 pts (54.5%) achieved DC (1 CR, 26 PR, 52 SD) and 66 pts (45.5%) had progressive disease (PD). DC was achieved in 41/79 (52%) pts treated by anti PD-1 and 38/66 (58%) pts treated by anti PDL-1: only 2/41 (5%) and 5/38 (13%) of them had high LDH levels at baseline, respectively. Among 107 pts in Group A, 72 (67.3%) achieved DC as compared to 7/38 pts (18.4%) with high levels of LDH (Group B-D) (p: 0.0001). In the latter groups, PD occurred in 17/23 (74%), 5/6 (83%) and 9/9 (100%) patients in the B, C and D group respectively.

CONCLUSIONS: According to this preliminary analysis high baseline LDH serum levels seems to predict a greater likelihood of obtaining worse clinical outcome in terms of response and PFS also in patients treated by anti PD-1 and anti PDL-1 mAbs. In order to confirm this interesting result, we are evaluating a greater number of patients treated with anti PD-1 and anti PDL-1 mAbs at our Institution.

#3944

Combining tumor glypican-3 expression and CD16 Expression on NK cells from peripheral blood to identify patients responding to codrituzumab/GC33/RO5137382.

Gong Chen,1 Ya-Chi Chen,1 Bernhard Reis,2 Anton Belousov,3 Lori Jukofsky,1 Christine Rossin,1 Axel Muehlig,1 Chao Xu,1 Laurent Essioux,2 Toshihiko Ohtomo,4 Laura Di Laurenzio,1 Oscar Puig,1 Ray Lee1. 1 _Roche Innovation Center New York, New York, NY;_ 2 _Roche Innovation Center Basel, Basel, Switzerland;_ 3 _Roche Innovation Center Penzberg, Penzberg, Germany;_ 4 _Chugai Pharmaceutical Co. Ltd., Tokyo, Japan_.

Background & Aims: Codrituzumab (GC33 or RO5137382), a monoclonal antibody targeting an oncofetal protein glypican-3 (GPC3) expressed on the cell surface of hepatocellular carcinoma (HCC), induces antibody-dependent cellular cytotoxicity (ADCC) and inhibits tumor growth in preclinical studies. Based on this mechanism of codrituzumab, two biomarkers GPC3 and CD16 of the NK cells, which are the effector cells of ADCC, were investigated to correlate with clinical efficacy of codrituzumab in patients with advanced HCC.

Methods: Data from a phase II clinical trial of codrituzumab were used for this analysis. GPC3 expression was determined by immunohistochemistry (IHC) analysis of tumor biopsies, and CD16 expression on NK cells were quantified by a flow cytometry analysis of peripheral blood mononuclear cells. Overall survival of patients with high exposure of codrituzumab or placebo (n = 113) was used to compare different patient subgroups stratified by high or low expression of GPC3 and CD16.

Results: In individual-biomarker analyses, longer survival after codrituzumab treatment correlates with either high expression level of GPC3 in tumors (n = 97, HR = 0.39, 95%CI = 0.21 - 0.70, p = 0.00081) or a relatively high level of CD16 expressed on NK cells (n = 80, HR = 0.44, 95%CI = 0.23 - 0.83, p = 0.0056) at baseline. Furthermore, joint analyses of the two biomarkers reveal that both high levels of GPC3 and CD16 are required for patients to benefit from codrituzumab treatment (n = 67, HR = 0.29, 95%CI = 0.13 - 0.62, p = 0.00074), and lack of either one of them leads to a loss of codrituzumab therapeutic effect.

Conclusions: The retrospective analysis supports the mechanism of ADCC, in which the combination of high GPC3 expression in tumors and high CD16 expression in NK cells from peripheral blood is associated with prolonged overall survival given treatment of codrituzumab. This result supports the usage of both GPC3 and CD16 as potential biomarkers to select HCC patients for codrituzumab treatment.

#3945

Antigen-specific immunity and regulation as predictive biomarkers for anti-tumor vaccination.

Laura E. Johnson, Brian M. Olson, Douglas G. McNeel. _Univ. of Wisconsin-Madison, Madison, WI_.

We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, PAP-specific proliferating and IFNγ-producing T cells developed in some patients, and persistent Th1-type immunity was associated with an increase in PSA doubling time. In the current study, we sought to determine if one or more measures of antigen-specific or antigen non-specific immunity present prior to treatment was associated with subsequent immune response as a possible predictive biomarker. Specifically, patients who had developed PAP-specific, IFNγ-secreting immune responses, detectable at least twice over one year after immunization, were defined as immune "responders". The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells did not differ between the immune responder and non-responder groups. PAP-specific immune responses, detected as cytokine secretion following antigen stimulation of peripheral blood mononuclear cells in vitro, were detected in many patients pre-immunization. PAP-specific expression of granzyme B, IFNγ, and IL-6 was not significantly different between immune responding (n=7) and non-responding patients (n=23), however non-responder patients tended to have higher PAP-specific IL-10 production (p=0.09). Using a trans vivo delayed-type hypersensitivity (DTH) assay to assess for antigen-specific immune regulation, responder patients tended to have preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen, a response previously demonstrated to be IL-35-mediated (p=0.049). While the number of patients analyzed was low, these results suggest that different measures of antigen-specific regulation might help predict outcome from vaccination. These will be prospectively evaluated in an ongoing randomized, placebo-controlled, phase II trial evaluating this vaccine (NCT01341652).

#3946

Stratification of metastatic colorectal cancer patients using DNA and RNA sequencing and in-silico prediction of tumor antigens for consideration in immunotherapy.

FangYin Lo,1 Sharon Austin,1 Kellie Howard,1 Mollie McWhorter,1 Heather Collins,1 Amanda Leonti,1 Lindsey Maassel,1 Christopher Subia,1 Tuuli Saloranta,1 Nicole Christopherson,1 Kathryn Shiji,1 Shradha Patil,1 Saman Tahir,1 Sally Dow,1 Evan Anderson,1 Jon Oblad,1 Kerry Deutsch,1 Timothy Yeatman,2 Steven Anderson,3 Anup Madan1. 1 _Covance, Seattle, WA;_ 2 _Gibbs Cancer Center, Spartanburg, SC;_ 3 _Covance, Durham, NC_.

Colorectal cancer (CRC) is the third most common type of cancer in the United States. Although chemotherapy, radiation and targeted therapies can improve survival rates, recent studies have shown the potential benefit of immunotherapies to improve outcomes for patients with advanced CRC. Targeted therapies that use monoclonal antibodies (mAbs) to EGFR have been shown to benefit some CRC patients. Until recently, KRAS has been the only predictive biomarker for anti-EGFR therapy for metastatic CRC. However, 40% to 60% of patients with wild-type KRAS do not respond to anti-EGFR therapy. Therefore, to accurately predict patients' response to treatments and improve clinical outcomes, additional prediction and treatment methods are imperative. One of the many efforts to improve prediction for CRC patient's response to the anti-EGFR therapy is the development of gene expression based RAS signature scores for identification of RAS activated tumors independent of mutations in the KRAS gene. Recently there have been major advances in immunotherapeutic approaches in a wide variety of cancers. In solid tumors such as melanoma and colon cancers, immune checkpoints have been shown to improve clinical outcomes. There is considerable effort being placed on combinations of targeted therapy and immunotherapies to improve responses for these cancers. Similarly, since no single treatment can apply to all CRC patients, we aim to stratify patients using a combination of three methods: 1. RAS signature score based on the expression profile of 18 genes. This RAS signature score enables measurements of mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway functional output independent of tumor genotype. 2. Expression profile of immune checkpoint inhibitor target genes, such as PD1 and PDL1, and 3. In-silico prediction of neo-antigens and peptide binding affinity between tumor antigens derived from mutations and human HLA alleles.

55 FFPE samples were selected from a cohort of 468 samples with matching FF samples. These 55 samples have about 1:1:1 ratio of high, medium and low RAS scores. Here we showed our ability to obtain RAS signature scores with concordant results using different platforms including RNA-seq, targeted RNA-seq, Nanostring and Affymetrix microarray. Samples that have RAS activating mutations such as KRAS and BRAF have significant higher RAS scores (p<0.001). Interestingly, expression of PD-L1 was significantly lower in tumor samples harboring mutations of genes such as MET, PTEN, NRAS, FBXW7, and GNAS. Kruskal-Wallis test showed that the expression of PD-L1 was significantly lower in samples with higher RAS signature scores (p<0.05). Combined with further prediction of tumor antigen derived from genes with missense mutations, we provide a combinatorial method for stratifying metastatic CRC patients.

#3947

Atypical chemokine receptor ACKR3 expression is associated with aggressive behavior and poor prognosis in gastric cancer.

Nayoung Kim, Seung-Woo Baek, Hyewon Ryu, Yoon Seok Choi, Ik Chan Song, Hwan Jung Yun, Deog Yeon Jo, Samyong Kim, Hyo Jin Lee. _Chungnam National Univ. Hospital, Daejeon, Republic of Korea_.

Chemokine and their receptors are key mediators of normal physiology and a large number of pathologic conditions such as cancer, and this family of receptors is the emerging therapeutic target in the field of cancer treatment. ACKR3 is an atypical chemokine receptor first cloned from a dog cDNA library as the orphan receptor and was initially named Receptor Dog cDNA 1 (RDC1). Shortly after demonstrating that RDC1 binds with its ligand, stromal cell-derived factor-1α (SDF-1α) and interferon-inducible T-cell α chemoattractant (I-TAC), RDC1 was officially deorphanized. Accumulating evidence of recent studies have suggested that expression of ACKR3 is augmented in most of tumor cells as compared to their normal counterparts and is involved in cell proliferation, survival, migration, invasion during the initiation and progression of cancer. However, there is little information regarding their expression and clinical relevance in gastric cancer. The expression status of ACKR3 was investigated in 221 specimens of primary gastric cancer using immunohistochemistry. The correlation of ACKR3 expression with the clinicopathological features and survival outcomes was analyzed as well. Immunohistochemical staining of gastric cancer tissue sections revealed diverse cytoplasmic and membrane staining patterns for ACKR3. One hundred-fourteen cases (51.6%) showed low ACKR3 expression according to an arbitrary scoring system (grade 0-1; grade 0, n=26; grade 1, n=88), and one hundred-seven cases (48.4%) showed high expression (grade 2-3; grade 2, n=58; grade 3, n=49). There were no significant differences in age, gender, histology, tumor location, lymphatic and venous invasion among the two groups. However, high CXCR7 expression in cancer cells tended to be associated with proportion of tumor size greater than 5 cm (P = 0.055) and was significantly correlated with depth of tumor invasion (P < 0.001), lymph node metastasis (P = 0.042), and higher stage (P = 0.002). Furthermore, patients with high ACKR3 expression showed worse overall survival rate of 47% compared to 56% in patients with low ACKR3 expression. In conclusion, ACKR3 is differentially expressed in gastric cancer cells, and high expression of ACKR3 is associated with aggressive tumor behavior and poor survival in patients with gastric cancer, suggesting that ACKR3 plays an important role during gastric cancer progression.

#3948

Lysosomal acid phosphatase 2 is an unfavorable prognostic factor associated with better survival in colorectal cancer patients receiving chemotherapy.

Chi-Long Chen. _Taipei Medical University, Taipei, Taiwan_.

Colorectal cancer (CRC) is one of leading malignancies worldwide. The therapeutic modality for advanced CRC including stage III and IV, is operation following by chemotherapy. For stage II CRC, surgery is the main therapeutic modality but the usage of adjuvant chemotherapy is still controversial and there is no reliable indicator for their purpose. In this study, we found that the expression of lysosomal acid phosphatase 2 (ACP2) in the tumor tissue of CRC was elevated and was correlated with poor outcome in stage II CRC patients, especially. In addition, in CRC patients with high ACP2 expression tended to have more sensitive for 5-FU treatment in our cohort. In CRC of stage II patients, multivariate Cox proportion hazard model analysis revealed that ACP2 expression and T stage are independent prognostic factor for overall survival (HR: 3.274, CI: 1.413-7.586, p=0.006; HR: 3.742, CI: 1.058-13.233, p=0.041). Our data indicated that high expression of ACP2 was a negative prognosticator and could predict 5-FU chemotherapy response in stage II CRC. Moreover, we used HCT116 cells transfected with ACP2 lentivirus to create stable shACP2-HCT116 clone. Our results showed knockdown ACP2 could induce chemoresistance and more mesenchymal-like phenotype of CRC cell line. Therefor, targeting ACP2 might be a novel chemotherapy factor and might a new strategy in stage II CRC patients.

#3949

Rapid 5-marker multiplex phenotyping of breast cancer subtypes & tumor-infiltrating leukocytes "in situ" in FFPE sections.

Matt Levin,1 Stephen J. Kron,2 David Schwartz,1 Helen Snyder1. 1 _Cell IDx, San Diego, CA;_ 2 _The University of Chicago, Chicago, IL_.

Breast cancer can be divided into four subgroups HER2+, Luminal A, Luminal B and triple negative using multiple clinically validated monoclonal antibodies. Each subtype presents a different prognosis and treatment option. It is also increasingly clear that immune subsets within the tumor microenvironment may also have a bearing on prognosis, with certain subsets being associated with increased patient survival. Determining the tumor and immunologic phenotype of initial tumor biopsies both in the composition of cells and their relative spatial distribution is becoming key in selecting optimal treatment. The aim of our research has been to develop a multiplex immunofluorescence technology that can rapidly and simultaneously detect 5+ markers on a single section, allowing multiparametric quantitative analysis of cell phenotype and tissue composition as well as providing important spatial information of cells relative to each other. Our multiplex immunofluorescence (mxIF), modified-hapten based technology allows the simultaneous detection of multiple biomarkers using a standard two-step procedure. The technology is antibody species independent and produces signals 3-4X stronger than direct fluor-labeled secondary antibodies. It can detect multiple co-localized markers and while it has been engineered to detect four biomarkers and DAPI, it has the potential for significantly higher multiplexing. In addition it is modular and can be readily modified for any multiplex panel. The data generated from a staining experiment can be readily exported into any image analysis program. In comparison to existing technologies from GE and PerkinElmer, our technology allows multiple targets to be interrogated rapidly and simultaneously without the need for multiple rounds of stripping and imaging. The breast cancer phenotyping panel (BC-1) provides rapid phenotyping information allowing identification of Luminal A, Luminal B, HER2+ or triple negative cancer. Our immuno-oncology panel (IO-1) identifies CD4 and CD8 T cells, CD20+ B cells and CD68+ macrophages. Application of each panel to one of two sequential sections and then image overlay allows both tumor and immunophenotyping. The modular and non-species specific nature of this technology means that panels can be modified and expanded to characterize multiple different tumors and immune cell subpopulations in a rapid, sensitive and quantitative manner.

#3950

Optimization of a method for FACS analysis of checkpoint receptor expression on tumor infiltrating lymphocytes in clinical tumor samples.

Norman Zhang,1 Qiyao Zhang,1 Yan Liu,1 Juntao Yu,1 Jingjing Wang,1 Jie Xu,1 Qunsheng Ji,1 Keith Wilcoxen,2 Jonathan Graves2. 1 _WuXi AppTec, Shanghai, China;_ 2 _Tesaro Inc., Waltham, MA_.

While immune checkpoint therapies against CTLA-4, PD-1 and PD-L1 have achieved promising clinical outcome recently, the dynamic therapeutic responses spanning individual patients and different cancer types warrant exploration and validation of additional therapeutic strategies. Since the expression of immune checkpoints in tumor infiltrating lymphocytes (TIL) has been of interest to the field, we attempted to analyze the expression of several checkpoint receptors on TILs in clinical tumor samples. Unexpectedly, a number of markers were subject to time-dependent degradation during the tissue disaggregation process. This degradation could be due to the proteolytic activity of a proteinase, which has been used commonly in various tissue disaggregation protocols. Analysis of the protein sequences verified the presence of the proteinase recognition sequences in the affected receptors. Using PHA-stimulated hPBMCs, we found and demonstrated that a specific protease was responsible for either partial or total degradation of these markers. To overcome this issue, we developed a protease-free tissue disaggregation method, which showed no adverse effect on protease affected cell membrane receptors, thus allowing for more reliable flow cytometric analysis of various TIL subpopulations. With this method, we analyzed these checkpoint receptors positivity of both CD4+ and CD8+ TILs and revealed a high degree of heterogeneity of these immune checkpoints in a number of tumor types, including lung, breast and gastric cancers. Analysis of the magnitude of lymphocyte infiltration to the tumor and expression of prominent immune checkpoints on CD4+ or CD8+ TILs will help understand the subtle interaction between tumor and the immune system in the tumor microenvironment and may provide guidance for clinical trials of novel combinatorial immuno-therapeutic strategies.

#3952

Label-free imaging identification of WBCs based on the features of quantitative phase microscope images for negative selection of CTCs.

Yusuke Ozaki,1 Hidenao Yamada,2 Hirotoshi Kikuchi,1 Tomohiro Murakami,1 Tomhiro Matsumoto,1 Toshiki Kawabata,1 Yoshihiro Hiramatsu,1 Manabu Ohta,3 Kinji Kamiya,1 Megumi Baba,1 Toyohiko Yamauchi,2 Kentaro Goto,2 Yukio Ueda,2 Shigetoshi Okazaki,4 Hiroyuki Konno1. 1 _Hamamatsu University School of Medicine, Second Department of Surgery, Hamamatsu, Shizuoka, Japan;_ 2 _Hamamatsu Photonics K.K, Central Research Laboratory, Hamamatsu, Shizuoka, Japan;_ 3 _Hamamatsu University School of Medicine, Oncology Center, Hamamatsu, Shizuoka, Japan;_ 4 _Hamamatsu University School of Medicine, Medical Photonics Research Center, Hamamatsu, Shizuoka, Japan_.

Background: Recent technological advances have enabled the reliable detection and characterization of circulating tumor cells (CTCs) in the blood of cancer patients. To quantify the amount of CTCs, various assays have been developed to facilitate the detection of epithelial cells in the blood by using cellular surface markers such as EpCAM and cytokeratins. However, recent studies have revealed the importance of CTCs undergoing epithelial-mesenchymal transition (EMT), which are difficult to detect by the conventional surface marker-based methods because those CTCs express low expression of epithelial markers. We previously reported a novel method for the identification of CTCs by removing WBCs from mononuclear cells in the blood, based on the features of Quantitative Phase Microscope Images (QPIs) of the cells obtained from machine-learning (AACR Annual Meeting 2015). Here, we report some progress of our study.

Methods: We analyzed WBCs and cancer cell lines using Quantitative Phase Microscope (QPM) that can image optical path-length of living cells with a high resolution of 1nm without staining in non-cytotoxic way. At this time, QPIs were reconstructed from line scanned images of the cells flowing in a chamber. Obtained QPIs were analyzed by a computer vision application which automatically identifies an object in digital images based on the features extracted from the dataset we had developed by machine learning.

Results: We imaged 325 WBCs obtained from healthy donors and 325 cell-line cells (from 5 cancer cell lines) with QPM. Then, we extracted certain features from the QPIs and used them as training images for machine learning. We employed 5-fold cross validation to create an algorithm to detect WBCs circulating in the blood. The algorithm successfully recognized WBCs among QPIs mixed in with WBCs and cancer cell lines. The ROC AUC value was 0.98, indicating that the algorithm was not a random selection. Furthermore, we successfully captured QPIs of the flowing cells, which makes it possible to sort living cells based on the algorithm we developed.

Conclusions: We successfully differentiated WBCs from cancer cell lines based on the features of QPIs. The object recognition method applying to QPIs of the cell is expected as a useful, non-cytotoxic, marker-free isolation of CTCs.

#3953

Highly accurate genetic profiling of circulating tumor cells using a label-free inertial microfluidic approach coupled with droplet PCR-based next-generation sequencing.

Yi Fang Lee,1 Mark Consugar,2 Kimberly Helzer,2 Mei Hui Tan,1 Lori Emrick,2 Ali Asgar Bhagat1. 1 _Clearbridge BioMedics, Singapore, Singapore;_ 2 _Raindance Technologies, Inc, Billerica, MA_.

Background: Circulating tumor cells (CTCs) could provide significant insights into cancer metastatic events and potentially even inform clinical decisions. However, the scarcity of CTCs, compounded by the abundance of circulating white blood cells, poses a major technical challenge in the isolation, yield and downstream molecular analysis. Moreover, next-generation sequencing (NGS) of CTCs has been challenged by low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we present a label-free microfluidic approach that utilizes curvilinear microchannel geometry for high purity CTC enrichment from 7.5 ml whole blood.

Methods: We showed high purity isolation of CTCs using a label-free inertial focusing microfluidics platform, ClearCell® FX. The lung cancer cell line, NCI-H1975, with known EGFR, CDKN2A and TP53 mutations, were spiked into 7.5ml healthy donor peripheral blood samples at varying concentrations (50, 100, 200 cells). DNA was extracted using the QIAamp Micro kit. The Thunderbolts Cancer Panel droplet digital PCR NGS library preparation method was used to enrich 230 loci in 50 known cancer genes from the isolated CTC gDNA. The libraries were sequenced on a MiSeq system.

Results: The CTCs were sufficiently enriched with an average recovery of 45.8% and purities ranging from 1% - 70%. All libraries were successfully generated and sequenced with ≥1400× mean coverage, even with as little as 2 ng of starting material. Known EGFR, CDKN2A and TP53 mutations in H1975 were successfully detected in all the samples (n = 9), with a significant variant frequency that correlates with CTC purity levels.

Conclusion: We have demonstrated a highly accurate and sensitive workflow utilizing a combination of inertial focusing and digital droplet microfluidics to detect low numbers of rare CTCs and interrogate their molecular profiles from as little as 10 cells/mL frequency. This workflow has the potential for further development and adoption as a liquid biopsy approach for non-invasive genomic profiling of metastatic cancers.

#3954

Prospective evaluation of circulating tumor cells (CTCs) in head and neck cancer patients receiving definitive radiotherapy with a nanotechnology based system.

Joseph M. Caster,1 Michael J. Eblan,1 Kyle Wang,1 Ja Hye Myung,2 Bhishamjit Chera,1 Seungpyo Hong,2 Andrew Z. Wang1. 1 _UNC Chapel Hill, Durham, NC;_ 2 _University of Illinois at Chicago, Chicago, IL_.

Background: Circulating tumor cells (CTCs) are an important biomarker in cancer. There has been substantial interest in utilizing CTCs to enable personalized treatment. However, there is limited data on CTCs in patients with head and neck cancers (HNC) where there is growing data that curative treatment needs to be risk-based. The purpose of this prospective, correlative study is to investigate the clinical significance of CTCs , as measured by a highly sensitive CTC capture technology UiChip, in HNC patients undergoing definitive treatment.

Methods: HNC patients (M0) undergoing definitive treatment (radiation+/- chemotherapy) were enrolled. Peripheral blood was collected prior to starting RT, at the first week, mid-point, final week of treatment, and every 4 to 12 weeks after completion. Quantification of CTCs was conducted using UiChip . The primary endpoint was change in CTCs pre- and post- RT.

Results: 35 patients are included in this analysis (median age 58, 34% non-smokers, 69% HPV/p16 positive, and 17% node (-)) and 90% received chemotherapy. CTCs were detected in all patients pre-RT (100%). There was no association between pre-RT CTC level and tumor or nodal stage. Median CTCs significantly decreased from 71 CTCs/mL (range, 7-849) before RT to 32 (2-209) at completion of RT and 27 (2-78) at 4 to 12 weeks post-treatment. Ten patients had persistent disease and/or distant metastasis at a median follow-up of 10.6 months (range, 3.7-17). CTCs declined throughout treatment in patients with complete clinical and/or radiographic responses, in contrast to an elevation in CTCs at mid or post-RT in the 7 patients with pathologic residual disease/distant failures.

Conclusions: We demonstrated that the novel UiChip can capture CTCs in a diverse population of head and neck cancer patients. Our pilot data suggest that individual patient CTC changes during and after treatment may be a predictive biomarker for radiotherapy response and clinical outcomes.

Table: Median CTCs/mL Peripheral Blood (Median % of Pre-Treatment Baseline Count)

---

|

Pre-RT | Mid-RT | Post-RT

Number (%) of CTCs in Complete Responders to RT (N=18) | 67

IQR: 39-117 | 37 (38%)

IQR: 22-79 (18-94%) | 21 (18%)

IQR: 7-40 (13-42%)

Number (%) of CTCs in Partial Responders to RT (N=7) | 120

IQR: 36-303 | 66 (123%)

IQR: 20-134 (30-172%) | 51 (45%)

IQR: 23-54 (17-63%)

#3955

Expression of cancer stem cell-associated proteins in exosomes derived from ascites of patients with advanced pancreatic cancer.

Takahiko Sakaue,1 Hironori Koga,1 Masaru Fukahori,2 Yasuko Imamura,3 Toru Nakamura,1 Yoshinobu Okabe,1 Yu Ikezono,1 Fumitaka Wada,1 Hideki Iwamoto,1 Atsutaka Masuda,1 Tomoyuki Ushijima,1 Keisuke Miwa,2 Tatsuyuki Kakuma,4 Osamu Tsuruta,1 Takuji Torimura1. 1 _Department of Medicine, Kurume University School of Medicine, Kurume City, Japan;_ 2 _Center for Multidisciplinary Treatment of Cancer, Kurume University Hospital, Kurume City, Japan;_ 3 _Liver Cancer Research Division, Kurume University Research Center for Innovative Cancer Therapy, Kurume City, Japan;_ 4 _Biostatistics Center, Kurume University, Kurume City, Japan_.

Background: The number of patients with pancreatic cancer is rapidly growing, and the disease is the fifth leading cause of cancer-related death in Japan. At end stage of the disease, the patients are prone to suffer peritonitis carcinomatosa with chemorefractory ascites. It is known that cancer cells surviving in ascites show cancer stem cell (CSC)-like features (PLoS One 2012). The CSC-like cells robustly secrete extracellular vesicle called exosome, which plays important roles in tumorigenesis, tumor growth, metastasis, angiogenesis, pre-metastatic niche formation, immunosuppression, drug resistance, and epithelial-mesenchymal transition. The AIM of this prospective study was to assess whether exosomes taken from malignant ascites of patients with advanced pancreatic cancer included the CSC-associated proteins, that might be predictive markers for chemoresistance and prognosis. Methods: The malignant ascites was collected from the cancer patients who underwent abdominocentesis and/or cell-free and concentrated ascites reinfusion therapy (CART) in Kurume University Hospital. Ascites derived from patients with benign diseases, including decompensated liver cirrhosis (d-LC), was used as control. Informed consent was obtained from all of the patients. Exosomes in ascites were purified by using ExoQuick Exosome Precipitation Solution (System Biosciences) according to the manufacturer's protocol. Western blot analysis was performed to detect CSC-associated proteins, including CD133, CD44, CD44v9, xCT, CD24, and Dclk1. Results: Successful purification of exosomes from both the malignant ascites and the benign one was confirmed by monitoring exosome-specific proteins such as CD68, CD9, CD81, and HSP70. Among the CSC-associated proteins examined, CD133 was predominantly expressed in exosomes obtained from ascites of the pancreatic cancer patients compared with those obtained from ascites of the d-LC patients. Other molecules were faintly expressed or absent even in the malignant ascites. Conclusions: We first demonstrated abundant expression of CD133, a human pancreatic CSC marker, in exosomes derived from ascites of patients with the disease. The finding suggests that exosomal CD133 might be a potential biomarker for chemoresistance and prognosis of the patients.

#3956

DLD microfluidic purification and characterization of intact and viable circulating tumor cells in peripheral blood.

Myra Koesdjojo,1 Zendra Lee,1 Christopher Dosier,1 Tanisha Saini,1 Khushroo Gandhi,1 Alison Skelley,1 Lee Aurich,1 Gregory Yang,2 Tony Ward,1 Roberto Campos-González1. 1 _GPB Scientific, Carlsbad, CA;_ 2 _Yuma Regional Cancer Center, Yuma, AZ_.

We have developed a microfluidic chip-to-chip approach to purify circulating tumor cells (CTC's). The first, a DLD (deterministic lateral displacement) microchip contains an array of microposts arranged in sub-arrays of different gap sizes. The "product" outlet of the DLD chip is connected with a second magnetic-separation chip. In the DLD chip, cells and particles are deflected or "bumped" based on their size, whereas the second chip is designed to remove any cell or component tagged with a magnetic particle (MNP). Air pressure is applied to maintain a constant flow of sample and buffer throughout the system in a vertical fashion. First, whole blood is mixed with biotinylated antibodies to CD45 and strepavidin-MNP's for 20min, diluted in buffer and passed through the DLD chip where the blood interacts with the posts and cells are bumped based on a deterministic lateral displacement principle, thus separating blood components. The "product" fraction from the DLD chip containing white blood cells and other larger cells is directed to the magnetic chip and the "waste" fraction containing red blood cells, platelets, debris and soluble blood components is discarded. The second chip has an array of magnets, capturing the sav-MNP/CD45+-tagged cells thereby allowing the gentle free flow of rare cells towards the final product fraction. With the chip-to-chip configuration we are able to process up to 8ml of blood, removing >99.8% of red blood cells and >98% of white blood cells, with the remaining purified cell populations suitable for further analysis and characterization. Validation of the chip-to-chip approach using tumor-derived cancer cells, including MDA-MB-231, PC-3 and SKBR-3 cells, confirms a linear recovery of >80% of the spiked-cells with a viability of ~90%. The minimum cell size captured by our approach is approximately 6µm ensuring the isolation of small-sized CTC's. Multi-color flow cytometry and imaging analysis of the chip-to-chip isolated cells from blood of breast cancer patients confirms the purification and identification of unique populations of HER2+/CD45-/EpCAM+ and CD146+/CD44+ cells characteristic of breast carcinomas-among other rare cell populations. Our chip-to-chip approach allows a gentle purification of intact and viable rare cells from cancer patients' blood, suitable for functional analysis, drug sensitivity tests and genetic characterization. Thus, we have developed a hands-free liquid biopsy capable of isolating and purifying rare circulating tumor cells.

#3957

Optimized plasma collection procedures for liquid biopsy analyses in cancer.

Sonya T. Parpart-Li,1 Bjarne Bartlett,2 Maria Popoli,2 Vilmos Adleff,2 Julie Brahmer,2 Nilo Azad,2 Sarah Bonerigo,2 Ilene Browner,2 Amy Ryan,2 Victor Velculescu,2 Mark Sausen,1 Luis A. Diaz2. 1 _Personal Genome Diagnostics, Baltimore, MD;_ 2 _Johns Hopkins Kimmel Cancer Center, Baltimore, MD_.

Analyses of genomic alterations in cell-free DNA (cfDNA) shed from tumors into the blood stream is rapidly advancing as a method to detect and genotype cancer. Liquid biopsy analyses are a favorable alternative to invasive tissue approaches, particularly for cancers in organs where tissue is difficult to obtain. As these methods become a clinical standard, the method for blood collection and plasma isolation will be an important consideration to ensure optimal assay performance with limited degradation of circulating tumor DNA (ctDNA). We compared blood collection and plasma isolation using both K2EDTA and cell-free DNA Streck™ BCT tubes to determine the stability of cfDNA and ctDNA in blood at room temperature or at 4°C. Blood was collected from nine cancer patients with KRAS mutations using K2EDTA and Streck™ BCT tubes. Over the course of seven days, cell lysis occurred in K2EDTA tubes at room temperature, releasing germline DNA into the blood (1300% increase over seven days), which significantly increased the number of total genomic equivalents. Additionally, we found that the volume of plasma collected from both tube types during blood fractionation decreased over time when tubes were at room temperature (an average of 3.5 mL vs. 1.0 mL, respectively). To evaluate the effect of storage conditions on performance of each tube type, blood was also collected from six cancer patients with KRAS or EGFR mutations and either maintained at room temperature or stored at 4°C for up to three days. Taken together, K2EDTA tubes stored at 4°C prevented cell lysis and preserved the ctDNA in a manner equivalent to Streck™ BCT tubes. These data demonstrate that liquid biopsies can be collected using either tube type with similar performance and appropriate consideration of storage conditions.

#3958

Tubulin hyper-acetylation in blood lymphocytes: pharmacodynamic (PD) biomarker for the selective histone deacetylase (HDAC) 6 inhibitors ricolinostat and ACY-241 in multiple myeloma (MM) patients.

David L. Tamang,1 Jeffery Supko,2 Kailash Bhol,1 Catherine Wheeler,1 Simon S. Jones,1 Min Yang1. 1 _Acetylon Pharmaceuticals, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA_.

INTRODUCTION: HDACs are a family of enzymes that remove acetyl groups from proteins and are involved in key cellular processes. Nonselective HDAC inhibitors have shown promise in treating MM in combination with standard therapies such as proteasome inhibitors (PIs) and immunomodulatory agents, but are limited by adverse effects. The selectivity profile of clinical HDAC inhibitors is critically important as the same drug may exert its effects via alternative mechanisms depending on the partner therapeutic. An example is HDAC6 inhibitors in combination with bortezomib, a proteasome inhibitor (PI). Delivery of protein-bearing vesicles to the aggresome involves HDAC6 through its role in vesicle trafficking, thus supporting a secondary survival pathway. In the case of lenalidomide or pomalidomide, inhibiting class I HDACs can suppress MYC protein levels, which potentially enhances activity in a clinical setting. Ricolinostat (ACY-1215) and ACY-241 that are currently in clinical trials have a selectivity profile tuned toward HDAC6 over HDACs 1, 2, and 3, which is hypothesized to enhance anti-cancer activity with multiple standard of care regimes while minimizing toxicity.

METHODS: Peripheral blood from patients that received escalating oral doses of ricolinostat or ACY-241 that range from 40 mg to 360 mg was collected for matched PK and PD assessment. Lymphocytes were examined for tubulin and histone hyper-acetylation by flow cytometry.

RESULTS & CONCLUSION: Plasma concentration for both ricolinostat and ACY-241 reach a maximal level (Cmax) 1-2 hr after administration. An exposure plateau was reached at dose levels ≥160 mg of ricolinostat, with Cmax observed to be ~2 µM. ACY-241 exposures reached higher levels, Cmax of ~3 µM and ~7 µM at 180 and 360 mg, respectively, and no evidence of an exposure plateau. Correlating to the PK levels, acetyl-tubulin increases to a maximal level by 1-2 h after dosing compared to predose, and declines to basal level by >4 hr. As plasma levels increased with dose from 40 mg to 160 mg for ricolinostat so do the number of patients that have an increase in acetyl-tubulin such that at dose levels ≥80 mg all patients have a measurable increase in acetylated tubulin. Levels of acetyl-tubulin were similar at dose levels of 180 and 360 mg of ACY-241. Acetyl-histone levels were low- moderate at the 160 and 180 mg dose level for ricolinosat and ACY-241, respectively, and for ACY-241 increased with increasing exposure at doses > 180 mg similar to results observed in vitro by immunoblot analysis of MM cells treated with ACY-241. In conclusion, the PK and PD results suggest that both ricolinostat and ACY-241 have reached a pharmacologically relevant level of HDAC inhibition as determined by acetylated tubulin and histone at the clinical dose levels examined.

#3959

Detection of somatic mutations at 0.1% frequency from cfDNA in peripheral blood with a multiplex next-generation sequencing assay.

Dumitru Brinza, Ann Mongan, Richard Chen, Dalia Dhingra, Jian Gu, Janice Au-Young, Fiona Hyland, Kelli Bramlett. _Thermo Fisher Scientific, South San Francisco, CA_.

Background:

Effective blood screening for tracking of recurrence and resistance of tumors may improve outcomes in the future. Research studies suggest that virtually all tumors carry somatic DNA mutations, and these may serve as biomarkers that also can be tracked in blood. One of the sources containing tumor DNA in blood is circulating cell-free DNA (cfDNA). Tumor DNA comes from different tumor clones, and its abundance in plasma can be very low at critical stages such as early recurrence or development of resistance. Hence, there is great interest in being able to detect mutation biomarkers at very low frequency from cfDNA for detection and characterization of tumor clones.

Method:

We present a research use only analysis workflow for peripheral monitoring that enables detection of low frequency DNA variants in blood.

We developed an analysis algorithm that models errors accumulated during amplification and sequencing, and accurately reconstructs sequence of original DNA molecules based on multiple next generation sequencing reads. The reads contain genomic sequence and an adaptor that allows identification of reads originated from the same DNA molecule. We then developed a variant calling method that uses accurately reconstructed sequences to enable sensitive and specific detection of somatic mutations to 0.1% allele ratio. We demonstrate the analysis in control and archived cfDNA research samples.

We used a next generation sequencing assay that allows interrogation of ~150 biomarkers relevant in lung from COSMIC and Oncomine™ databases, and de-novo variant detection at ~1,700 genomic positions in 11 genes implicated in non-small cell lung cancer (NSCLC).The assay delivers >95% on target reads and highly uniform amplification across targeted cfDNA molecules.

Results:

We tested the limits of variant detection in controlled dilution series and in cfDNA. First, we diluted engineered AcroMetrix™ Oncology Hotspot Control plasmids into background GM24385 genomic DNA down to 0.1% frequency, and then fragmented into fragments with average size of 170bp. The Acrometrix sample contains ~45 common tumor mutations interrogated by our assay. Next, we used 0.1% Horizon's (HD780) cfDNA reference sample that contains 8 mutations at our hotspot positions including two large insertion and deletion variants of size >10bp. Finally, we performed analytical verification of variant detection performance in cfDNA using a dilution series of normal blood samples.

We achieved >95% sensitivity with >20ng input DNA and >90% sensitivity with ~20ng input DNA and <1 false call per sample for variants in hotspot positions present at frequency 0.1%.

Conclusions:

This new lung cfDNA analysis workflow may facilitate researchers to study relevant NSCLC biomarkers at 0.1% frequency in cfDNA. Analysis is compatible with lower frequency variant detection, but will require higher input DNA amount and higher sequencing coverage.

#3960

Combined circulating tumour cell (CTC) and circulating tumor DNA (ctDNA) analysis of blood from patients with pancreatic cancer.

Ged Brady,1 Dominic Rothwell,1 Mahmood Ayub,1 Nigel Smith,1 Sumitra Mohan,1 Jakub Chudziak,1 Kyaw Aung,1 Richard Hubner,2 Crispin Miller,1 Alison Backen,2 Hui Sun Leong,1 Sakshi Gulati,1 Chang Sik Kim,1 Angela Lamarca,2 Mairéad McNamara,2 Juan Valle,2 Caroline Dive1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _The Christie NHS Foundation Trust, Manchester, United Kingdom_.

Background

The challenge to improve outcomes for patients diagnosed with advanced pancreatic cancer remains very real with only small improvements in median survival gained by the use of systemic chemotherapy and little improvement in 5-year survival over the past decades. The advent of next generation sequencing (NGS) of tumor nucleic acids has opened up the possibility of improving outcomes through personalized therapies selected on the basis of tumor genetics.

Whilst NGS biomarkers can be measured in tumor biopsies sampled shortly before and after treatment this is often not practical for ethical, logistical and simple lack of availability. To circumvent problems associated with tumor sampling we have developed and evaluated blood-borne nucleic acids biomarkers for patients diagnosed with advanced pancreatic cancer.

Approach

We developed a NGS circulating free DNA (cfDNA) analysis pipeline based on the generation of whole genome NGS libraries followed by sequencing of over 600 cancer-associated genes using Agilent SureSelect. Using whole blood collected with Streck cell free DNA blood collection tubes (cfDNA BCT) we optimised combined circulating tumor cell (CTC) enrichment and cfDNA isolation. Quantitative measurements of KRAS mutations using both NGS and droplet digital PCR (ddPCR) were used to compare the tumor component present in both CTCs and cfDNA. We evaluated the overall approach using External Quality Assessment (EQA) controls and over 50 advanced pancreatic cancer patient samples. For CTC analysis we also compared epitope dependent (CellSearch) and independent (Parsortix) enrichment.

Results

After applying our NGS pipeline to 5 EQA genomic controls we identified all 14 known mutations correctly indicating high sensitivity and specificity. Both ddPCR and NGS identified KRAS mutations in patient cfDNA with a higher success rate seen for ddPCR consistent with the higher sensitivity of this methodology. From the NGS output additional mutations were detected in samples which either harboured or lacked detectable KRAS mutations. Consistent with previous observations CellSearch CTCs were detected at low levels in around 20% of patients with a similar frequency seen in initial analysis of CTCs obtained by epitope independent enrichment (Parsortix). In general KRAS mutations were detected at a higher level in patient cfDNA compared to enriched CTCs although some patients showed detectable CTC KRAS mutations with no KRAS mutations detected in their cfDNA by either ddPCR or NGS. Ongoing analysis is aimed at establishing if the molecular observations correlate with clinical outcome.

Conclusion

Combined CTC enrichment and cfDNA isolation is readily achievable using a single Streck cfDNA BCT. Results indicate that combined CTC and cfDNA analysis is more sensitive than either approach alone.

#3961

Methodological considerations in the preparation of biomimetic reference materials for ctDNA assays.

Seth B. Harkins,1 Farol L. Tomson,1 Bharathi Anekella,1 Russell Garlick2. 1 _Seracare Life Sciences, Gaithersburg, MD;_ 2 _Seracare Life Sciences, Milford, MA_.

Clinical utilization of cell-free DNA (cfDNA) has become commonplace for NIPS testing and has recently been more widely deployed to quantitate cell-free circulating tumor DNA (ctDNA) for use in cancer patient care longitudinally across the treatment spectrum from subclinical, to diagnosis and through treatment. The scarcity and unstable nature of the cfDNA analyte in human plasma is problematic and thus the lack of robust detection methods has limited clinical validation. In this work we sought to avoid the inherent limitations of plasma-based patient reference materials and developed a strategy to prepare a biomimetic reference material which is sufficiently equivalent to the cfDNA found in patients with malignant cancers. Thus far the scarcity and physical form of ctDNA in human plasma has attenuated the validation of assays for clinical utility. To this end, we have generated a biosynthetic construct with short DNA fragments containing relevant cancer mutation sequences, including single-nucleotide variations, insertions and deletions in BRAF, EGFR, KRAS, NRAS, PIK3CA or HER/ERBB2. The mutant fragments were titrated into normal human reference DNA, GM24385, and ultrasonically sheared to ~160 bp. Precise allele frequency blends were verified by digital PCR (dPCR) prior to encapsulation to enhance stability and compatibility with commutable plasma. The reference material is formulated to 20 ng/mL of extractable DNA in modified Seracare Matribase™ to confer commutability in pre-analytic processes. These materials have been quantitated by dPCR and/or Next Generation Sequencing (NGS). A dilution panel was prepared at 10%, 5%, 2.5%, 1.25%, 0.1% allele frequency (mutant:normal DNA ). Linearity (R² = 0.9997) was observed by dPCR (measured vs. expected) and the NGS detected, Swift Accel-Amplicon 56G Oncology Panel (R² = 0.997). Due to the low abundance of ctDNA, a characterized reference set is critical to validate assays that are developed with extremely low limit of detection. Data will be presented to show the breadth of this methodology and its utility in validating NGS and dPCR assays.

#3962

Circulating tumor DNA (ctDNA) as an alternative to tumor biopsies for precision oncology: The Jules Bordet Institute experience.

Philippe G. Aftimos,1 Marion Maetens,1 Françoise Rothé,1 David Brown,1 Yacine Barèche,1 Alexandre Irrthum,2 Thierry Berghmans,1 Joseph Kerger,1 Alain Hendlisz,1 Michail Ignatiadis,1 Martine Piccart,1 Christos Sotiriou,1 Ahmad Awada1. 1 _Institut Jules Bordet - Université Libre de Bruxelles, Brussels, Belgium;_ 2 _Breast International Group Headquarters (BIG-aisbl), Brussels, Belgium_.

Background

In the era of precision medicine, data obtained from sequencing the cancer genome of a patient can be integrated into clinical decision-making. Liquid biopsy using ctDNA is a non-invasive tool, which can circumvent unfeasible or unrepresentative tumor biopsies and allow continuous monitoring of the disease thereby contributing to rational drug delivery.

Experimental procedures

Precision-f is a feasibility molecular screening study that enrolled 34 patients with metastatic melanoma, non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). Results from targeted gene sequencing (TGS) of metastatic biopsies were presented at AACR 2015. The patients had a blood draw concomitantly with the metastatic biopsy for plasma isolation. Similar to biopsies, somatic mutations (substitutions, small insertions and deletions) were assessed using the Ion AmpliSeq™ Cancer Hotspot Panel v2, designed to amplify 207 amplicons covering 2800 COSMIC mutations from the 50 most commonly reported oncogenes and tumor suppressor genes with a sensitivity of 1%. All mutations identified in the biopsy and/or plasma samples using TGS are currently being validated using Droplet Digital PCR (ddPCR) technology.

Results

Evaluable ctDNA-derived sequencing data were available for 32 patients. The sequencing results between biopsies and plasma were fully concordant in 63% (20/32) of the patients. TGS of ctDNA failed to detect clinically relevant mutations (EGFR, PIK3CA, KRAS and BRAF) identified in the metastatic biopsies in 19% (6/32) of the patients. In contrast, biologically relevant mutations (STK11, KRAS, PIK3CA and SMO) not detected in tumor biopsies were found in the ctDNA in 9% (3/32) of the patients. Interestingly, in one CRC patient a KRAS G13D mutation still potentially associated with benefit to anti-EGFR therapy was detected only in the tumor biopsy whereas a KRAS A146T mutation mediating resistance to anti-EGFR therapy was detected solely in ctDNA. Tumor biopsies and plasma are currently being tested using ddPCR to validate the findings. The results of these analyses will be presented at the meeting.

Conclusion

Although technical challenges remain, ctDNA profiling appears to be a promising tool for precision oncology by allowing the identification of genomic alterations associated with drug resistance and tumor progression.

#3963

Capture of EpCAM-negative mesothelioma cells with a "universal CTC-chip": Sensitivity test and comparison with EpCAM-based standard method.

Kazue Yoneda,1 Yasuhiro Chikaishi,1 Eri Kawashima,1 Takashi Ohnaga,2 Fumihiro Tanaka1. 1 _University of Occupational and Environmental Health, Kitakyushu, Japan;_ 2 _Toyama Industrial Technology Center, Takaoka, Japan_.

Backgrounds: Circulating tumor cells (CTCs), as a surrogate of distant metastasis, can be potentially useful in early diagnosis and monitoring therapeutic effects for patients with malignant tumors. Among a variety of systems for detection of CTCs, an epithelial cell adhesion molecule (EpCAM)-based immuno-magnetic separation system "CellSearch" is the only system approved for clinical use. Our previous study showed that CTC detected by CellSearch was useful in the diagnosis or the prognosis of epithelioid-type malignant pleural mesothelioma, however, the sensitivity was insufficient. Because EpCAM-negative tumor cells, such as those originating from non-epithelial cells and those undergoing epithelial-mesenchymal transition, cannot be captured using the "CellSearch". Therefore, we have developed a novel polymeric microfluidic device system "universal CTC-chip" that can capture a variety of CTCs with or without EpCAM expression (AACR 2015). In the present study, we assessed its sensitivity for capturing mesothelioma cells and compare with CellSearch.

Methods: PC-9, human lung cancer cell line and ACC-MESO-4, human mesothelioma cell line were used in this study. Antibodies were coated on the chip in two steps, first with anti-mouse IgG antibody ("base-antibody") and then as a "capture-antibody", either mouse anti-human EpCAM antibody to capture EpCAM-positive cells ("EpCAM chip") or mouse anti-human podoplanin antibody to capture podoplanin-positive mesothelioma cells ("podoplanin chip"). For sensitivity test, each cell line was suspended as concentration 500, 100, 50, 10cells/mL in phosphate buffered saline (PBS) or in blood. After 1ml of sample was flowed, we counted cells and calculated capture efficiency (number of capture cells /flowed cell). For comparison with CellSearch (CS), each cell line was suspended as concentration 50 or 10 cells /mL in blood and calculated detection efficiency (number of detected cells/ suspended cell).

Results: In sensitivity test, capture efficiency of PC-9 or ACC-MESO-4 was 101.1%, 90.7% (500 cells/mL), 94.8%, 95.9% (100 cells/mL), 104.7%, 97.9% (50 cells/mL), 114.3%, 113.75% (10 cells/mL), respectively in PBS. In the blood sample, capture efficiency of PC-9 or MESO-4 was 99.2%, 50% (50 cells/mL), 115.5%, 80.55% (10 cells/mL), respectively. On the other hand, detection efficiency of PC-9 (EpCAM chip, CS) was 110%, 121.3% at 50 cells/mL, 195%, 126% at 10 cells/mL in blood. In case of MESO-4, detection efficiency (podoplanin chip, CS) was 55%, 1.1% at 50 cells/mL, 70%, 0% at 10 cells/mL in blood.

Conclusion: The novel "universal CTC-chip" can be a promising CTC detection system.

#3964

Large scale noninvasive screening of EGFR mutations in advanced NSCLC patients: the Identify platform experience.

Clara de la Caridad Mayo de las Casas,1 Nùria Jordana Ariza,1 Mónica Garzón Ibañez,1 Ariadna Balada Bel,1 Jordi Bertrán-Alamillo,1 Ana Pérez Rosado,1 Santiago Viteri Ramirez,2 Daniela Morales Espinosa,2 Juan Carlos Monasterio,1 Niki Karachaliou,2 Miguel Ángel Molina-Vila,1 Rafael Rosell2. 1 _Hospital Quiron Dexeus, Barcelona, Spain;_ 2 _Instituto Oncológico Dr. Rosell, Barcelona, Spain_.

Background: Activating mutations in the epidermal growth factor receptor gene (EGFR) confer sensitivity to tyrosine kinase inhibitors (TKIs) in patients with advanced non-small-cell lung cancer (NSCLC). Starting in June 2012, a nationwide platform (the Identify Blood Platform) was implemented in Spain for large-scale screening of EGFR mutations at presentation in the peripheral blood of unselected, advanced NSCLC patients. The Platform was restricted to patients with no tumor biopsy available or those with insufficient tumor tissue for genetic analyses. In May 2015, the Platform was extended to analyze blood of EGFR-mutated patients progressing to TKIs.

Methods: Analysis of EGFR mutations was performed by a central laboratory using a sensitive multiplex TaqMan assay in the presence of a PNA clamp. Circulating free DNA (cfDNA) was isolated from both serum and plasma using an automatic extractor, and mutational analyses were performed in quadruplicates. Patients harbouring EGFR mutations were informed as eligible for TKI treatment.

Results: From June 2012 to November 2015, 765 NSCLC patients from 102 institutions in Spain were prospectively screened at presentation for EGFR mutations in peripheral blood. In five cases (0.7%), blood collected was insufficient for molecular analysis. Clinical characteristics of the remaining 760 evaluable patients were as follows: prevalence of male (58.55%), former smokers (48.16%) and adenocarcinoma (79.34%). Sensitizing mutations were found in 76 of 760 patients (10%) and were prevalent in females (61.84%), never smokers (59.21%) and adenocarcinomas (80.26%). Of the 76 EGFR-mutated patients at presentation, 48 (63.16%) were positive in both serum and plasma, 19 (25%) only in plasma, and nine (11.84%) in serum alone. Regarding the type of mutation, 50 (65.8%) had exon 19 deletions, the most common being 15 bp deletions; 25 (32.9%) had L858R substitution, while L861Q mutation was detected in one patient (1.3%). Additionally, the T790M resistance mutation in exon 20 was analyzed in 223 pretreatment samples and not detected in any (0%). Finally, blood of 84 patients progressing to TKI treatment with no biopsy available was screened and T790M detected in 28 patients (33.33%).

Conclusions: Our results demonstrate the feasibility of large-scale, nationwide screening of EGFR mutations in peripheral blood of NSCLC patients at presentation and after TKI progression. Mutational analysis in blood is useful to inform treatment decisions, particularly in patients with no biopsy available or after TKI progression

#3965

Rigorous validation of a clinical circulating tumor DNA assay for cancer molecular profiling.

Travis A. Clark,1 Mark Kennedy,1 Jie He,1 Geneva Young,1 Mandy Zhao,1 Mike Coyne,1 Virginia Breese,1 Lauren Young,1 Shan Zhong,1 Mark Bailey,1 Bernard Fendler,1 Erica Schleifman,2 Eric Peters,2 Phil J. Stephens,1 Geoff A. Otto,1 Doron Lipson1. 1 _Foundation Medicine, Inc, Cambridge, MA;_ 2 _Genentech, Inc, South San Francisco, CA_.

Background:

Profiling cell-free circulating tumor DNA (ctDNA) for genomic alterations which drive oncogenesis in patients with cancer promises to provide information important for understanding cancer biology, informing therapy selection when conventional FFPE biopsies are unobtainable and potentially monitoring response to therapy. To allow routine use of blood-based ctDNA molecular profiling with clinical samples we developed and performed analytic validation of an accurate, targeted NGS-based assay. The analytic validation included over 400 samples demonstrating ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with limit-of-detection below 1%.

Methods:

To ensure robust performance, the ctDNA assay was developed as part of an integrated workflow including sample collection, storage and transport, and ctDNA purification, followed by optimized construction of adaptor-ligated sequencing libraries and enrichment by solution hybridization and then sequencing to high depth (Illumina HiSeq). Computational methodologies were developed to enable sensitive and specific detection of base substitutions, indels, genomic rearrangements and high-level amplifications from ctDNA. Accuracy and reproducibility were analytically validated in a CLIA-certified laboratory using reference samples with known alterations (117 cell-line mixtures and synthetic constructs) and 268 clinical ctDNA samples. Many alterations found in clinical ctDNA samples were validated with orthogonal reference methods including a CLIA-validated NGS assay, droplet digital PCR and break-point PCR.

Results:

The ctDNA assay validation demonstrated ≥99% sensitivity and ≥99% positive predictive value for base substitutions, indels and rearrangements with a limit-of-detection below 1% and robust detection of high-level, focal amplifications when present at adequate tumor fraction. In addition, the assay accurately reports the allele frequency of alterations in the sample. In 48 clinical ctDNA samples, 95 alterations of all classes were 100% confirmed by orthogonal testing. As part of our extensive clinical utility study, we report results comparing alterations from patient-matched ctDNA and FFPE biopsies across more than 200 lung, breast and other cancer samples.

Conclusions:

Accurate clinical profiling of ctDNA enables detection of genomic alterations in patient plasma samples to provide rationale targeted therapeutic options. Our rigorous analytic validation study demonstrates high-sensitivity detection of alterations present in blood at low frequency with a very low rate of false positives, realizing the potential of ctDNA-based molecular profiling for the management of patients with cancer. This validated assay allows us to embark upon a rigorous investigation of clinical best-practices based on tumor-type specific assessment of matched ctDNA and solid biopsy specimens.

#3966

Effect of anticoagulant and sample storage conditions on circulating tumor cells enrichment by vortex.

Charles L. Wilkerson,1 Corinne M. Renier,1 Violet R. Hanft,2 Stefanie S. Jeffrey,2 Elodie Sollier1. 1 _Vortex Biosciences, Menlo Park, CA;_ 2 _Stanford University, Stanford, CA_.

Background

Circulating tumor cells (CTC) have recently emerged as powerful biomarkers for non-invasive diagnosis and prognosis of cancer [1]. The rarity and fragility of these cells has proven to be a significant impediment to their clinical use. Delay between phlebotomy and sample processing, together with blood sample storage, can impact the integrity of these cells and hamper their subsequent isolation. Addressing these issues could allow shipping between clinical facilities and significantly increase the use of CTCs. Here, we assessed the effects of various blood anticoagulants on the isolation of CTCs using Vortex technology. Vortex is a label-free, size-based technology that relies on the generation of laminar flow microvortices where CTCs can be trapped [2], and released off-chip, into a small volume compatible with various downstream assays.

Methods

To evaluate the effects of delayed processing on capture performances, samples from healthy donors were drawn into 4 different blood collection tubes (BCT): K3EDTA, ACD-B, CellSave and cell-free DNA Streck. Each BCT was spiked with MCF7 breast cancer cells and stored at room temperature for up to 3 days (72H). On days 0, 1, 2 and 3, aliquots of blood (0.5 ml; n=3) from each BCT were diluted 10X and processed through Vortex chip. Captured cells were stained (CK, CD45, DAPI) and enumerated. Capture efficiency and purity were determined for each time point and BCT. To simulate over-sea shipping and processing of patient samples, larger blood volumes (6mL in EDTA, CellSave and Streck BCT) were also tested after 48H. Capture performances and device clogging were evaluated.

Results

Significant differences were observed for capture performances between the BCTs. Best results were obtained for CellSave tube, with an efficiency of 19.8 ± 2.5%, stable for up to 3 days, and a purity ranging from 40.6 to 52.8%. In comparison, performances for standard K3EDTA were maximal at 24H, with 19.1% efficiency decreasing to 10% at 72H. ACD-B and Streck showed lower efficiencies, reaching 4.6 and 9.7% respectively. Purity for EDTA, CellSave and Streck BCT was in a similar range (48.4, 46.5 and 34.9% respectively). No significant device clogging was observed between EDTA, CellSave and Streck BCT when larger blood volumes were processed.

Conclusion

Here we demonstrate the ability to isolate cancer cells spiked in blood for up to 48H post-phlebotomy, with higher overall performances obtained with CellSave tube. Standard EDTA BCT gives optimal performances up to 24H and would be the option to select whenever viable cells are required. This suggests that Vortex technology is compatible with various anticoagulants and preservatives containing blood collection tube; the election would depend on the assay performed downstream. Future work will include shipping blood samples from remote sites and effect on CTC enrichment by Vortex technology.

[1] Ignatiadis et al. Clin Cancer Res 2015.

[2] Sollier et al. Lab Chip 2014.

#3967

Amplicon-based NGS detects targeted variants in paired tissue and ctDNA samples.

Adam H. Greer, Glenn Mills, Hong Yin. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Circulating tumor DNA (ctDNA) in blood stream has been recognized as an essential sample source for non-invasive biopsy of cancer patients. With next generation sequencing (NGS) technology it is possible to detect multiple cancer relevant mutation spots simultaneously. In order to understand the correlation of mutation spectrum between DNA from original solid tumor and DNA from plasma, we analyzed paired DNA samples with amplicon-based NGS. Paired frozen tissue and plasma samples were collected from 17 colon cancer patients under the IRB protocol. Tissue DNA was extracted with NucleoSpin Tissue kit of Clontech. The ctDNA was extracted from 0.5ml of plasma with Quick-cfDNA™ Serum & Plasma Kit from Zymo Research. The library was prepared with Accel-Amplicon 56G Oncology Panel of Swift Biosciences, Inc. This panel generates 263 amplicons covering 56 clinically relevant oncology-related genes. Input ctDNA from plasma was 0.5 to 10 ng depending on the sample yield. The sequencing was conducted on Illumina's Miseq with MiSeq Reagent Kits v2 (300cycles). FastQ files were generated by Miseq Reporter. Non-synonymous variant calls by the tissue DNA sequencing were defined by coverage depth (30), frequency (>5%), and forward/reverse balance (≠0). The defined non-synonymous variant calls were searched from variant calls by the paired ctDNA sequencing with the cutoff frequency at 0.1%. In 17 paired colon cancer samples, 86 non-synonymous variant calls were identified by tissue DNA sequencing. The variant calls of tissue DNA samples showed 56 of 86 variants matched variant calls in the paired ctDNA samples. Among 56 variant calls in ctDNA, 44 variant calls were assumed as germline variants according to the 1000 genome reference and similar frequency of the variant call between paired tissue DNA and ctDNA. There were 12 variant calls deemed somatic variant calls by the literature references and the differential frequency of the variant call between paired tissue DNA and ctDNA. The 12 detected somatic variant calls were clustered in seven cases. Twelve somatic variant calls with frequency (%) in tissue and plasma DNA samples are shown as APC S1456 frame shift (41.8 vs 6.17), KRAS G12D(37.0 vs 5.0),TP53 G105R(56.0 vs 11.3), HNF1A P291 frame shift(36.8 vs 2.41), HNF1A P291 frame shift(27.1 vs 1.2) KRAS G12D(23.1 vs 0.3), ERBB4 I377T(8.3 vs 0.5),PTEN L267 frame shift (12.4 vs 0.4), MAKP2K1 L57N(23.9 vs 0.7),TP53 R273C(25.8 vs 0.3), TP53 H178D(12.1 vs 1.1),TP53 T377 frame shift(11.2 vs 0.1). Remaining 30 somatic variant calls were detected only in tissue DNA samples. Our results indicated that the simultaneous detection of somatic variants of multiple genes in ctDNA samples with the amplicon-based DNA sequencing is applicable with variant frequency over 0.1%. The NGS analysis of paired tissue DNA and ctDNA is a reliable evaluation for cancer related mutations in ctDNA.

#3968

Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples.

Françoise Rothé, Ghizlane Rouas, Michelle Coureau, Marion Maetens, David Brown, Christos Sotiriou, Michail Ignatiadis. _Institut Jules Bordet, Brussels, Belgium_.

Background

Over the past years, tumor derived alterations in plasma has emerged as a promising cancer biomarker. The clinical applications envisioned with liquid biopsies range from monitoring of treatment response to disease progression and acquired resistance. However, the analysis of circulating tumor DNA (ctDNA) is challenging owing to the small fraction of tumor specific DNA relative to background levels of non-tumor derived DNA and therefore requires highly specific and sensitive techniques.

Experimental procedures

The Ion AmpliSeqTM Cancer Hotspot Panel v2, covering 2,800 COSMIC mutations from 50 cancer genes was used to analyze 31 primary tumor and metastatic lesions along with 19 matched plasma samples from 13 breast cancer (BC) patients receiving neoadjuvant treatment and 6 advanced stage metastatic BC patients. Digital PCR (dPCR) experiments were set up using mutation-specific assays (Biorad) for the detection of 13 hotspot mutations in PIK3CA, TP53, PTEN, AKT1, CDKN2A and STK11 genes previously identified in the solid tumor samples using next generation sequencing (NGS).

Results

The specificity and sensitivity of the mutation-specific dPCR assays were tested in dilution curves using synthetic oligonucleotides and tumor samples bearing the mutation of interest. Mutations could be robustly detected in plasma samples using dPCR with a sensitivity as low as 0.1% and a sensitivity of 0.5% using NGS. All mutations previously identified using NGS were validated using dPCR in tumor (n=27) as well as in plasma samples (n=5). Moreover, dPCR identified 4 additional mutations in 4 plasma samples in the neoadjuvant setting and 4 in the metastatic setting, 2 in tumor and 2 in plasma samples. The mutant allelic frequencies (MAF) of these newly identified mutations were very low (range 0.2%-1.8%) close to the detection limit of the technology. Through the use of dPCR, the detection rate of plasma ctDNA increased from 8% using NGS to 38% in the neoadjuvant setting and from 67% using NGS to 100% in the metastatic setting thereby highlighting the greater sensitivity of the dPCR technology. The MAF assessed by NGS and dPCR were highly correlated both in the neoadjuvant and metastatic (Spearman ρ=0.84 and ρ=0.97) settings, a higher concordance between NGS and dPCR being observed when the MAF are high.

Conclusions

The use of dPCR allows the detection of low-frequency tumor-specific mutations in plasma with a greater sensitivity compared to NGS providing the high accuracy and sensitivity essential for liquid biopsies.

#3969

CTCs and CTC clusters in breast cancer patient-derived xenograft models.

Debashish Sahay,1 Arturo B. Ramirez,2 Raksha R. Bhat,1 Lacey E. Dobrolecki,3 Agostina Nardone,3 Michael T. Lewis,3 C. Kent Osborne,3 Mothaffar Rimawi,3 Jackie L. Stilwell,2 Eric P. Kaldjian,2 Rachel Schiff,3 Meghana V. Trivedi1. 1 _University of Houston College of Pharmacy, Houston, TX;_ 2 _RareCyte, Seattle, WA;_ 3 _Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX_.

Breast cancer (BC) patient-derived xenograft (PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs). Using various BC PDX models, we describe the utility of CTCs and CTC clusters in detecting tumor-specific mutations and our preliminary results in understanding their predictive value for treatment response and long-term outcomes. CTCs were detected in 300-450 µl of blood of PDX-bearing mice using the RareCyte technology adapted for small blood volume. CTCs were isolated without cell surface marker-based enrichment and identified them as DAPI+, human Cytokeratin (CK)+, and mouse CD45-. Collective expression of cell surface markers (EpCAM, EGFR, and HER2) was assessed using a cocktail of target-specific antibodies in CTCs and primary PDX tumors. Individual CTCs and tumor cells from primary and metastatic tumors were isolated using CytePicker® for single cell analysis of tumor-specific mutations. Single CTCs (1-41 per mouse) and CTC clusters (1-2 per mouse) were detected in the blood of one ER+/PR+/HER2- (BCM-4888) and two triple-negative BC (TNBC, BCM-4272 and BCM-3887) PDX models. The PIK3CA T1035A mutation found in primary tumors of BCM-4888 was also detected in isolated CTCs and PDX primary and metastatic tumor cells. As a proof-of-principle experiment, we have evaluated numbers of single CTCs and CTC clusters at baseline and after treatment with 4 weekly cycles of vehicle (N=5-6) or chemotherapy regimens (N=2-3) [docetaxel or carboplatin or their combination] in TNBC PDX models BCM-4272 and BCM-3887. Preliminary analysis from these studies suggests dynamic and differential effects of chemotherapy regimens on single CTCs and CTC clusters, potentially reflecting the genetic characteristics of tumors and their unique response to the selected chemotherapy agents. Ongoing experiments in additional mice and PDX models will determine the predictive role of CTCs and CTC clusters in treatment response and long-term outcomes such as recurrence-free survival. In conclusion, we have demonstrated that RareCyte technology detects CTCs from small volumes of blood without the use of cell surface marker-based enrichment method. Furthermore, CTCs and CTC clusters can be used to assess the presence of tumor-specific mutations. Ongoing studies will fully reveal the potential of CTCs and CTC clusters as surrogate markers of treatment response and outcomes within PDX models.

#3970

Impact of endoscopic stent insertion on detection of viable circulating tumor cells for obstructing colon cancer.

Shinya Yamashita. _Nissei Hospital, Osaka, Japan_.

The obstructive colorectal cancer(CRC) was reported to be 7-20% of CRC. For the patients (pts) with obstructive colorectal cancer, self-expanding metallic stents (SEMS) have been useful as a palliative treatment. Insertion of SEMS for obstructive colorectal cancer is used as a bridge to surgery approach. But suspicion have been raised about whether shear forces that act on the tumor during expansion of the stent may result in a release of cancer cells into the circulation (i.e., CTCs). This study was conducted to clarify whether insertion of colonic stents results in increased levels of viable CTCs (v-CTCs). For detecting viable CTCs(v-CTCs) of CRC, we employed a novel method, TelomeScan F35 detection system. The system was constructed with a GFP-expressing attenuated adenovirus, in which the telomerase promoter regulates viral replication. 7.5 ml of peripheral blood samples were obtained before and after stent insertion and after operation. GFP-positive and CD45-negative cells were counted as v- CTCs. This study was a single institution trial, approved by the Kure Medical Center IRB. Surgical operation was scheduled within 1 month after stenting. Between October 2013 and June 2015,10 pts aged 58-89 years (5 males and 5 females) were enrolled. The surgical operation (R0) was performed in 9 cases. CEA was reduced to <50% of baseline value in 6 of 9 measurable pts after resection. 6 pts had no v-CTCs before stenting. Among them, 3 pts had increasing number of 1-3 v-CTCs after stenting and 1 of them developed multiple liver metastasis 12 month after resection. While, 4 pts had v-CTCs (1-19 cells) before stenting and 3 of these 4 patients had significant increase of number of v-CTCs (1-21 cells) after stent insertion. 2 of them had 12 and 14 v-CTCs, respectively after operation, despite the normalized value of CEA. The present study suggested that endoscopic stent insertion may result in tumor cell dissemination into the peripheral circulation and may induce early distant metastasis. In conclusion, v-CTCs may become useful for prognosis assessment, especially when SEMS insertion is considered.

### Novel Molecular Diagnostics and Imaging

#3971

Non-invasive assessment of prostate-specific membrane antigen (PSMA) expression as a prognostic marker in men with metastatic castration-resistant prostate cancer (mCRPC).

Kavya Pinto-Chengot, Yuliya Jhanwar, Jaspreet Batra, Beerinder Karir, Shankar Vallabhajosula, Paul Christos, Ana Molina, Himisha Beltran, David M. Nanus, Stanley J. Goldsmith, Neil H. Bander, Scott T. Tagawa. _Weill Cornell Medical College, New York, NY_.

Background: PSMA is nearly universally expressed by PC, has a correlation with androgen receptor (AR) pathway dysregulation, and some studies have pointed towards PSMA expression as a prognostic marker. Non-invasive measurement of PSMA expression may be biomarker of AR activity in animal models.[Evans et al, PNAS 2011] Anti-PSMA monoclonal antibody J591 has been utilized both for imaging as well as therapeutics and we have published semi-quantitative scoring in the setting of therapeutic clinical trials.

Methods: Patients with mCRPC underwent planar gamma camera imaging following radiolabeled (RL) J591 injection (177Lu- or 111In-J591). RL-J591 images were semi-quantitatively scored using a 5-point visual score (VS) system [Tagawa et al, Clin Cancer Res 2013]. Overall survival (OS) was calculated from the date of imaging to death or last follow up. As several life-prolonging treatments (docetaxel, sipuleucel-T, cabazitaxel, abiraterone (Abi), enzalutamide (Enza), radium-223) became available during the study time period, treatment following RL-J591 imaging was recorded and used in multivariate analysis.

Results: Between 2000 and 2015, 165 patients with mCRPC were imaged following RL-J591 and semi-quantitatively scored for PSMA expression by VS. Baseline demographics included median age of 71.3 years (44.5-85.9); bone metastases present in 86.7%, 52.7% with LN mets, 17% with lung mets, 7.9% with liver mets, 1.8 % other mets. Fifty three (32.2%) had low PSMA expression by imaging (VS 0-1) and 112 (67.8%) had high PSMA expression (VS 2-5). Post RL-J591 imaging, 41.2% (68/165) patients received life prolonging therapy, including taxane chemotherapy (33.9%), Abi/Enza (14.5%), sipuleucel-T (1.2%), or radium-223 (1.2%). At last follow up 12.7 % (21/165) patients were alive. Median OS was 22.6 months in patients with low PSMA expression and 16.6 mo with high PSMA expression (P=0.07). Those with subsequent receipt of life prolonging therapy had OS of 23.6 months vs 14.3 mo (P=0.002). On multivariable analysis controlling for life prolonging therapy, higher PSMA expression by imaging was a significant predictor for poorer OS (adjusted hazard ratio = 1.60; 95% CI = 1.10, 2.31; P=0.01).

Conclusion: Non-invasive measurement of PSMA expression may be evaluated on a semi-quantitative basis via planar imaging. The level of PSMA expression is inversely associated with survival when controlling for receipt of known life-prolonging therapy. With the introduction of newer quantitative molecular imaging (i.e. PET), quantitative PSMA imaging may prove to be a useful prognostic tool.

#3972

Total lesion glycolysis (TLG) as an imaging biomarker of regorafenib treatment in metastatic colorectal cancer (mCRC).

Yoojoo Lim,1 Ji-In Bang,1 Sae-Won Han,1 Jin Chul Paeng,1 Jeong Hee Yoon,1 Jeong Min Lee,1 Jae-Kyung Won,1 Gyeong Hoon Kang,1 Seung-Yong Jeong,1 Kyu Joo Park,1 Kyung-Hun Lee,1 Jee Hyun Kim,2 Tae-You Kim1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Seoul National University Bundang Hospital, Seong-Nam, Republic of Korea_.

Introduction: Regorafenib is an oral multikinase inhibitor with anti-angiogenic activity. Decrease in tumor density without significant decrease in tumor volume is often observed after treatment with the drug, suggesting the necessity of further means of response evaluation in addition to size criteria. This study was planned to evaluate whether metabolic imaging with 18-fluorodeoxyglucose (FDG) positron emission tomography (PET) could predict outcome in mCRC patient treated with regorafenib.

Methods: Patients were enrolled into a main study entitled Identification of Predictive Biomarker of Regorafenib in Refractory Colorectal Cancer: A Prospective Explorative Study (NCT01996969). mCRC patients (N = 117) were treated with regorafenib, 160mg orally once daily, on days 1-21 of a 28-day cycle, and treatment outcome was evaluated according to the Response Evaluation Criteria in Solid Tumors 1.1. For this imaging sub-study, the results of 18-FDG PET scans obtained at baseline and after 2 cycles of treatment were analyzed. Total lesion glycolysis (TLG) of each patient was calculated by multiplying the volume exceeding the threshold of 40% of maximum standardized uptake value (SUV) by the mean SUV of the lesion, and the percentage changes of TLG after treatment were calculated. The association between the changes of the metabolic imaging and treatment outcome was analyzed.

Results: A total of 44 patients were evaluable for 18-FDG PET scan results. The best overall response in the 44 patients were, partial response in 3 (6.8%), stable disease in 23 (52.3%) and progressive disease in 18 (40.9%). The median progression-free survival (PFS) and the median overall survival (OS) were 5.4 and 14.1 months, respectively. The median of TLG at baseline was 187.4 (range 18.3 to 8893.2). After 2 cycles of regorafenib, 50.0% of patients had a decrease in TLG. Median change in TLG was 1.7% (range, -75.7 to 251.6%). Changes in TLG were modestly associated with size changes (Pearson's r=0.51, p=0.001). Patients with lower baseline TLG (< 324.49) showed significantly longer OS (1 year survival rate 75.6 vs. 33.5%, p=0.008). Moreover, patients showing higher decrease in TLG (change < 1.7%) showed significantly longer PFS (median 8.4 vs. 1.9 months, p<0.001) and trend towards longer OS (1 year survival rate 67.6% vs. 50.9%, p=0.068).

Conclusions: Lower baseline TLG is associated with better OS and higher decrease in TLG after treatment predicted better PFS in mCRC patients treated with regorafenib.

#3973

Diffusion-weighted imaging of bone metastases as treatment response biomarker in prostate cancer.

Raquel Perez-Lopez,1 Matthew D. Blackledge,2 Helen Mossop,2 Joaquin Mateo,1 David Collins,2 Veronica A. Morgan,1 Alison McDonald,1 Shahneen Sandhu,1 Aurelius Omlin,1 Diletta Bianchini,1 Zafeiris Zafeiriou,1 Pasquale Rescigno,1 Michael Kolinsky,1 Daniel Nava Rodrigues,2 Penny Flohr,2 Berni Ebbs,2 Gemma Fowler,2 Nuria Porta,2 Emma Hall,2 Martin Leach,2 Johann S. de Bono,1 Dow-Mu Koh,1 Nina Tunariu1. 1 _The Institute of Cancer Research & The Royal Marsden NHS Foundation Trust, Sutton, United Kingdom; _2 _The Institute of Cancer Research, Sutton, United Kingdom_.

INTRODUCTION: Response assessment of bone metastases (BM) remains a challenge for drug development in metastatic castration resistant prostate cancer (mCRPC) as standard imaging techniques, computed tomography and bone scintigraphy, fail to characterize BM. Diffusion-weighted imaging (DWI) is a functional MRI technique that studies the motion of water molecules within a tissue informing on cellularity. We hypothesized that changes in whole body (WB) DWI informs on BM response to treatment in mCRPC.

METHODS: We conducted a phase II trial of the PARP inhibitor olaparib in mCRPC (TOPARP-A; CRUK/11/029); the primary endpoint was response rate defined using RECIST 1.1, PSA falls of ≥50% and conversion of circulating tumour cell (CTC) counts from ≥5 to <5. Optional WB-DWI in patients (pt) was performed prior to starting olaparib and after 12 weeks (w) of treatment on a 1.5 T MRI (Siemens Avanto). Images were analyzed with open-access imaging software (Osirix v5.6). Areas of DWI signal abnormality consistent with BM in the axial skeleton (C4 to mid-thigh) were delineated. We estimated disease volume and apparent diffusion coefficient (ADC), a parameter derived from DWI correlated with cellularity. BM volume and median ADC (mADC) changes between baseline and 12w were associated with binary response to treatment using Mann-Whitney tests and logistic regression; associations with best percentage PSA and CTC change after treatment were assessed using Spearman's correlation.

RESULTS: Overall, 33/42 pt consented to the WB-DWI substudy of whom 21 had WB-DWI at baseline and at 12w. Of these 29% (6/21) were classified as responders to olaparib as per the primary endpoint definition and had not progressed prior to 12w. At baseline, median CTC count was 46 CTC/7.5ml blood and median PSA was 411 ng/ml for this cohort. When assessing all the areas of DWI signal abnormality, median volume of BM per patient was 445ml and mADC was 782 x10-6 mm2/s. Baseline CTC counts and PSA were significantly associated with volume of BM (ρ=0.59, p=0.005; ρ=0.64, p=0.002 respectively). All pts who responded to olaparib showed a decrease in volume of BM (median -41.1%, range -58.8%, -6.3%), whilst in non-responders a decrease was not observed in any pt (median 20.7%, range 0.0%, 76.9%); the difference between responders and non-responders was statistically significant (p=0.001). Increases in mADC after 12 weeks of treatment were associated with increased odds of response (Odds Ratio: 1.08, 95% CI 1.00, 1.15, p=0.04). Additionally, we detected a significant positive association between changes in volume of BM estimated by DWI and best percentage change in PSA and CTC (ρ=0.63, p=0.002 and ρ=0.77, p<0.001 respectively).

CONCLUSION: Decrease in volume and increase in mADC of BM assessed by WB-DWI are indicators of response to olaparib in BM from mCRPC. These data require validation but support the use of WB-DWI for assessing BM during treatment.

#3974

18 **F-sodium fluoride PET/CT-guided dose escalation in stereotactic body radiotherapy for spine oligometastases from prostate cancer.**

Yu Kuang,1 Lili Wu,2 Sandi A. Kwee,3 Mei Li,2 Xun Peng,2 Liangxi Xie,2 Zhixiong Lin2. 1 _University of Nevada Las Vegas, Las Vegas, NV;_ 2 _Cancer Hospital of Shantou University Medical College, Shantou, China;_ 3 _The Queen's Medical Center, Honolulu, HI_.

Purpose: Recent clinical evidence has shown that SBRT using high dose with either a single fraction or a limited number of fractions can lead to excellent pain control as well as local tumor control in patients with spine oligometastases. However, as a dose limiting factor, proximity to spinal cord often precludes SBRT delivering the full prescription dose (PD) and/or escalating dose to the planning target volume (PTV) of spine oligometastases, thus compromising the therapeutic ratio. To investigate the technical feasibility of SBRT dose painting using ¹⁸F-NaF positron emission tomography (PET) scans guidance in patients with spine oligometastases from prostate cancer.

Methods: As a proof of concept, six patients with 15 spine oligometastatic lesions from prostate cancer who had ¹⁸F-NaF PET/CT scan prior to treatment were retrospectively included. GTVreg was delineated according to the regular tumor boundary shown on PET and/or CT images; and GTVMATV was contoured based on a net metabolically active tumor volume (MATV) defined by 60% of the SUVmax values on ¹⁸F-NaF PET images. The PTVs (PTVreg and PTVMATV) were defined as respective GTVs (plus involved entire vertebral body for PTVreg) with a 3-mm isotropic expansion margin. Three 1-fraction SBRT plans using VMAT technique along with 10 MV FFF beams (Plan24Gy, Plan24-27Gy, and Plan24-30Gy) were generated for each patient. All plans included a dose of 24 Gy prescribed to PTVreg. The Plan24-27Gy and Plan24-30Gy also included a simultaneous boost dose of 27 Gy or 30 Gy prescribed to the PTVMATV, respectively. The feasibility of 18F-NaF PET-guided SBRT dose escalation was evaluated by its ability to achieve the prescription dose objectives while adhering to organ-at-risk (OAR) dose constraints. The normal tissue complication probabilities (NTCP) calculated by radiological models were also compared between the plans.

Results: In all 33 SBRT plans generated, the planning objectives and dose constraints were met without exception. Plan24-27Gy and Plan24-30Gy had a significantly higher dose in PTVMATV than Plan24Gy (p < 0.05), respectively, while maintaining a similar OAR sparing profile and NTCP values.

Conclusion: The strategy of delivering differential doses using SBRT with a simultaneous integrated dose boost to MATV offers the option of irradiating host facilitating cells simultaneously with the cancer cells in the tumor ecosystem. Using VMAT with FFF beams to incorporate a simultaneous ¹⁸F-NaF PET-guided radiation boost dose up to 30 Gy into a SBRT plan is technically feasible. The relationship between local control and normal tissue toxicity in SBRT dose painting should be validated in clinical trials.

#3975

Matrix-depleting anti-hypertensives decompress tumor blood vessels and improve perfusion in patients with glioblastomas receiving anti-angiogenic therapy.

Kyrre E. Emblem,1 Elizabeth R. Gerstner,2 Gregory Sorensen,3 Bruce R. Rosen,2 Patrick Y. Wen,4 Tracy T. Batchelor,2 Rakesh K. Jain2. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Siemens Healthcare, Malvern, PA;_ 4 _Dana-Farber Cancer Institute, Boston, MA_.

Introduction: Impaired tumor perfusion is associated with poor drug delivery, hypoxia and metastatic behavior that effectively make cancers difficult to treat [1]. A main driver of impaired perfusion in permeable and impermeable tumor vessels is solid stress caused by proliferating tumor cells and the extracellular matrix that squeeze vessels shut [2]. Here, we demonstrate improved vascular function in glioblastoma patients who receive hypertension treatments with an anti-cancer cell, matrix-depleting effect as part of anti-angiogenic therapy.

Methods: We retrospectively assessed data from 40 patients with newly diagnosed glioblastomas (nGBM) and 30 with recurrent glioblastomas (rGBM) receiving cediranib, an oral pan-VEGFR inhibitor, with- (NCT00662506) and without (NCT00305656) chemoradiation, respectively. After cediranib onset, for nGBM, 12/40 patients received matrix-depleting angiotensin II receptor blockers (Valsartan), β1 receptor antagonists (Atenolol) or angiotensin-converting enzyme inhibitors (Lisinopril), while 8/40 patients received non-depleting calcium antagonists (Norvasc, Amlodipine) [3, 4]. For rGBM, 15/30 patients received Atenolol and 7/30 patients Norvasc. Perfusion and vessel caliber MRI were performed at baseline and repeated weekly (nGBM) or monthly (rGBM) [5, 6]. Tumor values were normalized to healthy tissue to account for patient-level systemic effects and variations in hypertension.

Results: In the total tumor bed (enhancing + edematous regions), the matrix-depleting alternatives significantly improved the perfused vessel fraction (% of tumor values >½ the value of healthy tissue) from 81% to 93% in nGBM (Wilcoxon: P<0.05) and from 70% to 78% in rGBM (P<0.05). In permeable tumor vessels (enhancing area), the increase in perfusion for nGBM was associated with a 19% higher fraction of small caliber vessels (<½ the size of healthy tissue) compared to baseline (P<0.01) and for rGBM a 20% higher fraction compared to cediranib alone (P<0.01). For matrix-depleting drugs only, the absolute number of small vessels increased relative to that of larger vessels, suggestive of vascular decompression of small caliber vessels by increased open lumen fractions. These effects were not observed with non-depleting calcium antagonists.

Conclusion: In line with theoretical [7] and pre-clinical [3] findings, we here present human data demonstrating that adding matrix-depleting anti-hypertensives, safe and inexpensive drugs with decades of use, improve vascular function in glioblastoma patients receiving anti-cancer therapy. Vascular MRI can help refine matrix-depleting drugs by directly assessing mechanisms of action in vivo.

References:

1: Science 2005; 307, 58-62

2: PNAS 2012; 109, 15101-8

3: Nat Commun 2013; 4, 2516

4: Blood Press 2006; 15, 198-206

5: PNAS 2013; 110, 19059-64

6: Nat Med 2013; 19, 1178-83

7: PNAS 2013;110:18632-37

#3976

Molecular imaging of epigenetic regulation mediated by HDACs 4, 5 using PET/CT/MRI with 18F-TFAHA in a rat model of human glioma.

Maxwell T. Laws,1 Robin E. Bonomi,2 Swatabdi Kamal,3 David Gelovani,3 Jeremy Llanguez,4 Vadim Popov,3 Xin Lu,2 Srinivasu Kallakuri,1 Thomas Mangner,3 Juri G. Gelovani2. 1 _Biomedical Engineering, Wayne State University, Detroit, MI;_ 2 _Karmanos Cancer Institute, Detroit, MI;_ 3 _Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 4 _School of Medicine, Wayne State University, Detroit, MI_.

Histone deacetylases (HDACs) are involved in the pathogenesis of cancer through modulation of the expression of various genes involved in cellular proliferation, migration, angiogenesis and apoptosis. HDAC class IIa enzymes interact with tumor suppressor proteins including HIF-1a, GATA-1, and PLZF-RARa. Given the importance of HDACs class IIa in epigenetic regulation of cancer development, progression, and maintenance, there is a pressing need for non-invasive imaging approaches for monitoring the expression-activity of HDACs in vivo. To address this need, our laboratory has developed 6-(tri-fluoroacetamido)-1-hexanoicanilide (18F-TFAHA) for imaging HDAC class IIa enzymes. Previous studies show that 18F-TFAHA is enzymatically cleaved specifically by HDAC class IIa, predominantly by HDACs 4 and 5. To quantitatively visualize the expression-activity of HDACs 4 and 5 in brain gliomas, immunocompromised rats (NTac:NIH-Foxn1rnu, Taconic Biosciences, NY) were implanted intracerebrally (i.c.) with U87-tdRluc cells (4x105 cells in 20 uL), that have been lentivirally-transfected to express GFP-luciferase and tdTomato fluorescent protein fusion reporter genes. Bioluminescence images (BLI) of GBMs were obtained after administration of luciferin (10 µl/g i.p.) using an InVivo Xtreme system (Carestream, Toronto, Canada) at 7 and 14 days post U87-tdRluc cell implantation. Gross tumor morphology and progression was evaluated with T2 MRI on day 14 after tumor implantation using a CliniScan 7T MRI system (Bruker, UK). Following 15-20 days, the rats were administered with 18F-TFAHA (500 µCi/animal, i.v.) and imaged using microPET R4 and Inveon CT (Siemens, TN). After PET/CT imaging, the animals were sacrificed and their brains extracted for immunohistochemical (IHC) and quantitative autoradiographic (QAR) studies (Typhoon 7000, General Electric, CT). The 18F-TFAHA accumulation in regions of interest (ROI) at 20-30 minutes post i.v. administration and was quantified using Logan graphical analysis, which demonstrated a significant increase in 18F-TFAHA accumulation in tumors versus surrounding normal cortex and white matter (p <0.05), outside the structures with normally-increased HDAC IIa activity (e.g., hippocampus, amygdala, periaqueductal gray, n. accumbens). PET/CT/MR imaging results were validated by IHC of brain tissue sections that demonstrated a significant hypoacetylation of histones H2A, H2B, and H4 in tumor tissue with increased 18F-TFAHA accumulation. The ongoing studies in brain tumor-bearing rats undergoing treatment with HDAC inhibitor vorinostat are aimed to assess the feasibility of PET/CT/MRI with 18F-TFAHA for pharmacodynamic monitoring therapies with HDAC class IIa inhibitors. Ultimately, we aim to translate 18F-TFAHA PET/CT/MR imaging into the clinic.

Support: NIH RO1 DA030333-06, NCI CCGS Core Grant CA 016672, NIH RC2 DA028912-01, NIH P30 CA022453

#3977

iKnife: Rapid evaporative ionization mass spectrometry (REIMS) enables real-time chemical analysis of the mucosal lipidome for diagnostic and prognostic use in colorectal cancer.

James Macalister Kinross, Laura Muirhead, James Alexander, Julia Balog, Cristina Guallar-Hoya, Abigail Speller, Ottmar Golff, Rob Goldin, Ara Darzi, Jeremy Nicholson, Zoltan Takats. _Imperial College London, London, United Kingdom_.

Background Real time electrospray ionization mass spectrometry (REIMS) enables detailed analysis of tumour lipid chemistry, based on real time analysis of electrocautery smoke plumes.

Methods: This was a prospective, observational study performed at St. Mary's Hospital, London, UK. Patients undergoing elective surgical resections for colorectal cancer were recruited and fresh samples were analyzed ex-vivo using a typical electrosurgery hand piece and monopolar diathermy. Sampling was performed using cutting mode with a standard generator and 30W of output power (ValleylabTM ). The hand piece was modified to allow aspiration of the electrosurgical aerosol to a Xevo G2-S iKnife QTof mass spectrometer (Waters Corporation). Raw mass spectrometric data were converted to imzML format (MSConvert) and imported into MATLAB (R2014a) for pre-processing. A prospective database of healthy, dysplastic and malignant colorectal tissues was built and multivariate analysis was performed using principal component analysis and linear discriminant analysis. Classification of each individual tissue type was performed using leave-one-patient-out cross-validation.

Results 40 consecutive patients were recruited (22 male, median age 68y, range 47-90). Of the 23 tumor samples 10 were rectal adenocarcinoma and 13 colonic adenocarcinoma. TNM staging of the tumour samples was as follows: T2 (8), T3 (11) T4 (4), N0 (12), N1 (6) N2 (5), M0 (22), M1 (1). Distinction of healthy and malignant colorectal tissue for the whole data set demonstrated an overall classification accuracy of 94.4% and a sensitivity of 92.4%, Specificity 96.8% (ROC AUC 0.98). The diagnostic accuracy for dysplasia was 93.7% (Specificity 95.1%, sensitivity 85.7%, AUC 0.97). Increases in glycerophospholipids (p<0.0027), and triacylglycerols (p<0.0004) were seen in healthy mucosa and increased prostaglandin D2 expression was found in malignant tissue (p< 0.0002). Adenocarcinoma arising in the rectum could be differentiated from more proximal tumors with an overall accuracy of 87.3%. Short and long course chemotherapy modified the lipid signature. Anatomically discrete chemical analysis of rectal cancers was able to provide accurate descriptions of established histopathological and molecular markers of poor prognosis: nodal status (AUC 0.97), KRAS mutation (AUC 0.93) and extramural vascular invasion (EMV) (AUC 0.96).

Conclusion REIMS chemical histology provides near real time diagnostic and prognostic information for stratifying oncological and surgical therapy.

#3978

Lipid tethering to enable real-time imaging of breast tumor cell cytoskeletal dynamics and rapid drug testing in free-floating metastatic microenvironments.

Kristi Riti Chakrabarti,1 James Andorko,2 Rebecca Whipple,1 Peipei Zhang,2 Elisabeth Sooklal,2 Christopher Jewell,2 Stuart Martin1. 1 _University of Maryland School of Medicine, Baltimore, MD;_ 2 _University of Maryland, College Park, College Park, MD_.

Free-floating tumor cells located in the peripheral circulation of cancer patients, known as circulating tumor cells (CTCs), play an important role in cancer and have become key targets for studying metastasis. The presence of CTCs in the bloodstream or lymphatics correlates with decreased cancer patient survival and provides a minimally invasive method to study disease progression and treatment response. Understanding the molecular characteristics and functional properties of CTCs have been impeded by the challenges of imaging CTCs under conditions that model the free-floating microenvironments of the circulation. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells within a microfluidic device while maintaining their free-floating character. We use cytophobic polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membrane. This approach enables high-resolution time-lapse microscopy of McTNs on viable free-floating tumor cells and real-time analysis of dynamic cellular features. In addition, tethering makes it now possible to rapidly measure drug responses of tumor cells in free-floating microenvironments to select effective therapies and avoid drugs that could inadvertently increase metastasis. The ability to image tumor cells in the absence of ECM attachment can immensely enhance our understanding of CTCs under conditions that better recapitulate the free-floating microenvironments that tumor cells encounter during metastasis.

#3979

Metabolic pathways associated with urinary metabolite biomarkers differentiate bladder cancer patients from healthy controls.

Won Tae Kim,1 Seok Joong Yun,1 Chunri Yan,1 Pildu Jeong,1 Ye Hwan Kim,1 Il-Seok Lee,1 Sunghyouk Park,2 Sung-Kwon Moon,3 Yung-Hyun Choi,4 Young Deuk Choi,5 Jayoung Kim,6 Wun-Jae Kim1. 1 _Chungbuk National University, Cheongju, Republic of Korea;_ 2 _Seoul National University, Seoul, Republic of Korea;_ 3 _Chung-Ang University, Seoul, Republic of Korea;_ 4 _Dongeui University, Pusan, Republic of Korea;_ 5 _Yonsei University, Seoul, Republic of Korea;_ 6 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Our high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA based on our previously identified urinary metabolome. Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n = 135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to adjacent normal bladder tissues (p < 0.05 all) of patients with non-muscle invasive bladder cancer, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive bladder cancer (p < 0.05), with no changes in IDO1 expression. In this study, alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, the most perturbed pathways in BCA, were determined. Our findings provide insight into the mechanisms underlying BCA-associated metabolic flux perturbation.

#3980

Combined detection of serum Dickkopf-1 and its autoantibodies to diagnose esophageal squamous cell carcinoma.

Yu-Hui Peng,1 Yi-Wei Xu,1 En-Min Li,2 Li-Yan Xu2. 1 _The Cancer Hospital of Shantou University Medical College, Shantou, China;_ 2 _Shantou University Medical College, Shantou, China_.

Esophageal squamous cell carcinoma (ESCC) can be treated effectively if diagnosed at an early stage. We evaluated whether measurement of Dickkopf-1 (DKK1) in combination of DKK1 autoantibodies in serum may benefit early diagnosis of ESCC. Serum DKK1 and DKK1 autoantibodies were measured by enzyme-linked immunosorbent assay in a training cohort (185 ESCC samples vs. 97 normal controls) and validated in a validation cohort (104 ESCC samples vs. 53 normal controls). Receiver operating characteristics (ROC) was applied to calculate diagnostic accuracy. The serum levels of DKK1 and DKK1 autoantibody were significantly higher in ESCC patients than in controls, respectively (P<0.0001). Measurement of serum DKK1 demonstrated an area under curve (AUC) of 0.709 [95% confidence interval (CI), 0.647-0.771], 37.3% sensitivity, and 90.7% specificity in the training set and an AUC of 0.697 (95% CI, 0.613-0.780), 41.3% sensitivity, and 84.9% specificity in the validation set. Testing of DKK1 and DKK1 autoantibodies together effectively improved the diagnostic accuracy for ESCC versus normal controls compared with either test alone (AUC 0.769, 95% CI 0.715-0.823, 50.3% sensitivity, and 90.7% specificity in the training cohort; AUC 0.752, 95% CI 0.675-0.829, 50.0% sensitivity, and 84.9% specificity in the validation cohort). Importantly, the diagnostic performance of the combination of DKK1 and DKK1 autoantibodies persisted in early ESCC patients (AUC 0.780, 95% CI, 0.699-0.862, 50.0% sensitivity, and 90.7% specificity in the training cohort; AUC 0.745, 95% CI, 0.626-0.865, 53.8% sensitivity, and 84.9% specificity in the validation cohort). Furthermore, the levels of serum DKK1 or DKK1 autoantibody after surgical resection were lower, respectively, compared with the corresponding preoperative samples (P<0.05). Our results suggest that measurement of DKK1 combined with DKK1 autoantibodies is a potentially valuable tool for the early detection of ESCC.

#3981

In situ **mass spectrometry using desorption electrospray ionization mass spectrometry (DESI-MS) can detect specific lipid profiles across breast cancer molecular subtypes.**

Victor P. Andrade,1 Adriana L. Santoro,1 Sabrina T. Moretti,1 Nicolas V. Schwab,2 Marcos N. Eberlin2. 1 _A.C. Camargo Cancer Center, Sao Paulo, Brazil;_ 2 _ThoMSon Mass Spectrometry Laboratory, UNICAMP, Campinas, Brazil_.

INTRODUCTION Lipid profiling as a source for biomarkers identification is an evolving field with some enthusiastic results in cancer. Alterations on lipid metabolism have been increasingly recognized as a hallmark of cancer cells and may help to understand breast cancer carcinogenesis. Analytical chemistry and Pathology have been recently combined in the development of chemical microscopy using in situ mass spectrometry (MS) and may help to decipher breast cancer molecular initiation and progression. OBJECTIVE We aimed to use in situ Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) to investigate lipid profiles across invasive breast cancer (IBC) molecular subtypes and related normal parenchyma (NBP) and ductal carcinoma in situ (DCIS). METHODS: We selected frozen samples from 9 IBC with various ER/PR/HER2 status (ER 0 to 100%, PR 0 to 90%, HER2 0 and 3+, Ki67 10 to 70%). Unstained slides obtained from frozen tissue at 10µm were submitted to DESI-MS analysis with no pretreatment and spatial resolution of 100µm. Molecular profiles (mass to charge [m/z] ratio versus signal intensity) were generated in triplicate directly from tissues to detect changes from areas of interest: IBC (and adjacent normal breast parenchyma and DCIS whenever present) regions were identified by a breast pathologist on HE-stained sequential slides. Experiment was conducted in full-scan mode and m/z range of 100-1200. DESI mass spectra were obtained using a Thermo ScientificTM Q ExactiveTM Hybrid Quadrupole Orbitrap Mass Spectrometer in the positive ion mode. The Biomap software was used to visualize the images and to export the spectrums to metaboanalyst, where the data were statiscally analyzed. RESULTS: The images obtained by lipid profiling presented a direct correlation with HE stained slides, with some ions m/z clearly overlaying areas of normal parenchyma, DCIS and invasive breast cancer. The unsupervised hierarchical clustering analysis showed all technical triplicates remained grouped. All NBP but one clustered together. Triple negative IBC and its related NBP formed a distinct cluster from all other ER+ samples. The ER+/HER2 + tumor segregated from other ER+/HER2- tumors. DCIS and IBC from ER+/PR+/HER2- tumor clustered together. The Ions with m/z 186.9 and m/z 303.2 were consistently related to NBP and IBC respectively. CONCLUSIONS: Chemical microscopy using DESI-MS is a powerful tool to identify specific lipid profiles across molecular subtypes of IBC and can contribute to understand breast cancer carcinogenesis.

#3982

Prediction and selection of cancer drug treatments using personalized tumor models or models with matching genomic profiles.

Jingjing Jiang,1 Ying Yan,1 Zhongguang Luo,2 Jia He,3 Tengfei Yu,1 Wei Du,1 Xuqin Yang,1 Jiali Gu,1 Xin K. Ye,1 Guanglei Zhuang,4 Jie Liu,2 Zhenyu Gu1. 1 _GenenDesign, Shanghai, China;_ 2 _Huashan Hospital, Shanghai, China;_ 3 _Peking Union Medical College Hospital, Beijing, China;_ 4 _Renji Hospital, Shanghai Jiao Tong University, Shanghai, China_.

The main purpose of precision medicine is to find the right drug for the right patient at the right time. Current progress in cancer drug target identification and development of new targeted drugs has presented many successful examples. However, for majority of patients, finding treatments based on the unique mutations in their own tumor cells are still hard. New approaches for precision medicine are in great needs and thus being investigated.

A mouse "avatar", also known as Patient-Derived Xenograft (PDX) model, is a personalized cancer model derived from a patient tumor sample and used to test different drugs for the patient. "GIFTS" (or Genomic Information Fitting based Therapeutics Selection) is a method with which a patient's cancer genomic information is compared to PDX model genomic profiles to find the best genomic fit and related therapeutic options in GenenDesign Drug Response Database and Genomic Information database.

So far we have successfully derived more than 1000 PDX models from patient tumor tissues of various cancer types including lung, gastric, liver, esophageal, colorectal, pancreatic and many other cancers. In some cases, both patients and their avatars were tested with same cancer drugs including targeted drugs and chemotherapies. By comparing drug responses in mouse avatars with patient clinical results, we found high correlations between them in both sensitivity and unresponsiveness.

We have also developed a bioinformatics algorithm to analyze PDX model genomic and drug response information. Both drug sensitivity and resistance biomarkers have been used in matching cancer patient genomic profiles to those of PDX models in GenenDesign database as a part of our GIFTS method. Our preliminary results show that there are high degree of similarities in drug response profiles between patients and their matched PDX models.

Avatar and GIFTS methods can provide predictions of therapeutic effectiveness on both targeted drugs and chemotherapies. Avatar is a drug screening based method, while GIFTS is a genomic profile matching based method. Both methods are especially useful for patients, whose tumors could not be treated with known targeted drugs.

#3983

Highly sensitive detection of MYB-NFIB fusion transcripts in adenoid cystic carcinoma.

Piotr T. Wysocki,1 Shizhang Ling,1 Chunbo Shao,1 Marietta Tan,1 David Sidransky,1 Patrick Ha,2 Mariana Brait1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University of California, San Francisco, San Francisco, CA_.

In recent years, the understanding of genomic events underlying head and neck cancer has significantly advanced. Several of the newly discovered genomic aberrations observed in salivary gland neoplasms were found to be recurrent and uniquely associated with specific disease entities. Adenoid cystic carcinoma (ACC), one of the most common salivary gland malignancies, demonstrates a particular challenge in head and neck oncology due its relentless growth, high local recurrence rate and poor long-term prognosis. Recent identification of the MYB-NFIB gene fusion as a recurrent and pathognomonic feature of ACC created new opportunities for the molecular diagnostics of this malignancy. Liquid biopsy techniques coupled with droplet digital PCR (ddPCR) platform may be feasible aiming for non-invasive detection of disease-specific genetic markers such as gene fusions. Given the prevalence of MYB-NFIB transcript in ACC, detection of the fusion event in patients' saliva may present a unique opportunity for an accurate diagnostics as well as for long-term patient monitoring. Our goal is to develop a highly-sensitive ddPCR-based assay that would precisely detect and identify MYB-NFIB transcripts in tumor tissue and patients' bodily fluids. RNA sequencing (RNAseq) performed in fresh frozen ACC samples identified MYB-NFIB fusion in 56% (10/18) of the cases and revealed large variability in MYB gene break sites and alternative splicing of NFIB component. 84% of ACC cases demonstrated high expression of 5' MYB fragment by ddPCR based RT-PCR, 62.5% of which were MYB-NFIB fusion positive in RNAseq. We developed a panel of RT-PCR assays to detect a range of different MYB-NFIB transcripts observed by our RNAseq data and/or previously reported. We identified all ACC cases harboring the most commonly reported MYB-NFIB fusions (9/9) and validated the fusion products with conventional Sanger sequencing. Additionally, our preliminary results indicate that the assays are feasible for PCR multiplexing retaining the high sensitivity and specificity observed in singleplex experiments. Evaluation of the assay performance in ddPCR and assessing the detection limit is currently in progress. Once the methodology is validated, we will be able to evaluate a cohort of salivary gland neoplasms and matched RNA samples isolated from saliva. The detection of MYB-NFIB fusion in bodily fluids using a highly sensitive ddPCR platform will open a new avenue for the diagnosis and the clinical management of patients with ACC. Furthermore, our work creates the possibility of a liquid biopsy-based detection of other disease-specific gene fusions increasingly identified in different solid tumors.

#3984

Novel t(X;21)(q26;q22) detected in a case of acute unclassifiable leukemia by application of anchored multiplex PCR-based next-generation sequencing.

Laura Johnson,1 Helen Wang,1 Katelyn Trifilo,1 Brian Kudlow,1 Peter R. Pappenhausen,2 Mohammad Hussaini3. 1 _ArcherDx, Boulder, CO;_ 2 _Labcorp, Durham, NC;_ 3 _Moffitt Cancer Center, Tampa, FL_.

In this study, we demonstrate real-time clinical application of novel anchored multiplex PCR (AMP) technology in hematologic disease. A 78-year-old man with a history of hairy cell leukemia treated with Cladribine (2013) and bladder cancer (2014) developed dyspnea in August 2015. Work up revealed bicytopenia and leukocytosis. Outside bone marrow biopsy was read as B lymphoblastic leukemia/lymphoma. Rebiopsy was performed at our NCI-designated cancer center with extensive work up resulted in reclassification as acute unclassifiable leukemia (rare) with deferral to molecular diagnostics for guidance regarding classification. MLL, BCR-ABL, and MDS FISH panel were negative. Next generation sequencing (NGS) interrogating more than 400 genes showed FLT3-ITD, truncation of RUNX1 exon 6, ASXL1 p.G646Wfs*12, MLL p.V297L, and EZH2 p.V674M and p.C443Y mutations. The molecular profile favored therapy-related AML. Interestingly, cytogenetics revealed 46,Y,t(X;21)(q26;q22)[20]. FISH confirmed RUNX1 rearrangement but the partner was unknown. The t(X;21)(q26;q22) appears to be novel. However, the rare t(3;21)(q26.2;q22) has been cited to segregate with therapy-related MDS/AML. Subsequent aberrant activation of RUNX1 and MECOM is postulated to be pivotal in pathogenesis. Therefore, it is of great interest to determine the translocation partner associated with RUNX1 in our case. This type of analysis is not easily amenable to either Sanger or conventional NGS approaches. Fortuitously, with the recent advent of AMP technology (Archer FusionPlex) novel fusion detection has become possible. AMP was key in the characterization of this sample and promotes discovery of novel fusions by targeting one (known) of the two genes involved in a translocation event. Application of this cutting edge technology lead to the discovery of the novel fusion RUNX1-G6PD in this case. The identity of the novel fusion partners was supported by the breakpoints originally observed by the karyotype analysis. G6PD has been previously reported to be upregulated in acute leukemia. This discovery raises the possibility convergent pathogenic pathways and carries potential biological, diagnostic, and therapeutic implications.

#3985

**First case of B LAL with** MLL - MAML2 **rearrangement.**

Fabrice Usseglio, Nathalie Beaufils, Régis Costello, Jean Gabert. _APHM, Marseille, France_.

Over 70 MLL partner genes have been reported so far. MLL gene (or 11q23) translocation is occurring in 7-10% of B acute lymphoblastic leukemias and AF4 being the most frequent partner gene. The prognostic is usually bad, specially for AF4 partner. A rare partner gene, mastermind like 2 (MAML2) gene has been reported in 4 cases of myeloid neoplasms after chemotherapy so far: 2 AML and 2 MDS. Here we report a case with MLL - MAML2 discovered by NGS analysis in front of an inv11 (q21q23) present in a 47 years old female previously treated for a sarcoma in 2014. It is, to our knowledge, the first case of B acute lymphoblastic leukemia (CD79a, CD22, CD19, CD45, CD10-) with this fusion gene. At the molecular level, the rearrangement consists in the fusion of exons 7 and 2 of the MLL and MAML2 genes respectively. We will discuss the biological, clinical, physiological and therapeutic consequences of this rare event. Its prognostic impact is unknown as the reported cases are too rare.

#3986

Diagnostic significance of cathepsin L and cathepsin B expression in human gallbladder cancer - A pilot study.

SIDDHARTH MEHRA, MANISH KUMAR, RAJESH PANWAR, NIHAR RANJAN DASH, RATNAKAR SINGH, RAJNI YADAV, SIDDHARTHA DATTA GUPTA, PEUSH SAHNI, SHYAM S. CHAUHAN. _ALL INDIA INSTITUTE OF MEDICAL SCIENCES, NEW DELHI, India_.

Background: Gallbladder cancer (GBC) is the most common biliary tract malignancy worldwide. Cancer of gallbladder usually refers to as an Indian disease as it is more common in Indian subcontinent especially in north and central India. Incidence rate of GBC in women in Delhi is as high as 21.5 per 100,000 population. It is an aggressive malignancy, which spreads locally to liver, adjacent organs and lymph nodes as well as to distant organs. Cathepsin L (CTSL) and B (CTSB) are lysosomal cysteine proteases implicated in protein turnover and tissue remodeling. Deregulated expression of these proteases has been associated with invasion and metastasis in several solid tumors.

Objective: To assess the expression and clinical significance of CTSL and CTSB in human gallbladder cancer.

Methods: We assayed the activities of CTSL and CTSB via spectroflurometrically in whole tissue lysates of gallbladder cancer patients (N=23) and normal gall bladder tissue samples with gallstones (N=31) / without gallstones (N=34), which served as controls. Activities of these proteases were correlated with several clinic pathological parameters in GBC patients. CTSL and CTSB mRNA levels were determined in tissues samples by Real time PCR. Furthermore serum levels of these proteases were also assayed using sandwich based ELISA in GBC patients and controls.

Results: Significant increase in the enzymatic activities of CTSL+B, CTSL and CTSB was observed in gallbladder cancer tissue samples in comparison to normal gall bladder both with and without gallstones (p < 0.001). ROC analysis confirmed the diagnostic relevance of CTSL and CTSB analysis with area under the curve 0.791 and 0.840 respectively for gallbladder cancer patients with respect to controls. Over expression of CTSB was also significantly associated with involvement of lymph node (p=0.05) in gall bladder carcinoma. Secretory levels of CTSL and CTSB in serum were significantly elevated in GBC patients (p <0.01). Increased expression of these cysteine proteases were also confirmed at the mRNA level by Real time PCR.

Conclusion: Our study for the first time demonstrated a significant increase in the levels of cathepsin L and B in GBC patients as compared to controls. Elevated levels of these proteases may serve as potential biomarkers for human gall bladder cancer.

#3987

Prospective serum metabolomic profiles of prostate cancer by size and extent of primary tumor.

Demetrius Albanes,1 Stephanie J. Weinstein,1 Alison M. Mondul2. 1 _U.S. National Cancer Institute, Bethesda, MD;_ 2 _University of Michigan School of Public Health, Ann Arbor, MI_.

Background: Recent studies have identified serum metabolites related to aggressive prostate cancer up to 20 years prior to diagnosis. Lipid and energy metabolites were significantly lower in cases vs. controls, including glycerophospholipids, inositol-1-phosphate, alpha-ketoglutarate, and citrate. To elucidate whether the findings represent an etiologic process or biomarkers of subclinical disease, we focused on differences in the prospective serum metabolomic profiles of men with T2, T3, and T4 prostate cancers.

Methods: Clinical data from a nested case-control analysis of prostate cancer in the ATBC Study cohort were examined, including 71 cases diagnosed with T2 primary cancer, 51 with T3, and 15 with T4 (all cases, ages 61-89 years at diagnosis). Median time from fasting serum collection to diagnosis was 10 yr, range 1-20 yr. Cases in each extent of disease category were compared to a common set of 200 non-case controls. Serum samples were analyzed on a high resolution accurate mass platform of ultrahigh performance liquid chromatography/mass spectroscopy and gas chromatography/mass spectroscopy (Metabolon, Inc.), and 654 known metabolites were identified. Unconditional logistic regression models estimated odds ratios (OR) and 95% confidence intervals (CI) for cancer associated with a one standard deviation (1-SD) increment in metabolite concentration.

Results: Among the strongest serum metabolite signals for men with T2 prostate cancers were N-acetyl-3-methylhistidine, 2-deoxyuridine, 3-methylhistidine, beta-hydroxyisovalerate, stearoylarachidonoyl-GPPE, and euricoyl-sphingomyelin [1-SD ORs 1.79, 1.71, 1.38, 1.48, 0.73, and 1.48 (p=0.0002, 0.005, 0.01, 0.02, 0.02, and 0.02), respectively]. Men diagnosed with T3 cancer exhibited a lipid metabolite profile most similar to that previously reported for aggressive disease, with top metabolite signals being for oleoyl-linoleoyl-GPPI, palmitoyl-linoleoyl-GPPI, cholate, inositol-1-phosphate, stearoyl-sphingomyelin, and 4-imidazoleacetate (ORs 0.49, 0.56, 0.57, 0.60, 1.54, and 1.59 (p=0.00002, 0.001, 0.002, 0.003, 0.01, and 0.01), respectively). Men with more extensive T4 cancers showed secondary bile acid and caffeine metabolite associations including orotate, taurodeoxycholate, glycodeoxycholate, caffeine, 4-imidazoleacetate, 1,3,7-trimethylurate, and deoxycholate (ORs 0.40, 2.62, 3.19, 2.40, 2.52, 2.31, and 3.31 (p=0.004, 0.007, 0.007, 0.01, 0.01, 0.02, and 0.02), respectively).

Conclusions: In this analysis, men with T2, T3, and T4 prostate cancer primaries exhibited qualitative differences in their serum metabolite profiles years in advance of the clinical diagnoses. Whether these differences represent novel biomarkers related to greater primary tumor mass that individual patients might "transition" through while still undiagnosed is unknown but testable through additional clinical series.

#3988

Detection of novel t(12;17)(p12;p13) in treatment-refractory/relapsed acute myeloid leukemia by anchored multiplex PCR(AMP)-based next-generation sequencing.

Laura Johnson,1 Katelyn Trifilo,1 Helen Wang,1 Brian Kudlow,1 Eric Padron,2 Peter R. Pappenhausen,3 Mohammad Hussaini2. 1 _ArcherDx, Boulder, CO;_ 2 _Moffitt Cancer Center, Tampa, FL;_ 3 _Labcorp, Durham, NC_.

Gene fusions are an important class of mutations and have been shown to drive many genetic diseases. While several technologies can be used to detect fusions, anchored multiplex PCR (AMP) next generation sequencing (NGS)-based detection offers the advantages of novel fusion detection and the ability to multiplex multitudinous genes. Here we report application of this technology in the evaluation of a 56-year-old man diagnosed with AML (2013). He was treated initially with Vidaza and later with CLAG-M induction (2014) with complete response and consolidation CLA (2015). He relapsed (4/2015) and was reinduced with 7+3 (Ida). He relapsed again and was reinduced with FCT but bone marrow biopsy showed persistent disease (75% blasts). Karyotype showed 46, XY, t(12;17)(p12;p13)[20]. It was postulated that ETV6 oncogene was involved based on 12p13 locus. The partner gene was unknown in this patient with multiply relapsed/refractory disease. AMP technology (Archer FusionPlex) confirmed involvement of the expected ETV6. However, more importantly, employing this technology lead to the discovery of a novel fusion partner, HIC1. The identity of the novel fusion partners was supported by the breakpoints originally observed by karyotype analysis. AMP promotes discovery of novel fusions by targeting one (known) of two genes involved in the fusion event. HIC1 regulates cellular growth and serves as a tumor suppressor gene. Inactivation of HIC1 has been associated with aggressive disease and poor survival in other cancer types. Interestingly, in vitro restoration of gene function by 5-aza-dC resulted in decreased cell proliferation and tumor aggressiveness in pancreatic cancer and head and neck squamous cell carcinoma suggesting the prospect of a therapeutic target. This discovery provides new insight into a possible leukemogenic pathway in AML and potential for targeted therapy.

#3989

Analysis of minute amounts of clinical biospecimen for the AXL receptor expression using real-time PCR and Simple Western size technology.

Florian T. Unger, Kristina Bernoth, Nicole Lange, Hartmut Juhl, Kerstin A. David. _Indivumed GMBH, Hamburg, Germany_.

Targeted therapy in personalized medicine is often affected by resistance mechanisms, such as activating mutations of signaling molecules and signaling pathway bypasses. It has become obvious that targeted therapy of patients has to be monitored by taking biopsies on a regular basis. Therefore, laboratory methods have to be optimized to be able to handle minute amounts of biospecimen, e.g. biopsies. In this study we are presenting the development of a Simple Western Size assay and a quantitative Real-time PCR assay for the analysis of AXL protein and gene expression in biopsies.

AXL, a tyrosine kinase receptor, is expressed in a variety of cancers and the most highly expressed gene in preclinical models with acquired resistance, and second most common alteration in EGFR (epidermal growth factor receptor) inhibitor-resistant tumors, behind the T790M mutation. Up-regulation of AXL is shown to be predictive for lack of response to ErbB family receptor-targeted inhibitors, e.g. to Her2-targeted agents. Protein expression was examined by Simple Western Size technology and gene expression was analyzed using quantitative Real-time PCR. For assay development, high and low AXL expressing breast cancer cell lines were used to establish "fit for purpose" assays. Subsequently, Her2-positive and Her2-negative clinical samples from breast cancer patients were analyzed by screening AXL gene and protein expression.

In this study, we developed and validated an assay for the detection of AXL protein expression using the Simple Western technology. This highly sensitive technology enables high-throughput screening of extremely small sample amounts such as biopsies or laser capture microdissected material. Therefore, the assay is well-suited for the monitoring of patients by using biopsies. Our results showed a broad linear dynamic range in both, high and low AXL expressing cell lines as well as a high reproducibility between multiple runs. The protein loading range suitable to detect AXL within whole cell lysates showed a lower limit of detection of 10 ng of total protein. AXL protein expression results were highly comparable to AXL gene expression results in analyzed breast cancer cell lines and breast cancer patient samples. This newly developed assay will allow us to analyze and quantify AXL protein expression profiles in breast cancer tissue and subsequently correlate them with e.g. Her-2 expression status.

The elucidation of networks and mechanisms underlying drug resistance will greatly improve the development of new drugs and promote personalized therapy. In order to gain scientific knowledge regarding gene and protein expression of resistance relevant marker, e.g. AXL, it is mandatory to establish assays that are very robust, sensitive and furthermore, if not most important, applicable to amounts of biospecimen being collected in the clinical setting.

#3990

FGFR3 mutations as novel oncogenic targets.

Gabriela Martinez Cardona,1 Dana Gaffney,1 Katherine Bell,1 Joseph Portale,1 Matthew Dunworth,2 Matthew Lorenzi,1 Suso Platero,1 Jayaprakash Karkera1. 1 _Janssen Research & Development, Spring House, PA; _2 _Gettysburg College, Gettysburg, PA_.

Fibroblast growth factors (FGFs) are a family of homologous secreted glycoproteins involved in signaling pathways responsible for embryonic development, cell proliferation, survival, and migration. FGF activity is mediated by four transmembrane fibroblast growth factor receptors (FGFRs) which are receptor tyrosine kinases. Typically, FGF binding induces FGFR dimerization, leading to phosphorylation of the intracellular tyrosine kinase domain. This leads to downstream activation of multiple signaling pathways, including the mitogen-activated protein kinase (MAPK), PI3K/AKT, signal transducer and activator of transcription (STAT), and phospholipase-C-γ cascades. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Aberrations in FGFR genes have been observed in several tumor types including bladder, gastric, colorectal, ovarian, and hematologic cancers. To date, several FGFR3 mutations have been identified in bladder cancer. In this study, we developed a TaqMan qRT-PCR-based approach to detect four FGFR3 mutations in formalin-fixed paraffin embedded tissue (FFPET) samples. Cell lines overexpressing FGFR3 mutations were generated to determine impact on cell signaling, and sensitivity to the small-molecule pan-FGFR inhibitor JNJ-42756493. To determine the role and significance of these FGFR3 mutations in cancer, mutation expression constructs were designed and individually transfected into normal rat kidney epithelial cells. Cells harboring the FGFR3 mutations exhibited anchorage-independent growth, increased proliferation, and showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro compared to parental lines. These findings underline the oncogenic potential of the FGFR3 mutation genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy.

#3991

Differentiating esophageal cancer cells from normal cells using ligand-conjugated microspheres.

Mahboubeh S. Noori, Sarah J. Bodle, Grady E. Carlson, David S. Drozek, Monica M. Burdick, Douglas J. Goetz. _Ohio University, Athens, OH_.

Cancer of the esophagus has a dismal overall prognosis and low 5 year survival rate due to its aggressive nature and the fact that it often presents at a late stage. Biochemical changes present on transforming tissue provide an opportunity for the early detection of cancer within the esophagus and thus the promise of a more favorable prognosis and a higher survival rate. Recently, there has been an increasing effort to detect cancer of the esophagus by introducing, during an endoscopic procedure, soluble molecules (ligands) cognate to moieties preferentially expressed on transforming tissue. The success of this approach depends on the selective binding of the ligand to transforming tissue relative to normal tissue. For soluble ligands, the factors that dictate the selective binding depend on a very small number of factors. In contrast, if the ligands are conjugated to particles, there are a large number of controllable factors that can be manipulated to "engineer" the detection scheme and thus optimize selective recognition of transforming tissue.

In this study, we utilized an in vitro system to investigate the feasibility of the ligand-conjugated particle approach. First, we explored the surface chemistry of an esophageal adenocarcinoma cell line, OE19, relative to a normal esophageal cell line, HEEpiC, using flow cytometric analysis. Among other differences, we found that the OE19 cell line expresses relatively high levels of the tetrasaccharides sialyl Lewis A (sLea) and sialyl Lewis X (sLex). sLea and sLex are known cognate molecules for the selectin family of adhesion molecules, in particular E-selectin. Thus, we conjugated an E-selectin construct to 10 μm diameter microspheres. The E-selectin construct consisted of the extracellular domain of E-selectin fused to the Fc domain of IgG. Flow cytometric analysis revealed that the E-selectin construct was conjugated to the microspheres and that the E-selectin portion of the molecule was available for binding. To roughly simulate the introduction of the conjugated microspheres during an endoscopic procedure, a parallel plate flow chamber was used. A planar substrate of either OE19 or HEEpiC cells was placed in the flow chamber and a suspension of E-selectin or IgG (negative control) microspheres were perfused through the flow chamber. We observed that the E-selectin microspheres exhibited significantly greater adhesion to the OE19 cells relative to the HEEpiC cells. In contrast, IgG microspheres exhibited negligible adhesion to the OE19 and HEEpiC cells. Combined, this study provides proof of concept for an assay approach that could be engineered to detect transforming tissue present within the esophagus.

## IMMUNOLOGY:

### Immune Modulation from Non-Immunotherapy: Preclinical

#3992

Allogeneic TCRαβ-deficient CAR T-cells targeting CD123 effectively eliminate myeloid leukemia cells in vitro and in vivo PDX mice.

Monica L. Guzman,1 Hongliang Zong,1 Mayumi Sugita,1 Luis A. Lara-Martinez,1 Laura M. Bystrom,1 Nicole M. Cruz,1 Roman Galetto,2 Agnès Gouble,2 Céline Lebuhotel,2 Alexander Bank,3 Julianne Smith,2 Gail J. Roboz1. 1 _Weill Cornell Medical College of Cornell University, New York, NY;_ 2 _Cellectis SA, Paris, France;_ 3 _Cellectis Inc, New York, NY_.

Acute myeloid leukemia (AML) is incurable in the majority of patients. While allogeneic stem cell transplantation remains the most effective therapy for AML to date, other types of cellular therapy have not yet been successful in this disease. The success of autologous T-cells expressing chimeric antigen receptors (CARs) in patients with advanced B cell leukemia and lymphomas has encouraged the investigation of CAR technology for the treatment of AML by targeting distinct tumor-specific antigens. We have developed an allogeneic CAR-T cell platform using T-cells from third-party healthy donors to generate T-cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed on blasts, leukemic progenitor and leukemic stem cells from the majority of patients with acute myeloid leukemia (AML).

Transcription Activator-Like Effector Nuclease (TALEN) gene-editing technology was used to inactivate the TCRα constant (TRAC) gene, eliminating the potential for engineered T-cells to cause graft versus host disease (GvHD). Using leukemia cell lines in both in vitro and in vivo models, we confirmed that TCR-deficient T-cells expressing an anti-CD123 CAR display significant antitumor activity.

We next evaluated the in vitro cytotoxic activity of TCR KO CD123-CAR T-cells (UCART123) in primary AML (n=6) samples and normal cells (normal bone and umbilical cord blood; n=3) using T-cell:AML cell ratios of 5:1, 2:1 and 0.5:1; and T-cell:normal cell ratio of 10:1 and 1:1. Degranulation and IFNγ release assays revealed potent activation of UCART123 cells when exposed to CD123 leukemia cells but not to normal hematopoietic cells. Cytotoxicity of UCART123 cells was observed as early as 4 hours upon initiation of the co-cultures. However, at 24 hours, more than 80% of leukemic cells (blasts, progenitors and stem cells) were eliminated at all ratios tested, whereas only approximately 20% CAR-independent cell death was observed with the TCR-deficient T-cells. Normal hematopoietic cells showed an average of 30% cell death when exposed to either UCART123 or TCR-deficient T-cells, demonstrating the selectivity of CD123 CAR-T cells toward leukemic cells.

Finally, we evaluated the in vivo activity of the CAR-T cells against established patient derived xenografts (PDX) using AML and normal CB CD34+ cells. We found no significant difference between PBS, TCR-deficient (10e6/mouse) and CAR T-cell treatments (10 e6/mouse) in PDX mice transplanted with normal CD34+ CB. Strikingly, 14 days of treatment eliminated most of the leukemic cells from the AML-PDX mice. T-cells were still detected at day 14 after treatment with UCART123 cells (mean 52% CD123 CAR and mean 1.5% TCR-), without evidence of GVHD. Efforts are underway to develop allogeneic CD123 CAR-T cells for clinical trials in AML.

#3993

Th1 cytokines regulate apoptotic cell death and HER family RTK expression in murine and human breast cancer lines.

Prachi M. Namjoshi,1 Lori Showalter,1 Brian Czerniecki,2 Gary K. Koski1. 1 _Kent State University, Kent, OH;_ 2 _University of Pennsylvania, Philadelphia, PA_.

A recent neoadjuvant vaccine trial to treat early breast cancer demonstrated powerful induction of Th1 immunity against the HER-2, complete pathologic responses in over 18% of subjects, and for many subjects, evidence of down-regulated HER-2 expression on residual disease. To explain these observations, we investigated the action of archetypical Th1 cytokines (TNF-α + IFN-γ) on both murine and human breast cancer cell lines that varied in the surface expression of HER-family receptor tyrosine kinases. We found that most tumor cell lines were sensitive to dual Th1 cytokines as evidenced by lower metabolic activity (alamar blue assay), lower proliferation, and enhanced apoptosis (AnnexinV/PI staining and TUNEL assay) as well as a reversible inhibition of surface expression of HER proteins. Apoptotic cell death was accompanied by demonstrated increases in activated caspase-3. Furthermore, the pharmacologic caspase-3 activator, procaspase-activating compound (PAC-1), mimicked both the killing effects and HER-2 suppressive activities of Th1 cytokines, while the caspase 3/7 inhibitor ((5-[(S)-(+)-2-(Methoxymethyl)pyrrolidino]sulfonylisatin), prevented cytokine-induced HER-2 downregulation. These studies therefore demonstrated that many of the in vivo effects of vaccination (apparent tumor cell death and loss of HER-2 expression) could be replicated in vitro using only the principle Th1 cytokines. These findings are consistent with the notion that IFN-γ and TNF-α work in concert to mediate some of the biological effects of therapeutic Th1-polarizing vaccination through the induction of a caspase 3-dependent cellular death mechanism.

#3994

Immune competent syngeneic models demonstrate additive effects of combination strategies using checkpoint immunotherapy and inducers of immunogenic cell death (ICD).

Andrew Mckenzie,1 Rajendra Kumari,1 Qian Shi,2 Nektaria Papadopoulou,1 Yinfei Yin,1 Simon Jiang,1 Jane Wrigley,1 Jason King,1 Neil Williams,3 Russell Garland3. 1 _Crown Bioscience UK Ltd, Loughborough, United Kingdom;_ 2 _Crown Bioscience Inc, USA, CA;_ 3 _KWS Biotest, Bristol, United Kingdom_.

INTRODUCTION: Recent progress in the field of cancer immunotherapy have made it possible to translate several emerging immunostimulatory strategies, e.g. anti-CTLA-4, and anti-PD-1 into the clinic resulting in promising clinical benefits. In addition, a number of treatment strategies such as radiotherapy (RT) oncolytic viruses and certain chemotherapeutic agents e.g. Doxorubicin, Bortezomib and Mitoxantrone have been highlighted as potential inducers of immunogenic cell death through a mechanism resulting in the increased presentation of cell-associated antigens to CD4+ and CD8+ T lymphocytes by dendritic cells. Thus combination strategies of ICD inducers with immunotherapy (IT) could provide opportunities to harness the immune system to extend survival, even among metastatic and heavily pre-treated cancer patients, and may increase the efficacy of immunotherapy in those cancer types to be of a low immunogenic status.

Here we compare the efficacy of immune checkpoint inhibitors in combination with documented ICD inducers to demonstrate an additive combination outcome in preclinical syngeneic models.

EXPERIMENTAL PROCEDURES: Bioluminescent CT26 mouse colon cells, 4T1 mammary carcinoma cells or H22 hepatoma cells were implanted subcutaneously or orthotopically into BALB/c mice. Subcutaneous tumour growth was monitored by calliper measurement and bioluminescent imaging (BLI) was carried out to confirm orthotopic and/or metastatic growth. Established tumours were treated with immunotherapy in combination with chemotherapy, or hypofractionated image-guided micro-irradiation (IGMI) using the small animal research platform (SARRP; Xstrahl Ltd; body weight and clinical condition of mice were monitored daily. At termination the tumours were collected and assessed for immune cell infiltration and/or ICD markers by FACS and IHC.

RESULTS: Response to treatment was evaluated by tumour growth inhibition or BLI following treatment of monotherapy or combinations of immunotherapy and ICD inducers (Oxaliplatin, Doxorubicin, and IGMI). Monotherapy with anti-CTLA4 exerted no statistically significant effect on primary or metastatic (4T1) tumour growth whereas ICD such as IR resulted in a modest tumour growth inhibition (TGI); When combined significant additive effect was observed (60% increase in TGI) on the primary tumour and reduction in tumour burden in the lungs indicating an abscopal effect. Details of modulation of immune cell infiltration and ICD markers observed in all models will be reported and correlated to response.

CONCLUSIONS: Combination of immune checkpoint immunotherapy with a known ICD inducer (IGMI) resulted in an additive TGI in both CT26 and 4T1 models and effectively demonstrates their applicability for further exploring combination strategies involving immunotherapy.

#3995

**pVAC-Seq: A genome-guided** in silico **approach to identify tumor neoantigens for personalized immunotherapy.**

Jasreet Hundal,1 Beatriz M. Carreno,2 Allegra A. Petti,1 Gerald P. Linette,2 Obi L. Griffith,1 Malachi Griffith,1 Elaine R. Mardis1. 1 _McDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO;_ 2 _Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO_.

Despite the recent surge in high-throughput sequencing of cancer genomes, the challenge of translating these data into clinically actionable information remains. One promising approach involves the identification of tumor-specific mutant antigens ('TSMAs' or neoantigens) via massively parallel sequencing and analysis of matched cancer and normal samples that can be used to create personalized vaccines. In the past, this effort has primarily focused on targeting selection of 'shared' tumor antigens, found across many patients. Here, we advocate a more 'personalized' approach.

These unique antigenic markers or TSMAs arise from numerous genetic changes, acquired somatically that are present exclusively in tumor (mutant) and not in normal (wild- type) cells. Vaccines incorporate these short, antigen-derived peptides (called epitopes) that aim to enhance the immune system's anti-tumor activity by selectively increasing the frequency of anti-tumor specific CD8+ T-cell antigens, and hence expand the ability of the immune system to recognize and destroy cancerous cells.

Selecting the best/most immunogenic epitopes from a large number of mutations is an important challenge, in particular in cases of high mutational load such as melanoma and lung cancer. To address this need, we have developed an in silico based sequence analysis method for identification and subsequent refinement of patient-specific antigens for use in personalized vaccines. This flexible and streamlined computational workflow for identification of personalized variant antigens by cancer sequencing (pVAC-Seq) integrates tumor mutation and expression data (DNA- and RNA-Seq) to shortlist candidate neoantigen peptides for a personalized vaccine. Harnessing existing class I prediction algorithms, high-affinity neoantigens over varying peptide lengths are evaluated.

To demonstrate the workings of pVAC-Seq, we applied it to four metastatic melanoma patients, the clinical results for three of whom were described previously. These patients were enrolled in a phase 1 vaccine clinical trial employing autologous, functionally mature, interleukin (IL)-12p70-producing dendritic cells (DC). Since melanoma patients harbor hundreds of mutations, it can be challenging to filter down and target the best set of potentially immunogenic neoantigens for vaccine design. By implementing the methods developed in pVAC-Seq, we were able to rapidly streamline the screening and identification of a smaller number of potentially immunogenic neoepitopes within the landscape of all neoepitopes.

#3996

KA2237 and KA2507: Novel, oral cancer immunotherapeutics targeting PI3K-p110β/p110δ and HDAC6 with single-agent and combination activity.

Stephen J. Shuttleworth. _Karus Therapeutics Ltd, Abingdon, Oxfordshire, United Kingdom_.

Two de novo-designed classes of orally-active immunotherapeutics - selectively targeting the PI3K-p110β/p110δ and HDAC6 enzymes - for solid and hematological cancer treatment will be described. The former, exemplified by the clinical candidate KA2237, are uniquely-selective inhibitors of the PI3K-p110β/p110δ isoforms, displaying immunotherapeutic activity and inhibiting primary tumor growth and metastasis. The latter, for which KA2507 is the candidate compound, are HDAC6-specific inhibitors, which inhibit tumor growth through regulation of aggresome formation, and inhibition of PD-L1 expression via decrease of STAT3 phosphorylation. In both cases, selectivity has been achieved over other enzyme superfamily members, with the aim being to minimize mechanism-related toxicity, thereby potentially providing opportunities to enable elevated single dose levels - and broad combination modalities not possible with pan-inhibitors of both enzymes - to be investigated. The PI3K-p110β/p110δ and HDAC6 inhibitors display potent oral activity as single agents in in vivo tumor syngeneic models, impacting on growth inhibition, PD and tumor marker responses; the PI3K-p110β/p110δ inhibitors also inhibit metastatic spread and, following surgical removal of primary tumor, confer promising inhibition of tumor regrowth. Crucially, the inhibitors work in combination with one another, making this co-therapy approach unique in oncology, and potentially important clinically in overcoming resistance mechanisms in both solid and hematological cancer. The rationale for this combination is based upon immunotherapeutic and cell signalling mechanisms, which will be described. KA2237 is entering Phase I studies in patients with lymphoma as a single agent in Q1 2016, with KA2507 scheduled to follow in Q3 to treat multiple myeloma. Further clinical investigations into solid tumor immunotherapy - both single agent- and combination-studies - are planned.

#3997

The impact of PD-L1 expression in patients with metastatic GEP-NETs.

Seung T. Kim,1 Won Lee2. 1 _Samsung Medical Center, Seoul, Republic of Korea;_ 2 _Gil Medical Center, Gachon University, Seoul, Republic of Korea_.

Programmed death-ligand 1 (PD-L1), which is expressed on many cancer cells, interacts with PD1 expressed on the surface of T cells, inhibiting the T cells and blocking the antitumor immune response. Expression of PD-L1 in gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has not been studied. We investigated the impact of PD-L1 expression in 32 patients with metastatic GEP-NET.

The expression of PD-L1 was evaluated using an anti-PD-L1 immunohistochemistry (IHC) antibody optimized for staining of formalin-fixed paraffin-embedded (FFPE) tissue samples. The correlation between PD-L1 and clinicopathological data including survival and response to systemic treatments was analyzed.

Primary sites were 24 foregut-derived GEP-NETs, including stomach (n=1), duodenum (n=2), biliary tract (n=7), and pancreas (n=14), and 8 hindgut-derived GEP-NETs of the distal colon and rectum. Among the 32 patients with metastatic GEP-NET analyzed in this study, 7 (21.9%) had expression of PD-L1 in tumor tissues. Expression of PD-L1 was significantly associated with high-grade WHO classification (grade 3) (p=0.008) but not with gender, primary site, and number of metastatic sites (p>0.05). The status of PD-L1 expression was statistically associated with progression-free survival (PFS) for first-line systemic treatment (p=0.047). Moreover, the status of PD-L1 expression could significantly predict overall survival (p=0.037).

The expression of PD-L1 was associated with higher WHO tumor grade (grade 3) in metastatic GEP-NETs. PD-L1 expression had both predictive and prognostic value for survival of patients with metastatic GEP-NETs.

#3998

Sequencing the B-cell and T-cell repertoire.

Fiona J. Stewart,1 Mehmet Karaca,1 Kris Adams,2 Chris Clouser,2 Bonny Patel,2 Sonia Timberlake,2 William Donahue,2 Lynne Apone,1 Salvatore Russello,1 Eileen T. Dimalanta,1 Theodore B. Davis,1 Francois Vigneault2. 1 _New England Biolabs, Inc., Ipswich, MA;_ 2 _AbVitro, Inc., Boston, MA_.

Immune sequencing allows for the study of complex immunological diseases by sequencing millions of V(D)J combinations from B-cell antibody and T-cell receptors. The popularity of this technique has increased due to recent throughput and read length improvements in next-generation sequencing technologies. However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging.

We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique identifier (UID) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. This makes the assay extremely accurate, by resolving PCR bias and sequencing errors as well as allowing quantitative digital molecule counting.

Immune sequencing libraries were generated from total RNA extracted from Peripheral Blood Mononuclear Cells in duplicate from a single patient. The use of UIDs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance, by avoiding the bias incorporated by PCR or sequencing when total reads only were measured. Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance. Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire.

#3999

Altering the balance between immune activation versus regulation in the skin to promote CD8 T-cell activity within epithelial cancers.

Jennifer A. Bridge, Nana H. Overgaard, Raymond J. Steptoe, Ian H. Frazer, James W. Wells. _The University of Queensland, Woolloongabba, Australia_.

The Human Papilloma Virus (HPV) 16 is a high-risk HPV known to be a causative agent in numerous cancers including cervical cancer. While prophylactic vaccines exist to combat the spread of HPV16, successful therapeutic vaccines to combat established HPV16-associcated disease remain elusive. The expression, in a mouse model ("E7"), of the HPV16 E7 gene in keratinocytes under the control of the K14 promoter, leads to a local immune suppressive environment, as evidenced by the lack of graft rejection when E7 skin grafts are placed on WT recipient mice. Furthermore, well healed (>30 days) E7 skin grafts are not rejected when mice are immunised with E7 peptide in combination with Quil A- or CASAC-based adjuvants. This is despite a substantial increase in E7 peptide/H-2Db pentamer staining in the blood, and marked killing of E7-peptide expressing TC-1 cells when injected i.v., confirming that CD8 T-cells respond to vaccination and differentiate into CTL capable of killing E7-expressing target cells. We hypothesised that the removal of regulatory T-cells (T-reg) might lead to E7 graft rejection in immunised mice. The co-administration of an anti-CD4-depeting antibody at the time of immunisation led to rejection of ~50% of grafts. To confirm a role for T-reg, E7-grafted T-reg-deficient Rag1-/- mice received purified donor CD8 T-cells from E7-vaccinated WT mice. FACS staining of Rag1-/- lymph nodes 30 days post CD8+ T-cell transfer confirmed the absence of classical CD4+FoxP3+ Treg, however the E7 grafts did not reject. As in the WT mice however, rejection could be induced through the co-administration of an anti-CD4 antibody. The data suggest that the removal of a CD4+, non T-reg cell, leads to CD8+ T-cell activity in the skin as evidenced by E7 skin graft destruction.

#4000

A DNA damage response deficiency (DDRD) group in breast cancer is associated with activation of the STING innate immune pathway and PD-L1 expression.

Eileen E. Parkes,1 Steven M. Walker,2 Nuala McCabe,1 Laura E. Taggart,1 Laura Hill,2 Niamh E. Buckley,1 Kienan I. Savage,1 Manuel Salto-Tellez,1 Stephen McQuaid,1 Mary T. Harte,1 Paul B. Mullan,1 D Paul Harkin,1 Richard D. Kennedy1. 1 _Queens University Belfast, Belfast, United Kingdom;_ 2 _Almac Diagnostics, Craigavon, United Kingdom_.

We have previously identified a DNA damage response deficient (DDRD) subgroup in breast cancer, associated with loss of the Fanconi Anemia/BRCA DNA repair pathway. The 44-gene DDRD signature developed to prospectively identify this subgroup has been validated as predictive of response to DNA damaging chemotherapy in the treatment of breast cancer. This subgroup is defined by immune signalling, with immune genes, such as the chemokines CXCL10 and CCL5, and immune checkpoints IDO1 and PD-L1 (CD274) overexpressed within the signature and subgroup. Here we report innate immune pathway activation in this subgroup, and association with PD-L1 expression.

Methods: We used isogenic cell line models to identify and model pathways constitutively active in DDRD cancer cells. CD4+, CD8+ T-cell infiltration and PD-L1 expression was identified by immunohistochemistry (IHC) on a previously described breast TMA of 191 breast tumor samples, of which 65 were in the DDRD subgroup. DNA microarray data from FFPE samples was used to identify other immune checkpoints associated with this subgroup.

Results: CD4+ and CD8+ T-cell infiltration, and PD-L1 expression were significantly associated with DDRD breast tumors on IHC analysis. In addition, expression of immune checkpoints such as IDO1, TIM-3 and LAG-3 were associated with the DDRD subgroup. Increased migration of peripheral blood mononuclear cells was identified into media conditioned by DDRD cells, this was dependent on chemokines secreted by DDRD cells. Endogenous activation of the innate immune pathway STING/TBK1/IRF3 was identified in DDRD cancer cells, and required for expression of chemokines CXCL10 and CCL5. This chemokine expression was associated with cytosolic DNA detected by cGAS and activation of the innate immune pathway STING/TBK1/IRF3. Importantly, this pathway was cell cycle related with upregulation of chemokine expression in S-phase of the cell cycle. Chemokine expression could be induced by S-phase DNA damaging chemotherapy but not by taxanes. Similarly, PD-L1 was induced by treatment with DNA damaging agents in cancer cells dependent on STING.

Conclusions: We have identified constitutive activation of the innate immune STING pathway in a DNA damage response deficient subgroup of breast cancer, which is associated with CD4+, CD8+ infiltration and PD-L1 expression. We propose activation of the STING pathway as an important signal for lymphocytic infiltration, independent of tumor-associated neoantigens. The DDRD signature could identify patients with innate immune activation in response to endogenous DNA damage, which may allow stratification for immune checkpoint targeted treatments. Moreover, S-phase DNA damaging agents activate PD-L1 expression, and a combination approach of immune checkpoint inhibitors and S-phase specific chemotherapy may be synergistic in the clinic.

#4001

The anti-CD47 antibody Hu5F9-G4 activates macrophages and inhibits ovarian cancer xenografts, alone and in combination with chemotherapy or immunotherapy.

Rishil J. Kathawala, David C. Mundy, George E. Duran, Jens-Peter Volkmer, Kevin S. Kao, Yan C. Wang, Aditi Bellary, Irving L. Weissman, Branimir I. Sikic. _Stanford University, Stanford, CA_.

Macrophage activation by inhibition of CD47 signaling is a new therapeutic approach to cancers. Binding of CD47 to its receptor SIRPα on macrophages initiates a signal cascade that inhibits phagocytosis. Blocking CD47 from interaction with SIRPα by the CD47 antibody Hu5F9-G4 results in phagocytosis of cancer cells, and anticancer activity in xenografts of human cancers. We developed taxane resistant variants from human ovarian carcinoma (OC) cell lines (ES-2, MES-OV and OVCAR-3) by step-wise exposure to paclitaxel (Ptx) and the transport inhibitor valspodar (6x resistance in ES-2/TP, 9x in MES-OV/TP, and 30x in OVCAR-3/TP). These 6 lines were transduced to establish stable luciferase expression for OC xenograft (OCX) imaging. All express CD47 by immunoblotting and flow cytometry, and all express calreticulin (CRT). CRT is a prophagocytic signal that promotes phagocytosis in the presence of CD47 blockade. CRT levels are lower in the Ptx-resistant variants (p<0.01). We are assessing the efficacy of Hu5F9-G4: (1) as a single agent in drug sensitive and resistant OCX; (2) combined with Ptx and carboplatin (Carbo); and (3) combined with cetuximab (Cetux) or trastuzumab (Trastuz) in OCX that express EGFR and/or HER2. We found that EGFR and HER2 are highly expressed in the ovarian cancer TCGA database. All 6 OC lines express EGFR by immunoblotting, and 4 express HER2. Blocking CD47 in vitro induces phagocytosis of parental and drug-resistant OC cell lines by murine macrophages derived from NOD/SCIDgamma (NSG) mice. Hu5F9-G4 has single agent activity in MES-OV OCX, with 89% growth inhibition at 9 weeks (T/C 11%) vs untreated controls (p=0.03). Ptx alone produced a T/C of 29%, and the combination of Ptx/Hu5F9-G4 3% (p=0.03 vs Hu5F9-G4; p=0.0002 vs Ptx). The Carbo experiment was terminated at 5 weeks because of toxicity in the Carbo groups, but at that time point the T/C for Carbo was 59%, Hu5F9-G4 20%, and the antibody combination/carboplatin combination 4% (p<0.005 vs either single agent). In the combined antibody experiment, Hu5F9-G4 T/C was 23%, Cetux 84%, Trastuz 37%, Hu5F9-G4/Cetux 13%, and Hu5F9-G4/Trastuz 12% (p=0.05 vs Hu5F9-G4 alone). Thus, Hu5F9-G4 has substantial efficacy in MES-OV OCX, and at least additive effects with chemotherapy (Ptx and Carbo) and immunotherapy (Cetux and Trastuz). Experiments with other xenografts are continuing. These results support CD47 as a therapeutic target in OC, and suggest that it may be successfully combined with chemotherapies and other antibodies.

#4002

Enhancing the antitumor efficacy of immunotherapy by using the topoisomerase I inhibitor MM398.

Jodi A. McKenzie, Rina M. Mbofung, Shruti Malu, Rodabe N. Amaria, Richard E. Davis, Li Zhang, Trang N. Tieu, Tim P. Heffernan, Patrick Hwu. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Melanoma is a highly aggressive form of skin cancer, whose rates of morbidity and mortality are increasing. The development of immunotherapies like anti-PDL1 and anti-CTLA4 antibodies has resulted in fundamental advances in the treatment of some cancers. However, long lasting responses are only observed in a subset of immunotherapy-treated patients. This shortfall highlights the need for a better understanding of the molecular mechanisms that govern tumor response to immunotherapy.

To address this need, autologous patient-derived tumor cell lines and tumor infiltrating lymphocytes (TILs) were utilized in an in vitro high throughput screen, to identify compounds that increase the sensitivity of melanoma cells to T cell mediated cytotoxicity. The screen consisted of an 850 compound library. One group of compounds that was most able to enhance T cell killing of melanoma cells was topoisomerase I (Top1) inhibitors such as topotecan and irinotecan.

Our results indicate that treatment of melanoma cells with a Top1 inhibitor prior to exposure to autologous T cells produced a synergistic increase in tumor cell death, as measured by intracellular staining of activated caspase 3. We have also recapitulated this finding in an in vivo model, where a better anti-tumor effect was observed in tumor bearing mice treated with an antibody against the co-inhibitory molecule PDL1 in combination with MM398 (nanoliposomal irinotecan), than in cohorts treated with either α-PDL1 or Top1 inhibitor alone. These findings suggest synergism between Top1 inhibitors and immune-based therapies in the treatment of melanoma.

Molecular changes elicited by inhibition of Top1 are now being investigated to identify the factors that mediate the effect of Top1 inhibitors on T cell-mediated killing of melanoma. We have identified a p53-driven gene signature in Top1 inhibitor-treated melanoma cell lines and are investigating the functional relevance of Tumor Protein p53 Inducible Nuclear Protein 1 (TP53INP1) in mediating increased T cell killing of Top1 inhibitor-treated melanoma cells. Our results indicate that TP53INP1 is a critical component of this apoptotic response, as overexpression of TP53INP1 in melanoma cells increased their susceptibility to T cell mediated cytotoxicity. Complementary to this observation, we have also found that knockdown of TP53INP1 by shRNA, impedes the sensitivity of Top1 inhibitor-treated melanoma cells to T cell mediated killing.

Understanding how Top1 inhibitors enhance melanoma killing by immunotherapy will allow for the development of predictive biomarkers, and also augment immune-based therapeutic strategies to ensure durable responses in a larger population of melanoma patients. By using melanoma as a model disease system, we can gain valuable insights into the dynamics of cancer immune response that may be applied to other cancers where effective treatment strategies are also lacking.

#4003

Metformin reduces intratumoral CD8+PD-1+ and Treg lymphocytes in orthotopic models of breast cancer and lymphoma, and has paradoxic effects on anti-PD-L1 treatment.

Stefania Orecchioni, Giovanna Talarico, Patrizia Mancuso, Valentina Labanca, Francesca Reggiani, Francesco Bertolini. _European Inst. of Oncology, Milan, Italy_.

Epidemiology studies have indicated that the administration of Metformin, a drug commonly used in the therapy of type 2 diabetes, is associated with reduced incidence and severity of several types of cancers. Interestingly, in most reports the effects of Metformin were observed in sites of neoplasia usually embedded in the white adipose tissue (WAT) such as the breast, the digestive tract, the pancreas and the prostate. We have reported that Metformin is active in preclinical models of breast cancer and can target neoplastic, endothelial and other tumor-associated WAT microenvironment cells. In our previous studies in orthotopic models of breast cancer, Metfomin reduced local and metastatic tumor progression, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels showed a dysplastic phenotype (Orecchioni et al, 2015). In these orthotopic breast cancer models we have more recently found that Metformin can also inhibit GM-CSF release from WAT progenitors, so that circulating GM-CSF levels, proposed to regulate WAT-embedded myeloid suppressor cells, are reduced. Few months ago Eikawa et al (PNAS 2015) have shown that Metformin enables normal but not T-cell-deficient mice to reject solid tumors, likely by increasing CD8+ PD-1-negative tumor-infiltrating lymphocytes (TILs) and protecting them from apoptosis and exhaustion. We report here that in immune competent, orthotopic models of triple negative breast cancer (4T1) and B-cell lymphoma (A20), treatment with Metfomin significantly reduced the number of intratumoral CD8+PD-1+ TILs, and of CD4+CD25brightCD127-low/neg intratumoral regulatory T cells (Tregs). In these models, anti-PD-L1 treatment was more effective in reducing tumor size when administered in the presence of concomitant Metformin treatment. Conversely, prolonged administration of Metformin after anti-PD-L1 treatment increased tumor growth. These effects of Metformin were observed only in intratumoral TILs and intratumoral Tregs, as the number of circulating CD8+PD-1+ T cells and Tregs were not reduced by Metformin administration. Taken together, our data indicate that, in addition to the direct effect on cancer cells, Metformin has a complex activity also on microenvironment cells and on the immune response against neoplasia. Further studies are ongoing to define a) whether the effects of Metformin on immune cells are related to the canonic targets of Metformin in cancer cells, namely AMPK and the Complex 1 of the respiratory chain, and b) the most effective schedule of administration of metformin and checkpoint inhibitors.

#4004

Development of a successful combination therapy for hepatocellular cancer by targeting Treg and PD-1.

Dai Liu,1 Guangfu Li,1 Timothy Cooper,2 Eric Kimchi,3 Xiaoqiang Qi,1 Ningfei Li,1 Don Rockey,1 Todd Schell,2 Kevin Staveley-O'Carroll3. 1 _Medical University of South Carolina, Charleston, SC;_ 2 _Penn State University College of Medicine, Hershey, PA;_ 3 _University of Missouri, Columbia, MO_.

We have established a clinically relevant murine model to reveal tumor-induced immunotoerance in hepatocellular carcinoma (HCC). Critical factors are targeted to develop immune-based therapies for HCC control.

Intrasplenic (ISPL) inoculation of oncogenic hepatocytes and intraperitoneal (IP) injection of carbon tetrachloride (CCl4) are combined to induce progressive HCCs in fibrotic livers of immunocompetent mice. We characterize the features of this model, examine tumor-antigen-specific (TAS) immunity during tumor initiation and progression, and identify the critical factors in tumor-induced immune tolerance. The established murine model recapitulates human HCC and reflects its typical features. TAS CD8+ T cells initially maintain a naive phenotype and function in early-stage tumor-bearing mice, then become profound exhaustion with the tumor progression to the advanced stage. The deep immunosuppression is associated with the significant upregulation of Programmed cell death protein 1(PD-1), Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and the increase of Tregs. While the changes in Tregs and PD-1 are systemic, they are particularly pronounced in the tumor microenvironment. Sunitinib-mediated reduction of Treg together with antibody-mediated blockade of PD-1 synergistically suppress tumor growth and activate anti-tumor immunity.

Our data provide evidence that oncogenic hepatocytes escape immune surveillance during tumor initiation via immune ignorance, while later-stage established HCCs evade immune destruction via tumor-induced immunotolerance. Synergy of sunitinib and anti-PD-1 Ab generates favorable effect on suppressing tumor growth and activating anti-tumor immunity. This combinational strategy has high translational potential in HCC treatment.

#4005

Understanding the mechanism of 2FF-induced immune modulation.

Jessica J. Field, Nicole M. Okeley, Weiping Zeng, Che-Leung Law, Peter D. Senter, Shyra J. Gardai. _Seattle Genetics, Bothell, WA_.

Fucosylation is the process of adding a fucose sugar to a glycan chain. The fucose analog, 2-fluorofucose (2FF), has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate, GDP-fucose, as well as direct inhibition of fucosyltransferases. 2FF has shown promising activity in a number of tumor models. For example 2FF enhanced the protective effect of a lymphoma vaccine (Okeley et al, PNAS 110, 2013), and this protection was determined to be immune dependent since depletion of CD4 and CD8 T-Cells reduced the 2FF/vaccine activity. Due to the complex nature of immune interactions in vivo we established an experimental system using human PBMCs to assess how 2FF-mediated immune changes may contribute to 2FF anti-tumor activity.

Human PBMCs were matured in the presence of 2FF for 10 days and phenotypic analysis was performed. Using lectin binding, we found that 2FF dose-dependently decreased cell surface fucosylation which correlated with decreased levels of GDP-fucose and formation of GDP-2FF. 2FF-treatment alone had minimal effect on immune cell phenotype and no significant changes in activation or lineage markers were identified.

Recently receptor fucosylation has been shown to be critical for BCR antigen recognition and antibody production (Li et al, J Immunol 194, 2015) as well as TLR recognition and signaling through the scavenger receptor DC-SIGN (Gringhuis et al, Nat Commun 5, 2014). As the TCR is reported to be fucosylated and basal signaling and activation of this receptor is regulated by a galectin-glycoprotein lattice we hypothesized that afucosylation following 2FF treatment may affect TCR functionality. During TCR engagement and activation the galectin-glycoprotein lattice is disrupted. 2FF-treated T cells show decreased galectin-3 levels and when activated have increased and more sustained TCR signaling, shown by increased levels of phosphorylated TCR associated proteins. Additionally, 2FF-treated T cells show increased tetramer specific binding. These data indicate that alteration in surface fucosylation on T cells impacts the regulatory glycoprotein lattice that negatively influences T cell action. Following up on this observation, in co-cultures 2FF-treated T cells activate dendritic cells in a contact dependent and antigen specific manner, shown by upregulation of maturation markers MHCII, CD83, CD86 and CD40.

Overall we hypothesize that 2FF-treated T cells are more easily activated due to decreased avidity of galectin-3 binding to glycoproteins which lowers the threshold of lattice disruption by peptide-MHC resulting in easier TCR engagement, increased TCR signaling, and dendritic cell maturation.

#4006

Oxaliplatin-induced immune factors and long-term disease control in rectal cancer - a reflection of immunogenic tumor cell death.

Sebastian Meltzer,1 Erta Kalanxhi,1 Svein Dueland,2 Kjersti Flatmark,2 Kathrine Røe Redalen,1 Anne Hansen Ree1. 1 _Akershus University Hospital, Nordbyhagen, Norway;_ 2 _Oslo University Hospital, Oslo, Norway_.

Purpose: In contemporary cancer therapy, the ability of the immune system to recognize and destroy tumor cells is increasingly acknowledged. Following cytotoxic damage of tumor cells, the priming of tumor-targeting T lymphocytes via presentation of shed tumor antigens by dendritic cells may ultimately result in systemic anti-tumor activity, a concept referred to as immunogenic cell death (ICD). Recent preclinical studies have highlighted oxaliplatin as an ICD-inducing agent. In a prospective study (NCT00278694) of curatively intended treatment of locally advanced rectal cancer (LARC), oxaliplatin-containing induction neoadjuvant chemotherapy (NACT; two cycles of the Nordic FLOX regimen) was administered prior to chemoradiotherapy (CRT) and surgery. Within the spectrum of possible ICD responses to NACT, we analyzed circulating osteoprotegerin (OPG) and fms-related tyrosine kinase 3 ligand (FLT3LG), both of which are factors implicated in activation of the antigen-specific interaction between dendritic cells and cytotoxic T lymphocytes.

Experimental procedures: Immunoassay measurements of OPG and FLT3LG were performed in serum samples collected from study patients at baseline and following the induction NACT. Estimated 5-year progression-free survival (PFS) and overall survival (OS) were assessed using the log-rank test.

Results: Fifty-six patients (38 T2-3 cases and 18 T4 cases) with median follow-up time of 65 months were included in the analysis. During follow-up, 18 patients (nine in each of the T2-3 and T4 groups) experienced metastatic progression as a PFS event. For the entire study population, significant increases in serum levels of both OPG and FLT3LG (from median baseline values of 46.1 pg/ml and 66.7 pg/ml to median post-NACT values of 53.6 pg/ml and 140 pg/ml, respectively) were observed. Patients were separated into cases with (n = 37) or without (n = 19) increase in circulating OPG levels and more (n = 30) or less (n = 26) than two-fold increase in circulating FLT3LG levels. When grouped together, patients with the designated increases in both factors (n = 25) or in either of OPG or FLT3LG values (n = 21) during NACT had significantly better PFS rates than the remaining cases (n = 10) (86%, 72%, and 20%, respectively; p < 0.001). Corresponding OS rates were 100%, 88%, and 60% (p < 0.001), suggesting that even patients who experienced metastatic progression had reasonably good OS.

Conclusion: Increase in circulating OPG and FLT3LG following oxaliplatin-containing induction chemotherapy was associated with favorable long-term outcome in LARC patients given curatively intended neoadjuvant treatment consisting of CRT and surgery. These immune effectors may have mediated systemic anti-tumor immunity invoked by ICD-inducing oxaliplatin effects.

#4007

A novel immunotherapy, INT230-6, is able to induce high rates of complete response in mice through a cytotoxic T-cell-dependent mechanism.

Ian B. Walters,1 Anja C. Bloom,2 Lewis H. Bender,1 Masaki Terabe,2 Jay A. Berzofsky2. 1 _Intensity Therapeutics, Westport, CT;_ 2 _National Cancer Institute, Bethesda, MD_.

INT-230-6 is a combination of cisplatin, vinblastine and an amphiphilic cell penetration excipient that when administered intratumorally (IT) can induce complete regression in drug-injected and bystander established colon26 tumors. INT230-6 also has the ability to induce long term protection in mice against intravenous (IV) or subcutaneous (SC) re-inoculation of the cancer cells. This long term protection is dependent on induction of both CD4 and CD8 cells as evidenced by in vivo depletion prior to tumor re-inoculation. Previous studies had not elucidated whether the initial drug-injected tumor regression in naive mice was mainly due to the formulation's direct cytotoxicity or whether early tumor regression is dependent also on immune- based cell killing. To address this question, BALB/c mice were inoculated with 1x106 Colon26 cells SC. Tumors were grown to a mean of 300mm3. INT230-6 was administered intratumorally daily for 5 days (day 0 to 4). At the same time mice were treated either with an IgG control, or with rat anti mouse anti-CD4, anti-CD8 or both anti -CD4/CD8 on days 0,1,5,8,15. T cell subset depletion was confirmed by flow cytometry. In preliminary data, INT230-6 treatment resulted in regression from baseline in all mice with a complete regression of the large tumors in 40-80% of mice in the INT230-6 arm alone or the arm with IgG control. The CD4 depleted mice had a similar CR rate of 70%. However, in the CD8 depleted or the dual depleted (CD4/8) mice, tumors regrew after initial regression and no CR was observed. These results suggest that intratumoral administration of INT230-6 in addition to improving the direct cytotoxicity of cisplatin and vinblastine (data shown previously), also induces a potent CD8 tumor-specific T-cell response which participates in regression of the injected lesion, and may be able to similarly target distant lesions such as metastases.

#4008

Pegylated liposomal alendronate: The impact of the drug cargo on carrier-induced immune modulation.

Robin Rajan,1 Manoj K. Sabnani,1 Laurence M. Wood,1 Vikram Mavinkurve,1 Alberto A. Gabizon,2 Ninh M. La-Beck1. 1 _Texas Tech University Health Sciences Center, Abilene, TX;_ 2 _Shaare Zedek Medical Center, Jerusalem, Israel_.

Introduction: Liposomes are commonly used as drug carriers and the majority of approved anticancer nanoparticles utilize this platform. We have reported that liposomes similar to those used in patients can enhance tumor growth through inhibition of the antitumor immune response. However, it is not clear how the drug cargo impacts the immune modulatory effects of the carrier. The primary purpose of this study was to determine how loading of alendronate, an immune modulatory drug, into liposomes affects tumor progression and functionality of immune cells in tumor and spleen.

Methods: Tumor free C57BL/6 mice (n=12) and mice bearing s.c. TC-1 tumors (n=24) were treated with up to 2 weekly i.v. injections of empty liposomes similar to the pegylated liposomal carrier used in Doxil, liposomes containing alendronate (PLA), or vehicle control. Tumor size was monitored biweekly and mice were sacrificed at endpoint to obtain tumors and spleens for single cell suspensions. To enumerate myeloid and lymphoid cell populations, cells were stained for CD11b, CD11c, Gr1, and F4/80, or TCR-β, CD8b and CD4 respectively. Separate aliquots of cells were also stimulated ex vivo with CpG (myeloid cells) or PMA/ionomycin (T cells), with monensin followed by intracellular staining for IL-2, IFN gamma, perforin and TNF alpha. All assays included Fc-blocking, CD45 stain, viability dye, and were analyzed by flow cytometry (BD LSR Fortessa).

Results: In tumor free mice, liposomes increased the infiltration of macrophages and MDSC's in the spleen with lowered iNOS production, and increased IL-2, IFN gamma, and perforin production in CD8+ and CD4+ T cells. Loading alendronate into the liposomes mitigated the splenic infiltration of both macrophages and MDSC's and potentiated the T cell cytokine responses (IL-2, IFNg, perforin and TNF alpha). In tumor bearing mice, liposomes increased the infiltration of TAMs and inhibited tumor CTLs. Loading Alendronate (PLA) decreased the iNOS and arginase producing TAMs and mitigated the liposome-associated inhibition of CTL infiltration. Importantly, we found that mean tumor volumes were significantly smaller in PLA treated mice (288.9 mm3) as compared with liposomes (885.7 mm3).

Conclusions: We found that loading alendronate into liposomes mitigates the tumor-promoting potential of the carrier through modulation of macrophage, MDSC, and T cell infiltration and functionality in spleen and tumor. Ongoing studies will identify the mechanisms underlying the interactions between the carrier, drug cargo, and tumor immunologic milieu. This will be especially critical to the successful translation of carrier-mediated immunotherapies into the clinic.

#4009

The expression of PD-L1 on human and murine pancreatic ductal adenocarcinoma is enhanced by anticancer agents via the JAK/STAT pathway.

Toshifumi Doi,1 Takeshi Ishikawa,1 Tomoyo Yasuda,1 Tetsuya Okayama,1 Kaname Oka,2 Naoyuki Sakamoto,3 Yuji Naito,1 Yoshito Itoh1. 1 _Kyoto Prefectural University of Medicine, Kyoto, Japan;_ 2 _Takeda Clinic, Kyoto, Japan;_ 3 _Hyakumanben Clinic, Kyoto, Japan_.

Background:

Pancreatic ductal adenocarcinoma(PDA) is the fourth most common cause of cancer-related death in Japan. Recently, new standard chemotherapies for PDA have been developed, but they are still largely unsatisfactory. Therefore, development of new treatment options has been required to improve the outcomes of patients with PDA. To break through this situation, blocking one of inhibitory immune checkpoints, Programmed death-1 (PD-1) /Programmed death-ligand 1 (PD-L1) pathway is considered to be a hopeful candidate for new treatment strategies for PDA.

In this context, for the future combination therapy of anticancer agents and immune check point inhibitors, we investigated how anticancer agents influence the expression of PD-L1 on pancreatic cancer cell lines. Additionally we analyzed the molecular mechanism by which PD-L1 expression on the pancreatic cancer cell lines are regulated.

Methods:

Human PDA cell lines MIA PaCA-2, AsPC-1 and murine PDA cell line Pan02 were used in this study. These cells were adjusted to 1.0 X 105 / ml and incubated with anticancer agents (i.e. gemcitabine, paclitaxel and 5-fluorouracil) at 37°C for 24-72h. Then, the expression level of PD-L1 was determined using qRT-PCR and flow cytometry.

For the blocking experiment of the JAK/STAT pathway, AG490 was used as a blocking agent. After 48h incubation of AsPC-1 cells at 37°C, cells were treated with various concentration of AG490 for 1h. Cells were stimulated with 5-fluorouracil, paclitaxel and gemcitabine, and then incubated in the presence or absence of AG490 for an additional 6-48h. The expression of PD-L1 was analyzed using qRT-PCR and flow cytometry. Stat1 and phospho-Stat1 protein were analyzed by western blotting.

Results:

In AsPC-1, MIA PaCA-2 and Pan02, the expression of PD-L1 was enhanced with all three anticancer agents in a concentration dependent manner.

The phosphorylation of STAT1 and the increase of total STAT1 was observed in AsPC-1 when stimulated by each anticancer agents. After the treatment of JAK/STAT inhibitor, the phosphorylation of STAT1 was attenuated, and the PD-L1 upregulation induced by anticancer agents was cancelled in a concentration dependent manner.

Conclusion:

The stimulation of anticancer agents leads to an enhancement of PD-L1 expression on PDA cell lines. The JAK/STAT pathway is reported to be involved in IFN-γ mediated PD-L1 upregulation in lung cancer and hepatocellular carcinoma cell lines. In the present study, the phosphorylation of Stat1 and the expression of total Stat1 were enhanced after the anticancer agents treatment. Moreover, JAK/STAT inhibitor attenuated anticancer agents-induced PD-L1 expression. Taken together, the JAK/STAT pathway may be responsible for the anticancer agents mediated PD-L1 transcription.

#4010

Identification of novel targeted and immunotherapy combinations by a high throughput assay of T cell-mediated cytotoxicity.

Leila Williams,1 Shruti Malu,2 Jodi McKenzie,1 Rina Mbofung,1 Marie-Andree Forget,1 Chantale Bernatchez,1 Patrick Hwu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Dana-Farber Cancer Institute, Boston, MA_.

Melanoma is the major cause of skin cancer-related deaths. However, the recent emergence of immune checkpoint inhibitors such as Ipilimumab (anti-CTLA-4) and Nivolumab (anti-PD-1), and adoptive cell therapy, using tumor infiltrating lymphocytes (TILs), has improved clinical outcomes and produced response rates of approximately 50%. Despite the progress made in melanoma immunotherapy, there is a large cohort of patients for which these treatments are currently not applicable. They are either inherently resistant to or acquire resistance to immunotherapy. As such, understanding mechanisms of resistance to immune response and ascertaining efficacious therapy combinations is important in overcoming immunoresistance and developing novel treatment regimens.

As proof of principle, preliminary work using a unique protocol for an in vitro T-cell mediated cytotoxicity screen was established. In this assay, intracellular staining of active caspase-3 in tumor cells, a marker of apoptosis, is measured by flow cytometry in a 96-well format. Using patient-derived tumor and TIL pairs, a screen of 850 compounds was performed to find candidate drugs that could modulate the sensitivity of tumor cells to T- cell mediated killing. Initial screens identified classes of compounds that inhibit Heat shock protein 90 (Hsp90), Aurora Kinase and Topoisomerase I, and studies to understand their mechanisms of action are currently underway.

However, as this screening process is very time-intensive, we are currently increasing the efficiency and feasibility of this screen by miniaturizing this workflow. This work presents the development of a screening method that is performed in a 384-well format, with a greater drug concentration range, and utilizing robotics to streamline and increase output. With this, we can specifically identify novel targeted and immunotherapy combinations that may be used in the clinic to treat melanoma. Additionally, by understanding the signaling pathways and molecular factors that regulate tumor response to T cell mediated killing, we can translate these findings into making immune-based cancer therapies applicable to other tumor types. This project aims to discover novel compounds that work synergistically with T-cell mediated tumor cell cytotoxicity, understand their mechanism of action and how they contribute to resistance and as a result identify innovative immune-based therapies that can be translated into clinical use.

#4011

**Effective** in situ **immunization via local radiation therapy (RT) and tumor-specific immunocytokine (IC): Suppression from distant tumor is blocked by RT or Treg-depleting CTLA-4 antibody.**

Zachary S. Morris,1 Emily Guy,1 David Francis,1 Monica M. Gressett,1 Eric A. Armstrong,1 Shyhmin Huang,1 Lauryn R. Werner,1 Stephen D. Gillies,2 Alan Korman,3 Jacquelyn A. Hank,1 Alexander L. Rakhmilevich,1 Paul M. Harari,1 Paul M. Sondel1. 1 _University of Wisconsin, Madison, WI;_ 2 _Provenance Biopharmaceuticals, Carlisle, MA;_ 3 _Bristol-Myers Squibb, Redwood City, CA_.

PURPOSE: Use "off the shelf" reagents to eradicate an established tumor and induce a tumor specific T cell response that destroys distant tumor.

PROCEDURES: We have identified a cooperative interaction between local tumor RT, intratumoral (IT) injection of IC hu14.18-IL2 (anti-GD2 hu14.18 mAb linked to IL2), and checkpoint blockade with anti-CTLA4 mAb. C57Bl/6 mice were implanted with 2x106 B78 (GD2+) melanoma in one flank (1-tumor model). In the 2-tumor model, mice received 2x106 B78 3 weeks (w) later in the opposite flank. After 5w the primary (1st) tumor was ~200mm3, and ~500mm3 after 7w. The 2nd tumor was ~ 50mm3 at 5w. At 5w or 7w, mice received single fraction RT (12Gy) to the 1st tumor and 6 days later received 5-daily 50μg injections of IC (IT-IC). When used, anti-CTLA4 was given i.p. on days 3, 6 and 9 after RT.

NEW UNPUBLISHED DATA: For mice bearing a single 200mm3 tumor, RT+ IT-IC results in complete response (CR) in 71% of mice and a tumor-specific memory T cell response. Mice with a single 500mm3 tumor showed slowing of tumor growth, but only 27% CR after radiation + IT-IC. Adding anti-CTLA-4 to RT + IT-IC improved tumor response (73% CR) and survival compared to doublet combinations of these 3 modalities. In contrast, in the 2-tumor model, providing RT + IT-IC to the 1st ~200mm3 tumor, but not to the distant ~50mm3 tumor, did not enhance 1st tumor shrinkage compared to RT alone and had no effect on the 2nd 50mm3 tumor. The presence of the 2nd B78 tumor resulted in systemic immune suppression that prevented the local RT and IT-IC from eliminating the 1st tumor. This was tumor specific, as local RT + IT-IC to the 1st ~200mm3 B78 tumor was still effective in treating the 1st tumor if the 2nd (~50 mm3 tumor) was the syngeneic but unrelated Panc02 tumor. Delivering RT to both the 1st + 2nd B78 tumors eliminated the inhibitory effect of the 2nd tumor, enabling IT-IC to the 200mm3 tumor to cause eradication of that tumor in 64% of mice. Preliminary PCR analyses of FoxP3 in the 1st tumor showed Tregs are depleted by 1st tumor RT only in mice with 1 tumor and not in mice with 2 tumors. In this 2 tumor model we combined RT + IT-IC of the 1st tumor with anti-CTLA-4. The IgG2b anti-CTLA-4 (which doesn't substantially deplete Tregs) had minimal effect on 1st tumor response to RT + IT-IC. In contrast the IgG2a anti-CTLA-4 (that depletes Tregs) rendered 60% of mice disease-free (durable CR of both the treated 200mm3 and the untreated 50mm3 tumors). Preliminary data, using DEREG mice that enable diphtheria toxin to deplete Tregs, show Treg depletion in the 2 tumor model also enables eradication of both 1st and 2nd tumors in 60% of mice after RT + IT-IC to only the 1st tumor.

CONCLUSIONS: Local RT+ IT-IC can result in long-term tumor eradication of macroscopic tumors via adaptive immunity to the "in situ vaccine", provided that Treg-associated immune suppression from distant tumor is eliminated by RT or Treg-depletion.

#4012

Improving radiotherapy abscopal effects with anti-PD1 and anti-CD137-based immunotherapy.

MariaE. Rodríguez-Ruiz,1 Inmaculada Rodriguez,1 Saray Garasa,1 Benigno Barbes,1 Jose Luis Solorzano,1 Jose Luis Perez Gracia,1 Sara Labiano,1 Arantza Azpilikueta,1 Elixabet Bolanos,1 Alfonso R. Sanchez-Paulete,1 M. Angela Aznar,1 Ana Rouzaut,1 Maria Jure-Kunkel,2 Iñaki Etxeberria,1 Carlos Alfaro,1 Carmen Oñate,1 Mariano Ponz,1 Ignacio Melero1. 1 _Clinica universidad de Navarra and centro de investigación medica aplicada, Pamplona, Navarra, Spain;_ 2 _Bristol Myers Squibb, Lawrenceville, NJ_.

Radiotherapy is considered an efficacious local tool to erradicate or at least control cancer progression. However, recent lines of preclinical and clinical evidence indicate that proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lessions and on distant, non-irradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favourable effects on distant non-irradiated tumor lesions as observed on transplanted MC38 (colorectal cancer), B16OVA (melanoma) and 4T1 (breast cancer) models. Immunotherapy and radiotherapy synergized both when irradiation was given using external beams or provided with brachytherapy. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. The irradiation regimen induced immune infiltrate changes in the irradiated and non-irradiated lesions featured by reductions in the content of effector T cells, Tregs, and myeloid-derived supresor cells (MDSC), while effector T cells were expressing more intracellular IFN gamma in both the irradiated and contralateral tumors. Importantly, 48h following irradiation CD8+ TILs showed brighter expression of CD137 and PD-1 thereby displaying more target molecules for the activity of the corresponding monoclonal antibodies. Likewise, PD-1 and CD137 were induced on tumor infiltrating lymphocytes from surgically excised of human carcinoma lessions that were irradiated ex-vivo. These findings advocate for clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompained by radiotherapy strategies on treatable lesions, even if leaving disease outside the irradiation field.

#4013

Transcriptionally mediated effects of radiation on the expression of immune susceptibility markers in murine and human melanoma.

Lauryn R. Werner, Monica Gressett, Maureen Riegert, Shyhmin Huang, Joseph G. Kern, Amy Erbe, Paul M. Harari, Paul M. Sondel, Zachary S. Morris. _University of Wisconsin Madison, Madison, WI_.

Radiation therapy (RT) may enhance tumor susceptibility to immune response. We and others have observed a cooperative interaction between RT and various immunotherapies, which appears dependent on the relative sequencing of each therapy. We reported a synergistic interaction between RT and the antibody-dependent cell-mediated cytotoxicity (ADCC) response to tumor-specific monoclonal antibodies (mAb) in murine melanoma. We reported enhanced cooperative effect when immunotherapy is administered 6-10 days after RT compared to administration on days 1-5 or 11-15. We hypothesized that this might reflect a delayed, transcriptionally-mediated effect of RT on the expression of tumor markers of immune susceptibility. In this study, we investigated the effect of RT on a variety of immune susceptibility markers at both transcriptional and post-translational levels to explore mechanisms behind the observed RT-induced changes.

We used flow cytometry to examine the time course of phenotypic changes in immune susceptibility markers in B78 murine melanoma cells following in vitro RT. We observed a time- and RT dose-dependent increase in the expression of specific death receptors (Fas, DR5), as well as T cell co-stimulatory/co-repressor ligands (PD-L1, CD80). The timing of these changes correlated with tumor susceptibility to ADCC immune response in vivo. All protein expression changes observed by flow cytometry were found to occur over a similar time course by quantitative polymerase chain reaction (qPCR), suggesting that RT-induced protein expression changes were mediated by changes in transcriptional activity. Using high throughput qPCR, we observed a similar time course of transcriptional effects in additional markers of tumor cell immune susceptibility, including Fas, MHC I, CD40, and others. We compared the effect of RT on these markers in human and mouse melanoma cell lines and observed a comparable time course for transcript-level changes across species.

This study sheds light on the mechanistic basis of time sensitivity in the interaction of RT with ADCC in vivo and suggests opportunities to enhance anti-tumor immune response by combining RT and immunotherapeutic agents. Such findings bear relevance for research investigating the potential role for RT in driving and optimizing the response to various cancer immunotherapies.

#4014

Combination of high-dose irradiation and local interleukin-12 treatment enhance tumor killing and have less toxicities than either treatment alone.

Ji-Hong Hong,1 Ching-Fang Yu,1 Fang-Hsin Chen,2 Chi-Shiun Chiang3. 1 _Chang Gung University / Chang Gung Memorial Hospital, Taoyuan, Taiwan;_ 2 _Chang Gung University, Taoyuan, Taiwan;_ 3 _National Tsing Hua University, Hsinchu, Taiwan_.

Immunotherapy such as anti-PD-1/PD-L1 is effective in several types of tumors, but in general the response rate is less than 30%. Systemic administration of cytokine such as IL-12 could create anti-tumor immunity but has unacceptable side effects. This study is to test our hypothesis that combination of high-dose local radiotherapy (RT) and local production of IL-12 inside tumors within short period could have potent anti-tumor effects but low systemic toxicity. TRAMP-C1 tumors derived from a murine prostate cancer were either no treatment, or irradiated with 25 Gy, or intra-tumorally injected with Ad-sc-IL12 virus (1×10sup8 pfu), or treated with both modalities at the tumor size of 4mm. RT alone and Ad-sc-IL12 alone could not shrink tumors and only caused tumor growth delay around 7 and 12 days, respectively. The combination of RT with Ad-sc-IL12 virus could shrink the tumors to the sizes of < 2mm and further extended the tumor growth delay more than 20 days. The percentage of lymphocytes and IFN-γproduction were evaluated by flow cytometry and ELISA. The percentages of CD4 cells in tumors were too low to be reliably measured in all groups, but the percentages of CD8 cells were increased in Ad-sc-IL12 virus-treated groups and even much higher in those combined with RT. For mice treated with Ad-sc-IL12 virus injection, the serum IL-12 levels were around 4.5 times increase at day7, but those combined with with RT had significantly lower levels than those treated with virus alone. The serum IFN-γ(Th1 response) levels were around 12 times higher in Ad-sc-IL12 groups at day7 as compared to control or RT only group, but had no significant differences between those treated with combined modality and with Ad-sc-IL12 virus alone. Liver damage is an index for IL-12 toxicity and the levels of GOT and GTP were also lower in those treated IL-12 combined with RT than those treated with IL-12 alone. This study supported our hypothesis and showed that combination of local IL-12 production and local RT are effective in increasing CD8 cells, exerting Th1 response, and enhancing tumor growth delay. The most important is that this combination has less systemic toxicity than local IL-12 production alone. This study provides evidence to develop new strategy for producing a strong anti-tumor and low toxicity for radioresistant tumors by combining RT and IL-12 immunotherapy.

#4015

Exposure of lymphatic endothelial cells to ionizing radiation increases the surface expression levels of integrin ligands.

Maria E. Rodriguez-Ruiz,1 Saray Garasa,2 Inmaculada Rodriguez,2 Alba Yanguas,2 Álvaro Teijeira,2 Ignacio Melero,2 Ana Rouzaut2. 1 _Clinica Universidad de Navarra, Pamplona, Spain;_ 2 _Center for Applied Medical Research, Pamplona, Spain_.

Radiotherapy has been proven an efficacious tool to combat cancer by directly destroying tumor cells. However, effects of radiotherapy have been also described that occur through the re-education of the immune tumor microenvironment, being leukocyte traffic regulation across the tumor vasculature of key importance.

Here, we address the effects that radiotherapy may be causing on the adhesive properties of the tumor lymphatic vasculature. To study this, we irradiated primary and immortalized human and murine lymphatic endothelial cells grown in vitro, and fresh tumor explants obtained from colon cancer patients. As a result, we observed dose- and time- dependent increases in the expression of the integrin counter-receptors ICAM-1 and VCAM on the surface of the tumor lymphatic endothelial cells. These effects are also observed in vivo on colon (MC38) and melanoma (B16) tumors transplanted into syngeneic mice and irradiated with a single dose of 20 Gy. These findings are consistent with increased ICAM-1 and VCAM expression levels on Lymphatic endothelial cells in tumor biopsies from cancer patients which were taken before and four weeks after fractionated radiotherapy, including head and neck, colon and oropharyngeal human carcinomas. While the TGFβ pathway was found to be involved in these effects, NF-κB inhibitors did not hamper ICAM-1 or VCAM induction.

In summary, these results show ICAM-1 and VCAM surface expression was enhanced on lymphatic vessels after radiotherapy and may explain differential leukocyte traffic following irradiation.

#4016

Personalizing the synergy of focal radiation and immunotherapy.

Jan Poleszczuk,1 Kimberly Luddy,1 Shari Pilon-Thomas,1 Jonathan Schoenfeld,2 Heiko Enderling1. 1 _H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _Brigham and Women's Dana Farber Cancer Center, Boston, MA_.

Tumors grow within a host tissue that both facilitates progression by supplying nutrients and growth factors, and inhibits it through physical constraints and immune surveillance. Transformed cancer cells are confronted with this innate and adaptive immune surveillance, and tumors that develop to become clinically apparent have evolved to evade the immune system. This complex ecological system may be tipped back in favor of effective immune surveillance and tumor regression through novel immunotherapeutic strategies, particularly when used in concert with focal cytotoxic agents such as radiotherapy.

We developed a multi-scale mathematical model framework to simulate local tumor-immune interactions as well as systemic distribution patterns of T cells activated locally in each metastatic site. We simulate focal radiotherapy-induced cytotoxicity and subsequent activation of an adaptive immune response, which may propagate systemically to cause regression of unirradiated metastases, known as the abscopal effect. We simulate concurrent anti-CTLA4 immunotherapy to investigate synergy.

Model simulations suggest that systemic distribution of locally activated T cells is dependent on (i) the anatomical distribution of metastatic sites, (ii) the tumor volume of each metastasis, (iii) the site of focal therapy, (iv) the local therapy protocol of immune activation, and (v) the timing of concurrent immunotherapy. We find that irradiation of different metastatic sites has different likelihoods of inducing systemic tumor regressions.

The developed framework can identify combination therapy protocols that optimally harness the synergy of focal therapy-induced immunity with immunotherapy, and identify optimum treatment targets on a per patient basis.

#4017

Anti-tumor activity of a BRAF inhibitor and IFNα combination in BRAF mutant melanoma.

Francesco Sabbatino,1 Yangyang Wang,2 Giosuè Scognamiglio,3 Elvira Favoino,4 Steven A. Feldman,5 Vincenzo Villani,2 Keith T. Flaherty,6 Sjoerd Nota,7 Diana Giannarelli,8 Ester Simeone,9 Anna M. Anniciello,3 Giuseppe Palmieri,10 Stefano Pepe,1 Gerardo Botti,3 Paolo A. Ascierto,9 Cristina R. Ferrone,2 Soldano Ferrone11. 1 _Department of Medicine and Surgery, University of Salerno, Salerno, Italy;_ 2 _Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 3 _Pathology Unit, I.N.T. Fondazione G. Pascale, Naples, Italy;_ 4 _Laboratory of Cellular and Molecular Biology, I.R.C.C.S. Saverio de Bellis, Bari, Italy;_ 5 _Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 6 _Department of Medical Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 7 _Department of Orthopedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 8 _Biostatistics Unit, Regina Elena National Cancer Institute, Rome, Italy;_ 9 _Unit of Melanoma, Cancer Immunotherapy and Innovative Therapy, I.N.T. Fondazione G. Pascale, Naples, Italy;_ 10 _Unit of Cancer Genetics, Institute of Biomolecular Chemistry, National Research Council, Sassari, Italy;_ 11 _Department of Surgery and of Orthopedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA_.

Background. BRAFV600E mediated-MAPK pathway activation is associated in melanoma cells with IFNAR1 down-regulation. IFNAR1 regulates melanoma cell sensitivity to IFNα, a cytokine currently used for the adjuvant treatment of melanoma patients. These findings in conjunction with limited therapeutic efficacy of BRAF-I prompted us to examine whether the efficacy of IFNα therapy of melanoma harboring BRAFV600E can be increased by its combination with BRAF-I. Methods. BRAF/NRAS genotype, ERK activation, IFNAR1 and HLA class I antigen expression were tested in 60 primary melanoma tumors of treatment naïve patients. The effect of BRAF-I on IFNAR1 expression was assessed in 3 melanoma cell lines and in 4 tumor biopsies of BRAFV600E metastatic melanomas. The anti-proliferative, pro-apoptotic and immunomodulatory activity of BRAF-I and IFNα combination was tested both in vitro and in vivo utilizing 3 melanoma cell lines, HLA class I antigen-MA derived peptide complex-specific T cells and immunodeficient mice (5 mice /group for mice survival and 10 mice/group for inhibition of tumor growth). All statistical tests were two-sided. Differences were considered statistically significant when the P value was <0.05. Results. IFNAR1 level was statistically significantly (P<0.001) lower in BRAFV600E primary melanoma tumors than in BRAF wild type tumors. IFNAR1 down-regulation was reversed by BRAF-I treatment in the 3 melanoma cell lines (P≤0.02) and in 3 out of 4 tumor biopsies from metastatic melanoma patients. IFNAR1 level in the melanoma tumors analyzed was increased as early as 10-14 days following the beginning of the treatment. These changes were associated with i) an increased susceptibility in vitro of melanoma cells to the anti-proliferative (P≤0.04), pro-apoptotic (P≤0.009) and immunomodulatory activity, including induction of HLA class I antigen APM component (P≤0.02) and MA expression as well as recognition by cognate T cells (P<0.001), of BRAF-I and IFNα combination, and ii) an increased mice survival (P<0.001) and inhibition of tumor growth of melanoma cells (P<0.001) in vivo by BRAF-I and IFNα combination. Conclusions. The results of this study provide a strong rationale for the novel clinical trials implemented in BRAFV600E melanoma patients with BRAF-I and IFNα combination

### Mechanisms and Applications of Immune-based Therapies

#4019

Inhibiting DNA methylation causes an interferon response in cancer cells via endogenous retroviruses and recruits immune cells to the tumor microenvironment to sensitize to immune therapy.

Katherine B. Chiappinelli,1 Meredith L. Stone,1 Michael J. Topper,1 Lauren Murphy,1 Pamela L. Strissel,2 Reiner Strick,2 Cynthia A. Zahnow,1 Stephen B. Baylin1. 1 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins School of Medicine, Baltimore, MD;_ 2 _Laboratory for Molecular Medicine, University-Clinic Erlangen, Erlangen, Germany_.

Therapies that activate the host immune system have shown tremendous promise for a wide variety of solid tumors, with patients exhibiting vigorous and durable responses. However, in most cancer types, fewer than half of patients respond to these immune therapies. We propose epigenetic therapy as a mechanism to sensitize these patients. DNA methyltransferase inhibitors (DNMTis) upregulate immune attraction, including the interferon response, in solid tumors. We have shown that in human epithelial ovarian cancer cells, DNMTis upregulate viral defense by cytosolic sensing of double-stranded RNA (dsRNA), triggering a Type I Interferon response and apoptosis. Demethylation and expression of bidirectionally transcribed endogenous retroviruses (ERVs) is a major component of the dsRNA that activates the response. Our work showed that treatment with the DNMTi 5-azacytidine (Aza) sensitizes mouse melanoma cells to subsequent anti-CTLA4 therapy, likely through activation of the interferon response and subsequent signaling to host immune cells. Our current work aims to verify this hypothesis. In addition, we observe that adding histone deacetylase inhibitors (HDACis) to DNMTis can augment the upregulation of specific ERVs and the resulting downstream interferon response in human cancer cell lines. Specifically, the ERV-K family as well as the Fc2 and ERV-9 families are increased by DNMTi treatment but further augmented by HDACi treatment, while HDACis alone have minimal effects on the ERVs and the downstream interferon response. We tested the hypothesis that epigenetic drugs sensitize to immune therapy by recruiting host immune cells in an immunocompetent mouse model of serous ovarian cancer. Treatment of this model with DNMTi and HDACi results in increased recruitment of (CD3+) T cells, including tumor-killing T Effector cells, to the tumor. This epigenetic therapy causes increased activation of CD8 T cells and natural killer cells, an increase in helper T cells, and a reduction in myeloid derived suppressor cells and macrophages. We observed upregulation of the immune checkpoint ligand PD-L1 on tumor cells by DNMTis and hypothesized that treatment of this mouse model with the above drug combination plus an antibody to the PD-L1 receptor (anti-PD-1) could reduce tumor burden. This combination does indeed significantly reduce tumor burden and increase survival. We thus define a major mechanism for how DNMTis and HDACis may induce cancer cells to increase attraction and activation of immune cells and sensitize patients to immunotherapy.

#4020

**Inhibition of KIT** in vivo **modifies immune cell populations to improve the efficacy of checkpoint inhibitors in syngeneic mouse tumor models.**

Andrew J. Garton, Lori Lopresti-Morrow, Scott Seibel, Theresa LaVallee, Richard Gedrich. _Kolltan Pharmaceuticals, Inc., New Haven, CT_.

The KIT receptor tyrosine kinase plays critical roles in GIST and a range of other tumor types, and when activated by somatic mutation serves as a potent oncogenic driver in these neoplasms. Whereas small molecule inhibitors of the tyrosine kinase domain of KIT have demonstrated considerable success in treatment of GIST patients with some KIT mutations, there remains a need to develop additional KIT-directed therapies for treatment of GIST patients that fail to respond to these agents, or that develop resistance during therapy. Furthermore, KIT is prominently expressed on mast cells, which promote an immune evasive environment within the tumor microenvironment by influencing the function of other immune cells, such as myeloid derived suppressor cells (MDSC's). However, the potential impact of KIT inhibition on anti-tumor immunity has not been studied extensively to date. KTN0158 is a humanized anti-KIT IgG1 monoclonal antibody that specifically binds to the extracellular domain of KIT, and is being developed as a potential therapy for cancer and other mast cell-related diseases such as neurofibromatosis type 1 (NF1). KTN0158 inhibits KIT signaling and function in vitro and in vivo and has demonstrated anti-tumor activity in dogs with mast cell tumors expressing either wild-type or mutant KIT. In order to explore the potentially broad anti-tumor benefit of KIT inhibition by an antibody, a series of syngeneic tumor models were evaluated for sensitivity and immune cell profile changes in response to a surrogate antibody that recognizes mouse KIT (ACK2), dosed as either a single agent or in combination with T cell checkpoint inhibitors. Within this panel of tumor models, ACK2 strongly enhanced the anti-tumor activity provided by an anti-CTLA4 antibody in the colon26 model, while exhibiting no anti-tumor activity when dosed as a single agent. The colon26 cell line does not express detectable levels of KIT, and does not exhibit sensitivity to ACK2 or SCF treatment in proliferation assays performed in vitro, suggesting that this effect of ACK2 is not due to a direct effect on the tumor cells. Strikingly, ACK2 treatment was associated with pronounced effects on immune cell populations, including a profound decrease in both intra-tumoral and splenic monocytic MDSC counts. These observations suggest that KIT inhibition in vivo has the potential to modulate immune cell populations including MDSC's and mast cells, thereby targeting immune suppressive innate immune cells in the tumor and sensitizing the tumor to immune checkpoint inhibitors. Furthermore, the data provide a rationale to investigate KTN0158 in combination with immune checkpoint inhibitors across a range of human tumor types.

#4021

The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations.

David Briere,1 Niranjan Sudhakar,1 Lars Engstrom,1 Jill Hallin,1 Ruth Tang,1 Harrah Chiang,1 Maryland Rosenfeld-Franklin,2 Peter Olson,1 James Christensen1. 1 _Mirati Therapeutics, Inc., San Diego, CA;_ 2 _Molecular Imaging, Inc., Ann Arbor, MI_.

Immunotherapy has led to major treatment breakthroughs for a number of cancers including non-small cell lung cancer (NSCLC). Although initial responses to immune checkpoint inhibitors are promising, a significant percentage of patients do not respond or rapidly acquire resistance. Although the mechanisms underlying intrinsic and acquired resistance remain largely unexplained; the expression of programmed cell death-ligand 1 (PD-L1), lack of tumor cell capacity to effectively present neoantigens, and presence of immunosuppressive cellular subsets have been implicated as potential mechanisms. Histone deacetylase (HDAC) inhibitors have emerged as a class of agents that may combat checkpoint inhibitor resistance by reversing immune evasion and eliciting an anti-tumor activity through a multi-faceted immuno-stimulatory mechanism of action. Mocetinostat is a spectrum-selective Class I/IV HDAC inhibitor specifically targeting HDAC-1, -2, -3 and -11. The present studies were designed to explore mocetinostat's effect as an immune-enhancer and ultimately, to evaluate its potential to be used in combination with immune checkpoint inhibitors (e.g., PD-1/PD-L1 antagonists). Specifically, we assessed mocetinostat's effect on the expression of various immunomodulatory factors by tumor cells as well as its effect on immune cell sub-populations in the tumor microenvironment in vivo. Mocetinostat elicited a concentration-dependent increase in PD-L1 mRNA expression which translated into increased PD-L1 surface protein expression in a panel of NSCLC cell lines. In addition, mocetinostat elicited a concentration-dependent increase in expression of MHC-class I related polypeptide-related sequence A (MIC-A) and MIC-B, and cluster of differentiation 86 (CD86). Furthermore, mocetinostat induced expression of several human leukocyte antigen (HLA) gene complex family members including HLA-A, -B, -DRA, and -DPA among others. To determine the effect of mocetinostat on systemic and tumor immune cell subpopulations we treated CT26 tumor-bearing mice. Mocetinostat increased splenic CD4-positive T effector cells and tumor mature cytolytic CD8-postive T cells and at the same time decreased tumor FoxP3-positive T regulatory cells and CD11b/Gr1-positive myeloid-derived suppressor cells (MDSC). These data provide evidence that mocetinostat modulates key immune regulators both in tumor cells as well as in relevant immune cell types in the tumor microenvironment and provides strong rationale for combination with immune checkpoint inhibitors.

#4022

PT2385, a novel HIF-2α antagonist, combines with checkpoint inhibitor antibodies to inhibit tumor growth in preclinical models by modulating myeloid cells and enhancing T cell infiltration.

Guangzhou Han, Christina Stevens, Zhaodan Cao, Shanhai Xie, Melissa Maddie, Barry Goggin, Eli Wallace, John Josey, Tai W. Wong. _Peloton Therapeutics, Dallas, TX_.

The majority of clear cell renal cell carcinoma (ccRCC) have deficiency in the gene encoding the von Hippel Landau (VHL) protein, as a result of DNA copy loss, non-sense mutations, and epigenetic silencing. Deficiency in VHL and its E3 ligase activity results in stabilization of the transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α. In ccRCC, HIF-2α has been proposed to function as an oncogenic driver, as its depletion in tumor cells results in inhibition of tumor growth.

We previously described the identification of a potent and selective small molecule antagonist of HIF-2α, PT2385 that disrupts the hetero-dimerization of HIF-2α with the aryl hydrocarbon receptor nuclear translocator. PT2385 inhibits growth of ccRCC tumor xenografts derived from cancer cell lines and from patients' tumors. Inhibition of tumor growth is mediated by suppression of HIF-2α target genes, such as cyclin D1 and VEGFA, that promote tumor growth.

In addition to a direct role in the transcription regulation of growth-promoting genes in ccRCC, HIF-2α has also been implicated in the pro-tumorigenic property of the tumor microenvironment. HIF-2α is expressed in cells of the myeloid lineage, and HIF-2α expression in tumor-associated macrophages was reported in a number of tumor types. A role for HIF-2α in the polarization of macrophages to the M2 phenotype has also been described. The availability of PT2385 provides an opportunity to confirm the involvement of HIF-2α in immune suppression by the tumor microenvironment, and to assess the therapeutic utility of HIF-2α antagonism in tumors other than ccRCC. PT2385 was evaluated for its ability to inhibit tumor growth in syngeneic mouse tumor models. The mouse tumor cell lines used in these models do not express HIF-2α and PT2385 has no single agent efficacy. However, the combination of PT2385 with antibodies to immune checkpoint control molecules (PD-1, PD-L1, and CTLA4) yielded additive or synergistic efficacy in a model-dependent manner. Immune phenotyping of the treated tumors was performed by immunohistochemistry and flow cytometry. The analyses revealed that tumor growth inhibition by the combination regimens is accompanied by an increase in T cell infiltration and changes in macrophage and myeloid-derived suppressor cell populations in the tumors. Changes in cytokine expression were also observed.

Results of our studies show that combinations of PT2385 and immune checkpoint inhibitor antibodies are well tolerated and efficacious in preclinical models. The results are consistent with the hypothesis that HIF-2α plays an immunosuppressive role in the tumor microenvironment, and its inhibition provides an additional approach to reverse immune evasion by tumors. The combination of PT2385 and immune checkpoint inhibitors is being further evaluated in clinical trials.

#4023

Combination therapy with immune checkpoint inhibitor and CCK-receptor blockade increases survival of pancreatic cancer.

Jill P. Smith, Shangzi Wang, Ajina Reham, Sandra A. Jablonski, Louis M. Weiner. _Georgetown University, Washington, DC_.

Introduction: Advanced pancreatic ductal adenocarcinoma (PDAC) has typically been resistant to chemotherapy and immune therapy; therefore, novel strategies are needed to enhance therapeutic response. Cholecystokinin receptors (CCKRs) are present on pancreatic cancer epithelial cells, fibroblasts of the microenvironment, and lymphocytes. We hypothesized that CCK receptor blockade would improve response to immune checkpoint blockade and survival by promoting influx of tumor infiltrating lymphocytes (TILs) and reducing fibrosis.

Methods: Subcutaneous tumors were established using Panc02 murine pancreatic cancer cells (1.0- or 2.0 x10E6) in a syngeneic immune competent mouse model. Three separate experiments were performed with 40 mice each. Mice (C57BL/6) were divided into 4 groups (N=10 mice each) and treated with PBS (control), a CCKR antagonist (L364,718 or proglumide), an immune checkpoint blockade antibody (PD-1 or CTLA-4; Im-Ab), or the combination of CCKR antagonist and immune checkpoint blockade antibody (combination). Tumor growth and animal survival were evaluated. Tumors were subjected to immunohistochemical staining for TILs, trichrome staining, and flow cytometry.

Results: On day 50 after inoculation of 10E6 cells all control mice had died while 42%, 42% and 71% of L364,718, CTLA-4 mice, or combination-treated mice, respectively were alive (p=0.02). By day 90 all mice died except one in the combination-treated group. In the experiment where mice were injected with 2x10E6 Panc02 cells, survival was also increased in mice receiving a proglumide and PD-1 antibody combination (p=0.0009). CD8+TILs increased by 3-,6.3-,and 7.5-fold in CCKR antagonist, Im-Ab, and combination therapy, respectively compared to controls (p<0.0001). CD4+ TILs also significantly increased (p=0.001) in the tumors of mice treated with the combination. Trichrome stain analysis revealed 50% less fibrosis in mice treated with CCKR antagonist compared to PBS controls (p=0.017).

Conclusions: The combination of CCKR antagonism and immune checkpoint blockade antibodies significantly improve survival in a murine model of PDAC. The mechanism is associated with decreased fibrosis in the microenvironment and an accompanying influx of CD8+ and CD4+ TILs.

Supported by NCI CA50633, CA51008, and CA194745

#4024

Epigenetic priming potentiates immune checkpoints inhibitors in ovarian cancer.

Yu Qing,1 Salvatore Condello,1 Katie J. Meyer,1 Andrea Caperell-Grant,1 Kenneth P. Nephew,2 Daniela Matei1. 1 _Indiana University, Indianapolis, IN;_ 2 _Indiana University, Bloomington, IN_.

Background: Ovarian cancer (OC) progression is accompanied by the establishment of stable and transcriptionally repressive epigenetic modifications. An important mechanism of immune evasion is represented by epigenetic silencing of tumor antigens (NY-ESO-1, Muc16 and MAGE). We hypothesize that by reversing DNA methylation, DNA methyl transferase inhibitors (DNMTIs) restore the expression of such antigens, potentiating anti-tumor immune response. The targeting of immune checkpoints regulated by programmed cell death protein-1(PD-1) signaling represents a novel therapeutic strategy in cancer, including in OC. Here we set out to measure the anti-tumor effects of epigenetic priming in combination with PD-1/PDL-1 blockade in OC preclinical models.

Methods: The ID8 intraperitoneal (ip) immunocompetent syngeneic mouse model was used to measure the effects of the novel DNMTI guadecitabine (SGI-110, Astex Pharmaceuticals Inc) and PDL1 blockade. The experimental groups consisted of non-specific IgG (control), guadecitabine 2mg/m2 sq bi-weekly, murine anti-PDL1 inhibitory antibody (10mg/kg) bi-weekly and combination of guadecitabine with anti-PDL1 antibody (n=6 mice/group, 3 week treatment). Immune cells collected from the ascites and spleens of tumor bearing mice were either directly processed or co-cultured with ID8 cells for 48 hours. Cells were immuno-phenotyped by flow cytometry. In OC cells treated with guadecitabine, changes in gene expression were analyzed by real time-PCR.

Results: In ID8 tumors bearing mice, the combination of PDL1 blockade with guadecitabine significantly decreased primary tumor formation (P<0.05) and malignant ascites accumulation (P<0.0001) compared with the control group. Treatments were well tolerated without detectable weight changes attributable to toxicity. CD8+ cell fractions expressing the exhaustion markers (PD1+ or CTL4+) isolated from the ascites and spleens of control mice were diminished by guadecitabine, PD-L1 inhibitory antibody, and the combination treatment. Guadecitabine and the combination regimen increased CD8+/MHC1+ and CD8+/CD40+ cell populations. Treatment with DNMTIs significantly increased the expression of tumor antigens Muc16, Mage A2, A11, and NY-ESO 1 (p<0.01) in several OC cell lines.

Conclusions: Guadecitabine in combination with anti-PDL1 antibody induced striking anti-tumor effects in an immunocompetent OC syngeneic model by activating cytotoxic T-cells. These data support clinical strategies utilizing epigenetic priming using DNMTI in combination with immune checkpoints inhibitors.

#4025

Impact of Kras mutant subtypes on PD-L1 expression in lung adenocarcinoma.

Marius Ilie,1 Laetitia Fazzalari,2 Nicolas Guibert,3 Nathalie Yazbeck,2 Laurie Signetti,2 Véronique Hofman,1 Elodie Long,1 Katia Zahaf,1 Julien Fayada,1 Virginie Lespinet,1 Olivier Bordone,1 Virginie Tanga,1 Kevin Washetine,1 Nicolas Vénissac,4 Jérôme Mouroux,4 Charles Hugo Marquette,5 Patrick Brest,2 Paul Hofman1. 1 _CHU Nice, Laboratory of Clinical and Experimental Pathology, Nice, France;_ 2 _Institute for Research on Cancer and Aging, Nice (IRCAN), University of Nice Sophia Antipolis, Nice, France;_ 3 _Université Paul Sabatier, CHU Toulouse, Thoracic Oncology Unit, Toulouse, France;_ 4 _CHU Nice, Thoracic Surgery Department, Nice, France;_ 5 _CHU Nice, Pneumology Department, Nice, France_.

Clinical responses to immune checkpoint blockade by anti-PD-1/PD-L1 monoclonal antibodies in non-small-cell lung cancer (NSCLC) may be associated with PD-L1 expression. This study was undertaken to determine the expression profile of PD-L1 in patients with Kras-mutant lung adenocarcinoma (LUAD) and to investigate the activation of Kras codon subtypes as a mechanism of PD-L1 regulation.

Immunohistochemistry analysis of PD-L1 expression (SP142 clone) on 117 LUAD (KrasWT, n=51; KrasG12D, n=25; KrasG12V, n=14; KrasG12C, n=27), showed significantly greater expression of PD-L1 in both tumor and immune cells compartments in Kras mutant LUAD compared with KrasWT wild-type tumors (37% vs. 18%; P=0.005). PD-L1+ tumors had greater CD66b+ neutrophil infiltrate and lower CD8+ T-cell infiltrate than PD-L1− tumors, notably in KrasG12D tumors.

To determine the effect of Kras codon subtypes on PD-L1 expression, stable cell lines were generated by transfection of KrasG12D, KrasG12V, KrasG12C and KrasWT plasmids into Beas2B bronchial cells. At basal level, mutant Kras led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, notably in KrasG12C and KrasG12V cells, suggesting transcriptional regulation. Moreover, there was differential activation of NF-kB, ERK and Pi3k/Akt pathways between Kras mutant subtypes. In addition, PD-L1 was upregulated 3-fold by stimulation with IFNγ, independently of the Kras codon subtypes. Instead, hypoxia significantly increased PD-L1 expression in KrasG12C and KrasG12D cells. Co-culture experiments with human PBMCs from healthy patients were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 expression by tumor cells induced by Kras mutations led to decreased PBMCs proliferation and increased apoptosis.

PD-L1 is expressed in 37% of Kras mutant LUAD, suggesting PD-L1 as a therapeutic target in this subset. According to the Kras mutation subtype, potential drugs targeting the NF-kB, ERK or Pi3k/Akt pathways may increase the antitumor adaptive immune responses.

#4026

Epigenetic changes in T cells in response to immune checkpoint blockade.

Sangeeta Goswami, Jianfeng Chen, Hao Zhao, Xuejun Zhang, Padmanee Sharma. _MD Anderson Cancer Center, Houston, TX_.

Immune checkpoint blockade therapy has led to clinical success and have established immunotherapy as one of the primary modality of cancer treatment. It leads to a durable response in around 20% of the patients (responders). Success of immune checkpoint blockade therapy relies on the increase of effector T cells and decrease of regulatory T cells in the tumor microenvironment. However, the underlying mechanism that govern the ratio of effector and regulatory T cells between responder and non-responder to checkpoint blockade is not understood. Understanding the process could reveal novel mechanisms to boost efficacy of immune checkpoint blockade. Differentiation of naïve T cells into effector and regulatory phenotype requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. One key epigenetic modification for T cell differentiation is trimethylation of lysine 27 on histone H3 (H3K27me3). Loss of H3K27me3 results in increased Th-1 plasticity whereas presence of H3K27me3 on Foxp3 locus is required to maintain the function of T-regulatory cells. We hypothesize that immune checkpoint blockade changes the epigenetic landscape in tumor infiltrating T cells that results in an effector phenotype and epigenetic modification of H3K27me3 could promote an anti-tumor immune microenvironment in tumor bearing mice.We showed that in-vivo inhibition of H3K27me3 in combination with anti-CTLA4 results in reduction of tumor size and also reduction in regulatory T cells in a B16-F10 melanoma mice model compared to anti-CTLA4 treatment alone, suggesting H3K27me3 inhibition could increase the efficacy of checkpoint blockade therapy. Future studies will aim to elucidate epigenetic changes in H3K27me3 and define an epigenetic signature as a result of immune checkpoint therapy by ChIP-sequencing. Data generated in this project will greatly enhance our understanding of epigenetic regulation of T cell differentiation is response to immune checkpoint blockade and aid identifying key changes that could be further modified to overcome resistance to immune checkpoint blockade in non-responders.

#4027

Recurrent gains of CD274 (PD-L1) and PDCD1LG2 (PD-L2) provide a genetic basis for PD-1 ligand expression in a subset of solid tumors.

Evisa Gjini,1 Christine Pak,1 Alyssa Kelly,1 Yuling Shi,1 Neal Lindeman,2 Frank Kuo,2 Gordon Freeman,1 Azra Ligon,2 Stephen Hodi,1 Margaret Shipp,1 Scott Rodig1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Brigham and Women Hospital, Boston, MA_.

Background: A subset of malignant tumors are immunogenic but avoid immune surveillance through ectopic expression of the programmed cell death 1 ligands, PD-L1 and PD-L2, that bind PD-1 on effector T-cells to inhibit T-cell activation, a phenotype that is reversible with PD-1 blockade. Co-amplification and co-gain of CD274 (PD-L1) and PDCD1LG2 (PD-L2) on chromosome 9p24.1 provide a genetic basis for PD-1 ligand expression by Reed-Sternberg cells in classical Hodgkin lymphoma, a tumor highly sensitive to PD-1 blockade. We sought to determine whether copy number alterations of CD274 and PDCD1LG2 are a mechanism of PD-1 ligand expression in other tumor types.

Methods: We surveyed data derived from a clinically-deployed next generation sequencing assay (OncoPanel) to identify tumors with evidence of selective copy gain of genes located at 9p24.1. Formalin-fixed paraffin-embedded biopsy specimens from available cases were reviewed for tumor content, immunostained for PD-L1, and analyzed with custom probes targeting the CD274 and PDCD1LG2 loci and a commercial probe targeting the centromere of chromosome 9 by flourescence in situ hybridization (FISH).

Results: Review of NGS data revealed 171 of 7,070 clinical samples (2.4%) with evidence for selective copy gain of CD274 and/or PDCD1LG2. Tissue was available for analysis in 73 cases, of which 55 were successfully analyzed. Co-amplification and co-gain of CD274 and PDCD1LG2 were observed for 20 (36%) and 18 (33%) of cases, respectively. Polysomy 9 and disomy 9 were observed among 9 (16%) and 2 (4%) cases, respectively. Genetic abnormalities associated with chromosome 9 loss were observed among the remaining cases 6 (11%). PD-L1 IHC revealed positive staining of tumor cells in 36 (65%) cases. Among cases with co-amplification/gain of CD274 and PDCD1LG2, a subset (Co-Amp: 36%; Co-Gain: 28%) exhibited moderate to high PD-L1 expression in significant percentages of tumor cells (Tumor H-score > 50). Tumor types with high PD-L1 expression in the context of co-amplification/gain included carcinomas of the head and neck, bladder, and cervix.

Conclusions: Review of over 7000 tumors analyzed by a clinically-deployed NGS platform identifies CD274 and PDCD1LG2 copy gain in a low percentage of a variety of solid tumor types, a subset of which has a corresponding increase of PD-L1 expression. These data suggest that copy gain of CD274 and PDCD1LG2 contributes to PD-1 ligand expression in a subset of solid tumors beyond classical Hodgkin lymphoma.

#4028

**Oncogenic** KRAS **mutations induce PD-L1 overexpression through MAPK pathway activation in non-small cell lung cancer cells.**

Yosuke Miura,1 Noriaki Sunaga,2 Kyoichi Kaira,3 Yusuke Tsukagoshi,1 Takashi Osaki,1 Reiko Sakurai,1 Takeshi Hisada,1 Luc Girard,4 John D. Minna,4 Masanobu Yamada1. 1 _Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Japan;_ 2 _Oncology Center, Gunma University Hospital, Maebashi, Japan;_ 3 _Oncology Clinical Development, Gunma University Graduate School of Medicine, Maebashi, Japan;_ 4 _Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, TX_.

The proto-oncogene KRAS is a frequently mutated oncogenic drivers in non-small cell lung cancer (NSCLC) and druggable molecular targets for KRAS-mutated NSCLC have been under investigation. Recent studies have reported promising results of nivolumab, a programmed death 1 (PD-1) immune-checkpoint-inhibitor antibody, for previously treated NSCLC patients and suggested that the programmed-death ligand 1 (PD-L1) is a candidate surrogate marker for the anti-PD-1 therapy. By microarray analysis, we found that short hairpin RNA (shRNA)-mediated stable knockdown of mutant KRAS downregulated the expression of the CD274 gene (encoding PD-L1) in the NSCLC cell lines, H358 (-2.68 fold-change) and H441 (-1.98 fold-change). Additionally, we confirmed that small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, reduced CD274 mRNA expression in these cell lines. As expected, the CD274 expression levels were higher in these cell lines when examining the expression in a panel of NSCLC cell lines by quantitative RT-PCR analysis. Furthermore, CD274 mRNA expression was downregulated by MEK and ERK inhibitors (U0126, FR180204, selumetinib) in H358 and H441 cells. On the other hand, the expression analysis also revealed that CD274 mRNA levels vary among KRAS-mutated cell lines. These results suggest that oncogenic KRAS mutations induce PD-L1 overexpression through activation of the MAPK pathway in NSCLC cells, whereas other unknown mechanisms that modulate PD-L1 expression underlie in different KRAS-mutated NSCLCs.

#4029

MEK pathway inhibition upregulates PD-L1 and MHC class I expression levels in head and neck squamous cell carcinoma.

Seong-Ho Kang,1 Bhumsuk Keam,2 Soyeon Kim,1 Miso Kim,2 Yong-Oon Ahn,1 Tae Min Kim,2 Dong-Wan Kim,2 Dae Seog Heo2. 1 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea_.

Background: Expression of programmed death-ligand 1 (PD-L1) and down-regulation of MHC class I are well known tumor immune evasion mechanisms. The purpose of this study is to investigate the regulation of PD-L1 and MHC class I expression in head and neck squamous cell carcinoma (HNSCC) cell lines by MEK inhibitor trametinib.

Methods: Six HNSCC cell lines (SNU-1041, SNU-1066, SNU-1076, PCI-13, Detroit 562, and FaDu) were used. Trametinib was purchased from Selleckchem. Growth inhibition of trametinib was measured using MTT assay. Trametinib was dosed at 10 - 640 nM for investigating their effect on PD-L1 and MHC class I molecule expression. Changes of PD-L1 and MHC class I (including HLA-A2) expression levels were analyzed by flow cytometry after treatment with trametinib (20 nM) and/or IFN-γ (1 - 10 ng/ml). Total and phosphorylated STAT1 and STAT3 were analyzed by western blot.

Results: Overall IC50 values of trametinib were over 10 μM in cell lines except SNU-1066 and Detroit 562 with IC50 values still higher than 100 nM. All the six cell lines have no upstream RAS or RAF mutation. Although trametinib treatment did not inhibit growth of these cells, it up-regulated MHC class I expression level in all cells. Furthermore, trametinib enhanced exogenous IFN-γ-induced MHC class I up-regulation in four out of six cell lines. Mechanistically, trametinib treatment increased phosphorylated STAT1 levels by IFN-γ in all cells, and subsequent total STAT1 levels in two out of six cell lines. Interestingly, trametinib treatment directly phosphorylated STAT3 although total STAT3 level was not changed. Either trametinib alone or with IFN-γ induce PD-L1 expression, but the effect was various in these cells. These results imply that blockade of MAPK pathway may alternatively activate JAK/STAT pathway in HNSCC cells, and MHC class I and PD-L1 expression levels were changed as the results

Conclusion: Our results suggest that MEK pathway is closely related with regulation of PD-L1 and MHC class I expression. Up-regulated levels of PD-L1 and MHC class I by inhibiting MEK pathway suggest a possible synergistic role of MEK inhibitor with anti-PD-1/PD-L1 immunotherapy in HNSCC.

#4030

Epigenetic induction of MHC-II pathway expression in murine ovarian cancer cell line.

Taylor B. Turner, Rebecca C. Arend, Mei Li, Troy D. Randall, Andres Forero-Torres, Albert F. LoBuglio, J Michael Straughn, Donald J. Buchsbaum. _University of Alabama Birmingham, Birmingham, AL_.

INTRODUCTION

This study evaluated the potential of epigenetic treatments to induce the expression of the MHC-II antigen presentation pathway in ovarian cancer. Ovarian cancer escapes immune response allowing it to spread in the peritoneal cavity; however, patients with greater immune response to their tumors at baseline have improved survival. Typically, exogenous antigens from tumors are processed and presented via MHC-II to CD4 T cells by antigen-presenting cells. However, if ovarian cancer cells could be induced to express the MHC-II pathway, they could be converted into antigen presenting cells and stimulate an anti-tumor response.

METHODS

Murine epithelial ovarian cancer cells (ID8) were treated for 72 h with the histone deacetylase inhibitors (HDACi): entinostat (MS-275) (1.25, 2.5, 5, 10 µM) and quisinostat (20, 40, 80, 160 nM). Cells were treated with 5-azacytidine (5-aza), a DNA methytransferase inhibitor, alone and in combination with MS-275 (5 µM) at 17.25, 37.5, 75, and 150 nM. After treatment, the expression of CD74 (an MCH-II pathway protein) and MHC-II was evaluated by flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to measure CD74 and CIITA, a transcriptional coactivator that controls MHC-II gene expression. RNA was extracted using RNEasy Mini Kit (Qiagen), cDNA was created with SuperScript VILO Master Mix (ThermoFischer), and a 109bp amplicon was created using JumpStart REDTaq ReadyMix (Sigma-Aldrich). For statistical analysis, a Welch ANOVA was performed using JMP Pro 12. Tukey-Kramer analysis evaluated individual differences. The mean fluorescence intensities (MFI) for the combination treatments were compared using an unequal variance F-test.

RESULTS

Increased protein expression of CD74 and MHC-II was seen after treatment with MS-275, quisinostat, and 5-aza compared to untreated control. Increased expression of CD74 and MHC-II was seen with the combination treatment of 5-aza (17, 37.5, 75, 150 nM) and MS-275 (5 µM) compared to individual treatments (p < .0001). A dose dependent increased expression of CIITA was demonstrated by RT-PCR with 5-aza treatment alone and in combination with MS-275. Increased mRNA expression of CD74 was also seen for all treatments compared to a negative control.

CONCLUSION

Treatment with HDAC inhibitors and a DNA methyltransferase inhibitor induces expression of MHC-II in murine epithelial ovarian cancer cells. The conversion of ovarian cancer cells into antigen presenting cells provides a potential therapeutic model to augment the immune response against epithelial ovarian cancer, a disease known to suppress anti-tumor immunity.

#4031

Different expression of programmed death 1 (PD1) and its ligand (PD-L1) in esophageal and gastric cancer.

Silvio Däster,1 Serenella Eppenberger-Castori,2 Raoul A. Droeser,1 Hannah M. Schaefer,1 Giulio C. Spagnoli,3 Luigi Terracciano,2 Luigi Tornillo,2 Urs von Holzen4. 1 _Department of Surgery, University Hospital Basel, Basel, Switzerland;_ 2 _Insitute of Pathology, University Hospital Basel, Basel, Switzerland;_ 3 _Institute of Surgical Research and Hospital Management; Department of Biomedicine, University of Basel, Basel, Switzerland;_ 4 _Indiana University School of Medicine South Bend, Indiana University Health Goshen Center for Cancer Care, Goshen, IN_.

Background: Prognosis of gastric and esophageal cancer remains poor. Improvements in gastric and esophageal cancer treatments are urgently needed. Programmed cell death 1 receptor (PD1) and its ligand 1 (PD-L1) are known to interact with T cells promoting epithelial cancer tolerance. Several therapeutic monoclonal antibodies blocking this interaction are reaching the clinical praxis. Therefore it is relevant to investigate the role of these biomarkers in different types of malignancy.

Methods: Four different copies of tissue microarrays (TMA), each including healthy mucosa (n=74) and a total of 241 clinically annotated malignancies of the esophagus (n=80) and stomach (n=161) were constructed and stained with PD1, PD-L1, and CD8 specific reagents.

Results: Interestingly, only two cancer samples out of the 241 specimens investigated weakly expressed PD-L1. None of the normal mucosa epithelial cells expressed PD-L1. Stromal PD1 expression correlated with infiltration by CD8+ lymphocytes (rho=0.4; P<0.0001). Elevated intraepithelial presence of CD8+ and/or PD1+ cells in gastric cancer was significantly correlated with longer patients' survival. No correlation could be found in esophageal cancer.

Conclusion: In contrast to other epithelial cancers, PD-L1 expression was virtually absent in the investigated malignancies of the esophagus and stomach. PD1 expression in the stroma surrounding such malignancies correlated with intraepithelial presence of T-cells CD8+ expression, and with better prognosis in gastric cancer patients.

#4032

Modulation of tumor PD-L1 expression by epithelial-mesenchymal phenotypic plasticity.

Justin M. David, Charli L. Dominguez, Jeffrey Schlom, Claudia Palena. _Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Epithelial-mesenchymal transition (EMT) is a molecular and cellular program in which epithelial cells lose their well-differentiated phenotype and adopt mesenchymal traits. This process occurs during the progression of cancer to metastatic disease; tumor cells undergoing EMT acquire invasive characteristics, and mesenchymal features can be highly enriched among circulating tumor cells. EMT has also been associated with the resistance to multiple therapeutic agents, and may ultimately lead to relapse. Emerging research indicates that tumor cells undergoing EMT may also exhibit resistance to killing by innate natural killer (NK) cells and antigen-specific cytotoxic T lymphocytes (CTLs). Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1. Given the contribution of tumor cell phenotypic plasticity to the susceptibility of tumor cells to immune attack, we have evaluated the role of EMT in the expression of PD-L1. Using several different cell line model systems, we devised an mRNA-based EMT scoring method to rank tumor cells according to their phenotypic characteristics. Our model systems included groups of isogenic cell lines in which phenotype modulation was achieved via 1) TGF-β1 treatment; 2) manipulation of EMT genes by transfection; 3) isolation of single cell clones; and 4) selection with chemotherapy. Scoring of the cell lines revealed the existence of distinct epithelial and mesenchymal phenotypes, as well as an intermediate (also termed 'hybrid' or 'metastable') phenotype in which markers of both differentiation states are co-expressed within the same cell line. Analysis of PD-L1 at the mRNA and protein level (antibody clone MIH1) demonstrated that significant changes in PD-L1 expression take place in response to tumor phenotypic modulation. Using multiple systems, it was observed that acquisition of a distinct mesenchymal phenotype associated with increased levels of PD-L1 expression, compared to isogenic tumor cells exhibiting an epithelial phenotype (for example, with tumor cells induced into the mesenchymal phenotype via TGF-β1 treatment or E-cadherin knockdown). In contrast, acquisition of an intermediate tumor phenotype characterized by co-expression of epithelial and mesenchymal features resulted in significant loss of PD-L1 expression. This phenomenon was observed in tumor cells exposed to chemotherapy in which acquisition of mesenchymal markers took place in the presence of sustained levels of epithelial markers (including E-cadherin). Altogether, our results highlight the importance of tumor phenotypic plasticity on PD-L1 expression. We plan to utilize these various models to understand the mechanism(s) of PD-L1 regulation by phenotype modulation and also to evaluate the potential predictive value of tumor cell phenotype, in conjunction with PD-L1 levels, on the susceptibility of tumor cells to killing by immune cells.

#4033

Adenosine regulates radiation therapy-induced antitumor immunity.

Erik Wennerberg,1 Aranzazu Mediero,2 Tuere Wilder,2 Silvia Formenti,1 Bruce Cronstein,1 Sandra Demaria1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _New York University Langone Medical Center, New York, NY_.

Radiation-induced immunogenic cell death (ICD) is a key mechanism whereby local radiation therapy (RT) can elicit anti-tumor immune responses and synergize with immune checkpoint inhibitors in enhancing tumor responses. ATP, which is an essential signal of ICD, activates tumor-resident dendritic cells (DCs) promoting their ability to cross-present tumor-derived antigens to T cells. Interestingly, while release of ATP by RT is dose-dependent (Golden et al., OncoImmunology 2014), a large RT dose of 20 Gy was not effective in inducing anti-tumor T cells and synergize with anti-CTLA-4 (Dewan et al., Clin Cancer Res 2009). Extracellular ATP is rapidly catabolized to adenosine (ADO) by ectonucleotidases CD39 and CD73, which are expressed on tumor cells and immune cells. ADO has immunosuppressive effects, inhibiting DC- and effector T cell-activation, while promoting regulatory T cells (Tregs). Here, we tested the hypothesis that conversion of ATP to ADO hinders generation of effective anti-tumor immunity by high dose RT.

Mice were inoculated s.c. with TSA breast cancer cells or MCA38 colon cancer cells on day 0 and assigned to treatment with: (1) control mAb; (2) anti-CD73 (TY/23); (3) RT (4) RT+TY/23. TY/23 (200 μg) was administered i.p. on day 11, 14, 17 and 20. RT was given locally as single 20 Gy dose on day 12. On day 18, some tumors were harvested for flow cytometry analysis of DC and T cells. Mice were monitored for tumor progression. HPLC was used to measure ADO levels in supernatants from 24 h co-cultures of bone marrow-derived DCs and irradiated TSA cells.In vitro, antibody blockade of CD73, the rate-limiting enzyme in the generation of ADO, reduced the levels of ADO in the supernatant and restored the activation of DCs cultured with irradiated TSA cells. In irradiated tumors, anti-CD73 reduced the percentage of Tregs within the tumor-infiltrating CD4+ T cell population (7.9±2.5% in RT+TY/23 vs 20±0.8% in RT, p<0.01) while increasing CD8\+ T cells (38.3±0.1% in RT+TY/23 vs 17.3±4% in RT, p<0.05). Among intratumoral DCs, the CD8a+ DC subpopulation was increased after CD73-blockade (37.9±15.7% in TY/23+RT vs 11.3±4.9% in RT, p<0.01). Importantly, in irradiated tumors, anti-CD73 enhanced expression of activation markers CD40 on CD8a+ DCs (MFI: 218±1 in RT+TY/23 vs 54±41 in RT, p<0.05) and CD69 on CD8+ T cells (MFI: 513±126 in RT+TY/23 vs 148±59 in RT, p<0.01). Furthermore, tumor-bearing mice treated with RT in combination with anti-CD73 had a significantly delayed tumor progression (p<0.05) and prolonged survival (p<0.01) compared to mice receiving RT alone. Anti-CD73 given alone had no effect on tumor growth.

Our data show that adenosinergic signaling regulates the ability of RT to induce anti-tumor immunity, affecting activation of both DCs and effector T cells. ADO blockade may represent a promising strategy to enhance the immunogenicity of irradiated tumors by improving the ability of RT to induce in situ tumor vaccination.

#4034

Immunotherapy generates selective pressure to create an escape tumor with increased susceptibility to treatment with T cell based therapies.

Jenny H. Pan, Suman Vodnala, Robert L. Eil, Zhiya Yu, Jared Gartner, David Clever, Rahul Roychoudhuri, Shashank J. Patel, Christopher A. Klebanoff, Madhu Sukumar, Tori Yamamoto, Nicholas P. Restifo. _National Institute of Health, Bethesda, MD_.

Host immune surveillance as a mechanism to promote tumor growth or to suppress tumor development is well studied. Less is known how immuno-selective pressure in the form of immunotherapies can instruct the immune system during this process. We hypothesized that the application of a T cell-dependent selection pressure in the form of a whole tumor vaccine would alter the immunogenic architecture in our transplantable murine melanoma model SB-3123. We generated an escape variant to test this hypothesis. We performed whole-exome and RNA-sequencing of the tumor line SB-3123 and two fresh tumors generated by subcutaneous implantation of SB-3123 to identify mutations and to assess levels of expression. There were 349 mutations shared between the tumor line and fresh tumors and less than 5 mutations were unique to each sample. We then investigated whether tumors that had undergone an additional round of host immunoediting would acquire different mutations. To test this, we generated a re-derived tumor cell line from a fresh SB-3123 tumor. Remarkably, the re-derived tissue culture line retained 333 mutations in common with the prior samples and produced only 2 unique mutations.

To test the immunogenicity of SB-3123, we vaccinated mice with irradiated SB-3123 and administered a live tumor challenge two weeks after vaccination. This resulted in a profound vaccination response with mice being completely protected from tumor for over 200 days. However, one vaccinated mouse developed a recrudesced tumor at the site of implantation 40 days after challenge and a tumor line was created (SB-3123-esc). We asked whether SB-3123-esc represented an escape variant by immunizing mice with SB-3123 and then challenging with either SB-3123 or SB-3123-esc. Vaccination with SB-3123 did not protect against SB-3123-esc. We also performed sequencing on SB-3123-esc and discovered that 441 nonsynonymous mutations were common to both tumor lines while 80 were unique to SB-3123 and 40 to SB-3123-esc. Furthermore, we were able to determine that SB-3123-esc lost 2 immunogenic neoantigens but had acquired 5. In addition, we identified a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor.

Our results demonstrate that the expressed mutations of SB-3123 tumor did not change, regardless of whether we sequenced the tumor line, fresh tumors, or a re-derived tumor cell line. However, the escape tumor had new unique mutations and at least one of these was demonstrably immunogenic; we were able to develop a murine T cell receptor with specific reactivity for the escape variant but not the parental tumor. In conclusion, we posit that these observations offer a proof of concept that the process of cancer immune evasion can create new opportunities to treat escape tumors with T cell based therapies.

#4035

High throughput validation of antibodies for cancer research.

Mark Shulewitz, Tim Hamilton, Kaiti Schwartz, Christine Thornton, Gerald Uy, Poulomi Archarya, Roumen Bogoev, Yan Wang. _Bio-Rad Laboratories, Inc., Hercules, CA_.

Antibodies are critical reagents both to study and treat a wide range of cancers. In addition to the relevance of antibodies in immunotherapy, their use in understanding cancer pathophysiology is one of the fastest growing applications. Such applications create a constant demand for high quality antibodies that can recognize a wide range of expression of a specific target that is involved in cancer and metastasis. Even though there are thousands of antibodies available, including those from commercial sources, only a small percentage is well characterized and demonstrated to be of dependable quality.

Establishing the ground rules for rigorous validation of antibodies used for immunodetection of cancer targets is critical for obtaining high quality data. In fact, a number of high profile preclinical study failures have been attributed to the use of inadequately characterized antibodies exhibiting non-specific binding or high background. While researchers typically rely on publications in peer-reviewed journals as absolute indicators of antibody quality, this approach has not proven to be very reliable.

In this poster we describe a robust and high throughput process for validating antibodies for use in western blot applications for cancer research. Some key cancer-related target proteins are detected by antibodies and the validation process is illustrated using these western blot data. In a nutshell, each antibody is screened against an entire panel of lysates from different cells expressing endogenous levels of the target protein. A rapid membrane transfer system coupled with a novel stain-free gel imaging technology is used to ensure a high efficiency of transfer and assess protein quality. Antibody sensitivity and binding specificity is determined based on quantitative data generated using an image analysis software. This software allows us to calculate the ratio of specific to non-specific binding accounting for background within the same lane from the western blot image. We have developed analytical parameters that, when applied to these data, allow us to identify high performing antibodies. The basic principles of this validation method are widely applicable for different types of antibodies and sample types. Overall, this approach ensures the selection of high-performing antibody reagents, thus enabling scientists to get dependable data for academic or preclinical research which, in turn, could significantly advance our understanding of cancer and help develop novel immunotherapies for cancer treatment.

#4036

Prognostic value of infiltrating immune cell profiling in lung adenocarcinoma.

Yutaka Kurebayashi,1 Katsura Emoto,1 Yuichiro Hayashi,1 Ikuo Kamiyama,2 Takashi Ohtsuka,2 Hisao Asamura,2 Michiie Sakamoto1. 1 _Department of Pathology, Keio University School of Medicine, Tokyo, Japan;_ 2 _Department of Surgery, Division of Thoracic Surgery, Keio University School of Medicine, Tokyo, Japan_.

Neoplastic cancer cells and cancer stroma (including infiltrating immune cells) determine the biology and prognosis of cancer. Various types of adaptive and innate immune cells are known to infiltrate the cancer stroma. However, the patterns and spatial distribution of immune cell infiltration as well as their association with tumor histology remain poorly understood. To address these issues, we comprehensively analyzed the infiltrating immune cells present in lung adenocarcinoma. The principal types of both adaptive and innate infiltrating immune cells were immunohistochemically evaluated in the predominant histological components of 111 lung adenocarcinomas. The same analysis was also carried out on 143 samples of histological subtypes making up more than 20% of tumors. As a result, plasma cells and B cells with interfollicular distribution were almost exclusively observed in invasive histological subtypes, while increased number of mast cells were observed in noninvasive histological subtypes. Cluster analysis revealed four distinct immunosubtypes: those characterized by high CD8/CD3 ratios with low stromal cellularity (CD8 subtype), those with increased mast cell numbers (mast cell subtype), those with increased macrophage/DC infiltration with variable amounts of T cell infiltration (mφ/DC subtype), and those with increased plasma cell infiltration in addition to increased macrophage/DC infiltration and variable increases in other infiltrating immune cells (T cells, Tregs, pDCs, and neutrophils) (plasma cell subtype). These immunosubtypes correlated with histological subtypes. Univariate and multivariate analyses identified the plasma cell subtype as an independent negative prognostic factor. Furthermore, we found that these plasma cells may be one of the major producers of immunosuppressive cytokine IL-35 in cancer stroma. Consequently, comprehensive immunohistochemistry-based profiling of infiltrating immune cells has potential as a powerful method for analyzing the cancer stroma of various cancer tissues.

#4037

Enhancement of efferocytosis by rhoA kinase inhibitor following doxorubicin treatment synergistically reduces tumor growth.

Gihoon Nam, In-San Kim. _Korea Institute of Science and Technology, Seoul, Republic of Korea_.

Massive dying of tumor cells after chemotherapy may promote chronic inflammation which favors tumor growth by uncleared secondary necrotic cells. Given that defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity, the prompt removal of apoptotic cells by phagocytes is important for anti-tumor immunity. To improve the anti-tumor effect of doxorubicin, we investigated the role of RhoA-kinase (ROCK) inhibitors on efferocytosis by macrophages, which is important for anti-tumor immunity and tissue regeneration. Treatment of bone marrow-derived macrophages (BMDMs) with ROCK inhibitor increased efferocytosis of dying tumor cells by doxorubicin in vitro. ROCK-blockade after doxorubicin treatment group showed effective inhibition of tumor growth and also T cell-mediated elimination of immunogenic tumors more than the doxorubicin monotherapy group in established tumor models. Furthermore, we also confirmed that macrophage depletion abrogated the therapeutic effect of ROCK inhibitor after doxorubicin treatment on tumor growth. Taken together, our findings indicate that ROCK inhibitor following doxorubicin treatment reduced tumor growth via efferocytosis enhancement.

#4038

Developmentof a murine tumor immunophenotyping platform to support drug discovery anddevelopment in immuno-oncology.

Brian Belmontes, Stephanie Matyas, Sarah O'Brien, Hong Tan, Kenneth Ganley, Kimberly Merriam, Jim Rottman, Jackson Egen, Pedro Beltran, Gordon Moody. _Amgen, Thousand Oaks, CA_.

Recent clinical data highlights the importance of immune cell localization, phenotype, and gene signature as it correlates to a productive anti-tumor response. Preclinically, multiple syngeneic mouse models have been used to study the effects of immunomodulation and define anti-tumor responses to transplanted "self" tumors. However, while the literature describes distinct aspects of many of these models, there is no comprehensive dataset comparing and contrasting their tumor-immune microenvironments across models. These data are critical for better understanding the role that various immune populations play in the anti-tumor response and interpreting observed changes in tumor clearance following treatment with immunomodulatory agents. We have therefore established a platform that 1) quantitates the types of immune cells within murine tumor models, and 2) describes the location of these cells within the tumor. In parallel to the immunophenotyping efforts we have benchmarked tumor models based on their response to antibodies against T cell checkpoint pathways.

We sought to use this immunophenotyping platform to identify specific immune modulation that occurs in syngeneic tumors post depletion of macrophages via CSF1R blockade. Tumor-associated macrophages (TAMs) are believed to help promote tumor survival through suppression of the adaptive immune response and the secretion of growth factors that promote tumor growth and angiogenesis. Therefore depletion of TAMs should lead to T-cell recruitment and bolster the antitumor T-cell response. Here we show that treatment of CT-26 and RENCA syngeneic tumors with a CSF1R antagonist leads to depletion of MHCII+ and F4/80+ expressing cells. Future experiments will seek to understand if CSF1R blockade improves the response to T-cell checkpoint immunotherapies. In summary, the development of a murine tumor immunophenotyping platform has allowed insight and evaluation of immune cells in the tumor microenvironment that can ultimately be leveraged to understand the synergistic effects of immunotherapeutics.

#4039

Mimotope variance analysis: A novel immunoprofiling method to monitor progression of cervical cancer.

Espen Enerly,1 Mari Nygård,1 Madleen Orumaa,1 Arno Pihlak,2 Susan Pihelgas,2 Hilde Langseth,1 Toomas Neuman,2 Kaia Palm2. 1 _Cancer Registry of Norway, Oslo, Norway;_ 2 _Protobios LLC, Tallinn, Estonia_.

The purpose of the study was to identify and describe cervical cancer specific changes in humoral immune response by using a novel technology platform Mimotope Variation Analysis (MVA) developed by Protobios.

The Janus Serum Bank is one of the world's oldest and largest population-based research biobanks established in 1973. The cohort is annually linked to the Cancer Registry of Norway using personal identification numbers. More than 1000 women have developed cervical cancer after donating sera. We identified sets of successive sera samples from ten patients with invasive cervical cancer, ten patients with pre-invasive cervical neoplasia and twenty cancer-free individuals (matched for age, gender, length of sample storage +3 months). For each subject we identified 4-10 samples donated to Janus over a period of up to 18 years, in total 213 samples. We applied MVA which combines phage display technology and high-throughput sequencing analysis to generate quantitative serologic profiles of millions of 12-mer peptide antigens called mimotopes from 2 μl of blood serum per analysis.

Hierarchical clustering analysis of the top 5000 mimotopes from each sample resulted in individual-specific immunoprofiles. Multiple samples from the same person clustered together. Moreover, clustering reflected the time the sera was drawn suggesting that part of the individual immunoprofile is stabile over time.

In conclusion, preliminary results suggests that Mimotope Variance Analysis generates individual-specific immunological profiles. This profile most likely reflect prior exposure environmental pathogens. Further, we aim to decode the differences in the immunological profiles between cancer patients and cancer-free subjects.

#4040

Validation of multiplex immunofluorescence for use in analysis of tumor infiltrating lymphocytes.

James Mansfield,1 Henry Galletta,2 Richard J. Byers,2 Kenneth Oguejiofor2. 1 _PerkinElmer, Hopkinton, MA;_ 2 _The University of Manchester, Manchester, United Kingdom_.

Multiplexed immunohistochemistry (IHC) has the potential to improve conventional IHC staining allowing for analysis of multiple cell phenotypes while maintaining spatial context. Automated multispectral image analysis and computer-based cell recognition make the process more attainable, but stringent validation of multiplex IHC is still required. Pertinently, multiplex IHC allows for the characterisation and enumeration of immune cell densities in the tumour micro-environment, of particular importance for analysis of tumour-infiltrating immune cells which require multiple co-localised markers for their identification. However, issues of antibody blocking, cross-reactivity and masking have caused concern that the results of multiplex staining may not accurately reflect those of single-plex staining. The Opal workflow from PerkinElmer uses a heat-induced epitope retrieval step between each antibody detected which aims to obviate these potential problems and enable single-species antibody use. In this study we systematically validated multiplex staining for a range of immune cell markers against single plex staining for each marker to determine the accuracy of the multiplex method.

Validation of multiplex IHC was undertaken using a multi-tissue TMA stained in multiplex for CD3, CD4, CD56 and CD20 in a 4-marker validation and for CD3, CD4, CD8, CD20 and FOXP3 in a 5-marker validation. The TMA was composed of 72 cores including normal lung, pancreas, breast, prostate and stomach and malignant prostate, lung and colon. Spearman correlations measured agreement of immune cell populations with those of singly stained serial sections on a per-core basis. Single-stain/single-stain comparisons of corresponding immune cell populations provided baseline variation of immune cells in subsequent sections.

All validation comparisons showed a highly significant correlation (P<0.0001), with strong correlation was observed between cores for the majority of multiplex stains. Specifically, for the 4-plex experiment, a high degree of correlation was observed for CD3, CD8 and CD20 with R2 of 0.835, 0.950 and 0.870 respectively (P=<0.001); CD56 showed a lower degree of correlation between the multiplex and single stains with an R2 of 0.584 (P=<0001). For the 5-plex experiment, a high degree of correlation was observed for high degree of correlation for CD3, CD8 and CD20 with R2 of 0.87, 0.95 and 0.87 respectively (P<0.0001). FOXP3 showed a poor correlation of 0.74 and CD4 showed a poor correlation of 0.58 (P<0.0001).

Multiplex IHC is comparable to single stain IHC preserving precious samples and reagents while enhancing the information gained.

#4041

Glioma cells display a prognostically significant hematopoietic phenotype.

Gerald Moncayo. _FMI/INDICASAT, Basel/Panama, Panama_.

Glioblastoma multiforme (GBM) is one of the most aggressive human cancers. Glioma cells expressed immunoreceptor tyrosine-based activation motif (ITAM) bearing molecules, such as Fc receptors, T cell receptor components and C-type lectins and expression was up regulated in two distinct tumor-derived prognostic signatures identified in GBM and low-grade gliomas. Targeting SYK, downstream of ITAM molecules, attenuated GBM tumor growth and invasiveness, and reduced B and CD11b+ cell mobility and infiltration resulting in improved survival. In addition, GBM tumor cells were found to display phagocytic characteristics in vitro and in vivo by flow cytometry and two photon imaging. Our data demonstrate that gliomas are hematopoietic-like tumors that express an immune signaling network important in glioma progression, and identify targets for therapeutic intervention.

#4042

Quantifying immune oncology markers across multiple tissue sections with digital image analysis.

Ciara A. Martin, Joshua Black, Nathan T. Martin, Logan Cerkovnik, Jasmeet Bajwa, Kristin Wilson, Daniel Rudmann, Carsten Schnatwinkel, AJ Milici. _Flagship Biosciences, Westminster, CO_.

Immuno-oncology (IO) approaches requires an understanding of the types of immune cells present in the tumor microenvironment (TME) as well as the precise localization of these immune cells. However, the acquisition of spatial IO information is technically challenging, due to the requirement for multiplex labeling of immune cells and the need to categorize their location and biomarker content simultaneously. Additionally, the multiplex biomarker panel must be engineered in advance based on a priori assumptions about the correct marker combinations and their location (such as the tumor epithelial nests, TME, or specifically the tumor-stroma interface). This limits the ability to implement novel, scientifically driven assessments into an existing clinical trial which already has defined immunohistochemistry endpoints. To meet the needs of IO clinical trials, Flagship Biosciences has developed a proprietary image analysis platform, FACTS (Feature Analysis on Complementary Tissue Sections), to deduce both the necessary multiplex staining information and the spatial context of IO markers based on the utilization of existing monochrome or multiplex stained slides from a clinical trial. In this study, we demonstrate the utility of this approach to deliver biologically relevant endpoints important for IO clinical trials. First, we performed single IO biomarker staining for CD4, CD8 and FoxP3 in colorectal cancer patient samples and developed a novel image analysis approach that allowed for the accurate quantification of multiple IO markers within specific margins of the tumor microenvironment across multiple tissue sections. For each biomarker the positive cell populations were binned based on distance from the tumor-TME boundary (i.e. % positive cells within 100µM, 200µM or 500µM of the tumor-TME boundary). Next, we used computational alignment for evaluating the co-localization of multiple immune cell biomarkers from serial sections with single stains for CD4, CD8 or FoxP3. We demonstrate that the information extracted from multiple single stained serial sections can be used to generate co-localization information for multiple IO markers in a given patient sample. Lastly, we developed multiplex chromogenic assays for these same markers, analyzed the multiplex-stained slides with image analysis, and derived the same analysis endpoints for the multiplex slides to the digitally aligned individually labeled sections. The study found that similar interpretation of the inflammatory landscape was possible with multiplex-stained slides and digital alignment of monoplex-stained slides. In summary, this study demonstrated a novel image analysis approach that allows for quantification of IO markers utilizing clinically relevant wet assay technologies and existing IHC stained slides derived from an immune-oncology clinical trial.

#4043

Establishment of a mouse skin squamous cell carcinoma allograft model for in vivo pharmacological analysis of immunotherapy.

Gavin Jiagui Qu, Annie Xiaoyu An, Jinping Liu, Davy Xuesong Ouyang, Likun Zhang, Jie Cai, Jean Pierre Wery, Henry Q. X. Li. _Crown Bioscience, Inc., Santa Clara, CA_.

The recent clinical success of novel therapeutics blocking immune checkpoints cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed cell death 1 (PD-1) has fueled an intense interest around immuno-oncology. However, the lack of relevant animal models is a major bottleneck for understanding the mechanism of action and evaluating the efficacy of such therapeutics. Syngeneic mouse tumor models, despite being widely used as experimental models for efficacy studies, are limited by the restricted number of models available and responsive to current checkpoint inhibitors. Genetically engineered mouse models (GEMMs) of human cancer are effective tools for mechanistic analysis, but they are often not suitable for efficacy studies due to usually un-synchronized tumor progression. Syngeneic allografts of spontaneous mouse tumors derived from GEMMs (MuPrime®) may be used as a new type of immuno-oncology models with the following advantages: 1) their primary nature of "stem cell diseases" and relevant tumor microenvironment mirror the patient derived xenograft (PDX) models; 2) the availability of diverse cancer types and oncogenic drivers deriving from a wide range of GEMMs. We set out to build a library of allografts of spontaneous mouse tumors, including those derived from GEMM, in an immunocompetent host to support pharmacological investigation, particularly of immuno-oncology agents.

Recently, we observed the growth of spontaneous cutaneous tumors on the neck of the C57BL/6J-ApcMin/J GEMM, which is heterozygous for the Apc (APCMin/+). Histopathology suggests the tumors are well-differentiated skin squamous cell carcinoma. We subcutaneously transplanted these skin tumors into the syngeneic C57BL/6J mice. The allografts grow well, are serially transplantable and maintain the histopathology of the primary tumor. Transcriptome sequencing revealed that the allografts maintain the original ApcMin mutation, express high levels of HER2 (also confirmed by immunochemistry, or IHC), and present frameshift mutations in both c-met and EGFR. In vivo pharmacological assessment indicate that tumors respond to 5-FU, paclitaxel, gemcitabine, docetaxel, and cisplatin chemotherapy. They is also sensitive to anti-mouse CTLA-4 antibody, and the response is associated with an increased number of tumor infiltrating immune cells, including TILs. Alltogether, here we show that we have established an allograft model suitable for in vivo efficacy analysis of immunotherapy using surrogate anti-mouse antibodies.

#4044

Rapamycin targets Interleukin 6 (IL-6) expression and suppresses endothelial cell invasion stimulated by tumor cells.

Oleksandr Y. Ekshyyan, Xiaohua Rong, Tara Moore-Medlin, Xiaohui Ma, Jonathan Steven Alexander, Cherie-Ann O. Nathan. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Background: Previously we demonstrated that mTOR inhibitors have potent antiangiogenic and anti-lymphangiogenic effects in addition to their growth inhibitory effects in head and neck squamous cell carcinoma (HNSCC). Lymphatogenous spread is a significant problem and much more predominant in HNSCC than hematogenous spread. In this study we evaluated the effects of rapamycin on targeting tumor-stroma crosstalk in HNSCC.

Methods: HNSCC tumor cells (FaDu), human blood microvascular endothelial cells (HMEC-1), and human lymphatic endothelial cells (HMEC-1A) were co-cultured in various combinations using transwell cell culture inserts to study tumor-stroma crosstalk and the effects of mTOR inhibitor rapamycin. Cell invasion was assessed using transwell inserts with an 8.0 μm pore size. Levels of various growth factors and cytokines in cell culture media were measured using Milliplex bead immunoassay (EMD Millipore) and ELISA assay (R&D Systems).

Results: We found that conditioned media collected from tumor cells or co-culture with tumor cells significantly increased the invasiveness of lymphatic and blood vascular endothelial cells (p < 0.05), while there was no effect of conditioned media collected from endothelial cell cultures or co-culture with endothelial cells on tumor cell invasiveness. Conditioned media collected from tumor cell cultures increased the invasiveness of endothelial cells by almost two-fold.

Next we evaluated the effects of rapamycin on the baseline and tumor cell stimulated (co-culture or conditioned media) invasiveness of endothelial cells. There was a significant effect of rapamycin on both baseline and tumor cell stimulated invasiveness of endothelial cells. While rapamycin had a moderate effect on the levels of several growth factors and cytokines tested (sVEGFR-1, HGF, FGF-2, TNF-alpha) there was a significant effect of the drug on the levels of IL-6. Importantly the level of IL-6 secreted in media increased significantly in tumor-endothelial cell co-culture compared to monocultures. Rapamycin showed only modest effect on the invasiveness of endothelial cells transfected with rapamycin-resistant mTOR as expected. There was no significant effect of rapamycin on secretion of IL-6 by tumor cells and endothelial cells transfected with rapamycin-resistant mTOR.

Conclusions: HNSCC cells produce chemotactic stimuli that promote endothelial cell invasion toward tumor cells that can stimulate angio- and lymphagiogenesis. Rapamycin effectively reverted the stimulatory effect of cytokines secreted by tumor cells on endothelial cell. IL-6 is being evaluated as an important target in HNSCC and a potential biomarker of response to mTOR inhibitor therapy.

## TUMOR BIOLOGY:

### Chemical and Viral Carcinogenesis

#4045

Urban-rural disparity of overweight/obesity distribution and its potential association with breast cancer among Chinese females.

Ying Gao,1 Yubei Huang,2 Hongji Dai,2 Peishan Wang,2 Haixin Li,2 Fengju Song,2 Hong Zheng,2 Henglei Dong,2 Jiali Han,3 Yaogang Wang,1 Kexin Chen2. 1 _Tianjin Med. Univ, Tianjin, China;_ 2 _Tianjin Med. Univ. Cancer Inst. & Hospital, Tianjin, China; _3 _Simon Cancer Center, Indiana University, Indianapolis, IN_.

Background: Disparity of overweight/obesity distribution had been investigated in developed countries, but rarely been investigated in developing countries, especially for China with rapid socio-economic development in the past decades.

Methods: A total of 1 210 762 participants were recruited from the Chinese National Breast Cancer Screening Program. Overweight and obesity were defined as body mass index (BMI) ranged 24.0-27.9 kg/m2 and BMI ≥ 28.0kg/m2, respectively.

Results: The prevalence of overweight/obesity for Chinese rural women (35.2%, 29.2% for overweight and 6.0% for obesity) was significantly higher than that for Chinese urban women (33.4%, 27.7% for overweight and 5.7% for obesity) (P <0.001). For either rural or urban women, the prevalence of overweight/obesity was highest in north region, followed by east region for rural women and north-east region for urban women. For rural women, higher prevalence of overweight/obesity was significantly positively associated with elder age, Han nationality, low level of education, no occupation, high family income, less number of family residents, insurance, and elder age at marriage. Similar positive associations were also found for urban women, except negative associations for high family income, less number of family residents, and elder age at marriage. A non-significant positive association between overweight/obesity and breast cancer was found for rural women [odds ratio (OR) = 1.06; 95% confidence interval (CI): 0.87-1.29], but a significant positive association for urban women (OR = 1.55; 95%CI: 1.19-2.02).

Conclusions: There was an obvious urban-rural disparity of overweight/obesity distribution among Chinese females, which could also lead to an obvious disparity of breast cancer distribution.

#4046

The role of TFPI2 and FGF2 in asbestos-induced mesothelial to fibroblastic transition.

Joyce K. Thompson, Jill Miller, Maximilian B. MacPherson, Arti Shukla. _University of Vermont, Burlington, VT_.

Mechanisms involved in the tumorigenesis of the devastating cancer, malignant mesothelioma (MM) are poorly understood. We have recently shown that interleukin-1β (IL-1β), an inflammatory cytokine is upregulated by asbestos via the activation of the inflammasome (a molecular platform that assembles for the activation of caspase-1) in mesothelial cells. Furthermore we have demonstrated that IL-1β secretion may lead to the activation of downstream signaling cascades involved in malignant transformation of mesothelial cells.

Preliminary data from our lab indicate that in addition to IL-1β, asbestos exposure upregulated the secretion of basic fibroblast growth factor (bFGF/FGF2) and tissue factor pathway inhibitor 2 ((TFPI2) a kunitz type protease inhibitor). These factors were also regulated by the inflammasome and have never before been implicated in asbestos-induced mesothelial to fibroblastic transition (MFT). Based on our preliminary data, we hypothesized that upregulation of IL-1β by asbestos-induced inflammasome activation increases FGF2 secretion and signaling. Furthermore, we hypothesize that FGF2 together with increased TFPI2 secretion induces transition of mesothelial cells to a fibroblastic phenotype that facilitates MM carcinogenesis. In the proposed study we will delineate the role of TFPI2 (siRNA) and FGF2 (pan FGFR inhibitor, BGJ398) in the process of asbestos-induced MFT.

Data from this study will provide added insight into the mechanisms involved in the initiation of MM and indicate whether TFPI2 and FGF2 can serve as drugable targets for combination therapy against MM. This work is supported by NIH grant, RO1 ES021110.

#4047

Salmonella protein AvrA activates the STAT3 signaling pathway in colon cancer.

Rong Lu,1 Shaoping Wu,2 Yong-Guo Zhang,1 Yinglin Xia,1 Zhongren Zhou,2 Ikuko Tako,3 Hui Dong,4 Marc Bissonnette,5 Jun Sun1. 1 _UIC, Chicago, IL;_ 2 _University of Rochester, Rochester, NY;_ 3 _Wayne State University School of Medicine, Detroit, IL;_ 4 _The Third Military Medical University, Chongqing, China;_ 5 _The University of Chicago,, Chicago, IL_.

Salmonella infection in humans can become chronic which leads to low grade persistent inflammation. These chronic infections increase the risk of several gastrointestinal diseases, including cancer. A recent study has shown that antibody against Salmonella flagellin was higher in colorectal cancer and pre-cancer cases than controls in two distinct populations in US and the Netherlands and that dietary intake is the one of the mediating factors, suggesting a potential link of Salmonella to colorectal cancer. Salmonella AvrA is a multifunctional protein that influences eukaryotic cell pathways by regulating ubiquitination and acetylation. In an animal model, we have demonstrated that infection with AvrA expressing Salmonella induces beta-catenin signals and enhances colonic tumorigenesis. Beta-catenin signaling is a key player in intestinal proliferation and tumorigenesis. The relative contributions of AvrA-induced proliferation and inflammation on tumorigenesis, however, are unknown. STAT3 is activated in chronically inflamed intestines in human inflammatory bowel diseases and in colitis-associated colon cancer. In the current study, mice were colonized with Salmonella AvrA-sufficient or AvrA-deficient bacterial strains. Then, inflammation-associated colon cancer was induced through the use of azoxymethane/dextran sulfate sodium. We determined that AvrA expressing bacteria activated the STAT3 pathway, which is predicted to increase inflammation, enhance proliferation, and promote tumorigenesis. Transcriptional activity of STAT3 and its target genes were up-regulated by Salmonella expressing AvrA, thus promoting proliferation and intestinal tumorigenesis. Our current findings provide new insights regarding a STAT3-dependent mechanism by which the specific bacterial product AvrA enhances the development of infection-associated colon cancer. Our observations also raise a note of caution regarding the use of mutant Salmonella organisms as vectors for ant-cancer therapy. Finally, these studies could suggest biomarkers (such as AvrA level in gut) to assess cancer risk in susceptible individuals and infection-related dysregulation of STAT/β-catenin signaling in colon cancer.

#4048

Stem cell properties of normal human keratinocytes influence transformation responses to HPV16.

Yvon Woappi, Kim E. Creek, Lucia Pirisi. _University of south Carolina, Columbia, SC_.

Human papillomavirus (HPV) infection is the primary cause of cervical cancer. While HPV infection is very common, cervical cancer is a rare outcome. Why some women develop cervical cancer and others do not is not well understood, but likely involves host cell-specific factors and a woman's ability to get rid of HPV-infected cells. We have previously reported marked variability of epidermal growth factor receptor (EGFR) expression in normal human epidermal tissue from different individuals and demonstrated that increased immortalization by HPV16 E6 and E7 oncogenes correlated with lower basal levels of EGFR. We now report a significantly greater transformation response in stem cell-like keratinocytes after transfection with the full length HPV16 genome. Primary normal human keratinocyte strains (NHKc) from 59 individuals were cultured in 3-D anchorage-free suspension to assess spheroid forming ability and to determine resistance to anchorage-independent cell death. Only about 40% of NHKc strains survived and formed spheroids in 3-D culture and grew when plated back in monolayers as spheroid-derived NHKc (SD-NHKc). Fluorescence-activated cell sorting analysis found that SD-NHKc were enriched in cells expressing known keratinocyte stem cell markers: low levels of EGFR and high levels of integrin α6, while spheroid-non forming strains (SNF-NHKc) had dramatically reduced numbers of EGFRlo/ITGα6hi cells. Isolation and cultivation of EGFRlo/ITGα6hi keratinocytes isolated from primary monolayer NHKc cultures revealed this cell population to behave like SD-NHKc with respect to anchorage-independent survival, proliferative potential, cell morphology, and clonogenicity. Upon transfection with HPV16 DNA, most NHKc strains acquired anchorage-independence and exhibited increased expression of EGFR. Importantly, EGFRlo/ITGα6hi keratinocytes and SD-NHKc displayed exuberant growth responses after transfection with HPV16 DNA and significantly higher transformation efficiency by HPV16. In contrast, SNF-NHKc grew more sluggishly and were relatively resistant to HPV16-mediated immortalization. Our study indicates that stem cell-like keratinocytes are preferentially transformed by HPV16 DNA and that stemness is a phenotypic determinant of susceptibility to HPV16-mediated transformation in primary human keratinocytes.

#4049

Conserved region 2 of Human Papilloma Virus 18 protein E7 is required for E7 binding to centromere protein C.

Aoi Tokuda, Shoko Takahashi, Masafumi Yoshimoto, Yuji Yaginuma. _Graduate School of Health Sciences, Kumamoto University, Kumamoto, Japan_.

The molecular genetics of cervical cancer have not been thoroughly elucidated, despite the fact that cervical cancer is responsible for the deaths of over 250,000 women annually worldwide. Infection with human papillomaviruses (HPVs) is linked to the pathogenesis of a variety of human diseases, including genital warts, laryngeal papillomas, and most notably, cervical cancer. Recently HPV infection was reported to be associated with head and neck cancers.High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. HPV infection leads to aneuploidy, a numerical chromosomal aberration caused by dysregulation of chromosomal segregation. We found that high-risk HPV18 and HPV58 E7 proteins bind to centromere protein C (CENP-C), a component of the kinetochore that is essential for proper chromosomal segregation. Low-risk HPV4, HPV6, and HPV11 E7s do not bind to CENP-C. The PxDLLCxE sequence in conserved region 2 (CR2) of HPV18 E7 is required for E7 binding to CENP-C. Our results indicate that differences in the ability of high- and low-risk HPV E7s to bind to CENP-C reflect the different oncogenic potentials of high- and low-risk type HPVs.We demonstrated that the E7 proteins from low-risk HPVs do not bind to CENP-C, and that the PxDLLCxE sequence in conserved region 2 of the HPV18 E7 protein is required for E7 binding to CENP-C. CENP-C is a component of the inner kinetochore, and as such, it plays an essential role in proper chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Therefore, we hypothesize that differences in the capability of various E7s from low- and high-risk type HPVs to bind to CENP-C reflect the different oncogenic potential of high- and low-risk type HPVs.

#4050

Bovine leukemia virus small noncoding RNAs are functional elements that regulate replication and contribute to oncogenesis in vivo.

Nicolas Gillet,1 Malik Hamaidia,1 Alix de Brogniez,1 Geronimo Gutierrez,2 Nathalie Renotte,1 Michal Reichert,3 Karina Trono,2 Luc Willems1. 1 _Univ. of Liège, Liège, Belgium;_ 2 _Instituto de Virología, INTA, Castelar, Argentina;_ 3 _National Veterinary Research Institute (PIWet), Pulawy, Poland_.

Bovine leukemia virus (BLV), a retrovirus closely related to the human T-lymphotropic virus type 1 (HTLV-1), induces lymphocytosis and B-cell leukemia/lymphoma in ruminants. Although the provirus undergoes transcriptional silencing during transformation, BLV-infected tumor cells massively express viral miRNAs from internal Pol III promoters. To understand the mechanisms involved, a provirus isogenic to a wild type molecular clone but devoid of miRNAs was inoculated into calves. High-throughput RNA-sequencing and bioinformatic analyzes indicated that BLV miRNAs modulate expression of genes involved in B-cell receptor signaling, apoptosis, cell activation and immune response (GZMA, FOS, PPT1, ANXA1, MAP2K1 and PIK3CG ). Direct and indirect interactions within these regulatory pathways were validated by luciferase reporter assays. In vivo, the deletion of BLV miRNAs reduced proviral loads in the natural host, indicating their biological relevance in viral replication and persistence. Furthermore, oncogenesis was abolished in the ovine experimental model. These observations thus show that BLV miRNAs affect key signaling pathways, promote viral replication and drive oncogenesis.

#4051

Lipid peroxidation-derived DNA adduct formation in obesity-related hepatocarcinogenesis.

Heidi Coia,1 Hongyi Guan,2 Ying Fu,1 Marcin Dyba,1 Fung-Lung Chung1. 1 _Georgetown University, Washington, DC;_ 2 _Thomas Jefferson High School for Science and Technology, Alexandria, VA_.

In this study we are investigating the role and mechanism of formation of Lipid-peroxidation (LPO)-derived DNA adduct formation in obesity-related hepatocarcinogenesis. Obesity has been implicated as a risk factor for many types of cancer, particularly hepatocellular carcinoma (HCC). HCC incidence correlates with the increasing prevalence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in the US. The mechanisms underlying this increased risk of liver cancer in the obese population are still unclear. Liver tissue damage from fat accumulation in NAFLD and NASH produce inflammation and increased levels of reactive oxygen species (ROS). ROS induce LPO of polyunsaturated fatty acids (PUFAs) in cell membranes leading to the formation of reactive aldehydes, such as acrolein and 4-hydroxy-2-nonenal (HNE), which react with DNA forming the DNA adducts γ-OHPdG and DHH-ԑdA, respectively. γ-OHPdG is derived from ω-6 and ω-3 PUFAs, while DHH-ԑdA is specifically from ω-6 fatty acids. Previous data has indicated that mutational hotspots targeted by γ-OHPdG and DHH-ԑdA may be within key cancer driver genes, such as p53. Research has shown that B-6 mice fed a high fat diet (HFD) develop fatty liver disease and eventually HCC. The livers of B-6 mice fed a HFD showed an increased ω-6/ω-3 PUFAs ratio. We have detected and quantified γ-OHPdG and DHH-εdA by LC-MS/MS in livers from six healthy individuals and six NAFLD patients and found that levels of DHH-εdA in the DNA of the fatty liver samples were nearly three-fold higher than that in normal liver samples. Preliminary in vitro data using primary human hepatocytes has indicated that treatment with oleic and palmitic acid promote the formation of γ-OHPdG. Similarly, treatment of these cells with the epoxide of HNE, 2,3-epoxy-4-hydroxynonanal (EH), and the omega-6 fatty acid, arachidonic acid, induce the formation of DHH-εdA, supporting the proposed mechanism of adduct formation. In an 80-week tumor bioassay using C57Bl/6J mice on a HFD, we have observed through live-animal MRI imaging and immunohistochemistry, an increase in pro-inflammatory white adipose tissue accumulation, infiltration of fat into the liver and an increase in overall body weight compared to a low fat diet control. In addition, in mice fed a HFD combined with the green-tea derived antioxidant Theaphenon E, we observed through MRI, reduction in body weight gain, white adipose tissue, and lipid accumulation in liver. Theaphenon E has the potential to decrease fat accumulation and inflammation within the liver that lead to decreases in LPO-derived adduct formation and consequently, the mutations critical for HCC development.

#4052

The role of the phosphorylated RXRα on diethylnitrosamine-induced liver tumorigenesis in mice.

Hiroyasu Sakai, Yohei Shirakami, Masahito Shimizu. _Gifu Univ. School of Medicine, Gifu, Japan_.

We have previously reported that a malfunction of RXRα due to aberrant phosphorylation at serine 260 by MAPK is associated with the development of hepatocellular carcinoma (HCC). The phosphorylated RXRα impairs normal receptor functions of either RXR or RAR, which are respective homodimeric or heterodimeric partner of RXRα, in a dominant-negative manner. Besides, these impaired receptor functions result in the downregulation of their target gene expression and inducing hepatocarcinogenesis by stimulating cell proliferation and inhibiting apoptosis, suggesting that phosphorylated RXRα plays a crucial role in the development of HCC. However, these findings were revealed primarily using HCC cell lines that express the phosphorylated-RXRα (p-RXRα) proteins and it still remains unclear whether p-RXRα may impact in the process of hepatocarcinogenesis in vivo. Therefore, in order to investigate the biological functions of p-RXRα on hepatocarcinogenesis in vivo, we generated doxycycline-inducible ES cell line and transgenic mouse, both of which overexpress phosphomimic mutant of RXRα (T82D/S260D). Interestingly, the transcriptional activities of Retinoid X Receptor Response Element (RXRE) were significantly reduced in the Mouse Embryonic Fibroblasts (MEFs) derived from the transgenic mice, suggesting that the retinoid signaling via RXRα were impaired in the mice. Moreover, the development of liver tumors induced by liver carcinogen diethylnitrosamine (DEN) was enhanced in the transgenic mice that overexpress T82D/S260D protein in the liver. Notably, the increased liver tumors in the transgenic mice resulted from the promotion of cell cycle progression, not the inhibition of apoptosis. Interestingly, expression of β-catenin and its target genes, cyclin D1 and c-myc, were increased in transgenic liver tumors, although the expression of retinoid-related genes, such as RARβ, p21 and p27, were not altered compared with control mice. These results suggest that the phospho-modification of RXRα may promote the development of DEN-induced liver tumors through the activation of β-catenin signaling pathways in mice.

#4053

A unique nutrient mixture suppresses ovarian cancer growth of A-2780 by inhibiting invasion and MMP-9 secretion.

M. Waheed Roomi, Matthias Rath, Aleksandra Niedzwiecki. _Dr. Rath Research Institute, Santa Clara, CA_.

Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. The American Cancer Society estimated that it would claim 14,250 lives in 2013. Despite the advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a unique nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n=12) were inoculated by I.P. with 2x106 cells in 0.1ml PBS and randomly divided into two groups. Group A (n=6) was fed a regular diet and group B (n=6) a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed and processed for histology. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with NM in triplicate at concentrations between 0-1000 μg/ml. Cell proliferation was measured by MTT assay, MMP-9 secretion by gelatinase zymography, invasion through Matrigel and morphology by H&E staining. All control mice (Group A) developed large ovarian tumors, whereas 5 out of 6 mice in group B developed no tumors, and one, a small tumor. Zymography demonstrated only MMP-9 expression, which NM inhibited in a dose dependent fashion, with virtual total block at 250 μg/ml concentration. NM significantly inhibited invasion through Matrigel with total block at 250 μg/ml concentration. MTT showed dose dependent inhibition of cell proliferation with NM and H&E staining showed no morphological changes below 500 μg/ml NM. These results suggest that NM has therapeutic potential in treatment of ovarian cancer by significantly suppressing tumor growth and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.

#4054

Efficient identification of integration sites of human papilloma virus (HPV) genome in HPV-related head and neck squamous cell carcinoma cell lines with next-generation sequencing-based novel approach.

Takashi Hatano,1 Daisuke Sano,1 Hideaki Takahashi,1 Hiroshi Hyakusoku,1 Yasuhiro Isono,1 Shoko Shimada,1 Kae Sawakuma,1 Yoshiyuki Watanabe,2 Hiroyuki Yamamoto,2 Fumio Itoh,2 Jeffrey N. Myers,3 Nobuhiko Oridate1. 1 _Yokohama City University, Yokohama, Japan;_ 2 _St. Marianna University, Kawasaki, Japan;_ 3 _The University of Texas M. D. Anderson Cancer Center, Houston, TX_.

Background: The incidence of human papillomavirus (HPV)-related oropharyngeal cancers (HPV-OPC) is increasing remarkably these days, in contrast to other head and neck cancers with use of tobacco and/or alcohol as major risks. While the detail mechanism of HPV integration on the host genome for the development of cervical cancer has been well characterized, less is known about the interactions between HPV integration and the human genome in HPV-OPC. The purpose of this study is to reveal genome-wide maps of HPV integration loci in HPV-related head and neck squamous cell carcinoma (HNSCC) cell lines. Methods: Three HPV 16-related HNSCC cell lines (UPCI:SCC090; tongue base, UPCI:SCC152; hypopharyngeal recurrence of a tongue base cancer, UPCI:SCC154; tongue) were prepared for this study. To detect integrated HPV sequences in the genome of these 3 HPV-related HNSCC cell lines, we used technique of target enrichment for next-generation sequencing analysis A total 427 of custom RNA probes were designed to specifically target the HPV genome for this study. After capture and amplification, the templates were sequenced using next-generation sequencer (NGS). Reads that aligned perfectly to the human or HPV 16 genome were removed. Reads aligned partially to human genome and HPV 16 genome were reserved. The integration sites, exact joint position of human and HPV 16 sequence, were then efficiently detected from the reserved reads. PCR and direct sequencing were used to verify the detected HPV 16 integration sites. Results: The average read length was 414.18 bp. The average read quality was 31.30, corresponding to >99.9% accuracy. The HPV integrants mapped to chromosomes 2q23, 3p12, 6p21, and 9q22 in UPCI:SCC090, chromosomes 2q23, 3p12, 9q22, and 9q31 in UPCI:SCC152, and chromosome 21q21 in UPCI:SCC154 were detected. Conclusion: In the present study, we identified novel integration sites in HPV-related HNSCC cell lines with our unique approach with deep and high-throughput sequencing which enabled enrichment of viral fragments for sequencing using custom-made probes based on the sequence of the HPV genome, without conducting unnecessary sequencing of regions where the HPV genome is not integrated.

#4055

Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors.

Ram Prasad, Tripti Singh, Santosh K. Katiyar. _University of Alabama at Birmingham, Birmingham, AL_.

Ultraviolet (UV) radiation has variety of deleterious effects on mammalian skin, and triggers several causative factors such as DNA damage, inflammation, immune suppression and genetic and epigenetic alterations which leads to the development of skin cancer. Earlier, we have shown that the deficiency of interleukin (IL)-12, an immunoregulatory cytokine, enhances photocarcinogenesis in mice through inhibition of repair of damaged DNA. In further studies we have tried to explore and examine the effect of IL-12 on epigenetic regulators in UV-exposed skin and UV-induced skin tumors using IL-12 knock out (IL-12 KO) mice as an in vivo animal model. The exposure of IL-12 KO mice with UVB (200 mJ/cm2) radiation resulted in higher levels of DNA methylation, DNA methyltransferase (Dnmt) activity, Ten-eleven translocations (TETs), a family of methylcytosine dioxygenase, histone deacetylases (HDAC), and lower levels of histone acetylase (HAT) activity than their wild type counterparts. To verify that IL-12 deficiency is associated with DNA hypermethylation and higher HDAC activity in UVB exposed skin, IL-12-KO mice were treated with endotoxin-free murine rIL-12 (50 ng/mouse/100 µl PBS; subcutaneous injection) 3 h before UVB exposure. Our results indicate that significant reduction in the levels of global DNA methylation, Dnmt and HDAC activity were observed compared with non-rIL-12 treated mice. Conversely, UVB exposed IL-12 wild type mice were treated with anti-IL-12 antibody (500 ng/mouse/100 µl PBS; subcutaneous injection). This treatment resulted in increased levels of DNA methylation, Dnmt and HDAC activities. These observations were also noted and compared between skin tumors from IL-12 KO mice and their wild type counterparts. To further explore the role of IL-12 deficiency on epigenetic modification-mediated photocarcinogenesis, we determined its effect on methylated and unmethylated levels of tumor suppressor genes, such as p16INK4a and RASSF1A, in UV-exposed skin and UV-induced skin tumors. It was observed that the levels of methylated tumor suppressor genes were higher in IL-12 KO mice skin than their wild type counterparts. Together, these findings indicate that IL-12 deficiency promotes: (i) DNA hypermethylation, (ii) HDAC activity and (iii) silencing of tumor suppressor genes in UV exposed mice and that makes the mice more susceptible to UV radiation-induced skin cancer risk. Together, these observations suggest that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of UV radiation-induced skin cancer risk.

#4056

Development of Kras mutant lung adenocarcinoma in tobacco carcinogen exposed mice with knockout of the airway lineage-specific G-protein coupled receptor Gprc5a.

Junya Fujimoto,1 Sayuri Nunomura,1 Wenhua Lang,1 Yihua Liu,1 Jinsong Wei,1 Joshua Ochieng,1 Yasminka Jakubek,1 Edwin Ostrin,1 Jason Petersen,2 Gareth Davies,2 Nadine Darwiche,3 Erik Ehli,2 Jerry Fowler,1 Paul Scheet,1 Humam Kadara1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Avera Institute for Human Genetics, Sioux Falls, SD;_ 3 _American University of Beirut, Beirut, Lebanon_.

Lung adenocarcinoma (LUAD) represents the most common lung cancer subtype in smokers. Despite the urgency for prevention in high-risk smokers, we still do not know the earliest changes that drive LUAD development and would thus be ideal targets for chemoprevention. Earlier work revealed that the retinoic acid-inducible G-protein coupled receptor, Gprc5a, is abundantly expressed in mouse normal lung relative to other normal tissues. Moreover, mice with knockout of Gprc5a develop spontaneous late onset lung tumors, including LUADs, suggesting a tumor suppressor role for this gene. In the present study, we sought to better understand the impact of Gprc5a expression on LUAD pathogenesis. We first examined the expression of human GPRC5A in publicly available datasets and found that GPRC5A was highest in human normal lung relative to other normal samples and was down-regulated in human LUADs. We then assessed expression patterns of Gprc5a within different mouse airway compartments and found that the gene was predominantly expressed in alveolar type I (AT1) cells pointing to potential airway lineage-specific functions for Gprc5a. Next we sought to analyze tobacco carcinogen-mediated lung oncogenesis as an attempt to emulate smoking-induced lung cancer in humans. In contrast to wild type littermates, Gprc5a-/- mice treated with the nicotine-specific carcinogen NNK developed LUADs by six months after carcinogen exposure. Following immunofluorescence analysis, these LUADs were found to express surfactant protein C (Sftpc) and lack the clara cell marker Ccsp suggesting that the cells of origin for these LUADs are alveolar type II (AT2) cells. We were then prompted to understand genomic alterations in the pathogenesis of the Gprc5a-/- LUADs. Using the Illumina HiSeq 2500 platform, we performed whole exome sequencing (WES) of five carcinogen-induced LUADs as well as two Gprc5a-/- tail veins and normal lung tissues to infer somatic variants. Of note, all LUADs exhibited somatic activating Kras mutations (p.G12D or p.Q61R), the same variants purported to act as drivers of human LUAD in smokers. The Kras mutations were also observed in premalignant lesions that developed prior to LUAD onset. Moreover, WES analysis revealed additional driver genes that were mutated (Apc, Atm, Kmt2d, Nf1, Trp53) or exhibited copy number alterations (CNAs) (gain of Kras, Met, Braf, Ezh2) in multiple Gprc5a-/- LUADs. Our data suggest that Gprc5a loss coupled with NNK exposure leads to AT2-derived Kras mutant LUADs with co-occurring mutations and CNAs in other driver oncogenes and tumor suppressors. Our findings also imply that the tobacco carcinogen exposed Gprc5a-/- model emulates the molecular pathology of human LUAD and offers unique opportunities to map the temporal evolution of Kras mutant LUAD and identify targets for chemoprevention of this fatal malignancy.

#4058

DNA damage and tumour burden in mouse colon is increased in response to carcinogen exposure after induction of chronic inflammation - a more disease relevant model of colitis-associated colorectal cancer.

Tamsin RM Lannagan,1 Mark T. Mano,1 Richard J. Head,1 Trevor Lockett,1 Matthias Ernst,2 Leah J. Cosgrove1. 1 _CSIRO Food and Nutrition, Adelaide, Australia;_ 2 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia_.

Chronic inflammation (CI) such as inflammatory bowel disease increases the risk of developing colorectal cancer (CRC). Tumorigenesis is supported by inflammatory cytokines through increased cell proliferation and inhibiting apoptosis of neoplastic cells. The classic mouse model of colitis-associated CRC (CA-CRC) requires treatment with a mutagen (azoxymethane, AOM) followed by induction of inflammation through ingestion of a luminal irritant (dextran sulfate sodium, DSS). AOM is metabolized by colonic epithelium causing DNA damage through formation of mutagenic DNA adducts. Although the AOM + DSS model of CA-CRC is a valuable research model, it "reverses" events that take place in humans where CI typically precedes CRC. We hypothesise that CI sensitises colonic epithelium to DNA damage and predisposes the epithelium to neoplastic transformation.

In order to investigate this we reversed the AOM + DSS model to more accurately represent the disease process in humans. Using two experimental approaches we treated wildtype (WT) mice with AOM alone or with DSS then AOM and collected colonic tissue after 6 and 48 hours (short-term) or colonic tumours (long-term). Tumour number and size were quantified. Immunohistochemistry was used to analyse DNA damage, apoptosis, and proliferation. Formation of the DNA adduct O6MeG was measured by a modified method of HPLC, and qPCR was used to analyse expression of DNA damage signalling pathway components.

Compared with an AOM challenge alone, prior CI induction by DSS treatment increased DNA damage, reduced apoptosis, and increased tumour number and tumour size. This data reveals how prior CI modulates the colonic epithelial response to mutagen exposure in the short-term and how it promotes tumour formation in the long-term. In addition, this data demonstrates that the "reverse" model of CA-CRC can be successfully used as a more appropriate experimental framework to better understand the molecular mechanisms underpinning CA-CRC and will allow us to further investigate the relationship between prior CI, DNA damage, and tumour development.

#4059

Molecular role of cytochrome P450 (CYP)1A2 in the regulation of hepatic and pulmonary CYP1A1 expression in mice exposed to 3-Methycholanthrene.

Bhagavatula Moorthy, Paramahamsa Maturu, Lihua Wang, Weiwu Jiang. _Baylor College of Medicine, Houston, TX_.

3-Methylcholanthrene (MC) is one of the most potent polycyclic aromatic hydrocarbons (PAHs) carcinogens known to date. PAHs are present in cigarette smoke, diesel exhausts, and charcoal broiled meats, etc. Cytochrome P450 (CYP) 1A enzymes play key roles in the activation of PAHs to carcinogenic metabolites. In this investigation, we tested the hypothesis that CYP1A2 plays a mechanistic role in the regulation of hepatic and pulmonary CYP1A1 by MC. Wild type (WT) (C57B6) and Cyp1a2_null mice were divided into two groups. Group I was treated with vehicle corn oil (CO) (8 ml/kg) and group II was treated with four doses of MC (100 µmol/kg), i.p. once daily for 4 days. Four animals from each group were sacrificed at 6, 12, 24, and 48 h, 8 and 15 days after MC withdrawal. The mRNA levels, protein content and enzyme activities of CYP1A1 and 1A2 were determined at different time points. In addition, the binding of MC-AHR-AHR nuclear translocator (ARNT) to the AHREs on the CYP1A1 promoter region was determined by chromatin immunoprecipitation (ChIP) assay in liver and lung tissues. In WT mice, ChIP experiments indicated that transcriptional activation of CYP1A1 was most pronounced at 6 h, followed by 12 h, but declined at later time points. In Cyp1a2_null mice, on the other hand, transcriptional activation of CYP1A1 persisted till 48 h. In lungs, AHR complex binding peak was observed at 12 h and declined later, but Cyp1a2 null mice showed more persistent binding till 48 h with a peak at 24 h. On the other hand, the expression of CYP1A1/1A2 at the mRNA, protein, and enzyme levels persisted for up to 48 h both in lung and liver tissues. These results suggest a mechanistic role for CYP1A2 in the molecular regulation of CYP1A1, and that transcriptional activation of CYP1A1 at 6-12 h is sufficient to result in sustained induction of CYP1A mRNA and protein expression for up to 48 h. These studies have important implications for carcinogenesis mediated by PAHs.

#4060

The role of p62-dependent regulation of COX-2 in UVA response and skin tumor progression.

Ashley Sample,1 Lei Qiang,1 Baozhong Zhao,2 Yu-Ying He1. 1 _University of Chicago, Chicago, IL;_ 2 _Bio-Synthesis Inc, Lewisville, TX_.

Skin cancer is the most common type of cancer in the US, with an estimated 3.5 million cases diagnosed each year. Exposure to ultraviolet (UV) radiation, namely UVA (320-400 nm) and UVB (290-320 nm), is the major risk factor for the development of skin cancer. UVA is 20-fold more abundant in sunlight than UVB and is the major component of tanning beds. However, the mechanism of UVA's contribution to skin cancer remains unclear. One putative effector of UVA in skin cancer is p62, a selective autophagy cargo protein and substrate. We have found that UVA upregulates p62 expression, independent of autophagy, by inducing transcription of p62. p62 is upregulated in many types of cancer, including squamous cell carcinoma (SCC) and melanoma, and can facilitate the activation of a number of pathways to promote cell proliferation, invasion, inflammation, and survival. We have previously shown that p62 stabilizes Twist1 to promote invasion and proliferation, indicating p62 can function independent of autophagy to promote tumor progression. However, the function of p62 in UVA-induced tumor progression is unknown. Using a candidate gene approach, we identified a novel interaction between p62 and cyclooxygenase-2 (COX-2), which suggests a putative function for p62 in UVA-induced skin cancer. COX-2 is a prostaglandin synthase often overexpressed in cancer, where it correlates with poor prognosis. COX-2 promotes cell proliferation and survival, and inhibition of COX-2 prevents skin cancer development. COX-2 protein expression is induced concomitantly with p62 in response to UVA and knockdown of p62 prevents induction of COX-2 by UVA. As COX-2 transcription is not induced by UVA in skin cancer cells along with p62, we assessed a possible protein-level regulatory mechanism. Co-immunoprecipitation of endogenous p62 and COX-2 in skin cancer cells reveals a novel physical interaction between these proteins. Therefore, we hypothesize that p62 transcription is induced by UVA to promote stability of cyclooxygenase-2 (COX-2) and consequently, tumor progression. This project aims to further understand how UVA regulates p62 and thus COX-2 availability, and the functional significance for skin tumor progression. As both p62 and COX-2 are critical for skin tumor progression, understanding the link between these proteins will unravel a signaling axis central to skin cancer progression.

#4061

Origanum majorana **organic extract induces senescence and autophagic cell death in breast cancer cells through influencing mitochondrial metabolism.**

Mohannad Garoub, Jayanta Das, Stanislaw Wnuk, Deodutta Roy. _Florida International University, Miami, FL_.

Excess 17β estradiol (E2) is a risk factor of breast cancer. Previously, we have reported that E2 through influencing mitochondria by unknown mechanism contribute to the estrogen induced breast carcinogenesis. The aim of this study was to evaluate the mitotoxic and cytotoxic effects of normal O. Majorana organic extract (OME) as well as PEGylated nanoconjugate of OME with triphenylphosphonium (P-OME) against human breast epithelial and cancer cell lines. Origanum majorana, commonly known as marjoram, is a perennial herb, which is widely used in the Middle East as a spice. It has been shown to possess extensive range of biological activity, including antioxidant, anti-inflammatory, and anti-tumor growth effects. Interestingly, the anticancer potential of O. Majorana against breast cancer remains largely unexplored. Here, we investigated the anticancer effect of O. Majorana on three breast cell lines. We also used triphenylphosphonium (TPP) cation to check whether an imbalance in mitochondrial bioenergetics, in part, may be responsible for estrogen induced growth of breast cancer. Determination of cell density (cell survival) by SRB and mitochondrial metabolic activity by MTT assays showed that both OME and P-OME reduced growth of MDA-MB-231 and MCF-7 cells, but no effect on normal breast epithelial MCF-10A cells. OME and TPP blocked E2-induced increases of cell survival, metabolic activity and proliferation of breast cancer MCF-7 cells. E2 treatment increased MitoTracker® Red 580 labeling, indicating E2 treatment increased MCF7 cells mitochondrial mass. We observed a several fold increase in mitochondrial transcription factor A (TFAM) DNA binding activity as early as 3 hrs in treated MCF7 cells. DNA synthesis was inhibited in E2-exposed MCF7 cells by the silencing of TFAM. To discern whether a decrease in ATP production may be responsible for the E2-induced growth signaling, we measured the ATP present in the MCF7 cells. Our data showed that the ATP levels in E2 treated cells were very similar to control cells. E2 treatment of MCF-7 cells increased mRNA and protein levels of BNIP3 involved in mitophagy/autophagy. Together these data suggest that a carcinogenic concentration of E2 may modify mitochondrial dynamics, mitophagy, biogenesis and metabolism. In summary, our results demonstrated for the first time that OME was able to inhibit estrogen-induced growth of MCF-7 cells in a time- and concentration-dependent manner. Our results also demonstrated that P-OME nanoconjugate compared to OME was far more effective in exerting its cytotoxic effect through the induction of growth arrest, mitochondrial metabolic activity, senescence, apoptosis and autophagic cell death in the highly metastatic triple negative MDA-MB-231 cells. Our findings offer a new perspective on the utility of O. Majorana plant extract to be developed as a new alternative medicinal therapy against breast tumors.

#4062

Human mammary tumor virus (HMTV) is a breast cancer pathogen.

James F. Holland,1 Stella Melana,1 Teiko Nartey,1 Joseph Tripodi,2 Shabnam Jaffer,2 Anupna Nayak,2 Beatriz GT Pogo3. 1 _Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 3 _Tish Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY_.

Human mammary tumor virus (HMTV) a betaretrovirus 90-98% homologous to Mouse Mammary Tumor Virus (MMTV), the causative agent for breast cancer in mice, has been detected in approximately 40% of American women's breast cancers. Mouse DNA contamination has been definitively excluded by contamination-free guarantee techniques. In Western European countries and their former colonies, age standardized rates (ASR) of breast cancer incidence (47 to 92 per 100,000 per year) are higher than Asian incidence (29 to 43 ASR/y). This difference could be explained by excess HMTV-related breast cancer incidence. In 7 West European, American and Oceania countries, 30 to 60% contain HMTV, while in 5 Asian nations HMTV associated breast cancers range from 0 to 22%. Different indigenous murine species with disparate MMTV burdens parallel these findings: mus domesticus in the West with much MMTV in its genome, and mus castaneus or mus musculus in the East with less. HMTV is found in cells in milk of 8% of unselected American mothers and in milks of 21% of women previously biopsied for unconfirmed cancer suspicion.

HMTV isolated from primary cultures of metastatic breast cells is able to infect In vitro, human mammary epithelial cells, B and T lymphocytes and dendritic cells. MCF10A breast cell line infected with HMTV shows molecular changes associated with epithelial-mesenchymal transition, such as up-regulation of vimentin, and down-regulation of E-cadherin.

HMTV sequences were also shown to be more prevalent in inflammatory, gestational and familial breast cancers than in unselected sporadic samples, consistent with association with more aggressive breast cancer phenotypes. To establish a direct connection between the presence of HMTV sequences and metastatic breast cancer, we have found HMTV sequences in 94% of primary cultures of metastatic breast cells obtained directly from effusion fluids. HMTV present in primary breast tumors has been demonstrated in their metastatic axillary nodes.

Taken together the results suggest an association between the presence of HMTV sequences and breast cancer aggressiveness. HMTV poses a new and challenging dimension in breast cancer research involving causation, diagnosis, molecular mechanisms, epidemiology, therapy and prevention.

#4063

Effects of electronic cigarettes (E-cigs) and hookah on the metabolic activation of the tobacco carcinogen, benzo(a)pyrene (BaP), and cellular proliferation in human oral leukoplakia cells.

Joseph B. Guttenplan,1 Yuan-Wan Sun,2 Kun-Ming Chen,2 Wieslawa Kosinska,1 Karam El-Bayoumy2. 1 _New York University, New York, NY;_ 2 _Penn State College of Medicine, Hershey, PA_.

The possible carcinogenic effects of E-cigs on the oral cavity are uncertain because their recent availability and usage precludes long-term epidemiological studies. Also of concern is the use of hookah, which has recently increased in popularity. In addition, many, E-cig and hookah users are tobacco smokers or individuals trying to quit or cut down on smoking. Such individuals likely have precancerous cells and/or lesions in the oral cavity, as do former smokers or oral cancer survivors. The use of E-cigs and hookah then, may affect the initiation and promotion/progression stages of carcinogenesis, and be of particular concern to dual users. Here we have investigated the effects of E-cigs and to a lesser extent, hookah on the metabolism of BaP to its ultimate carcinogenic metabolite, BaP diol-epoxide (BPDE), and also the effects of E-cigs on proliferation, and expression of proliferation-related genes in leukoplakia cells. Tobacco-flavored E-cigs, and hookah were prepared from local products using a mechanical smoking device to simulated smoking, and the resulting vapor collected in a liquid N2-cooled trap to generate E-cig or hookah vapor condensates (VC). To determine the effects of E-cigs and hookah on BaP metabolism, cells from a leukoplakia-derived cell line, MSKLeuk1, were pretreated for 16 hr with E-cig or hookah VC at levels yielding nicotine concentrations of 0.5 - 8 uM (similar to plasma nicotine levels in smokers) and then treated with one uM BaP. After 8 hr the concentrations of BaP-tetrols released into the medium were measured using HPLC. BPDE hydrolyzes into BP tetrols, and their concentration serves as a surrogate for BPDE concentration. Cells pretreated with a tobacco smoke extract (TSE) from the reference cigarette, 2R4F, were included, as were untreated cells. Pretreatment with E-cig or hookah VC increased the concentration of BaP tetrols in the cell medium from 22 to 165 and 64 pM, resp. at the highest dose of each. For comparison, pretreatment with TSE at a nicotine level of 4uM increased the concentration BaP tetrols to 252 pM. The effects E-cigs on cell proliferation were assayed using an MTT assay. The cells were plated with and without E-cig VC and 24 hr later cell proliferation was assayed. Exposure to E-cigs significantly increased the rate of cell proliferation by 61%. The increase maximized at one uM nicotine. We also monitored the effects of 2 different E-cigs on the expression of the certain proteins related to cell proliferation-related genes (PCNA and pAKT) by immunoblotting. Both E-cigs increased the levels of these proteins to various extents. Our results demonstrate, for the first time, that E-cig or hookah VC enhanced metabolism of BaP to its ultimate carcinogen, and E-cig VC stimulated cellular proliferation. These results suggest that "vaping", or hookah usage may contribute to oral cancer risk. (Support: CA173465).

#4064

The identification of UBR5-ZNF423 recurrent fusion gene in EBV-associated nasopharyngeal carcinoma.

Pok Man Hau,1 Grace Chung,1 Raymond Lung,1 Samantha Lun,1 Chit Chow,1 Alice Wong,2 Fei-Fei Liu,3 George Tsao,2 Kevin Yip,1 Ka Fai To,1 Kwok Wai Lo1. 1 _The Chinese University of Hong Kong, Hong Kong, China;_ 2 _University of Hong Kong, Hong Kong, China;_ 3 _University of Toronto, Ontario, Canada_.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer arising from the epithelium of nasopharynx. While the incidence rate of NPC is low in Western countries, it is prevalent in southern China, south-east Asia and northern Africa. Generally, it is believed that multiple factors, including genetic predisposition, environmental carcinogens and Epstein-Barr virus infection, contribute to the tumor initiation and progression of NPC. Similar to other solid tumors in which genetic rearrangements play causal role in the genesis of tumors, our earlier cytogenetic and spectral karyotyping have demonstrated the prevalence of chromosomal translocations in NPC tumors. These evidences suggest that novel fusion gene product arises from chromosomal translocation may contribute to the pathogenesis of the disease.

Using paired-end whole-transcriptome sequencing, we discovered a novel fusion transcript composed of the exon 1 of the ubiquitin protein ligase E3 component n-recognin 5 (UBR5) on 8q22.3 and exon 7-9 of zinc finger protein 423 (ZNF423) on 16q12.1 from the NPC cell line C666-1. Moreover, FISH analysis using break-apart probes validated the UBR5-ZNF423 fusion. Furthermore reverse transcription-PCR (RT-PCR) confirmed the expression of the corresponding fusion transcript. Intriguingly, the fusion transcript was recurrently detected in 12/144 (8.3%) of primary tumors by RT-PCR, which indicates the UBR5-ZNF423 fusion gene may contribute to the genesis of a subset of NPCs.

The growth of C666-1 cells with UBR5-ZNF423 rearrangement is dependent on expression of this fusion protein. Knock-down of UBR5-ZNF423 by RNA interference inhibited the cell proliferation of C666-1 cells. The transforming ability of UBR5-ZNF423 fusion was also confirmed in NIH3T3 mouse fibroblasts by soft agar colony formation assay. Moreover, NIH3T3 cells stably expressing the fusion protein induced tumor formation in nude mice. Furthermore, using an inducible shRNA (Lac operon-driven) targeting the fusion transcript, the growth of C666-1 tumor was suppressed in nude mice. These findings suggest that expression of UBR5-ZNF423 fusion protein contributes to the transformation of a subset of NPCs. Currently, we are working on the functional role(s) of the fusion protein in C666-1 cells. Preliminary results demonstrated that the fusion protein interacts with the Early B-cell Factor 3 (EBF3) and may deregulate EBF3 transcriptional activity. Since EBF3 is a tumor suppressor gene, deregulating EBF3 downstream gene expression by UBR5-ZNF423 may confer to its oncogenic function on NPCs.

In conclusion, we have successfully identified the UBR5-ZNF423 fusion transcript in a subset of NPC tumors. By understanding the molecular mechanisms of this fusion protein on NPCs, novel therapeutic interventions may be implemented on treating a subsets of NPC tumors expressing this oncogenic fusion protein.

#4065

Mechanistic role of heavy metal cadmium exposure in the etiology of pancreatic cancer.

Wei Yu, Yiming Ma, Rakesh Srivastava, Sharmila Shankar. _Kansas City VA Medical Center, Kansas City, MO_.

Persistent and cumulative human exposures to toxic metals lead to various health concerns and chronic illnesses. Cadmium is a heavy metal of considerable occupational and environmental concern, and has been shown to have an epidemiological association with pancreatic cancer. In this pursuit we have demonstrated that cadmium metal is carcinogenic and can induce transformation and reprogramming of normal pancreatic epithelial cells. In this study we have shown that exposure of normal pancreatic ductal epithelial HPNE cells to low levels of Cd+2 induce cellular transformation and also induce the expression of a transcription factor SATB2 which is oncogenic in pancreatic cancer. During cadmium-induced cellular transformation, cells gain the phenotype of cancer stem cells (CSCs) / progenitor cells and express pluripotency maintaining factors. SATB2 is a transcription factor and its role in cadmium-induced cellular transformation in pancreas has never been examined. SATB2 (special AT-rich binding protein-2), a transcription factor and epigenetic regulator that binds DNA in nuclear matrix attachment regions influences gene expression both by orchestrating chromatin structure and by functioning as a transcriptional co-factor. Our data have demonstrated that human pancreatic normal ductal epithelial (HPNE) cells do not express SATB2, whereas it is highly expressed in pancreatic cancer cell lines and cancer stem cells (CSCs). The CSCs / tumor initiating cells are believed to be the cause of cancer initiation, progression, and metastasis. During cadmium-induced cell transformation, cells gain the phenotype of cancer stem cells (CSCs) / progenitor cells and express pluripotency maintaining factors. This suggests that overexpression of SATB2 may induce cellular transformation. Interestingly, our data also reveal that chronic exposure of HPNE cells to low levels of cadmium results in SATB2 induction and cellular transformation. Using ChIP assay, we have demonstrated that SATB2 can directly bind to Bcl-2, Nanog, Bsp, Klf4, Myc, Xiap, and Hoxa2, and regulate the expression of EMT genes, pluripotency maintaining factors and drug resistance genes. Based on these observations, we conclude that the SATB2, induced by cadmium exposure or through over-expression studies, play a role in cell transformation of human pancreatic normal ductal epithelial cells. It can thus serve as a potential novel target for treatment of pancreatic cancer. To evaluate the human health risks associated with chronic exposure to cadmium we develop a mechanistic understanding of the cadmium induced human pancreatic normal ductal epithelial cell transformation. The results shown set the stage that cadmium toxicity enhances pancreatic carcinogenesis by up-regulating SATB2 which may thus be a potential target for development of targeted therapeutic interventions.

#4066

The chemokine CXCL13 in lung cancers associated with environmental polycyclic aromatic hydrocarbons pollution.

Gui-Zhen Wang, Xin Cheng, Guang-Biao Zhou. _Institute of Zoology, Chinese Academy of Sciences, Beijing, China_.

More than 90% of lung cancers are caused by cigarette smoke and air pollution, with polycyclic aromatic hydrocarbons (PAHs) as key carcinogens. In Xuanwei City of Yunnan Province the lung cancer incidence is among the highest in China attributed to smoky coal combustion-generated PAH pollution. Here we screened for abnormal inflammatory factors in non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used, and found that a chemokine CXCL13 was overexpressed in 63/70 (90%) of Xuanwei NSCLCs and 44/71 (62%) of smoker and 27/60 (45%) of non-smoker CR patients. CXCL13 overexpression was associated with the region Xuanwei and cigarette smoke. The key carcinogen bezo(a)pyrene (BaP) induced CXCL13 production in lung epithelial cells and in mice prior to development of detectable lung cancer. Deficiency in Cxcl13 or its receptor, Cxcr5, attenuated BaP-induced lung cancer in mice, demonstrating CXCL13's critical role in PAH-induced lung carcinogenesis.

#4067

Cell phone use is associated with an inflammatory cytokine profile of parotid gland saliva.

Ricardo S. Gomez,1 Elisa C. Siqueira,1 Fabrício Tinôco A. Souza,1 Efigênia F. Ferreira,1 Renan P. Souza,1 Eitan Friedman,2 Marcus V. Gomez,1 Carolina C. Gomes1. 1 _Federal Univ. of Minas Gerais, Belo Horizonte, Brazil;_ 2 _Tel-Aviv University, Tel-Aviv, Israel_.

There is controversy on the effects of the non-ionizing radiation emitted by cell phones on cellular processes and the impact of such radiation exposure on health. Conflicting results were reported on the association between cell phone use and parotid tumor development. Chronic inflammation is associated with an increased risk for cancer, such as seen in ulcerative colitis. To evaluate the effect that cell phone use has on the parotid gland, cytokine expression profile was determined in the saliva produced by the parotid glands in healthy volunteers, and correlated with self-reported cell phone use and laterality. The following parameters were determined, in 83 Brazilian individuals in saliva produced by the parotid glands comparing the saliva from the gland exposed to cell phone radiation (ipsilateral) to that from the contralateral parotid: salivary flow, total protein concentration, IL-1β, IL-6, IL-10, IFN-γ and TNF-α by ELISA. After multiple testing correction, decreased IL-10 and increased IL-1β salivary levels in the ipsilateral side compared with the contralateral side (p<0.05) were detected. Subjects who used cell phones for more than 10 years presented higher differences between IL-10 levels in ipsilateral versus contralateral parotids (p = 0.0012). No difference was observed in any of the tested parameters in correlation with cell phone monthly usage in minutes. We conclude that exposure of parotid glands to cell phones can alter salivary IL-10 and IL-1β levels. These findings suggest a pro-inflammatory profile on parotid naturally exposed to cell phones, which can be associated not only to the radiation emitted by the device but with the warming caused by radiation and the battery of the cell during use.

Supported by: FAPEMIG (Fundação de Amparo à Pesquisa de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil

#4068

Role of cytochrome P450 (CYP)1A2 in the molecular regulation of CYP1A1 by the carcinogen, 3-methylcholanthrene (MC) in mouse heptoma cells (hepa-1).

Sudha Kondraganti, Chun Chu, Bhagavatula Moorthy. _BCM, Houston, TX_.

Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. We reported earlier that MC causes persistent induction of hepatic and pulmonary CYP1A1 in mice for several weeks after MC withdrawal, and that the phenomenon of sustained hepatic CYP1A1 induction is lost in Cyp1a2-null mice. In this study, we tested the hypothesis that MC elicits induction of persistent CYP1A1 induction in hepa-1 cells, and that CYP1A2 contributes mechanistically to this phenomenon. Hepa-1 cells were treated with the MC (2.5 µM), or dimethylsulfoxide (DMSO) as control, and at selected time points, CYP1A1 promoter activity, CYP1A1 enzyme activities, contents, and CYP1A1 mRNA levels were studied. A 24 h treatment induced the CYP1A1 promoter activity, CYP1A1 mRNA, and ethoxyresorufin O-deethylase (EROD) activity by 2-, 1000-, and 15-fold, respectively. The induction was sustained for 4-5 days. MC persistently induced CYP1A1 apoprotein level as well. Electrophoretic mobility shift assay indicated that MC induced a nuclear protein that bound to aryl hydrocarbon response elements in the CYP1A1 promoter region. Transfection of CYP1A2 siRNA resulted in knockdown of CYP1A2 mRNA by 70%, but a statistically significant increase of basal CYP1A1 mRNA by 35-40%. The induction of CYP1A1 promoter activity, CYP1A1 mRNA, CYP1A1 protein, and EROD activity by MC were not affected by CYP1A2 siRNA at the 24 h time point, but was significantly attenuated by CYP1A2 siRNA on day 5. These results suggest that CYP1A2, possibly via a metabolite, contributes to the sustained induction of CYP1A1 by MC in hepa-I cells. Further investigations into the mechanisms of persistent induction of CYP1A1 by MC could lead to novel preventative/therapeutic strategies against PAH-mediated carcinogenesis in humans.

#4069

The carcinogenic effects of electronic cigarettes in oral cancer.

Avinaash Korrapati, Vicky Yu, Maarouf A. Saad, Mehran Rahimy, Yinan Xuan, Angela Zou, Aswini Krishnan, Kevin Brumund, Weg M. Ongkeko. _UC San Diego, San Diego, CA_.

Alcohol consumption, tobacco use, and human papillomavirus infection have been well-established as the primary risk factors for head and neck squamous cell carcinoma (HNSCC). However, the surge in popularity of e-cigarettes (e-cigs) has prompted speculation of e-cig use potentially emerging as a new risk factor. With previous research on the safety of e-cigs still collectively inconclusive, a comprehensive study of the carcinogenicity of these devices remains urgently necessary. We therefore investigated the potential genotoxic, cytotoxic, and invasive and migratory effects of e-cig vapor exposure on human epithelial cells. A panel of normal epithelial and head and neck cancer cell lines was treated with 0.5%, 1.0%, and 2.0% by volume e-cig vapor from two popular e-cig brands, over periods ranging from 24 hours to 4 weeks. Neutral comet assay and immunostaining for γ-H2AX foci revealed significant induction of DNA double-stranded breaks (up to 3-fold increase, p < 0.05) in cell lines incubated with both nicotinized and nicotine-free e-cig vapor. To evaluate the cytotoxicity of e-cigarettes, trypan blue exclusion and clonogenic assays were performed following short-term e-vapor exposure. Our results indicate that in epithelial cells, short-term treatment induces up to a 5-fold increase in cell death without nicotine, and up to a 10-fold increase with nicotine as compared to untreated controls (p < 0.001). We subsequently assessed the effects of e-cigarettes on HNSCC progression and metastatic potential through exposure of established HNSCC cell lines to e-cigarette vapor. Wound healing assays revealed increased migration of HNSCC cells following e-cigarette treatment, with significant upregulation of key EMT-promoting genes observed via qRT-PCR. We will next examine the effects of long-term (6-9 months) e-cig vapor exposure in vivo using mouse models. Mouse oral epithelium will be harvested for histologic analysis and sequenced to determine the mutagenic potential of long-term e-cig vapor exposure. Nevertheless, our present findings based on short-term e-vapor exposure alone already pose alarming implications for the carcinogenic effects of e-cigarette use.

#4070

Carcinogen 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis is accelerated in Smad3 heterozygous mice compared to Smad3 wild type mice.

Zhengxue Liu,1 Tanima R. Kundu,1 Isao Matsuura,2 Guannan Wang,1 Yong Lin,1 You-Rong Lou,1 Nicola J. Barnard,1 Xiao-Fan Wang,3 Mou-Tuan Huang,1 Nanjoo Suh,1 Fang Liu1. 1 _Rutgers University, Piscataway, NJ;_ 2 _National Health Research Institutes, Taiwan;_ 3 _Duke University Medical Center, Durham, NC_.

Smad3 plays an important role in inhibiting cell proliferation and promoting apoptosis. Previous studies based on cell culture and xenograft animal models suggest that Smad3 has tumor suppressor function for breast cancer during early stages of tumorigenesis. Here we show that DMBA (7,12-dimethylbenz[a]anthracene), a chemical carcinogen, induces mammary tumor formation at a significantly higher frequency in the Smad3 heterozygous mice than in the Smad3 wild type mice. This is the first genetic evidence showing that Smad3 inhibits mammary tumorigenesis in a mouse model. Our findings provide strong support that Smad3 has important tumor suppressor function for breast cancer.

#4071

Targeted disruption of T-cell protein tyrosine phosphatase in mouse epidermis reveals its critical role in chemically-induced skin carcinogenesis.

Hyunseung Lee,1 Mihwa Kim,1 Liza D. Morales,1 Thomas J. Slaga,2 John DiGiovanni,3 Dae J. Kim1. 1 _Department of Biomedical Sciences, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX;_ 2 _Department of Phamacology, School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX;_ 3 _College of Pharmacy, University of Texas at Austin, Austin, TX_.

Environmental factors such as chemical toxicants contribute to the development of skin cancer by creating mutations in housekeeping genes and proto-oncogenes which disrupt intracellular signaling mechanisms. One vital signaling mechanism is tyrosine phosphorylation signaling. Phosphotyrosine signaling is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Recently we reported TC-PTP (PTPN2) is critical regulator of STAT3 signaling in mouse keratinocytes. To assess the role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TC-PTP-deficient (K14Cre.PTPN2fl/fl) transgenic mice. Loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency in epidermis resulted in a significant increase in epidermal thickness and hyperproliferation (assessed by bromodeoxyuridine (BrdU) labeling index) compared to control mice following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Consistently, primary keratinocytes derived from TC-PTP-deficient mice showed a faster growth rate. To further investigate the role of TC-PTP in skin carcinogenesis, TC-PTP-deficient mice were subjected to two-stage skin carcinogenesis analysis. TC-PTP-deficient mice showed a shortened latency of tumor development and significantly increased numbers of tumors compared to control mice, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. The data reveals TC-PTP may be a novel potential target for the prevention of skin cancer due to its role in the regulation of proliferation and apoptosis in epidermis.

#4072

Molecular cytogenetic characterization of HPV types 16 and 18 cervical cancers: Acquired genomic instability by E6 and E7 oncoproteins.

Karen Tate,1 Lucy Tran,1 Melanie Baker,1 Kamilah Evans,1 Mechelle Rouse,1 Seyung Chung,1 Duy Nguyen,2 Juri Kim,1 Enijah Smith-Joe,1 Jay Vadgama,1 Mengtao Li,3 Yu-Ling Lin,3 Roland Patillo,4 Eva McGhee1. 1 _Charles R. Drew University of Medicine and Science, Los Angeles, CA;_ 2 _University of California Berkeley, Berkeley, CA;_ 3 _UCLA School of Dentistry, Los Angeles, CA;_ 4 _Morehouse School of Medicine, Atlanta, GA_.

Human Papillomavirus (HPV) high-risk types 16 and 18 are directly associated with approximately 90% incidence of invasive cervical carcinoma. Epithelial cells infected with HPV become transformed and acquire genomic instability. As a result of this transformation, E6 and E7 oncogenes are perpetually expressed. E6 degrades the tumor suppressor p53; E7 inhibits tumor suppressor pRB, leading to disproportionate target cell growth and proliferation. While there have been studies exploring genomic instability induced by HPV oncoproteins E6/E7, the full scope of the genomic damage has not been clearly characterized. Three HPV positive cervical cancer cell lines, HeLa (HPV18), SiHa (HPV16) and CaSki (HPV16), were studied using Spectral Karyotyping (SKY) and Giemsa banding (G-banding). Our results using SKY and G-banding analysis showed HeLa cells exhibited frequent translocations on chromosomes 4 and 11, and deletions on chromosomes 11 and 20. The SiHa cells exhibited translocations primarily on chromosomes 9 and 20, with deletions on chromosome 10. CaSki revealed translocations on chromosomes 10 and 4, with deletions on chromosome 21. All three cell lines gained copies on chromosome 5. The aforementioned aneuploidy is demonstrative of the induced genomic instability acquired from HPV infection, which include tumor suppressor genes; p53 and pRB disruptions caused by E6 and E7 oncoproteins. HeLa cells showed extensive genomic instability on chromosome 11q22-23, where the ATM gene is located. Acquired genomic instability to the ATM chromosome region may further increase the rate of cervical cancer.

#4073

The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation.

Sukanya Gayan,1 Hong Li,1 Rashmi Bhuyan,1 Sora Sandhya,2 Joyeeta Talukdar,2 Bidisha Pal,1 Jaishree Garhyan,1 Wael Tasabehji,1 Manaf Muhammad Alkurdi,1 Heidar Zohrehei,1 Seema Bhuyan,2 Anupam Sarma,2 Gayatri Gogoi,1 A.C. Kataki,3 Rakesh Bhatnagar,4 Debabrata Baishya,5 Bikul Das1. 1 _Forsyth Institute, Cambridge, MA;_ 2 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India;_ 3 _B Borooah Cancer Institute, Guwahati, India;_ 4 _Jawaharlal Nehru University, New Delhi, India;_ 5 _Gauhati University, Guwahati, India_.

Oral cancer presents a major health burden across the globe, specially in developing countries. Kamrup, a district of India, where the KaviKrishna laboratory is located has the highest incidence of oral cancer in the entire world. The mechanism of oral cancer carcinogenesis process is not clearly known. Previous studies indicate the potential role of HPV-16 virus as well as tobacco consumption as major contributing factors of oral cancer. In mouse, the carcinogen agent 4-NQO was found to induce oral cancer having similar phenotype as human oral mucosa cancer. 4-NQO may exert tobacco like oxidative stress toxicity to oral mucosa. Hence, developing both in vitro and in vivo models of HPV-16 and 4-NQO mediated oral carcinogenesis might help to understand the initiating event of oral carcinogenesis. In this study, we propose that oral mucosa stem cells (OMSCs) could be the target of HPV-16, and 4-NQO induced carcinogensis. Thus, in vitro treatment of oral mucosa cells of healthy human volunteers with HPV-16 protein E6, and 4-NQO led to expansion of CD271+ cells, which are enriched in OMSCs. Importantly, the expanded CD271+ cells exhibited high HIF-2alpha, low p53, and high glutathione secretion, a phenotype of stem cell altruism that we recently described in human embryonic stem cells (Das B et al. Stem Cells, 2012). In human ES cells, the altruistic reprogramming served as an initiating event of malignant transformation by altering p53/MDM2 oscillation, in an abnormal state of low p53 and high MDM2. Therefore, we performed a complete evaluation of E6 protein and 4-NQO treated CD271+ oral mucosa cells for altruistic behavior, including low p53 and high MDM2 state. For this purpose, the carcinogen treated oral mucosa cells were subjected to immunomagnetic sorting for CD271+ cells. We found that post-carcinogen treated CD271+ cells exhibited in vitro self-renewal activity, high GSH secretion, and importantly activation of a HIF-2alpha/MYC co-operation. ChiP assay revealed the MYC binding to HIF-2alpha, and Sox-2, an stemness associated transcription factor. Importantly, HIF-2alpha was important for the reversible but prolonged suppression of p53 for more than two weeks. We also found that the high HIF-2alpha and low p53 expressing CD271+ cells could be enriched in a ABCG2+ population. Thus, we were able to enrich a CD271+/ABCG2+ cell population in oral mucosa cells of both human and mouse. Based on these findings we propose to use HPV-16 and 4NQO derived carcinogenesis models to study the potential role of altruistic reprogramming in the malignant transformation of oral mucosa stem cells to oral cancer stem cell like cells. Our study may unravel a novel mechanism of malignant transformation, the failure of altruistic stem cells to sacrifice its fitness (altruism) as a potential initiating event of malignant transformation of stem cells to cancer stem cells.

### Host-Tumor Interactions

#4074

Macrophages promote the progression of premalignant mammary gland lesions through activation of the Axl signaling cascade.

Emily C. Carron, Heather L. Machado, Samuel Homra. _Tulane School of Medicine, New Orleans, LA_.

Infiltrating inflammatory cells, including tumor-associated macrophages (TAMs), have been shown to promote breast cancer cell invasion and have been correlated with metastasis and poor prognosis. While it is well-established that macrophages are recruited to the invasive fronts of established tumors to exert pro-tumor signals, their role in premalignancy remains poorly understood. Using a novel p53-/- syngeneic transplantable model of premalignancy, we show that inflammatory cells, including macrophages, are indeed recruited to early lesions prior to invasion. Microarray analysis was performed on two different pre-invasive lines, termed PN1a, (high tumor-forming potential) and PN1b (low tumor-forming potential). Interestingly, several pro-inflammatory chemokines, macrophages markers, and the pro-inflammatory cytokine growth arrest specific 6 (Gas6) were increased in PN1a lesions as compared to PN1bs. In a 3-D co-culture system, macrophages were recruited to PN1a acini and induced invasion, while PN1b cells remained non-invasive, supporting our in vivo data. Depletion of macrophages in vivo with clodronate containing liposomes significantly delayed progression of PN1a lesions to invasive cancer, indicating a critical role for macrophages in early progression. Gas6 and its receptor tyrosine kinase, Axl, were highly expressed in PN1a preinvasive lesions, but decline in invasive tumors, suggesting that this pathway is a key driver of the transition from pre-invasive to invasive cancer. Further studies using our 3-D co-culture system with Gas6-/- macrophages demonstrated that macrophages promote the formation of non-polar disorganized structures by activating Axl. Finally, transplantation of PN1a cells into Gas6 +/- mice resulted in a delay in tumor formation, indicating that stromal-derived Gas6 (and potentially macrophage derived) may promote the progression of early stage lesions through the paracrine activation of Axl. As a plethora of Axl inhibitors have been developed and are currently in clinical trial, these studies have critical implications for the prevention and treatment of invasive breast cancer.

#4075

Composition and distribution of tumor-infiltrating lymphocytes in breast cancer.

Liang Guo,1 Yayun Chi,1 Jingyan Xue,1 Shyamal Goswami,2 Bingqing Zhou,1 Chunmei Cao,1 Zhimin Shao,1 Xiaoming Zhang,2 Jiong Wu1. 1 _Fudan University Shanghai Cancer Center, Shanghai, China;_ 2 _Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China_.

Backgroud: High tumor-infiltrating lymphocytes(TILs)are known to be associated with response to primary systemic therapy (PST) in breast cancer patients especially in Her2+ and triple negative patients. However, the composition of TILs in the high infiltration group is largely unknown. Uncovering the immunodynamic composition of TILs may be useful for better understanding the mechanism how the TILs exert effect. And the immune reservoir data for extensive analysis of altered TIL subsets after chemotherapy is still lacking.

Methods: The tumor specimens of 56 breast cancer patients who did not receive PST and 16 breast cancer patients who underwent surgical resection after anthracycline- or anthracycline/taxane-based PST were analysed. We used the multi -parametric flow cytometric platform (optimized for 20 colors for each panel of T cells, B cells, NK, and other Lineage markers detection) to accomplish the purpose. The result is more objective and estimated parameter is more abundant compared to the histological methods (hematoxylin-eosin staining and immunohistochemical staining) . Breast cancer patients can be divided into low, medium and high infiltration group according to the CD45+ number in the tissue and weight of the tumor specimens. TIL subset composition is compared among different groups (Low vs Medium vs High, Chemotherapy vs non Chemotherapy ).

Result: In patients with ER- and high histological grade patients, there are more TILs infiltration compared to the ER+ and low histological grade patients (P=0.038 and P=0.039 respectively). And we found that the percentage of CD4+T cells in total CD3+T cells, Treg in CD4+T cells and Plasmablasts in total B cells is much higher in the high infiltration group compared to the low infiltration group (56.03% vs 44.21%, P<0.01; 18.64% vs 9.37%, P<0.001; 16.75% vs 1.88%, P<0.001 respectively). By contrast, percentage of CD8+T in total CD3+T cells and NK cells in the total lymphocytes is much lower (35.26% vs 46.71%, P<0.001; 2.74% vs 5.88%, P<0.01 respectively). After PST both the percentage of CD8+T in total CD3+T cells and effector memory CD8+T cells increase heavily (53.82% vs 41.14%, P<0.001; 79.49% vs70.88%, P<0.05 respectively) and percentage of CD4+T cells in total CD3+T and naïve CD8+T cells decrease (31.81% vs 50.35%, P<0.001; 2.81% vs 4.21%, P<0.05 respectively). Functional markers such as HLADR,CD28 and PD1 in the T cells decrease dramatically and CD127 increases noticeably.

Conclusion: The inner composition of TIL subsets in the high infiltration is largely different from low infiltration group and this might help explain the reason why high TILs could predict pathological complete response. Chemotherapy can lead to the heavy change of TIL subsets and this may be the part of immunological mechanism that why chemotherapy has deep influences on the tumor.

#4076

Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells.

Yu-Chih Chen,1 Shamileh Fouladdel,1 Zhixiong Zhang,2 Ebrahim Azizi,1 Euisik Yoon,2 Max S. Wicha1. 1 _Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI;_ 2 _Dept. of Electrical Eng. and Computer Science, University of Michigan, Ann Arbor, MI_.

Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis. Though functional CSCs can be identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in a dish-based format. Tumor-stromal interaction is another key regulator of cancer micro-environments, affecting tumorigenesis and metastasis. To enable tumorsphere assays that incorporate interaction with stromal cells, we developed a microfluidic dual adherent/suspension co-culture device. This device combined a suspension environment for single-cell tumorsphere assay and an adherent environment for co-culture of stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allowed the use of small samples (<100 cells), affording the potential to study rare cells such as circulating tumor cells (CTCs). As a proof-of-concept, we co-cultured single T47D breast cancer cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-cultured T47D cells, co-cultured T47D have significantly higher tumorigenic potential (sphere formation rate) by three times and proliferation rates (larger sphere size). In addition to the phenotypic observation, the platform allows the retrieval of mono-cultured and co-cultured spheres for downstream analyses. Multiplexed single-cell gene expression analyses using C1 and BioMark HD platforms were performed to compare the expression of 96 target genes in co-cultured and mono-cultured T47D cells. Co-cultured cells expressed significantly higher MKI67, which suggests a possible mechanism for the elevated proliferation rate. Additionally, lower BAX (pro-apoptotic marker) expression matches well with higher sphere formation rate in the co-cultured cells. Interestingly, we noticed the down-regulation of CDH1, KRT8, and KRT18 in the co-cultured cells, indicating possible epithelial-mesenchymal transition induced by CAF. Single cell resolution, while to comparing between groups, also allows examination of cellular heterogeneity within one population. While cells in the mono-cultured group were more homogeneous co-cultured cells displayed clear bimodal gene expression distribution including, ANXA3, MTOR, MKI67, BAX, and PGR. This is suggestive of variable responses to the same stimuli at the single cell level. Combining the dual adherent/suspension co-culture platform presented here with the single cell gene expression analysis, we may successfully identify functional CSCs, investigate the phenotypic and transcriptional effects induced by tumor-stromal interactions, and study cellular heterogeneity in greater detail.

#4077

Crucial factors of the inflammatory microenvironment (IL-1β/TNF-α/TIMP-1) promote the selection of highly malignant hemopoietic clone of myelofibrosis.

Daria Sollazzo, Dorian Forte, Nicola Polverelli, Marco Romano, Margherita Perricone, Sofia Fatica, Francesco Lapi, Emanuela Ottaviani, Martinelli Giovanni, Nicola Vianelli, Michele Cavo, Maria Pantaleo, Francesca Palandri, Lucia Catani. _University of Bologna, Bologna, Italy_.

Myelofibrosis (MF) is a clonal disorder of the hemopoietic stem/progenitor cells (HSPCs). Together with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of MF pathogenesis. It is argued that the up-regulated production of pro-inflammatory cytokines selects for the malignant clone. However, the key players linking inflammation and cancer in MF are still poorly known. Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived HSPCs.

Plasma TIMP-1, TNF-α and IL-1β were measured by ELISA assay. Circulating CD34+ cells from MF patients (JAK2V617F (10 cases) and CALR (10 cases) mutations) or cord blood (CB; 8 samples) were functionally characterized after incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), cell cycle and clonogenic capacity. Migration was assessed towards a C-X-C motif chemokine 12 (CXCL12) gradient in the presence or absence of the pro-inflammatory factors. MF-derived CD34+ cells were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence of the pro-inflammatory factors and then evaluated for survival.

Regardless mutation status, the plasma levels of IL-1β, TNF-α and TIMP-1 are increased in MF patients and the presence of pro-inflammatory cytokines confers a survival advantage of MF-derived CD34+/CD34+CD38- cells. In addition, MF-derived CD34+ cells promoted cell-cycle progression to the S-phase. Whereas in the JAK2V617F mutated group, the addition of IL-1β or TNF-α ± TIMP-1 impaired the erythroid clonogenic output of the CALR mutated patients. Of note, IL-1β + TNF-α ± TIMP-1 highly promoted the in vitro migration of MF-derived CD34+ cells in the presence of CXCL12. Intriguingly, MF-derived CD34+ cells revealed increased clonogenic ability after migration toward CXCL12 and IL-1β + TNF-α ± TIMP-1 , suggesting a pivotal role of inflammation in the selection of the malignant clone. Finally, co-cultures of normal MSCs with MF-derived CD34+ cells sustained the survival effects of IL-1β+TNF-α±TIMP-1.

Overall, our data indicate that the behavior of the MF-derived HSPCs can be influenced by regulatory signals provided by the microenvironment through the cooperation between various pro-inflammatory factors. The interplay of these pro-inflammatory factors promotes and selects the circulating MF-derived HSPCs with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach.

#4078

KRAS mutation status is associated with stromal inactivation in colorectal cancer and predicts poor response to neoadjuvant chemoradiotherapy.

Raphael Pelossof,1 Moshe Elkatebts,2 Oliver Chow,3 Lauren Fairchild,4 Kevin O'Rourke,1 Jesse J. Smith,1 Chin-Tung Chen,1 Samuel Brook,1 Maurizio Scaltriti,1 Jinru Shia,1 Philip Paty,1 Christina Leslie,1 Scott Lowe,1 Jose Baselga,1 Julio Garcia-Aguilar1. 1 _Memorial Sloan Kettering, New York, NY;_ 2 _Ben Gurion University, Be'er Sheva, Israel;_ 3 _Beth Israel Deaconess Medical Center, Boston, MA;_ 4 _Weill Cornell, New York, NY_.

Background: Treatment for locally advanced rectal cancer (LARC) consists of neoadjuvant chemoradiotherapy (NCRT) followed by radical excision. Patients with tumors carrying a mutant KRAS are less likely to respond to NCRT compared to KRAS wild type tumors. We hypothesized that an RNA-based signature differentiating KRAS mutant and wild type patients could serve as an indicator of the biological process associated with response to NCRT. We found that the RNA-based signature is enriched for stromal and immune genes. Furthermore, the stromal component of the signature is a predictor of response to NCRT.

Methods: Tumors from 120 LARC patients enrolled in a multicenter phase 2 trial studying response to NCRT were tested for KRAS status by Sanger Sequencing or Memorial Sloan Kettering (MSK)-IMPACT assay and gene expression was quantified by sequencing. Colorectal cancer (CRC) patients from MSK (n=95) and TCGA (n=261), previously annotated for KRAS mutation status and gene expression, were used for validation. A KRAS-inducible mouse model and CRC patient-derived xenografts (PDXs) were utilized to determine the cell of origin for the gene expression signature. Stromal enrichment was assessed with the ESTIMATE stromal gene signature. Immunohistochemistry (IHC) was completed for Periostin (POSTN), a stromal marker from the RNA-signature. Variant Allele Frequency (VAF) was used to measure the abundance of KRAS, TP53 and Adenomatous Polyposis Coli (APC) mutant alleles in tumors, and was quantified by targeted exome sequencing with the MSK-IMPACT assay.

Results: Analysis of the KRAS-associated gene signature showed significant stromal inactivation in KRAS mutant patients. The signature was validated in the MSK and TCGA cohorts. The stromal signature was recapitulated in a KRAS inducible mouse model. Human CRC PDXs in mouse indicated that the signature arose from murine stroma and not human epithelium. Consistent with the stromal signature, IHC for POSTN, a stromal marker, was significantly lower in the KRAS mutant tumors compared with the KRAS wild type tumors (p<0.05) and was absent from the epithelium. The stromal enrichment in mutant KRAS tumors was inversely correlated with the KRAS VAF (p<0.01). This finding was not observed for TP53 or APC VAF indicating specificity for stromal inactivation in KRAS mutant tumors. Furthermore, the stromal component of the signature is associated with poor response to NCRT in LARC.

Conclusions: This study shows that a KRAS mutation in CRC is associated with a lower expression of a stromal signature and that this signature is derived from the tumor microenvironment. This study indicates that CRC KRAS mutant tumors and a stromal subtype are closely related. Understanding this relationship may play a key role in elucidating the mechanism by which a KRAS mutant tumor is resistant to standard therapy.

#4079

IL-6 promotes radiation-induced macrophage infiltration to tumors via upregulation of CCL2 and CCL5.

Xin Wang, Ying Tsai, Kwang-Hsiang Chuang, Peter Keng, Soo Ok Lee, Yuhchyau Chen. _University of Rochester Medical Center, Rochester, NY_.

Background: Radiotherapy is a major treatment modality for non-small cell lung cancer (NSCLC), and it is controversial if radiotherapy contributes to metastasis. We hypothesize that radiotherapy increases lung cancer cell recruitment of macrophage-infiltration, and the infiltrated macrophages may accelerate the metastatic potential of cancer. We examined the role of intracellular IL-6 in NSCLC cells in promoting the recruitment of macrophages after irradiation and explored the molecular mechanisms that govern the increase of macrophages.

Methods: By utilizing the IL-6 knocked down (IL-6si) and scramble control (sc) A549 and H157 NSCLC cell lines, we investigated whether the intracellular IL-6 level affected macrophage migration into cancer cells. We also developed orthotopic human xenograft models in nude mice using the luciferase tagged-H157IL-6si and H157sc cells and investigated IL-6 effects on macrophage infiltration to tumor sites after irradiation. Tumors were irradiated and the irradiated mice were divided into two groups: (1) 1st group, the endogenous macrophage-infiltration into tumors was examined; and (2) in the 2nd group, mice were injected with the GFP-labeled THP-1 cells through tail veins after irradiation, and the recruitment of exogenous macrophages (THP-1) to tumor site was investigated 3 days after the injection. We also studied the molecular mechanisms by which IL-6 mediated the increase of macrophage-infiltration upon irradiation.

Results: We found a decrease of macrophage recruitment by A549/H157 cells upon radiation when IL-6 was knocked down. Consistent with the in vitro results, we observed the contribution of IL-6 signaling to promote macrophage-infiltration after irradiation in vivo in human tumor xenografts. In the mechanism dissection studies, we found that CCL2 and CCL5 molecules were the critical IL-6 downstream target molecules, which mediated the radiation-induced increase of macrophage infiltration. We confirmed the contribution of these molecules by demonstrating the effects of neutralizing antibodies to these target molecules in reducing the radiation-induced enhancement of macrophage migration to tumors.

Conclusion and Impacts: Study results suggest that IL-6 signaling, through the up-regulation of CCL2/CCL5, mediated the increase of macrophage infiltration to NSCLC cells after irradiation. Molecular studies suggest that blocking CCL2/CCL5 pathways can potentially be applied to conventional radiotherapy to intercept the process of radiation-promoted macrophage-infiltration.

#4080

Analysis of murine stromal components in patient-derived xenograft (PDX) models of pancreatic cancer.

Diana Behrens,1 Ulrike Pfohl,1 Britta Büttner,1 Jens Hoffmann,1 Wolfgang Walther,2 Iduna Fichtner2. 1 _EPO GMBH, Berlin, Germany;_ 2 _ECRC - Charité University Medicine Berlin and Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany_.

Pancreatic cancer remains a lethal disease with only 3 - 8% of patients surviving 5 years after initial diagnosis (WHO, 2012). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models of high clinical relevance.

Since the desmoplastic stroma has impact on the progression and treatment of pancreatic cancer, we investigated the attributes of the murine stroma in patient-derived xenografts that completely replaced the human surrounding tissue within a few months after primary transplantation. We elucidated the functionality of murine tumor microenvironment for growth and therapeutic response in a cohort of well-characterized pancreatic cancer (PDAC) PDX. PDX are a valuable tool for the prediction of therapy response, the identification of new biomarkers and therapeutic targets or pancreatic cancer specific pathways.

In this study, 57 patient tumors were collected and immediately transplanted into immunodeficient mice. So far, 14 out of 57 samples were established as passageable pancreatic cancer xenografts (PDX). All engrafted PDX are poorly or moderately differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations revealed K-ras mutations in 13 and additionally p53 mutations in 9 out of 14 PDX. Furthermore, chemosensitivity to standard of care (SoC) drugs was determined by using clinically relevant and optimized schedules and doses. The testing revealed that the response to Gemcitabine (1/10 responder) was moderate within the PDX panel, while the most efficient drug was Abraxane with 5 out of 10 responders. In general, the response profile of all PDX closely reflected patient's situation in the clinic. Cryo- and formalin-preserved tumor tissues of these chemosensitivity studies were investigated for markers of desmoplastic stroma (SPARC, alpha-SMA, FAP and collagen I). Immunohistochemistry and real-time PCR revealed, that even the replacing murine stroma is characterized by a distinct reactivate nature. Semi-quantitative analysis of stromal components showed that the tumor surrounding tissue mass was not significantly reduced due to therapeutic intervention. Though the tumor burden was diminished under SoC, the mRNA expression level of SPARC and FAP was unaffected in corresponding samples of the treatment groups compared to vehicle-treated control. The same effect was found for alpha-SMA and collagen I in immunohistochemically stained specimens.

In summary, this study revealed a functional tumor environment of murine origin in patient-derived xenografts of pancreatic cancer and furthermore an apparently inherent resistance of this stromal tissue towards conventional therapy. Thus, targeting the tumor microenvironment should be implicated into clinical decisions.

#4081

Stromal autophagy is required for the growth of cutaneous melanoma.

Ran Ellen Zhang,1 Natalie J. Guo,1 Nithya Krishnan,1 David Jelinek,2 Gizem Karsli Uzunbas,3 Xiaoqi Xie,3 Arek Gertych,4 Janice M. Mehnert,3 Beatrice Knudsen,4 Eileen White,3 Hilary A. Coller2. 1 _Princeton University, Princeton, NJ;_ 2 _University of California, Los Angeles, Los Angeles, CA;_ 3 _Rutgers University, New Brunswick, NJ;_ 4 _Cedars-Sinai Medical Center, Los Angeles, CA_.

Through degrading bulk cytoplasmic materials into intermediate building blocks, autophagy refuels metabolism and maintains homeostasis under starvation and stress conditions. The context-dependent role of autophagy in cancer cells during tumorigenesis and tumor progression has been explored over the past decade; however, studies of autophagy in relation to the tumor microenvironment are still lacking. In this study, we investigated the role of autophagy in the cancer-associated fibroblasts (CAFs), a major component of the microenvironment that is important for the growth and development of cutaneous melanoma. Analysis of clinical biopsies revealed high levels of autophagy in fibroblasts that surrounded melanoma cells, but not in the non-tumor adjacent area. Fibroblasts co-cultured with any of multiple melanoma cell lines, but not melanocytes, also showed increased autophagic flux, which requires transforming growth factor beta (TGF-β) signaling. Knocking down autophagy-related (ATG) genes ATG5 or ATG7 in fibroblasts did not impair their proliferation in vitro, but inhibited the growth of co-engrafted melanoma cells in vivo. Further, by introducing intradermal melanoma allografts into mice that harbored fibroblast-specific Atg7 deletion, we observed remarkable tumor growth retardation. In order to mimic anti-autophagy therapy for patients, we generated mice that allowed conditional Atg7 depletion in their entire bodies, and found significant tumor growth suppression. Atg7 knockout mice contained elevated plasma levels of immune cell attractants, including CXCL9, CXCL10 and MCP-1. By inhibiting these cytokines/chemokines with atorvastatin, tumor growth in Atg7 knockout mice was completely rescued; while, there was no effect of atorvastatin on control mice. Taken together, our data show that through modulating the tumor microenvironment, stromal autophagy plays an important role during melanoma growth and development. Our findings indicate that stromal autophagy may represent a valuable therapeutic target that might induce less side effects and systemic toxicity.

#4082

Epithelial-stromal interactions are altered at the earliest stages of colon cancer development.

Allen Mo,1 Stephen Jackson,1 Kamini Varma,1 Alan Carpino,1 Charles Giardina,2 Thomas J. Devers,1 Daniel W. Rosenberg1. 1 _UConn Health, Farmington, CT;_ 2 _University of Connecticut, Storrs, CT_.

While progression of nonmalignant colonic cells carrying somatic mutations to early malignancy is dependent upon interactions between mutated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here we report the development of an ultra-sensitive laser capture microdissection/RNA-seq approach for profiling the epithelial and stromal compartments of mutated aberrant crypt foci (ACF). ACF are the earliest detectable pre-neoplastic lesion found in human colon and were identified using high-definition endoscopy with contrast dye-spray. We focused on epithelial and stromal cells of ACF carrying somatic mutations to either KRAS, BRAF or APC, and compared these to control samples from each patient. By comparing the gene expression from each group, we identified an increase in a number of pro-inflammatory NF-κB target genes that are specific to ACF epithelium (including TIMP1, RELA and RELB). We confirmed distinct transcriptional changes associated with each somatic mutation and demonstrate that a subset of ACF display a BRAFV600E-mediated senescence-associated transcriptome, characterized by increased expression of CDKN2A (p16). Furthermore, ACF-associated stroma is transcriptionally distinct from adjacent normal stroma and genes related to immune cell infiltration and activation of fibroblasts are up-regulated. Immunofluorescence analysis confirms the abundance of activated fibroblasts in ACF stroma regardless of mutational status. These results provide new insight into the cellular interplay that occurs at the very early stages of colon cancer development, highlighting the role of activated stromal fibroblasts and inflammation.

#4083

Tumor associated macrophages mediates the oncogenic effect of FGF18 in ovarian cancer.

Wei Wei, Michael Birrer. _Massachusetts General Hospital, Boston, MA_.

Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. Effective management of this deadly disease still remains scanty due to the inadequate understanding of the molecular mechanisms associated with the tumor progression and prognosis. We have previously reported the tumor-promoting role of Fibroblast growth factor 18 (FGF18), which have been identified through genomic analysis of ovarian tumors. Initial studies suggested FGF18 promotes ovarian tumor progression through NF-κB pathway mediated elevation of proinflammatory cytokines, which mediate the increased tumor burden, angiogenesis and infiltration of macrophages in FGF18 expressing xenografts. Based on these findings, the present study aims to delineate the mechanism by which FGF18 modulates the phenotype of tumor associated macrophages (TAMs) as well as the reciprocal tumor-promoting feedback from the TAMs which potentially contributes FGF18 mediated ovarian tumorigenesis.

Immunohistochemical staining of a tissue array comprising 216 archived high-grade advanced stage serous ovarian tumor specimens revealed a significant correlation between the FGF18 expression and infiltration of M2-polarized macrophages (by CD163). In addition, the density of intratumoral M2 TAMs inversely associated with patients' overall survival as a negative prognostic factor independent to age, tumor grade and debulking status. These findings suggest intratumoral TAMs may play an important role in ovarian tumorigenesis. In vitro co-culture studies further demonstrated that human monocyte cell line THP-1 or murine peritoneal macrophage cell line IC-21 could significantly activate the NF-κB pathway and increase the production of various proinflammatory cytokines in A224 ovarian cancer cells. Co-culture also increased the migratory potential of A224 cells. Addition of an IKKβ inhibitor into the co-culture system nullified the cytokine production and migration of tumor cells promoted by monocytes/macrophages, implicating the involvement of canonical NF-κB signaling in the crosstalk. Conversely, liposome clodronate mediated in vivo macrophage depletion significantly shrunk the tumor burden of the intraperitoneal xenograft derived from FGF18 overexpressing SKOV3 cells, while minimal effect was observed over the control RFP overexpressing xenograft. Besides, macrophage depletion also markedly decreased the intratumoral microvessel density induced by FGF18 overexpression. Taken together, our data potentially suggest TAMs may mediate the FGF18 related ovarian tumorigenesis by augmenting the proinflammatory status of the tumors.

#4084

Crosstalk between TNBC and stromal components via secreted factors enhances cell motility that can be attenuated by a CXCR1 inhibitor.

Kideok Jin, Niranjan B. Pandey, Aleksander S. Popel. _Johns Hopkins Univ. School of Medicine, Baltimore, MD_.

Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Unfortunately reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotropic tumor inoculation. We have discovered that the TNBC tumor cells secrete large amounts of interleukin-6 (IL-6) that "educates" lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis.

In this report, using a human antibody array, we identified factors secreted by fibroblasts (HGF, IL-6, IL-8, CCL7, MIF, GDF-15, EMMPRIN and VEGF) and by macrophages (CXCL5, IL-8 and uPAR) upon induction by MDA-MB-231 TCM. We ranked the expression level of each factor by real time qRT-PCR and determined that interleukin 8 (IL-8) was the top candidate. We confirmed by ELISA that IL-8 secreted from either fibroblasts or macrophages treated with MDA-MB-231 TCM was upregulated compared to treatment with serum free media (SFM). Our data showed that the proliferation of MDA-MB-231 cells incubated with conditioned media (CM) from TCM-induced fibroblasts or TCM-induced macrophages was enhanced compared to SFM treatment. Furthermore, MDA-MB-231 cell migration, a key step in tumor metastasis, was promoted by CM from TCM-induced fibroblasts or macrophages. Significantly, inhibition of the IL-8 signaling pathway by Reparixin, an inhibitor of the IL-8 receptor CXCR1, abrogated the upregulation of MDA-MB-231 cell proliferation and migration induced by TCM-induced fibroblasts or macrophages.

These findings implicate IL-8 signaling as a critical event in TNBC cell proliferation and migration via crosstalk with stromal components. Further, these studies suggest that IL-8 acts as a key regulator orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional antagonism of the IL-8 signaling pathway has the potential to circumvent TNBC breast cancer growth and metastasis.

#4085

Notch1 signaling determines melanoma-regulating role of tumor stromal fibroblasts.

Hongwei Shao,1 Ranran Kong,1 Mecker Moller,1 Massimiliano L. Ferrari,1 Leiming Zhang,1 Freddy Radtke,2 Anthony J. Capobianco,1 Zhao-Jun Liu1. 1 _University of Miami, Miami, FL;_ 2 _Ecole Polytechnique Fédérale de Lausanne, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland_.

Objectives. Tumor stromal fibroblasts are crucial in regulating tumor growth, invasion and metastasis, yet the molecular mechanisms that determine the tumor regulatory role of stromal fibroblasts remains unknown. Here, we uncover the Notch1 signaling pathway as a molecular determinant that controls the tumor regulatory role of tumor stromal fibroblasts in melanoma growth and invasion. Methods. Murine melanoma cells B16-F10, stably transduced with Luciferase 2 (Luc2)/lentivirus, were xenografted on the skin of two new mouse lines: the Gain-Of-Function of Notch1 (GOFNotch1: Fsp1.Cre+/-;ROSALSL-N1IC+/+) and Loss-Of-Function of Notch1 (LOFNotch1: Fsp1.Cre+/-;Notch1LoxP/LoxP+/+), respectively. GOFctrl (FSP1.Cre-/-;ROSALSL-N1IC+/+) and LOFctrl (FSP1.Cre-/-; Notch1LoxP/LoxP+/+) mice were used as control. Tumor growth was measured based on tumor weight and melanoma local invasion were examined by H&E staining of resected tumor tissue.

Results. Using a mouse melanoma model in which exogenous melanoma cells were grafted on the skin of two lines of mice where the genetic activation or inactivation of Notch1 signaling specifically occurs in natural host stromal fibroblasts, we demonstrated that Notch1 pathway activity could determine the tumor-promoting or tumor-suppressing phenotype in tumor stromal fibroblasts. Tumor stromal fibroblasts carrying elevated Notch1 activity significantly inhibited melanoma growth and invasion, while those with a null Notch1 promoted melanoma invasion.

Conclusions. These findings identify the Notch1 pathway as a molecular determinant that controls the regulatory role of tumor stromal fibroblasts in melanoma skin growth and invasion, unveiling Notch1 signaling as a potential therapeutic target for melanoma and potentially other solid tumors.

#4086

Critical roles of FRS2beta, a feedback inhibitor of ErbB-ERK signaling, for communication between cancer stem cells and their niche during mammary tumorigenesis.

Yukino Machida,1 Natsuko Kimura,2 Arinobu Tojo,3 Ko-ichi Akashi,4 Hideyuki Saya,5 Issay Kitabayashi,6 Noriko Gotoh7. 1 _Division of Molecular Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Japan;_ 2 _Institute of Medical Science, University of Tokyo, MInato-ku, Japan;_ 3 _Institute of Medical Science, University of Tokyo, Minato-ku, Japan;_ 4 _Kyusyu University, Fukuoka City, Japan;_ 5 _Keio University, Shinjuku-ku, Japan;_ 6 _National Cancer Center Research Institute, Chuo-ku, Japan;_ 7 _Cancer Research Institute, Kanazawa University, Kanazawa City, Japan_.

Inflammatory microenvironment contributes to tumorigenesis. Although it is thought that breast cancer stem cells (CSCs) appear and grow in the inflammatory microenvironment that is called CSC niche, it remains largely unclear how it occurs. Here, we uncovered FRS2beta, a feed-back inhibitor of ERK activity in progenitor cells, plays critical roles for various cytokine production to increase progenitor cell- and CSC- state, and create the CSC niche. Expression levels of FRS2beta were increased in mammary tumors of MMTV-ErbB2 mice than in normal mammary tissues. Deficiency of FRS2beta greatly delayed mammary tumorigenesis, decreased mammosphere-forming ability and tumor stroma, a component of the CSC niche. Expression levels of various cytokines, including IGF1 and CXCL12, stemness- and stroma-inducing cytokines, respectively, were reduced in FRS2beta-mutant mammospheres, pre-cancerous mammary and tumor tissues. Furthermore, expression levels of FRS2beta were higher in CD44highCD24low CSCs-enriched population in patient-derived breast cancer tissues. Thus the mechanisms to maintain progenitor cell-state by prevention of excess ERK activity may permit production of various cytokines to create CSC niche, and allow appearance and growth of CSCs. These results suggest that treatment with MEK inhibitors may allow survival of CSCs, raising the risk of recurrence. We provide a rationale of combination therapy targeting both FRS2beta and MEK inhibitors to eradicate tumors.

#4087

Mammary fibroblasts exert divergent effects on the survival of invasive breast cancer cells.

Kimberly Hagel, Kelsey Weigel, Meaghan Boyd, Luke McCormack, Matthew Champion, Zachary Schafer. _University of Notre Dame, Notre Dame, IN_.

Cancer development and progression are inherently inefficient processes riddled with anti-tumorigenic obstacles. Anoikis, or apoptosis induced by detachment from the extracellular matrix (ECM), represents one prominent hindrance to cancer progression that transformed cells must overcome in order to survive, particularly during the ECM-bereft metastatic cascade. Far from acting in isolation, we report that one mechanisms of anoikis evasion by invasive breast cancer cells involves the exploitation of carcinoma-associated fibroblasts (CAFs) from the surrounding tumor microenvironment. CAFs secrete insulin-like growth factor-binding proteins (IGFBPs) that facilitate non-canonical activation of integrin-linked kinase (ILK), activation of ERK/MAPK, and ultimately stabilization of the anti-apoptotic protein Mcl-1 to promote cancer cell survival during ECM detachment. Further investigation into the influence of the tumor microenvironment on cancer cell survival revealed that, contrary to the role of CAFs, normal mammary fibroblasts (NMFs) appear to exert anti-tumorigenic effects on ECM attached cancer cells. Utilizing conditioned media from NMFs, we have found that NMFs secrete factors that increase caspase activation in multiple cancer cell lines, suggestive of NMF-mediated cancer cell death. Because cancer progression necessitates the intermingling of cancer cells with normal stromal cells, particularly at early time points in primary tumor and metastases formation, we hypothesize that this NMFs pose yet another obstacle to disease progression that may compliment the process of anoikis by capping the metastatic cascade with environments that are inhospitable to cancer cell survival. Similar to anoikis evasion, our data suggests that cancer cells must evade NMF-induced death stimuli in order to survive, possibly through the accrual of CAFs in the tumor microenvironment to combat or replace the pro-apoptotic NMFs. Using traditional and 3D mammalian cell culture in combination with in vivo studies, we aim to delineate the roles of both CAFs and NMFs on cancer cell survival under conditions of differential ECM availability. A more complete understanding of the nuances dictating cancer cell-fibroblast interactions could yield informative data relevant to chemotherapeutic research and development.

#4088

Adipocytes-mediated autophagy activation and metabolic reprogramming promotes colon cancer survival.

Yang-an Wen, Xiaopeng Xiong, Jennifer Harris, Yekaterina Zaytseva1, Tianyan Gao. _Markey Cancer Center, Lexington, KY_.

Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism underlying the functional interaction between obesity and cancer remains elusive. Here, we demonstrated that adipocytes isolated from adipose tissues of colon cancer patients play an important role in promoting tumor cell survival and progression by altering cellular metabolism. In stage IV colon cancer cases, abundant adipocytes were found in close association with invasive colon cancer cells. Co-culture of adipocytes with colon cancer cells led to direct transfer of free fatty acids (FAs) released by the adipocytes to colon cancer cells. Uptake of FAs allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation, suggesting that FAs from adipocytes were used as an energy source by the cancer cells. In addition, we found that co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize FAs and blocked the growth promoting effect of adipocytes. Furthermore, we found that adipocytes induced dedifferentiation of tumor cells in primary colon cancer cells and mouse tumor organoids. Taken together, these results identify adipocytes as active contributors to the tumor microenvironment that promote tumor growth and survival by serving as an energy provider and a metabolic regulator for the embedded colon cancer cells.

#4089

Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells.

Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy.

#4090

Metabolic and microenvironment changes in the mammary of calorie restricted, normal weight, and obese mice throughout the lifespan.

Laura A. Smith,1 Emily L. Rossi,1 Laura W. Bowers,1 Emma H. Allot,1 Sarah Dunlap,2 Liza Makowski,1 Bentley Midkiff,1 Melissa Troester,1 Stephen D. Hursting1. 1 _University of North Carolina Chapel Hill, Chapel Hill, NC;_ 2 _University of Texas at Austin, Austin, TX_.

Background: Breast tissue remodeling occurs with age and is marked by a progressive increase in stromal tissue. Advancing age is also associated with increased senescence among stromal cells that promote a protumorigenic microenvironment by adopting a senescence-associated secretory phenotype (SASP). In previous studies, calorie restriction has been effective in cancer prevention through inhibiting cellular senescence and SASP. However, obesity has shown to be cancer promoting with inconsistent findings on its role in cellular senescence.

Purpose: We tested the hypothesis that calorie restriction is protective to the protumorigenic, age-related changes that occur within the mammary microenvironment while diet-induced obesity accelerates these changes.

Methods: Six week old female mice were randomized to receive either a low-fat control regimen (10 kcal% fat), a 30% calorie restricted (CR) regimen relative to control, or a high-fat diet induced obesity (DIO, 60 kcal% fat) regimen, resulting in control, CR, and DIO mice respectively. A subset of mice was sacrificed at 1, 3, 5, 12 and 20 months following diet initiation with mammary fat pads (MFP) harvested and serum stored for further analysis. H&E staining was analyzed using a digital algorithm to quantify the composition of adipose, epithelia and stoma in the MFP tissue. RNA was extracted from MFP sections and rt-PCR was preformed to analyze gene expression of CDKN2a, the gene encoding p16(INK4a) a well known marker for senescence.

Results: CR significantly decreased body weight and decreased serum IGF-1, insulin, leptin and estradiol and significantly increased adiponectin relative to control and DIO mice at all time points. MFP composition remained relatively stable from 1 and 12-month time points. However, there was an apparent non-significant trend from 12 and 20-month time points, with increasing stromal tissue and a decrease in adipose tissue, which was most drastic in CR mice. Gene expression analysis showed an increase in CDKN2a expression in control mice relative to CR mice at 5 and 20-month time points. Additionally, control and CR mice demonstrated an increase in CDKN2a expression with age from 5 months to 20-months.

Conclusions: Stromal composition of murine MFP displayed an age-related increase that is consistent with findings in human breast tissue. Despite this uniform increase, we see a decrease in the age-related acquisition of a senescence phenotype in CR mice relative to control supporting the notion that CR is protective to this protumorigenic change that occurs with advancing age. Ongoing analysis of gene expression in DIO mice will determine the role of obesity in MFP cellular senescence and confirmation of senescent phenotypes via β-galactosidase staining and analysis of SASP associated gene expression.

#4091

Mesenchymal stem cells drive paclitaxel-resistance in erbB2-overexpressing breast cancer cells via paracrine of NRG-1.

Ling Zhu,1 Jin Wang,1 Weimin Zuo,1 Rong Lin,1 Tingting Lin,1 Yan Lei,1 Bingshuang Ren,1 Jun Lu,1 Huiyue Dong,1 Lingjing Lin,1 Lianghu Huang,1 Qinghua Wang,1 Yujie Ma,1 Hui Lyu,2 Bolin Liu,2 Jianming Tan,1 Shuiliang Wang1. 1 _Fujian Key Laboratory of Transplant Biology, Fuzhou General Hospital, Xiamen University, Fuzhou, Fujian Province, China;_ 2 _Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO_.

Breast cancer remains the most frequently diagnosed cancer and the leading cause of cancer death in women, both worldwide and in less developed countries. Surgery in conjunction with adjuvant chemotherapy is the main treatment of choice for patients with locally advanced breast cancer, leading to reduce cancer-related symptoms and prolong survival. Paclitaxel as a critical drug in the treatment of breast cancer patients, intrinsic and acquired resistance to paclitaxel represents a significant clinical problem. We had previously demonstrated that increased expression of erbB3 is required for erbB2-mediated paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway-dependent upregulation of Survivin. Mesenchymal stem cells (MSCs) are emerging as an important component of tumor microenvironment, which may play an essential role in regulating cancer cell growth, motility, invasion and therapeutic resistance. In the present study, we have explored the possible role of MSCs in regulating the chemo-sensitivity of erbB2-overexpressing breast cancer cells. We show that both human umbilical cord and bone marrow-derived MSCs express significantly higher level of Neuregulin-1 (NRG-1, also Heregulin-β1) as compared with erbB2-overexpressing breast cancer cells themselves. Coculture or treatment with conditioned medium of MSCs not only decreases the anti-proliferation effect of paclitaxel on erbB2-overexpressing breast cancer cells, but also significantly inhibits paclitaxel-induced apoptosis. We further demonstrate that this MSCs-drived paclitaxel-resistance in erbB2-overexpressing breast cancer cells could be attributed to paracrine effect of NRG-1/erbB3 signaling, as specific nutralizaion of NRG-1 or blocking of erbB3 resensitizes erbB2-overexpressing breast cancer cells to paclitaxel treatment. Moreover, overexpression of erbB3 enhances, while knockdown expression of erbB3 abrogates MSCs-drived paclitaxel-resistance. Taken together, our current data indicate that paracrine of NRG-1 by MSCs induce resistance of paclitaxel in erbB2-overexpressing breast cancer cells through activation of erbB3 signaling. Our findings suggest that simultaneously targeting mesenchymal stem cells in tumor microenviroment may be a novel strategy to overcome chemotherapeutic resistance.

#4092

High mast cell density is associated with improved prognosis in colon cancer patients.

Anita Sjölander. _Lund Univ., Malmö, Sweden_.

Background. Inflammatory cells and inflammatory mediators play an important role in colon cancer. However, the role of mast cells (MCs) in colon cancer progression remains unclear. Pro-inflammatory cysteinyl leukotrienes (CysLTs) have been associated with colorectal cancer (CRC) development. In particular, previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have poorer prognoses, and Cysltr1-/- mice display fewer intestinal polyps. The aim of the present study was to investigate the clinical and prognostic significance of mast cell density (MCD) in patients with CRC and to determine the significance of CysLTR1 on MCD in a mouse model of colitis-associated colon cancer (CAC).

Materials and methods. A tissue microarray (TMA) of primary colon cancers from 72 patients was stained with MC anti-tryptase and anti-chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCD in cancerous and normal areas of patient tissues and in polyp/tumour, adjacent and normal areas of mouse tissues.

Results. In patient samples, the mean MCD was significantly higher in normal tissue than in cancer tissue (p = 0.0009). Patients with a high MCD in cancer tissue showed better overall survival compared with those with a low MCD (hazard ratio (HR) 0.575; 95% confidence interval (CI), 0.293 - 1.130). The same result was observed in patients aged 55-75 years (HR 0.332; 95% CI 0.121 - 0.911) and among patients without distant metastasis at diagnosis (HR 0.311; 95% CI 0.118 - 0.821). In the CAC mouse model, Cysltr1-/- mice had fewer intestinal polyps and significantly higher MCD in polyp/tumour areas compared with controls.

Conclusion. A high MCD in cancer tissue correlates with longer patient survival. In Cysltr1-/- CAC mice, a high polyp/tumour MCD also appears beneficial. Thus, these results indicate that a high MCD is a good prognostic factor.

#4093

The impact of the prostate cancer microenvironment on the expression and regulation of aldehyde dehydrogenases.

Maria Sadiq,1 Simon J. Allison,2 Fiona Frame,3 Mark Sutherland,1 Roger M. Phillips,2 Norman J. Maitland,3 Klaus Pors1. 1 _University of Bradford, Bradford, United Kingdom;_ 2 _University of Huddersfield, Huddersfield, United Kingdom;_ 3 _University of York, York, United Kingdom_.

Introduction:

Aldehyde dehydrogenase (ALDH) enzymes are involved in the detoxification of specific endogenous and exogenous aldehyde substrates. High ALDH activity is used to identify stem cells and has also been associated with poor prognosis in cancer. In prostate cancer (PCa) high ALDH activity has been associated with elevated clonogenicity, migratory behaviour, tumor progression and metastasis. Despite these observations there exists a poor understanding of the role of selected ALDH isoforms in PCa. In an attempt to understand if the tumor microenvironment impacts on ALDH function, we investigated their expression under hypoxia.

Methods:

Gene and protein expression analysis of ALDH isoforms -1A1, -1A2, -1A3, -1B1, -2, -3A1 and -7A1 in normal prostate cell line PNT2C2, and a panel of PCa cell lines with different stages of cancer was carried out by quantitative PCR and western blot under normoxic and hypoxic conditions at 24 and 48 hours. Gene expression of the ALDHs in primary prostate cultures was assessed following retinoic acid treatment. siRNA knockdown technology was used to study functional roles of selected ALDH isoforms.

Results:

Gene expression of ALDH1A3 was increased in Bob cells at 24h under hypoxia, at 48h this was less apparent but the expression of most ALDHs in Bob cells including ALDH1A2, -2, -3A1 and -7A1 was reduced at 48h. In SerBob cells, the gene expression of ALDH1A1 and -1B1 was reduced at both time points, whereas the expression of ALDH1A2, -2, and -7A1 was increased under hypoxia. At protein level, the expression of ALDH1A3 appeared low under hypoxia at both time points in SerBob cells while the expression of ALDH3A1 also appeared low after 24h exposure to hypoxia. In DU145 cells, there was an increase in ALDH1A1, -1A2, 1A3, -2, and -3A1 gene expression under hypoxia at 48h. In LNCAP cells, the gene expression of ALDH2 was increased at 48h under hypoxia whereas the expression of ALDH7A1 was reduced at 48h. The protein expression of ALDH7A1 was higher in PC3 cells under hypoxia at 24h. A significant increase was observed in ALDH1A3 and ALDH3A1 expression in response to retinoic acid. ALDH1A3 knockdown showed a significant reduction of cell viability in PC3 cells. Other investigations underway that will be reported at the conference are focused on how prostate cancer stem-like cells expressing ALDHs are responding to hypoxia.

Conclusion:

Our data suggests that the expression of certain ALDHs is affected by hypoxia and ALDH1A3 potentially is involved in cell survival in PCa. Future work will investigate if any of these isoforms can be used as biomarkers to distinguish indolent from malignant PCa.

#4094

Exosomes secreted from the noncancerous microenvironment promote proliferation, migration, and invasion in breast cancer cells.

Letitia A. Yearby, Terri Cunningham, Jacenta Matthews, KiTani Parker Lemieux. _Xavier University of Louisiana, New Orleans, LA_.

Introduction: Triple negative breast cancer (TNBC) is one of the most aggressive forms of five major types of breast cancer. Because TNBC mortality disproportionately affects premenopausal African American women, a health disparity exists within this group. Our research has provided insight on how the noncancerous microenvironment plays a role in TNBC proliferation, migration, and invasion. Exosomes, extracellular vesicles (30-300 nm) produced ubiquitously by mammalian cells, are secreted by noncancerous breast epithelial cells, and have been noted to enhance proliferation, migration and invasion in TNBC. Therefore, the purpose of this study is to determine if exosome-free conditioned media (EFCM), which contains noncancerous MCF10A exosomes, enhances proliferation, migration and invasion in TNBC.

Methods: MDA-MB-231 and MCF10A breast epithelial cells were used as cell line models. Proliferation was measured by the alamar blue proliferation assay. The transwell migration assay was used to assess migration, and the Matrigel invasion assay was used to measure migration. ExoELISA assays were also used to detect exosomes in exosome-free conditioned media (EFCM) from MCF10A cells. MCF10A cells were serum-starved for 24 hours, using DMEM/F-12 with 0.5% standard or exosome-depleted FBS (edFBS), to produce exosomal conditioned media (CM) and EFCM. This media was harvested and sterile filtered before use. MDA-MB-231 cells were treated with EFCM in each of the bioassays, and RPMI-1640 was used as a control.

Results: MCF10A exosomes introduced via EFCM enhanced proliferation, migration and invasion of MDA-MB-231 cells.

Conclusions: Taken together, our findings demonstrate that secreted factors from noncancerous MCF10A cells that have been stressed as a direct result of serum starvation are exosomes. These exosomes recruit TNBC cells to increase proliferation, migration, and invasion. The role of these exosomes in the noncancerous microenvironment is poorly understood, but these data suggest that exosomes from the noncancerous microenvironment have potential to be used in targeted therapies against triple negative breast disease.

#4096

Fibroblast growth factor-2-derived from cancer-associated fibroblasts stimulates proliferation and migration of human breast cancer cells.

Jinyoung Suh, Do-Hee Kim, Young-Joon Surh. _Seoul National University, Seoul, Republic of Korea_.

Cancer-associated fibroblasts (CAFs) constitute a major compartment of the tumor microenvironment. CAFs produce a variety of cytokines, growth factors and extracellular matrix proteins, thereby stimulating tumor progression. CAFs are distinct from normal fibroblasts for their overexpression of α-smooth muscle actin and fibroblast specific protein-1. Recent studies suggest that CAFs play an important role in proliferation and migration of cancer cells through crosstalk with them.In the present study, we investigated the role for CAFs in proliferation and migration of breast cancer cells and underlying molecular mechanisms.When triple-negative human mammary MDA-MB-231 cells were treated with the conditioned medium (CM) collected from cultured CAFs, the cell viability and migration were significantly elevated. Furthermore, these cells manifest a more proliferative phenotype, exhibiting enhanced mRNA expression of cycinD1, c-Myc and PCNA as well as increased phosphorylation of Akt and STAT3.In addition, MDA-MB-231 cells exhibited elevated expression of proliferative and invasive genes including MMP2 and MMP9 when incubated with CAFs in the indirect co-culture system. Notably, mRNA levels of fibroblast growth factor-2 (FGF2), stromal-derived factor-1, interleukin-6 and interleukin-8 detected in CAFs were higher than those in normal fibroblasts of the same patients. In contrast, FGF2 was expressed at a relatively low level in breast cancer cells. FGF2 exerts its biological effects by binding to and activating FGF receptor (FGFR), a subfamily of cell surface receptor tyrosine kinases. Notably, the expression of FGFR1 was up-regulated in triple-negative breast cancer cells including MDA-MB-231 cells while another major FGF2-corresponding receptor FGFR2 was rarely expressed. Therefore, we focused on FGF2-FGFR1 signaling in the context of paracrine communication between breast cancer cells and CAFs. CAF-stimulated MDA-MB-231 cell migration as well as FGFR1 expression was abolished when FGF2-neutralizing antibody was added to the CAF-CM. In addition, treatment of MDA-MB-231 cells with FGF2 induced the phosphorylation of FRS2 and Akt. FGF2-induced cell migration and up-regulation of cyclinD1 expression were abrogated by siRNA-mediated FGFR1 silencing. Furthermore, FGF2 promotes nuclear localization of FGFR1. Taken together, above findings suggest that secretion of FGF2 by CAFs stimulates proliferation and migration of breast cancer cells through interaction with FGFR1 which may contribute to human breast cancer progression.

#4097

Significance of glypican-1 expression on tumor stromal cells in gastric carcinoma.

Yuichiro Miki, Masakazu Yashiro, Kishu Kitayama, Hiroaki Kasashima, Go Masuda, Naoshi Kubo, Katsunobu Sakurai, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. _Osaka City University, Osaka, Japan_.

Background: Glypican1 (GPC1) is a cell surface heparan sulfate proteoglycan that acts as a co-receptor for heparin-binding growth factors, and it plays a crucial role in cancer growth. Recently, it has been reported that GPC1 enriched on cancer-derived exosomes, and GPC1-positive exosomes in the circulation was frequently found in the patients with early pancreatic cancer. However role of GPC1-positive exosomes derived from tumor stromal cells remains to be unclear. The aim of this study is to clarify the significance of GPC1 expression on stromal cells in gastric carcinoma.

Methods: A total of 597 patients with gastric cancer was enrolled in this study. An anti-GPC1 antibody was used for immunohistochemical staining. GPC1 expression was scored by the intensity of staining and the percentage of positive cells. GPC1 expression on cancer cells or stromal cells was evaluated. The corelation between GPC1 expression and clinicopathological features was statistically analyzed.

Results: GPC1 expression on cancer cells was found in 473 (79 %) of 597 cases. GPC1 expression on stromal cells was found in 147 (75 %) of 597 cases. A Kaplan-Meier survival curve showed that overall survival of patients with GPC1-positive stromal cells was significantly better (log-rank p= 0.0028) than that with GPC1-negative stromal cells (5-year survival rate; 72.5 % vs 57.9 %, respectively). In contrast, overall survival was not statistically different (log rank p= 0.090) between patients with GPC1-positive tumor cells and GPC1-negative tumor cells (5-year survival rate; 71.8 % vs. 64.6 %, respectively). GPC1 expression on stromal cells was significantly correlated with T stage, lymph node metastasis, lymphatic vessel invasion, and clinical stage.

Conclusions: GPC1 expression on stromal cells was associated with the good prognosis of patients with gastric cancer. GPC1-positive exosomes in stromal cells might be correlated with the progression of gastric cancer.

#4098

SPARC expression stimulates paracrine inhibitory response from bone marrow stroma during dormancy of prostate cancer in the bone.

Sambad Sharma, Fei Xing, Kerui Wu, Yin Liu, Kounosuke Watabe. _Wake Forest University School of Medicine, Winston-Salem, NC_.

Recurrent disease is the most daunting aspect of cancer treatment; however, how tumor cells become dormant and later recur even years after "successful" treatment is poorly understood. However, studies to decipher mechanisms responsible for dormancy have been hampered due to lack of appropriate model. In this study, we isolated two syngeneic cell lines (Indolent and Aggressive), which recapitulates dormancy and recurrence of prostate cancer in the bone microenvironment. We found that Indolent cells retained dormant phenotype whereas Aggressive cells grew rapidly in the tibial bone in vivo whereas the in vitro cell proliferation, invasion, migration and self-renewal properties of both cells in culture were not altered, suggesting the role of microenvironment in regulation of dormancy and recurrence. The expression profile by our microarray analysis revealed that SPARC and Noggin (a known inhibitor of BMP7) were significantly upregulated in Indolent and Aggressive cells, respectively. SPARC secreted by Indolent cells was found to stimulate BMP7 expression in bone stromal cells that in turn inhibited cancer cells by activating the dormancy-associated p38 MAPK pathway and its downstream cell cycle inhibitors, p21 and p18. In addition, BMP7 increased senescence and diminished stem-cell phenotype of cancer cells, which was rescued by addition of recombinant Noggin. We also found that BMPR2 plays a crucial role in SPARC-induced paracrine inhibition of tumor cells residing in the bone. Accordingly, BMPR2 knockdown rescued the BMP7-mediated decrease in stemness. Importantly, the BMPR2 correlative signature was enriched in patients who did not experience recurrence for a long period of time, which further verified the role of BMPR2 downstream signaling in dormancy. When primary tumor samples were examined by immunohistochemistry, both SPARC and BMPR2 were found to be significantly downregulated in patients with bone metastasis. Moreover, patients who did not experience bone metastasis were found to express high level of both SPARC and BMPR2. Importantly, we also observed elevated expression of DNA methylase genes, DNMT1 and -3B in Aggressive cells. Treatment of Aggressive cells with NS398, a COX-2 inhibitor downregulated DNMTs and concomitantly augmented SPARC expression in vitro. Therefore, recurrence of cancer cells in the bone microenvironment involves epigenetic regulation of SPARC and disruption of inhibitory crosstalk of cancer cells with the stroma. These findings suggest that SPARC plays a critical role in maintaining dormancy of prostate cancer cells in bone microenvironment.

#4099

Oligodendrocyte stimulates the invasion ability of glioblastoma cells.

Toshiyuki Kawashima. _Osaka City University Medical School, Osaka, Japan_.

Background & Aims: Glioblastoma (GBM, WHO Grade IV) is characterized by rapid cancer cell infiltration, and is one of the most lethal neoplasms among solid cancers. GBM might arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interaction between GBM cells and glial cells.

Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived from surgical tissues of each patient with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived from surgical tissues of each patient with GBM. The culture medium was composed of DMEM with 10% fetal calf serum. We examined the effects of glial cells on the invasion ability of GBM cells by wound-healing assay and invasion assay. Using IncuCyte invasion assay and matrigel invasion assay, the invasiveness of T98G and U251 cells was analyzed in the presence or absence of conditioned medium from oligodendrocyte cells or fibroblasts. The effect of the proliferation of T98G and U251 cells was examined by MTT assay or cell count.

Results: ODC1 and ODC2 cells were positive for Olig2 staining but not for GFAP staining, suggesting that ODC1 and ODC2 were oligodendrocyte cells. GF1 and GF2 cells were spindle-shape and were negative for both Olig2 staining and GFAP staining, suggesting that GF1 and GF2 cells were fibroblasts. Oligodendrocyte cells, ODC1 and ODC2 cells, from grade II glioma significantly (p<0.01) increased the migration and invasion ability of GBM cells, T98G and U251 cells. In contrast, GF1 and GF2 fibroblasts from GBM did not affect on the migration and invasion ability of GBM cells. Both oligodendrocyte cells and fibroblasts did not stimulate the proliferation of T98G and U251 cells.

Conclusion: Oligodendrocytes might up-regulate the invasiveness of malignant glioma cells. An invasion-stimulating factor(s) from oligodendrocyte cells might be partially associated with the progression from lowe-grade gliomas to GBM.

#4100

Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones.

Richard Steinman,1 Jingjing Huang,2 Paul Woods,1 Daniel Normolle,1 Julie Goff,1 Christine Stehle1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Tsinghua University, Beijing, China_.

Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. Bone marrow is the most common site for luminal breast cancer recurrence. We explored whether the HS5 bone marrow stromal cell line that is known to arrest co-cultured breast cancer cells could suppress estrogen receptor alpha (ER) expression during arrest, and whether it could prime the emergence of estrogen-independent breast cancer clones. We demonstrated that paracrine signaling from HS5 cells downregulated ER at the mRNA level and also decreased ER protein stability in T47D and MCF7 breast cancer cells. Conditioned medium (CM) from HS5 arrested the cells in G0/G1 in part through interleukin-1 (IL1), inhibiting mammosphere formation and 2D-growth despite activation of proliferative pathways (Erk and AKT) by the CM. Reversal of stromal-induced growth arrest by blockade of signaling by IL1 in HS5-CM enabled the outgrowth of ER-negative breast cancer cells that were fulvestrant-resistant and estrogen-independent. Outgrowth depended on AKT signaling. Our results describe pro- and anti-proliferative paracrine signals arising from a bone marrow stromal cell line. Blockade of the growth suppressive signaling unmasked stromal facilitation of ER conversion and anti-estrogen resistance in breast cancer cells.

#4101

Stromal cell-induced alterations in the response of colorectal cancer cell to demethylating agents.

Viswanath Das,1 Svetlana Skolekova,2 Khushboo Agrawal,1 Jan Gursky,1 Lucia Kučerová,2 Marián Hajdúch1. 1 _Institute of Molecular and Translational Medicine, Olomouc, Czech Republic;_ 2 _Cancer Research Institute, Bratislava, Slovakia_.

Epigenetic mechanisms such as DNA hypermethylation, that results in gene silencing, is closely associated with resistance to platinum-based chemotherapy for colorectal cancer (CRC). DNA methyltransferase inhibitors (DNMTi) that reactivate and induce reexpression of silenced gene have the potential to improve the outcome of platinum-based and other therapies in CRC. In fact, non-cytotoxic concentrations of azacitidine, a clinically used DNMTi, has been reported to resensitize platinum-resistant ovarian cells to carboplatin. Hypomethylating agents, such as azacitidine and decitabine have shown significant promise in hematologic malignancies; however, studies on their application to CRC and other solid tumors have yielded ambivalent results potentially due to the complexities of the tumor microenvironment that result from the numerous cell type interactions.

In this study, we analyzed the impact of different tumor-stroma ratios on cancer cell response to azacitidine, decitabine, and two lead compounds identified by in vitro screens in two-dimensional (2D) and three-dimensional (3D) co-cultures of colorectal HCT116 carcinoma demethylation reporter cells and adipose-derived mesenchymal stromal cells (MSCs) isolated from healthy individuals undergoing elective lipoaspiration. Each subject provided informed consent. Activity of hypomethylating drugs was examined in 2D and 3D cultures of different tumor-stroma ratios using confocal microscopy, flow cytometry, and MTT analyses.

Our preliminary flow cytometry results show an altered response of hypomethylating agents in cocultures of HCT116 and MSCs compared to monocultures of HCT116 cells alone. There was an increase in demethylation of HCT116 cells in 3D cultures containing a higher ratio of stromal cells following treatment with low concentration of decitabine. Furthermore, confocal analysis 3D co-cultures at various z-plane heights revealed increased demethylation at low concentrations of all drugs in cultures with high stromal cell numbers. These results indicate that intratumoral stroma may alter the sensitivity of cancer cells to demethylating agents and warrant further studies.

#4102

Effect of HGF concentrations on c-Met inhibition investigation.

Veronica S. Hughes, Dietmar W. Siemann. _Univ. of Florida, Gainesville, FL_.

Tumor cells frequently harbor abnormalities in signaling pathways, leading to increased migration, invasion, survival, angiogenesis, and proliferation. C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair, and protein levels are often elevated in a large variety of tumors. C-Met is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and stimulates the c-Met protein, leading to a variety of downstream signaling pathways that result in increased migration, proliferation, survival, and angiogenesis.

While many tumor cells express the c-Met receptor, a number of tumor types, including glioblastoma and osteosarcoma, have been observed to co-express both HGF and c-Met. The purpose of the current study was to measure tumor cell HGF secretion in vitro, to determine HGF concentrations in vivo, and study how HGF concentrations affect tumor cell response to c-Met inhibition. Over forty cell lines were investigated for HGF secretion via ELISA of conditioned media. The small molecule c-Met inhibitor BMS-777607 was used to interrupt the c-Met axis in a select number of tumor cell lines. C3H/HeJ, nude, and Balb/c mice, were measured for serum levels of HGF.

Data obtained using ELISA of conditioned media show that about one quarter of the cell lines tested secreted HGF. Cell lines tested include, but are not limited to, glioblastoma, prostate, breast, fibrosarcoma, and osteosarcoma. Migration assays were performed on several of these cell lines to determine sensitivity to BMS-777607. HGF-secreting cell lines, including the KHT, U87, U118, and OS156, are sensitive to BMS-777607 at 0.1, 1, and 10 µM. Non-HGF-secreting cell lines, such as PC-3 and MDA-MB-231, are insensitive to BMS-777607 at the same concentrations. A migration assay was performed with the PC-3 cells, with cells exposed to 0.1, 1, and 10 uM BMS-777607 while in the presence of 0, 5, 10, and 25 ng/mL HGF. These cells are not sensitive to the c-Met inhibitor at low concentrations of HGF, yet become sensitive at higher concentrations. These results indicate that sensitivity to c-Met inhibition may be dependent on both HGF-secretion status and levels of exogenous HGF. Furthermore, our findings suggest that in vitro investigations of c-Met inhibition should be done at physiologically relevant HGF concentrations.

In order to properly perform c-Met inhibition experiments in vitro, a better understanding of in situ HGF concentration is needed. Serum levels of HGF were measured in the C3H/HeJ, nude, and Balb/c mice. The average serum HGF concentration in these mice was between 3 and 5 ng/mL. Still, it is ultimately the local concentration of HGF within the tumor microenvironment that is critical, but no intra-tumor values for HGF have been reported. Consequently measurements of HGF levels within tumors by microdialysis are ongoing.

#4103

Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo.

Roberto Lugo,1 Marta Gabasa,1 Francesca Andriani,2 Marta Puig,1 Federica Facchinetti,2 Josep Ramírez,3 Abel Gómez-Caro,3 Ugo Pastorino,2 Pere Gascón,3 Albert Davalos,4 Noemí Reguart,3 Luca Roz,2 Jordi Alcaraz1. 1 _University of Barcelona, Barcelona, Spain;_ 2 _Fondazione IRCCS Istituto Nazionale dei Tumori INT, Milano, Italy;_ 3 _Hospital Clínic de Barcelona, Barcelona, Spain;_ 4 _Buck Institute for Age Research, Novato, CA_.

Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors.

### Imaging and Therapeutics of Metastasis

#4104

Characterization and validation of an implantable chronic lung imaging window.

David Entenberg, Allison Harney, Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis. _Albert Einstein College of Medicine, Bronx, NY_.

Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. Recent advances in intravital microscopy have enabled visualization of the live intact lung, but are limited in the duration over which they can be utilized (hours) and require major invasive surgeries. Both of these limitations reduce their usefulness in the study of single cell events in the lung and are susceptible to introducing artefacts. We report the development of a novel, minimally invasive surgical protocol for the implantation of a long-lasting lung imaging window. Once implanted, this window allows for the repeated, non-invasive visualization of living, breathing lung tissue over a period of two weeks without the need for ventilation, vacuum plates, or gated imaging equipment. Here we characterize and validate the use of this window in living mice. The window is well tolerated, does not cause infection or inflammation, and offers a 4mm view of the left lung. We utilize it to visualize, with single cell resolution, the progression of experimental metastasis from the arrival of a single cell to the formation of micro-metastases. The development of this novel optical window will allow visualization of, with single cell resolution, tumor microenvironments and metastatic progression within the lung over extended periods of time. This approach to imaging live lung tissue has the potential to reveal the mechanisms underlying many diseases affecting the lung as well as the means to directly evaluate responses to therapeutics in in real time and within a single animal.

#4105

Dissecting the mechanisms of hepatocyte replacement in breast cancer liver metastases.

Mark R. Nathan, Lefteris Kostaras, Victoria Bridgeman, Shane Foo, Peter Vermeulen, Andrew Reynolds. _Institute of Cancer Research, London, United Kingdom_.

The liver is a common site of metastasis in metastatic breast cancer and is associated with significant mortality. Histopathological examination of human breast cancer liver metastases (BCLMs) reveals that the majority of these tumours present with a 'replacement growth pattern.' In this growth pattern, the breast cancer cells freely invade the liver parenchyma and replace the resident hepatocytes. In advanced BCLMs, breast cancer cells can eventually replace a significant volume of the liver parenchyma which, ultimately, leads to organ failure and death. However, the mechanism through which breast cancer cells replace the resident hepatocytes is unknown.

Here we set out to establish how this replacement of hepatocytes by breast cancer cells occurs in BCLMs. To do so, we established two in vivo models of advanced BCLM: (a) an ER+PR+HER2- model using the MCF7 cell line, and (b) a triple negative patient-derived xenograft model using breast cancer cells isolated from a pleural effusion. We have also established an in vitro co-culture system, where MCF7 cells are co-cultured with HepG2 cells, which is designed to mimic the breast cancer cell-hepatocyte interaction observed in vivo.

Both in vivo models exhibit a histology that closely mimics the replacement growth pattern of human BCLMs, where breast cancer cells replace hepatocytes at the tumour-liver interface. To address whether hepatocytes are killed by adjacent breast cancer cells in vivo, we evaluated hepatocyte death by apoptosis using appropriate markers (cleaved-PARP and cleaved-caspase-3). Surprisingly, although extensive apoptotic death of breast cancer cells in the tumour mass could be observed, no evidence for apoptotic death of hepatocytes could be found. However, we observed extensive cell-in-cell invasion at the tumour-liver interface, where live breast cancer cells enter the cytoplasm of adjacent hepatocytes. We also observe a similar process of cell-in-cell invasion within in vitro cultures of MCF-7 cells with HepG2 cells.

Although cell-in-cell invasion has been described as a mechanism of non-apoptotic cell death in breast cancer cells, the role of cell-in-cell invasion between breast cancer cells and hepatocytes in BCLMs has not been investigated. Our current studies are focused in two areas: (a) we are using intra-vital microscopy and time-lapse microscopy to investigate whether cell-in-cell invasion between breast cancer cells and hepatocytes mediates non-apoptotic death of hepatocytes both in vivo and in vitro, and (b) we are investigating the molecular mechanisms that mediate cell-in-cell invasion between breast cancer cells and hepatocytes.

In conclusion, we present preliminary evidence for a novel mechanism via which breast cancer cells could replace hepatocytes in BCLMs. Further studies aimed at elucidating the molecular basis of this mechanism may reveal novel targets for preventing the replacement of the liver parenchyma by metastatic breast cancer cells.

#4106

Modified RECIST for monitoring hepatocellular carcinoma with computed tomography: Inter-reader variability of the response.

hubert Beaumont,1 Sophie Egels,2 Antoine Iannessi,3 Eric Bonnard,3 Christopher Foley,4 Olivier Lucidarme2. 1 _MEDIAN Technologies, Valbonne, France;_ 2 _Hopital la Pitié Salepétriére, Paris, France;_ 3 _Centre Antoine Lacassagne, Nice, France;_ 4 _Glaxo Smith Kline, Uxbridge, United Kingdom_.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. In clinical trials, computer tomography (CT) is a widely used imaging technique for the monitoring of HCC patients, and modified RECIST (mRECIST) have been suggested as appropriate criteria in part because they distinguish the viable part from the necrotic part of the tumor.

Based on longitudinal changes of tumor burden, this study investigates the causes of inter-reader variability in evaluating the therapeutic response of HCC when relying on mRECIST.

24 patients with advanced (unresectable and/or metastatic) HCC enrolled in a phase I/II multicentre international study were retrospectively reviewed. From the originally selected target lesions, 41 were randomly selected. Original mRECIST expert evaluations were aggregated to the retrospective readings of three radiologists having different expertise (non mRECIST experts). All the 150 readings performed by each readers were made in using an electronic calliper. Precision of measurements between couple of readers was analysed by assessing standard deviation (SD) and reproducibility coefficient (RDC). The agreement of readers responses were compared by using Kappa coefficient statistic. The variability between non-experts and the variability between mRECIST expert and non-experts were analysed. Reader's disagreement at declaring progressive (PD) and responding (PR) patient, were visually classified between variability of the measure, difference in the perception of tumour boundaries and differences in using either RECIST or mRECIST criteria for a given lesion.

SD of measurements between non-experts ranged [24.9%; 36.3%] and RDC was 16.6 [13.85; 23.95]. Kappa coefficients was 0.41 [0.28; 0.55]. SD of expert against non-experts ranged [33.2%; 41.1%] and RDC was 18.8 [16.02; 24.53]. Kappa coefficients was 0.20 [0.06; 0.35].

Pooling the four readers together, rate of discrepancy at declaring respectively PD or PR by patients was 47.8% (11/23) and 34.8% (8/23). 90.9% (10/11) of discrepancies at declaring PD were due to different perceptions of tumour boundaries. Discrepancy at declaring PR was, in 62.5% of the cases, correlated to the application of mRECIST, 25% was correlated to different perception of tumours boundaries.

Inter-reader variability of HCC measurements was large which leads to poor agreement in the response. The main cause of discrepancy at declaring PD originated from the complexity of HCC patterns and the poor definition of tumours boundaries while discrepancies at detecting PR come from the variability of readers at selecting the viable part of the tumours. In the context of HCC clinical trials, reliability of endpoints depend on the criteria involved. Present study must be completed with an inter-reader analysis of the variability between mRECIST experts.

#4107

CD44 expressing cancer associated fibroblasts guide collagen 1 fibers in breast cancer.

Samata Kakkad, Desmond Jacob, Balaji Krishnamachary, Zaver M. Bhujwalla. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Cluster of differentiation 44 (CD44) has been identified as a cancer stem cell marker in breast [1], pancreatic, prostate, ovarian, and colon cancer [2] making it a significantly important molecule for investigating and targeting. CD44 is abundant in triple negative breast cancer (TNBC), and its expression is a poor prognosticator for outcome [3]. Stromal cells play a critical role in tumor progression [4]. Amongst these, cancer associated fibroblasts (CAFs) significantly influence the proliferation, invasion, and metastasis of cancer cells [5]. CAFs are a major source of collagen 1 (Col1) fibers in the tumor stroma, play a role in cancer cell dissemination, and contribute to the reactive desmoplastic tumor stroma, and the high density and stiffness of the tumor extracellular matrix (ECM) [6]. Here we investigated the relationship between CD44 expression and Col1 fibers in a human breast cancer tissue microarray (TMA). We found that CD44 was expressed by cancer cells and CAFs in the TMA. CD44 expressing CAFs were seen guiding Col1 fibers.

A breast tissue microarray containing stage I-IV human breast cancers and normal adjacent biopsy samples was used in this study. CD44 immunostaining was performed on 5μm thick sections following which Col1 fiber distribution was obtained from the same section using second harmonic generation (SHG) microscopy. Z-stacks of SHG tile images were acquired on Olympus FV1000MPE multiphoton microscope using a 25x lens. A high-resolution digital scan of the CD44 stained slide was acquired. CD44 and Col1 fiber images were co-registered and overlaid to evaluate co-localization patterns. 8/12 stage I cancers showed high (3+) CD44 staining intensities, 45/62 stage II cancers showed high (3+) staining intensities, 14/16 stage III cancers showed high (3+) staining intensities and 1/1 stage IV cancer showed high (3+) staining intensities. Col1 fibers patterns changed from short and thick in normal adjacent tissue to long, thin and straight fibers in malignant tissue. We observed strong expression of CD44 in breast cancer cells and in CAFs that were closely associated with straight long Col1 fibers that are typical of an invasive metastatic ECM phenotype. CD44 expressing CAFs were found attached to the Col1 fibers and appeared to guide fibers through the cancer cells. We are currently performing advanced texture analysis to characterize the differences in distribution patterns. Our data highlight the importance of CD44 as a target in TNBC to eliminate cancer cells and CAFs. CD44 targeting may also change Col1 fiber patterns to a less aggressive ECM phenotype.

1. Al-Hajj M et al., PNAS 2003; 2. Kim GR et al., Oncology letters. 2015; 3. Idowu MO et al., Hum Pathol. 2012; 4. Quail DF et al., Nat Med. 2013; 5. Kalluri R et al., Nature reviews Cancer. 2006; 6. Byun JS et al., Am J Pathol. 2013.

#4108

In vitro **vascularized model for tumor growth and progression.**

Ashley Smith,1 Charles Garson,1 Shantanu Pradhan,2 Elizabeth Lipke,2 Robert Arnold,2 Balabhaskar Prabhakarpandian,1 Kapil Pant1. 1 _CFD Research Corporation, Huntsville, AL;_ 2 _Auburn University, Auburn, AL_.

Introduction: Tumor metastasis is the primary reason for mortality of cancer patients. The ability to model the microenvironment of the primary tumor and the secondary and tertiary sites is critical for advancing the treatment options for the invasive cancers. There is currently no 3D tumor model that mimics the in vivo vascular geometry and the microenvironment for monitoring progression of tumors. In this study, we report on the development of a 3D tumor model comprising of vascular cells in communication with tumor cells leading to invasion and metastasis at secondary and tertiary sites.

Materials and Methods: Vascularized tumor networks comprising primary, secondary and tertiary tumor sites were developed using in vivo images and fabricated using soft lithography. Human mammary microvascular endothelial cells (hMMEC) were cultured in the vascular channels while a GFP-labeled metastatic breast cancer cell (MDA-MB-231) and a GFP-labeled non-metastatic cell (MCF-7) mixed with and without human fibroblast cell line BJ-5ta was cultured in the primary tumor site in a 3D environment using Matrigel™. The tumor networks were perfused with endothelial cell media and the growth, migration, invasion and metastasis of the tumor cells from the primary site to secondary and tertiary site was monitored for 28 days using time lapse microscopy.

Results and Discussion: The aggressively metastatic MDA-MB-231/BJ-5ta tumor at the primary site was found to proliferate rapidly resulting in breakdown of the Matrigel and invasion across the endothelial cells. Secondary sites were localized with pockets of tumor colonies within 48 hours and by 120 hours had metastasized to the tertiary site. By 14 days, the primary site tumor formed a necrotic core while the tumor cells at secondary and tertiary were viable. In contrast, the non-aggressive MCF-7/BJ-5ta tumors were able to proliferate within the Matrigel scaffolding but did not break down beyond the primary site until 14 days. Culture of tumor cells without the presence of fibroblast or endothelial cells resulted in a significant difference in the invasion and metastasis pattern highlighting the importance of the native tumor microenvironment.

Conclusions: We have developed a 3D, heterogenic model of invasive tumor growth and metastasis which closely mimics the in vivo microenvironment of solid tumors. This model can be used to investigate tumor progression and their underlying mechanisms using a combination of real-time techniques as well as 'omic' methodologies for screening and evaluation of the therapeutics.

Acknowledgements: We gratefully acknowledge financial support from NIH (#HHSN261201400037C).

References: B. Prabhakarpandian et al., J Control Release 2015; 201:49-55

#4109

Unbiased detection of disseminated tumor cells in murine bone marrow.

Kenneth C. Valkenburg, James R. Hernandez, Sarah R. Amend, James E. Verdone, Michael A. Gorin, Kenneth J. Pienta. _Johns Hopkins School of Medicine, Baltimore, MD_.

Approximately 20-30% of prostate cancer patients develop disease recurrence and metastasis years after initial therapy. This is thought to be largely due to the presence of growth-arrested and chemoresistant disseminated tumor cells (DTCs) in secondary sites, such as bone. Bone metastasis is found in 90-100% of prostate cancer patients who succumb to the disease. There are still many gaps in knowledge about the biological mechanisms by which DTCs home to bone, resist chemotherapy, become dormant, and escape dormancy to grow into clinical metastases. As such, it is important to be able to detect, quantify, and study bone marrow DTCs. In particular, it must be possible to do this in metastatic cancer mouse models, which are critical to study the process of tumor dissemination. DTC detection techniques currently exist, usually as either a positive selection or negative selection methodology. Positive selection techniques use markers or cell size to isolate and purify tumor cells out of the bone marrow. Positive selection markers are generally epithelial-specific, such as EpCam, E-cadherin, or Cytokeratin, and therefore may miss cells that lose epithelial marker expression and may gain mesenchymal markers. DTCs can also be as small as or smaller than white blood cells, meaning that positive selection based on size may miss some DTCs. Negative selection enriches for DTCs by removing blood and bone marrow cells from the population, usually using cell-specific markers. A popular strategy is CD45-based depletion, which removes white blood cells, and theoretically leaves behind DTCs. In our hands, this strategy causes loss of DTCs in the depletion process. To capture these heterogeneous and rare DTCs, we have developed a strategy to detect DTCs in murine bone marrow in an un-biased manner. The procedure entails removal of the bone marrow via centrifugation from the long bones (femur and tibia) of mice that have been injected with cancer cells (the injection site may vary depending on the experimental setup). The bone marrow then undergoes red blood cell lysis, and further centrifugation. The white blood cells are then counted, and the bone marrow is spread onto glass slides. The cells on the slide are fixed, permeabilized, and stained (immunofluorescence and RNA fluorescent in situ hybridization can be used). The staining can include any type of marker, including epithelial, mesenchymal, disease-specific, species-specific, or other biologically interesting markers, such as cell cycle markers. The unbiased nature of this procedure is based on the lack of positive or negative selection based on cell size or protein expression. Some DTC loss is noted in this protocol, due to the centrifugation and staining steps, but the cell population on the slide should include all DTC types. Notably, this protocol can be used to detect human or mouse cells in the mouse bone marrow and can thus be used in immune-compromise and immune-competent mouse models of metastasis.

#4110

Color-coded imaging of the tumor microenvironment of malignant lymphoma in a syngeneic mouse model.

Takuro Matsumoto,1 Atsushi Suetsugu,1 Kosuke Hasegawa,1 Miki Nakamura,1 Hitomi Aoki,1 Takahiro Kunisada,1 Hisashi Tsurumi,1 Masahito Shimizu,1 Robert M. Hoffman2. 1 _Gifu University Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer, Inc., San Diego, CA_.

EL4-RFP lymphoma cells were injected in the spleen of C57/BL6 GFP transgenic mice. EL4-RFP metastasis was observed in the lymph nodes of the supra mediastinum and abdomen and in the liver 28 days later. The EL4-RFP tumors in the spleen (primary injection site), liver, supra mediastinum lymph nodes, abdominal lymph nodes, bone marrow, and lung were visualized with the Olympus FV1000 scanning laser confocal microscope. EL4-RFP tumors in the liver, lymph nodes, and bone marrow in C57/BL6 GFP mice were rich in GFP stromal cells such as macrophages, fibroblasts, dendritic cells, and normal lymphocytes derived from the host animal. Phagocytosis of EL4-RFP lymphoma cells by macrophages, as well as dendritic cells and fibroblasts, were observed in culture. Small tumors were observed in the spleen (primary injection site), but the stroma cells in the spleen tumors were abundant. We could observe that in the lung, no mass formation of lymphoma cells occurred, but lymphoma cells circulated in lung peripheral blood vessels.

#4111

Imaging of colon cancer-cell mitosis within liver metastasis in nude mice.

Kosuke Hasegawa,1 Atsushi Suetsugu,1 Miki Nakamura,1 Takuro Matsumoto,1 Hitomi Aoki,1 Takahiro Kunisada,1 Masahito Shimizu,1 Robert M. Hoffman2. 1 _Gifu University graduate school of medicine, Gifu, Japan;_ 2 _AntiCancer, Inc,, San Diego, CA_.

Colon cancer frequently results in metastasis to the liver, when it becomes the main cause of death. The tumor cell cycle in primary tumors and metastases are poorly understood. We developed a liver metastasis mouse model using a human colon cancer cell line (HCT-116), which expresses GFP in the nucleus and RFP in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP (2.0 × 106) cells were injected in the spleen of a BALB/c nude mouse. The cells subsequently formed tumor colonies in the liver and retroperitoneum 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, mitosis of the dual-colored colon cancer cells could be clearly imaged in the liver and other metastases. Multi-nucleated cancer cells, in addition to a mononuclear cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nuclear cells were also observed in cell culture of HCT-116 GFP-RFP cells. Multi-nuclear HCT-116 cells may participate in the tumor growth in vivo. A significant difference in mitotic cells count in the primary tumor and metastasis was not observed.

#4112

Geminin overexpression-dependent recruitment and crosstalk with mesenchymal stem cells enhances aggressiveness in triple-negative breast cancers.

Theresa Bonner,1 Suryatheja Ananthula,1 Janene Nordan,1 Simran Batth,1 Gailen D. Marshall,1 Lauren H. Gardner,2 Yoshiko Shimizu,2 Wael M. ElShamy1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _University of Hawaii, Honolulu, HI_.

Introduction: Tumor resident mesenchymal stem cells (MSCs) promote cancer progression. Pathways involved in recruitment of MSCs to breast tumor cells remain largely undefined. We aimed at defining the mechanism whereby geminin overexpressing breast tumor cells recruit and interact with MSCs to increase their own aggressiveness.

Experimental Procedures: We used co-culturing assay, immunohistochemistry, in vitro invasion assay with or without cytokines neutralization, western blot analysis, immunoprecipitation, immunofluorescence, ELISA, exposure to hypoxic conditions, cytokines receptor inactivation, in vivo orthotopic tumor formation assay in the presence or absence of inhibitors to the proposed signaling pathways, analysis of publically available data sets for the association of selected factors with overall survival, distant metastasis free survival and recurrent free survival to define the mechanism geminin overexpressing cells employ in vivo to recruit and initiate bi-directional interactions with MSCs in order to enhance their own aggressiveness.

Results: Here we show that geminin-dependent acetylation of chromatin HMGB1 (Ac-HMGB1) leads to its release to the extracellular space. Extracellular Ac-HMGB1 promotes geminin overexpressing (GemOE) cells survival by binding to RAGE receptors. Extracellular Ac-HMGB1 also triggers expression and activation of RAGE in the non-expressing MSCs leading to induction of CXCR4 expression and migration of MSCs towards SDF1/CXCL12-expressing GemOE cells. Inhibiting c-Abl, RAGE or CXCR4 prevented MSCs recruitment to GemOE cells in vitro and in vivo. Reciprocal interactions between GemOE cells and MSCs elevate the TIC, basal and EMT traits in GemOE cells leading to enhanced aggressiveness. Indeed, faster, larger and more aggressive tumors develop when GemOE cells are co-injected with MSCs in orthotropic tumor model. Concurrently, inhibiting c-Abl and thus geminin function or RAGE or CXCR4 activity in the in vivo model not only suppressed MSCs recruitment to the orthotopic breast tumors, but also led to decrease in TIC, basal and EMT phenotypes in these tumors.

Conclusion: We suggest that GemOE generates metastatic tumors in part through recruiting and interacting with MSCs.

#4113

Pre-metastatic niche formation in palmitic acid and Caco2 BBE secreted exosomes treated HepG2 cells by the CD98-integrin-FAK pathway.

Jenniffer L M Stetler, Brandon S. Canup, Hamed Laroui. _Georgia State University, Atlanta, GA_.

Up to 60% of patients with colorectal cancer (CRC) will develop metastatic CRC. This study will use an in-vitro Non-Alcoholic Fatty Liver Disease (NAFLD) model in HepG2 cells to determine how CD98 and colon cancer secreted exosomes initiate a pre-metastatic niche. CD98 is a glycoprotein that mediates integrin signaling, amino acid transportation, inflammatory cytokines, and possibly endocytosis of exosomes. The pathway of CD98-integrin-FAK may mediate uptake of exosomes as well as the formation of pre-metastatic niche in the liver. Exosomes secreted from colon cancer and NAFLD have been implicated to initiate a pre-metastatic niche in the liver. This combinatorial study will mimic in-vivo conditions of metastatic CRC. HepG2 cells are treated with 0 µM or 7 µM of palmitic acid (PA) for 5 days as well as stimulated with and without 10 µg/mL LPS to mimic chronic inflammation in-vitro. After the 5 days of PA and LPS, then the cells are treated for additional 2 days with Caco2BBE secreted exosomes. The treatments were assessed for NAFLD and inflammatory markers FAS, ABCA1, PPARα, PPARϒ, IL-1β, IL-6, TNF-α, and CD98 by Oil-Red staining, Western blot, RT-qPCR, and immunocytochemistry (IMC). The same set experiments with a knock down of CD98 and creation of pre-metastatic niche were examined through CD98-integrin-FAK pathway. This model showed that low doses of PA formed lipid vacuoles and increased NAFLD markers FAS, PPARα, PPARγ, and ABCA1. The cytotoxicity test of the 0-7 uM of PA with and without LPS as well as exosomes indicated low toxicity. IMC of HepG2 cells showed a unique co-localization patterns with no treatment and PA causing association CD98 with integrin β1 and CD98 with caveolin-1, while the LPS and knock down of CD98 disrupted these interaction and allowed an interaction between CD98 and actin. The addition of Caco2BBE secreted exosomes with PA and LPS altered the inflammatory markers and cellular location of CD98. In conclusion, low doses of palmitic acid produced lipid vacuoles, little cytotoxicity, and increased NAFLD markers. The loss of CD98 led to cellular re-location of CD98 to the nucleus and could explain the decrease uptake of Caco2 BBE secreted exosomes and decrease in inflammatory markers. These data suggest that a pre-existing condition such as NAFLD and Caco2 BBE secreted exosomes play an integral role in the development of pre-metastatic niche by the CD98-integrin-FAK pathway. The identifying the nuances of CD98-integrin-FAK pathway will elucidate future nano-particle therapeutics for metastatic CRC.

#4114

Blocking Ezrin can inhibit HGF/Met signaling-induced metastasis.

Yanlin Yu, Liping Huang, Glenn Merlino. _NCI-CCR, Bethesda, MD_.

Hepatocyte Growth Factor (HGF) binds its receptor Met and induces autophosphorylation of MET, which in turn activate appropriate signaling pathways. Aberrant HGF/Met signaling promotes tumor progression by facilitating cell proliferation, migration, invasion, and metastasis. Previously, we found that constitutive HGF/Met signaling promotes melanoma metastasis through a nonautocrine mechanism in engineered mouse models (Cancer Res, 2002); however, the molecular mechanisms by downstream of HGF/Met signaling that induce metastasis are not known. We recently identified the cytoskeletal organizer Ezrin as a key metastatic regulator using an HGF transgenic mouse model (Nature Medicine, 2004), implicating Ezrin is in HGF/Met signaling-induced metastasis. We hypothesize that HGF/Met signaling promotes metastasis by regulating expression of Ezrin. To test hypothesis, we are: 1) evaluating the relationship between ezrin expression and HGF/Met signaling; 2) validating the role of Ezrin in HGF/Met signaling -induced metastasis; 3) elaborating mechanisms underlying this HGF/Met signaling-mediated regulation of Ezrin expression; 4) evaluating the clinical therapeutic utility of blockage of Ezrin function in preclinical mouse models. We found that HGF is able to stimulate the expression of Ezrin. Moreover, analysis of the established model systems with HGF and Met alone or HGF with Met demonstrated that Ezrin expression correlated with activated HGF/Met signaling, suggesting that HGF/Met signaling regulates the expression of Ezrin. Interestingly, we showed that HGF/Met signaling could activate transcription factor Sp1 through the MAPK pathway. We further found that activated Sp1 could directly bind to the promoter of the Ezrin gene and regulate the transcription of Ezrin. Notably, knockdown of Ezrin expression by shRNAs inhibited the metastasis induced by both HGF/Met autocrine and notautocrine signaling in syngeneic host and HGF GEM hosts. We also used small molecule drugs in preclinical mouse models to confirm that Ezrin is at least one of downstream molecules mediating HGF/Met signaling-induced metastasis in melanoma. We conclude that Ezrin is a key downstream substrate involved in the regulation of HGF/Met signaling-induced metastasis. We demonstrate a link between Ezrin and HGF/Met/MAPK/Sp1 activation in the metastatic process. Our data indicate that Ezrin represents a promising therapeutic target for patients with activated HGF/Met signaling.

#4115

Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling.

Yeon-Jin Kwon,1 Boris A. Leibovitch,1 Nidhi Bansal,1 Lutecia Pereira,2 Edgardo V. Ariztia,1 Kevin Petrie,3 Arthur Zelent,2 Ming-Ming Zhou,1 Eduardo F. Farias,1 Samuel Waxman1. 1 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 2 _University of Miami Miller School of Medicine, Florida, FL;_ 3 _Institute of Cancer Research, London, United Kingdom_.

Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poor survival in TNBC patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need for TNBC treatment and for other tumor types. We reported that decoys with the SID sequence designed to bind and inhibit the function of PAH-2 domain of Sin3A protein markedly prolong survival in the adjuvant setting due to inhibition of metastatic dissemination to the lungs and bone marrow in TNBC mouse models. Here, we show that TNBC cell lines treated with SID decoys (peptides) display a strong in vitro inhibition of migration and invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin, and inhibition of proteolytic enzymes (MMP9; MT-MMP1 and uPA) involved in extracellular matrix degradation. DNA microarray and Ingenuity pathway analysis (IPA) showed that the SID decoys inhibit Wnt and TGFβ signaling that is associated with epithelial to mesenchymal transition (EMT). Treatment with SID decoy peptide downregulated WNT/β-catenin-driven transactivation as measured by decreased promoter H3K4me3 and decreased expression of Wnt target genes like LEF1 and TCF7L2. We also show that SID decoys induce translocation of nuclear β-catenin to the cytoplasm in TNBC at 24 hours. Wnt/β-catenin is critical for EMT, cancer stem cell self-renewal, and early invasion in TNBC. TGIF1, a transcription factor that modulates TGFβ and Wnt signaling pathways and known to to interact with the PAH2 domain of Sin3A, can be dissociated from Sin3A complex by SID decoy treatment as measured by co-immunoprecipitation (Co-IP) and proximity linked assay. DNA microarray of SID peptide treated TNBC cells shows inhibition of TGFβ signaling evidenced by downregulation of MMP9, MT1-MMP and PLAU, known target genes of this pathway. This is in line with inhibition of the EMT program predicted by the IPA analysis in SID peptide treated TNBC. Taken together, the results indicate that SID decoys have potential value as therapeutic agents to revert the EMT program in TNBC that should translate into the inhibition of metastasis dissemination and eradication of residual disease in TNBC. To test this in clinic future investigations will involve the use of our previously identified small molecule mimetic of SID peptide, selamectin that is also a FDA approved drug. Use of a recently constructed cyclic stapled peptide that inhibits PAH-2 binding and invasion at <10nM is also anticipated.

#4116

Anti-tumor and anti-metastasis efficacy of a novel Notch inhibitor (PF-03084014) in hepatocellular carcinoma.

Wu Chuanxing, Wang Xiaoqi. _The University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular Carcinoma (HCC) is well-known for its aggressive growth and highly metastatic potential. Efficacy of current therapy is limited by fast tumor growth and rapid metastasis. Cancer stem cells (CSCs) represent a distinct population that can be prospectively isolated from tumor tissues and have long-term clonal repopulation and self-renewal capacity. They are highly tumorigenic, metastatic, and resistance to conventional radiation and chemotherapy. Therefore, it is essential to identify effective therapy to target CSCs. In our prior study, Notch deregulation was correlated with poor outcome in HCC. In this study, we investigated anti-tumor and anti-metastatic efficacy of a novel Notch inhibitor (PF-03084014) in liver cancer spherical cells in vitro and sphere cell-derived orthotropic model in vivo. PF-03084014 significantly suppressed liver CSCs spheroid formation (derived from 97H HCC cells), with survival rate less than 20% by higher dose of PF-03084014. In accordance, CD90+, EpCAM+ and Oct4+ liver CSC populations significantly decreased, so as down-regulation the stem cell genes expression such as Sox2 and Nanog. These results suggest a suppressive effect of PF-03084014 on liver CSC self-renewal ability. Low dose PF-03084014 induced cancer spheroid cell differentiation leading to chemosensitization. Moreover, Notch inhibitor (PF-03084014) altered liver CSCs stemness properties and blocked spherogenesis of CSCs in vitro, leading to decrease tumorigenicity in vivo. In HCC spherical cell- and non-spherical cell-derived orthotropic model, PF-03084014 effectively inhibited tumor growth, with tumor size was 2570±690 mm3 in vehicle group vs. 800±500 mm3 in Notch inhibitor group (p < 0.001). More importantly, under PF-03084014 treatment, HCC tumor metastasis to lung was significantly reduced (87.5% metastasis rate in vehicle group vs. 18.75% in PF-03084014 group). To explore the potential mechanism, we found PF-03084014 was able to suppress phosphorylation of STAT3 and AKT, the kinases involving in self-renewal of stem cells. PF-03084014 also reversed endothelial mesenchymal transition (EMT) shown by down-regulation of Snail-1 and up-regulation of E-cadherin. Together, PF-03084014 shows a strong anti-cancer and anti-metastasis effect on HCC spherical cell derived in vitro and in vivo model, which provides a preclinical rational of the PF-03084014 to serve as a new novel drug in liver cancer therapy.

#4117

AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system.

Makoto Kinouchi,1 Daisuke Uchida,1 Nobuyuki Kuribayashi,1 Takahiro Wakui,2 Kyoko Kuribayashi,1 Maki Okubo,3 Masahiro Saito,1 Eri Masuyama,1 Hitoshi Kawamata1. 1 _Dokkyo Medical University School of Medicine, Tochigi, Japan;_ 2 _Kamituga General Hospital, Tochigi, Japan;_ 3 _Sanokosei General Hospital, Tochigi, Japan_.

We have demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in metastatic processes in oral cancer. Moreover, we have also reported that blocking CXCR4 with AMD3100, a CXCR4 antagonist, may be a potent anti-metastatic therapy for CXCR4-related head and neck cancer. Recently, AMD070 has been described as a novel orally bioavailable inhibitor of CXCR4, being minimally invasive than AMD3100. In this study, we examined the effect of AMD070 on the SDF-1/CXCR4 dependent-metastases in oral cancer cells, B88-SDF-1, which express high levels of SDF-1 and CXCR4.

We examined the effect of CXCR4-specific antagonists, AMD3100 and AMD070 on the cell growth, cell migration and cell invasion of B88-SDF-1 cells. Both antagonists did not affect the anchorage-dependent growth of B88-SDF-1 cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. Moreover, AMD070 significantly suppressed anchorage-independent growth and invasion of B88-SDF-1 cells. Next we examined the effect of AMD070 to distant metastasis in vivo. Daily administration of AMD070 per os significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results indicated that AMD070 might be a novel orally bioavailable inhibitor on the metastases of oral cancer by the SDF-1/CXCR4 system.

#4118

Honokiol inhibits lung cancer brain metastasis through inhibition of STAT3 signaling pathway.

Yongik Lee, Jing Pan, Qi Zhang, Yian Wang, Ming You. _MEDICAL COLLEGE OF WISCONSIN, milwaukee, WI_.

Lung cancer is the leading cause of cancer death in the United States, and metastasis to lymph-node (LN) and distal organs especially brain leads to sever complications and are the major cause of death. Preventing lung cancer development and metastases is important strategy to reduce lung cancer mortality. Honokiol (HNK), a natural compound present in magnolia bark extracts, has a good bioavailability profile and recently has also been shown to readily cross both the blood-brain barrier (BBB). We previously demonstrated that HNK can prevent and inhibit squamous lung cancer cell carcinoma both in-vitro and in-vivo. In the current study, we evaluated the anti-metastatic effects of HNK in both LN and brain murine models of lung tumor metastasis. The H2030-BrM3 (BR-brain seeking) lung tumor cells stably transfected with luciferase and GFP were orthotopically injected into lung for LN metastases, or directly to the left ventricle of mouse heart for brain metastases, LN or brain metastasis was monitored using a non-invasive IVIS bio-luminescent imaging system. To test the efficacy of HNK in preventing tumor cells migrating to LN or brain, mice were given 10 mg/kg per body weight HNK by aerosol (for lung to LN metastasis model) or oral gavage starting the day after tumor cell injection, our results reveal that HNK significantly prevented the metastasis of lung cancer cells to LN and brain. Our results demonstrate that HNK decreased LN metastases incidence to 30% compare to 100% in control mice, and inhibited brain metastasis near 70% compare to control. Furthermore, analysis of HNK's mechanism of action by utilizing both RNA sequencing and receptor tyrosine kinase assay suggests that its effect is primarily mediated by inhibiting STAT3 pathway. HNK specifically inhibits STAT3 phosphorylation, and knock-down STAT3 totally abrogated the anti-metastasis effects of HNK in brain metastatic lung cancer cell lines, PC9-BrM3 and H2030-BrM3. These finding suggest that HNK could provide novel chemopreventive or therapeutic options for preventing both lung tumor progression and lung cancer brain metastasis.

#4119

Inhibition of PORCN prolongs metastasis free survival in a mouse model of Ewing sarcoma.

Masanori Hayashi, Alissa C. Baker, Seth D. Goldstein, Catherine M. Albert, Kyle W. Jackson, David M. Loeb. _Johns Hopkins University, Baltimore, MD_.

Metastatic disease is the most important factor in determining the survival of patients with Ewing sarcoma (ES). Although intensive cytotoxic chemotherapy has improved the survival of patients with localized disease, the survival rate for patients with metastatic disease remains dismal, and the most pressing unmet need in ES is to prevent and treat metastasis better. The Wnt signaling pathway is a key regulator of a number of cellular functions associated with metastasis, including proliferation, motility, and stem cell self-renewal. A single enzyme, Porcupine (Porcn), mediates post-translational palmitoylation of Wnt ligands, and it's activity is required for secretion of all 17 Wnt ligands, making it an attractive target for a pan-Wnt inhibitor. We have studied the effect of LGK974, a potent, selective, Porcn inhibitor, on Ewing sarcoma metastasis. In vitro, we have observed that LGK974 does not affect proliferation or sarcosphere formation, but inhibits migration of ES cell lines through the suppression of expression of multiple transcriptional regulators of Epithelial-Mesenchymal Transition (EMT). In our orthotopic implantation/amputation mouse model, single agent LGK974 treatment leads to significant delay in formation of lung metastasis following amputation of the affected leg, without a significant effect on primary tumor growth. This delay leads to a significant post-amputation survival benefit in multiple xenografts. In LGK974 treated xenografts, the expression of multiple EMT related transcription factors were observed to be suppressed. Furthermore, in tail vein injection models, LGK974 did not produce a survival benefit, suggesting that LGK974 effects Ewing sarcoma metastasis by inhibiting early steps in the metastatic cascade, such as migration and invasion. Our findings strongly implicate Wnt signaling in the early steps of ES metastasis and demonstrate that LGK974 has the potential to significantly improve the survival of ES patients through the specific inhibition of metastasis.

#4120

c-Met inhibition blocks MEK-induced tumor cell invasion in uveal melanoma.

Oliver Surriga,1 Bryan D. Smith,2 Grazia Ambrosini,1 Daniel L. Flynn,2 Richard D. Carvajal,1 Gary K. Schwartz1. 1 _Columbia University Medical Center, New York, NY;_ 2 _Deciphera Pharmaceuticals LLC, Cambridge, MA_.

Uveal melanoma is the most common primary intraocular malignant tumor in adults. About half of patients with uveal melanoma will develop metastatic disease to the liver and the lung. These tumors are characterized by mutations in G-proteins (GNAQ and GNA11), activation of MAPK, and over-expression of c-Met. We have previously reported that c-Met inhibition inhibits tumor cell invasion and metastasis formation in uveal melanoma. Altiratinib is a selective inhibitor for c-Met as well as TIE2, VEGFR2 and TRK kinases. We found that this agent had no effect on inhibiting cell growth, consistent with our previous findings with crizotinib. However, it did inhibit invasion of uveal melanoma cells through matrigel in a concentration dependent fashion (25 nM to 250 nM) with inhibition of phospho-Met noted at concentrations as low as 25 nM. Selumetinib, a MEK inhibitor, is currently in clinical trial in patients with this disease. We found that treatment with 250 nM selumetinib inhibited cell proliferation but unexpectedly induced a marked increase in cell invasion of GNAQ and GNA11 mutant cell lines. This was associated with an increase in c-Met RNA and protein expression, as well as receptor phosphorylation, after 24 hours of selumetinib treatment. Combining selumetinib with altiratinib inhibited cell invasion to the level of altiratinib alone and continued to inhibit cell proliferation to the level of selumetinib alone. This effect was recapitulated by the knockdown of c-Met by siRNA prior to treatment with selumetinib. In a uveal melanoma xenograft model, the combination treatment of 15 mg/kg altiratinib and 25 mg/kg selumetinib significantly delayed tumor growth compared to vehicle control, altiratinib and selumetinib alone. Western blot analysis of tumor tissue confirmed target inhibition of p-Met and p-ERK in animals treated with altiratinib and selumetinib, respectively. Furthermore, tumor metastasis was inhibited by altiratinib, selumetinib and combination treatments in a uveal melanoma mouse model. These results indicate that the combined inhibition of MEK and c-Met by selumetinib and altiratinib, respectively, may be sufficient to suppress uveal melanoma tumor growth and metastasis. This strategy can potentially be used as therapy for patients with primary uveal melanoma who are at high risk for the development of metastatic disease.

#4121

Chaperonin containing-TCP-1 protein level in breast cancer cells predicts therapeutic application of a cytotoxic peptide.

Annette R. Khaled,1 Amr S. Khaled,2 Rania Bassiouni,1 Priya Vishnubhotla2. 1 _University of Central Florida, Orlando, FL;_ 2 _Orlando VA Medical Center, Orlando, FL_.

Metastatic disease is a principal cause of death from breast cancer. This is due in part to the development of resistance to current therapeutics and the often debilitating side effects that impair quality of life. The challenge is to therapeutically target an essential physiological function of cancer cells not found in normal, non-transformed cells. To this end, we discovered CT20p, a therapeutic peptide that causes cancer-specific death in human breast tumor cells and tumor regression in xenograft models of breast cancer. Using a proteomics approach, we found that CT20p directly binds to multiple subunits of a type II chaperonin called chaperonin containing T-complex or CCT. CCT forms a large macromolecular complex composed of 8 subunits. Inhibition of CCT by CT20p depletes the pool of native actin and tubulin (obligate clients of CCT), impairing the polymerization of cytoskeletal elements needed to support cell adhesion and motility. As a result cancer cells lose the ability to migrate and die from loss of substrate survival signals. We found that expression levels of CCT varied among different triple negative breast cancer (TNBC) cell lines, with the highest expression occurring in those of the mesenchymal stem-like (MSL) subtype with metastatic potential. Lowest levels of CCT were found in normal breast epithelial cells. Sensitivity to killing by CT20p thus correlated with levels of CCT, with cancer cells expressing high amounts of CCT being the most susceptible. Using tissue microarrays (TMAs) of breast cancer progression, we developed an immunohistochemistry staining procedure for CCT. Results were interpreted on a scale of 1 to 4 (with 4 being the strongest staining). CCT expression was statistically higher in invasive ductal carcinoma (IDC) as compared to normal and cancer adjacent tissue (CAT) (p<0.0001). Within the types of IDC, CCT was highest in tumors that exceeded 5 cm across (T3), grew in chest wall or skin and in inflammatory breast cancer (T4) (p<0.05). Examining CCT levels in different molecular types showed little correlation with estrogen receptor (ER) positivity but strong correlation with ER and progesterone receptor (PR) positivity or PR alone (p>0.001). Statistical correlations were also observed with Her2 positivity (p>0.05). However, no statistically significant correlations were observed with TNBC tissues, with CCT staining ranging from the strongest staining (4) to lowest (1). These results are similar to that observed with the TNBC cell lines and indicate that CCT expression may reflect the heterogeneity of TNBC. These results suggest that CCT is a promising target for therapeutic intervention due to its increased expression in advanced stage breast cancer, independence of molecular identity and dependence by cancer cells to support essential cytoskeletal changes associated with the metastatic stem-like phenotype.

#4122

Ceramide is a key factor that regulates the crosstalk between TGF-ß and sonic hedgehog signaling at the basal cilia to control cell migration and tumor metastasis.

Salih Gencer, Natalia Oleinik, Mohammed Dany, Besim Ogretmen. _Medical University of South Carolina, Charleston, SC_.

Recent studies indicate that ceramide species with different fatty-acid chain lengths play diverse biological functions in various cellular processes, highlighting the importance of ceramide synthases (CerS) in these processes. Migration and cell mobility, a part of these processes, also are effected by ceramide metabolism. However, the molecular mechanism of CerS/and ceramide involved is unknown. Here, we investigated the effect of CerS/and ceramide on migration and its related signal pathways in situ and in vivo model. Interestingly, our data show that among CerS only CerS4/ceramide is involved to cell migration and tumor metastasis. Here, we also have generated CerS4-/- mice for in vivo studies. Interestingly, we observed that genetically loss of CerS4 resulted in severe irreversible alopecia, which was associated with hyper-proliferation and migration of keratinocytes. Mechanistically, we show here that genetic loss or shRNA-mediated knockdown of CerS4 enhances cell migration by which ligand-independent signaling of TGF-beta receptors I and II in various cell types, including keratinocytes, mouse embryonic fibroblasts and cancer cells. Moreover, we found that ceramide directly interact with Smad7 and this interaction was decreased by shRNA-mediated knockdown of CerS4. Thus, ceramide-Smad7 binding modulates plasma membrane association of TGF-ßR1 at primary basal cilia, and inhibits its signaling through Sonic-Hedgehog (Shh) for migration. Furthermore, Ceramide accumulation at the primary basal cilia was decreased by knockdown of CerS4. Thus, these data revealed that CerS4/ceramide signaling plays key roles in the regulation of cell migration and metastasis via controlling the TGF-ßR and Shh axis at primary basal cilia.

#4123

Thymoquinone inhibits bone metastasis in a breast cancer mouse model by modulating CXCR4/CXCL12 signaling axis.

Muthu K. Shanmugam,1 Annie Hsu,1 Kam Man Hui,2 Benny K.H. Tan,1 Gautam Sethi1. 1 _National University of Singapore, Singapore, Singapore;_ 2 _National Cancer Center, Singapore, Singapore_.

Several lines of evidence(s) indicate that CXCR4 overexpression has been correlated with distant site metastasis and poor overall survival rate in patient with breast cancer. The tumor metastasis promoting molecule CXCR4 is considered as a potential therapeutic target for inhibiting breast cancer metastasis. Thus, novel agents that can down-regulate CXCR4 expression have potential against breast cancer metastasis. In the present report we investigated the effect of thymoquinone (TQ), derived from the seeds of medicinal plant Nigella sativa, on the expression and regulation of CXCR4 in breast cancer cells. In addition, we evaluated the effect of TQ in a metastasis mouse model established by intracardiac injection of luciferase tagged MDA-MB-231 breast cancer cells that metastasize to the bones. We observed that TQ could inhibit the expression of CXCR4 in MCF-7 and MDA-MB-231 cells in a dose and time dependent manner. TQ (2 mg/kg or 4 mg/kg) treatment for four weeks significantly inhibited tumor growth and significantly reduced metastasis to multiple vital organs, including lungs, brain and bone. Immuno-histochemical analysis of the lung and brain tissue showed significant reduction in CXCR4, Ki67, MMP9, VEGFR2 and COX2 expression compared to control tissues. TQ treatment also reduced the overall bone tumor burden. Overall, our results show that TQ exerts its antitumor and anti-metastatic effects by downregulation of CXCR4 expression both in vitro and in vivo thus may have possible potential for the treatment of breast cancer.

Acknowledgement: This work was supported by NUHS Basic Seed Research grant to Prof. Benny Tan.

#4124

Sub-cytotoxic concentrations of a novel polyphenol fatty acid ester derivative arrest breast cancer cell proliferation and metastasis.

Wasundara Fernando,1 Melanie R. Power Coombs,1 David W. Hoskin,1 H. P. Vasantha Rupasinghe2. 1 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 2 _Dalhousie University, Truro, Nova Scotia, Canada_.

In this study, the antiproliferative and antimetastatic properties of phloridzin docosahexaenoate (PZ-DHA), a novel polyphenol fatty acid ester derivative, were explored using in vitro and in vivo models of mammary carcinoma. PZ-DHA combines phloridzin (PZ, a dihydrochalcone found in apple peels) with docosahexaenoic acid (DHA, an omega-3 fatty acid). The regioselective acylation reaction was catalyzed by lipase B enzyme from Candida antarctica. PZ-DHA-induced selective cytotoxicity towards breast cancer cells (triple-negative mammary carcinoma cells, MDA-MB-231, MDA-MB-468, and 4T1; estrogen receptor-positive mammary carcinoma cells, MCF-7 and T-47D) was observed compared to human mammary epithelial cells (HMECs and MCF 10A) using the MTS assay. Significantly less (p<0.05) cytotoxicity of PZ-DHA for normal cells was also confirmed using flow cytometric analysis of annexin-V-FLUOS/propidium iodide-stained MCF 10A and human dermal fibroblasts compared to MDA-MB-231 cells. Flow cytometric analysis of Oregon Green 488-stained MDA-MB-231 cells showed an antiproliferative effect of PZ-DHA at sub-cytotoxic concentrations (10 to 30 µM). Cell cycle analysis showed that MDA-MB-231 replication was arrested at the G2/M phase of the cell cycle following treatment with PZ-DHA at sub-cytotoxic concentrations. PZ-DHA suppressed the migration of MDA-MB-231 and 4T1 cells in vitro in wound healing and cell migration assays. Reduced expression of proteins (β-catenin, slug, Zinc finger E-box-binding homeobox 1, vimentin and matrix metalloproteinase-2) involved in the epithelial-to-mesenchymal transition was demonstrated by western blot analysis of PZ-DHA-treated MDA-MB-231 cell lysates. Finally, 4T1 tumor bearing-female BALB/c mice that received intra-peritoneal injections of PZ-DHA showed a significant reduction (p<0.05, n=9) in primary tumor volume at the mammary fat pad and fewer metastatic lesions in the lungs compared to the saline-treated control mice, suggesting an in vivo antimetastatic effect of PZ-DHA. In conclusion, findings of this study reveal that PZ-DHA suppresses mammary carcinoma cell proliferation and metastasis, suggesting a potential clinical application to prevent breast cancer progression in patients.

#4125

Targeting metastasis and cancer stem-like cells in triple negative breast cancer through inhibition of focal adhesion kinase.

Samuel Jones, Christopher Smith, Stephen Hiscox, Wen G. Jiang. _Cardiff University, Cardiff, United Kingdom_.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer closely associated with an early pattern of metastasis and lack of targeted therapies. As such the prognosis for TNBC is poor. Whilst targeting signalling pathways known to promote metastasis-supporting behaviours may well provide an important therapeutic opportunity for such cancers, recent evidence points to an association between cancer stem-like cell (CSC) populations and the aggressive behaviours of TNBC, including their chemoresistance.

Aberrant activity and/or expression of the non-receptor tyrosine kinase FAK (focal adhesion kinase) is known to contribute to the development and progression of cancer due to its central role as a mediator of cell attachment, migration and survival. More recently, FAK has been suggested to influence anoikis resistance, thus implying its activity may contribute to the phenotype of CSCs. Here we have explored the role of FAK in TNBC cells as a putative therapeutic target to suppress the metastatic phenotype.

FAK activity was inhibited in MDA-MB-231 (TNBC) and MCF7 (luminal breast cancer) cells pharmacologically using PF-562,271 (PF271), a competitive-inhibitor of the ATP-kinase domain or through siRNA-mediated knockdown to probe scaffolding function of FAK. Functional assessment of TNBC proliferation, migration and invasion was performed using cell counting and Boyden chamber assays respectively, whilst stem-like behaviours (anchorage-independent growth, self-renewal) were investigated using stem-cell enrichment (mammosphere-based assays). The surface marker profile of CSCs was investigated using FACS, while changes in intracellular signalling pathways in response to FAK inhibition was revealed through Western blotting and immunofluorescent microscopy.

MDA-MB-231 and MCF7 cells displayed comparable levels of total and activated (Y397/Y861) FAK which were depleted in response to PF271 in a dose dependent manner: however, TNBC cells were significantly more sensitive. Cellular migration in TNBC cell models occurred in a FAK-dependent manner with both PF271 and FAK siRNA suppressing endogenous and fibroblast-stimulated migration/invasion (p<0.001 versus vehicle control). Inhibition of FAK also caused a significant decrease in the growth rate of TNBC cells. FACS analysis of MDA-MB-231 cell cultures revealed a significant subpopulation of cells exhibiting the CSC surface marker profile CD24-/CD44+. Moreover, TNBC cells were able to form self-renewing mammospheres in non-adherent culture. PF271 treatment or siRNA-mediated knockdown of FAK significantly attenuated mammosphere-forming ability and self-renewal of these cultures.

These data point to a role for FAK as a mediator of metastatic behaviour in TNBC cells, whilst also showing the capacity to influence self-renewal of a CSC population. FAK may therefore represent an emerging therapeutic target in such cases.

#4126

An anti-CD4 antibody protects mice from anti-PD-1/PDL-1 antibody-induced fatal anaphylaxis and shows potent anti-metastatic activity in the 4T1 spontaneous metastasis model.

Satoru Ito,1 Shoji Yokochi,1 Yoshiro Ishiwata,1 Satoshi Ueha,2 Francis Shand,2 Kouji Matsushima2. 1 _IDAC Theranostics Inc, Tokyo, Japan;_ 2 _The University of Tokyo, Tokyo, Japan_.

[Introduction]

We previously reported that an α-CD4 depleting monoclonal antibody (mAb) has very potent anti-tumor effects in several murine tumor models, particularly when combined with α-PD-1/L1 mAbs (Cancer Immunol. Res. 3: 631-640; 2015). Here we examined the effect of this α-CD4 mAb on 4T1 murine mammary tumors, which undergo spontaneous metastasis from an orthotopically transplanted site to the lung.

[Methods]

4T1 tumor cells (1 x 105) were injected into the mammary fat pad of female BALB/c mice and primary tumor volume was measured every 2-3 days. Spontaneous lung metastasis was quantitated by removing the lungs, fixing them in Bouin's fixative, and counting the number of metastatic nodules on the surface of lung.

[Results]

Administration of α-CD4 mAb (clone GK1.5; days 5 and 9 after tumor inoculation) significantly inhibited 4T1 lung metastasis (n=10) without affecting primary tumor size. α-PD-1 and α-PD-L1 mAbs had minimal effects on primary tumor growth and metastasis. However, administration of α-PD-1 or α-PD-L1 mAbs triggered an IgG1-mediated fatal anaphylactic reaction upon the third or fourth administration. Interestingly, the anaphylactic reaction was completely prevented by co-administration of the α-CD4 mAb.

[Discussion]

Based on these results, α-CD4 mAb may be useful for preventing metastasis associated with advanced cancer. In addition, α-CD4 mAb appeared to be protective against the IgG-mediated anaphylactic reaction and autoimmune-like side effects that are associated with α-PD-1/α-PD-L antibody administration-induced enhanced B-cell immunity. Increased myelopoiesis due to high G-CSF production by 4T1 cells may have contributed to the anaphylactic reaction.

#4127

Blockade of TGFβ signaling enhances anticancer effect of chemotherapeutics in gynecologic cancers.

Haiyan Zhu, Junhua Yang, Xiang Gu, Luzhe Sun. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Chemoresistance is a significant clinical challenge in the management of gynecologic malignancies. Previous studies have showed that TGF-β signaling pathway can mediate chemotherapy resistance. However, the underlying mechanisms of how TGF-β pathway is activated and causes chemoresistance are not well understood. Here, we found that ovarian cancer patients displayed an increased RNA transcripts of genes associated with TGF-β signaling after chemotherapy. In vitro, treatment with four commonly used drugs for gynecologic malignancies, including cisplatin, doxorubicin, paclitaxel and camptothecin, activated TGFβ signaling by stimulating phosphorylation of Smad2 and Smad3, two intracellular mediators of TGFβ signaling pathway in ovarian and cervical cancer cell lines. Furthermore, cisplatin and doxorubicin can induced epithelial-mesenchymal transition (EMT) and promote migration. Blockade of TGFβ signaling abrogated chemotherapeutics-stimulated Smad phosphorylation, epithelial-mesenchymal transition, and migration. These observations suggest that chemotherapy-mediated activation of TGFβ signaling may be a mechanism of chemoresistance, and that TGFβ pathway inhibitor may prevent the development of chemoresistant. We are currently testing the anti-tumor efficacy of the combination therapy with a novel pan-TGFβ inhibitor and cisplatin in a mouse xenograft model of ovarian cancer and whether the treatment with TGFβ inhibitor can alleviate symptoms of nephrotoxicity, which is known to be caused by cisplatin. Our studies may reveal new strategies for the treatment of gynecologic malignancies.

#4128

Anti-angiogenic and anti-metastatic activities of juglone in pancreatic cancer cells.

Namrata Karki,1 Frank Greenway,2 William Hansel,2 Sita Aggarwal,2 Jack N. Losso1. 1 _Louisiana State University, Baton Rouge, LA;_ 2 _Pennington Biomedical Research Center, Baton Rouge, LA_.

Pancreatic cancer is the fourth leading cause of cancer related deaths in the US. The late diagnosis, early metastasis and poor survival rate make this disease lethal. This current study was designed to examine the effect of juglone, 5 hydroxy-1, 4-naphthoquinone in modulating the angiogenic and metastatic potential of pancreatic cancer cells in vitro, using the MIA Paca-2 human pancreatic cancer cell line. Juglone is a natural quinoid abundantly found abundantly in plants of Juglandacea family. To understand the anti-angiogenic and anti-metastatic properties of juglone, expression of markers related to angiogenesis were measured by Western blot or ELISA and the effect on ability of cancer cells to migrate and invade after treatment was analyzed by transwell invasion and migration assay, wound healing assay, and in vitro tube formation assay in human umbilical vein endothelial cells. The Akt pathway is highly upregulated and is responsible for the regulation of various biological processes in pancreatic cancer cells including proliferation, growth, survival and angiogenesis. Akt activation is also associated with regulation of key angiogenic markers such as, VEGF (Vascular Endothelial Growth Factor) and HIF-1 (Hypoxia Inducible Factor). Upon treatment of pancreatic cancer cells with juglone, reduced expression of serine-phosphorylated Akt expression was observed. Juglone inhibited metastasis of pancreatic cancer cells as indicated by the results of cell migration, invasion and wound healing assay. Juglone treatment also downregulated the levels of VEGF and HIF-1α. Significantly, it was found that juglone inhibited pancreatic cancer cell motility, migratory and invading capabilities. These findings suggest that Akt inhibitors such as juglone possess ability to attenuate the aggressiveness of pancreatic cancer cells and can be used as a novel anti-cancer agent to prevent metastasis of highly invasive pancreatic adenocarcinomas.

#4129

Genomic targets of BAP1 in uveal melanoma.

Michael D. Onken, Matthew Yen, John A. Cooper. _Washington University School of Medicine, St. Louis, MO_.

Uveal melanomas, cancers arising from the pigmented uveal layers of the eye, are highly metastatic despite the eye's lack of lymphatics and anatomical barriers that make local spread rare. Almost half of patients develop distant metastatic disease, most often to the liver, even after the tumor-bearing eye is completely removed. Uveal melanoma tumors with loss of function mutations in the histone deubiquitinase BAP1 lead to deadly metastatic disease, whereas those with normal BAP1 rarely metastasize. Despite this clear clinical link, the cellular mechanisms by which BAP1 control metastatic behavior are still poorly understood. We have identified the genomic targets of BAP1 in uveal melanoma, using BAP1 fused to a transposase that inserts engineered transposons into genomic regions visited by BAP1. This "calling card" technique results in a spatial and temporal map of transcription factor activity that is more dynamic and sensitive than fixation-based techniques like ChIP-seq. Among the genes targeted by BAP1, we have identified regulators of differentiation, cell cycle progression, cell adhesion, and cell motility. We have used the CRISPR genomic editing system to develop BAP1-null uveal melanoma cell lines. We are knocking down our BAP1 genomic target genes in BAP1-null and BAP1-wildtype cell lines, and we are testing for compensation of the effects of loss of BAP1 function on growth, morphology, and invasion. Transendothelial migration is a key step in hematogenous metastasis regulated by BAP1. Using a novel assay that we developed, we model the blood vessel wall using primary human microvascular endothelial monolayers grown on polyacrylamide substrates that mimic the physiological stiffness of normal tissue. To these monolayers, we add uveal melanoma cells, and then collected time-lapse movies using spinning-disc confocal microscopy to follow the transmigration of the uveal melanoma cells. We have shown previously that BAP1-deficient uveal melanoma cells transmigrate through endothelial monolayers more quickly than BAP1-wildetype cells, and we are using this assay to identify the BAP1 genomic target genes that are involved in transendothelial migration. Using these techniques, we are characterizing the cellular function of BAP1 as a metastasis suppressor, and connecting BAP1 to specific metastatic pathways that can be targeted therapeutically.

#4130

A novel tumor-suppressor role for squalene epoxidase in human colorectal metastasis.

Soo Young Jun, Hyun-Soo Cho, Jeong-Ju Lee, Ji-Yong Yoon, Jun-Ho Ahn, Ju-Sik Min, Sujin Jeon, Jae-Hye Lee, Min Hyuk Choi, Nam-Soon Kim. _Korea Research Institue of Bioscience and Biotechnology/UST, Daejeon, Republic of Korea_.

Malignancy cancer cells are reported to develop mechanisms to resist anoikis (apoptotic cell death from deprivation of cell-cell/matrix interactions), thereby enhancing the survival of cancer cells and secondary tumor formation in distant organs, which is responsible for 90% of cancer-related fatalities. A high serum level of cholesterol (hypercholesterolemia) in patients with colorectal cancers (CRCs) metastasis is reported, but the underlying relationship were never elucidated. Human CRC tissues revealed that the expression of squalene epoxidase (SqE), the rate-limiting enzyme in cholesterol synthesis, is significantly reduced in the advanced stages of CRC. Here we report that the knockdown/degradation of SqE by either the application of small-interfering RNA against SqE or a high-level of cholesterol significantly potentiates CRC cell survival/metastasis via the induction of an epithelial-mesenchymal transition as well as of anoikis resistance. We identified the interactions of SqE with glycogen synthase kinase 3β (GSK3β) and/or p53: SqE knockdown induced the dissociation of GSK3β and p53, thereby resulting in the downregulation of E-cadherin via the inhibition of GSK3 activity and of -catenin degradation, as well as the p53 degradation by the hdm2 upregulation. Overall, the present study demonstrates SqE is a key negative regulator of cancer cell survival and of CRC metastasis.

#4131

Blocking endogenous TBX2 abrogates prostate cancer bone metastasis through WNT signaling.

Srinivas Nandana,1 Manisha Tripathi,1 Peng Duan,1 Gina Chu,1 Haiyen E. Zhau,1 Robert J. Matusik,2 Leland W.K. Chung1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Vanderbilt University, Nashville, TN_.

Bone metastasis is a crucial turning point in the progression of PCa and is seen in > 90% of the lethal cases. Identifying factors that mediate the metastatic spread and subsequent remodeling of the bone is highly relevant to successful therapeutic intervention in advanced human prostate cancer (PCa). TBX2, a transcription factor that belongs to the T-box family negatively regulates p21 cell cycle inhibitor and is known to play critical roles during embryonic development and cancer progression. Interestingly, a recent report found TBX2 over-expression in the bone metastases from patients with castrate resistant prostate cancer. We found TBX2 over-expression in human prostate cancer specimens and in aggressive androgen independent human PCa cell lines. Further, we found TBX2 over-expression in bone metastases in multiple xenograft mouse models of human PCa progression. Blocking endogenous TBX2 in PC3 and ARCaPM PCa cells through the use of a dominant negative construct resulted in decreased proliferation, colony formation and invasion in vitro. Utilizing various pre-clinical xenograft mouse models, we found that blocking endogenous TBX2 in PCa cells leads to decreased invasion and abrogation of bone and soft tissue metastasis in vivo. Further, utilizing the intra-tibial mouse model of growth in the bone microenvironment, we found that blocking endogenous TBX2 in PCa cells dramatically reduces bone remodeling. Additionally we found that TBX2 acts upstream of the canonical WNT (WNT3A) signaling in PCa and positively regulates its transcription by binding to its promoter. Further, rescue of WNT3A levels in PCa cells in the context of blocking endogenous TBX2 rescues the metastatic ability of the cells in vivo. Taken together, our findings highlight a novel and critical role of TBX2 in PCa progression, metastasis and the subsequent bone remodeling, and that TBX2 function in PCa is mediated through the canonical WNT signaling.

#4132

Multiple drug resistance-associated protein 4 (MRP4): Role in triple negative breast cancer.

Tyler Kochel, Xinrong Ma, Namita Kundu, Jocelyn Reader, Olga Goloubeva, Amy M. Fulton. _University of Maryland Greenebaum Cancer Center, Baltimore, MD_.

Introduction: MRP4 is responsible for the active export of prostaglandin E2 (PGE2) from cells. PGE2, derived from cyclooxygenase-2-mediated synthesis, contributes to tumor growth and metastasis of many epithelial tumors; however, the role of MRP4 in breast cancer has not been investigated. Methods: We employed publicly available breast cancer gene expression datasets and a panel of breast cancer cell lines to examine the expression and function of MRP4 as well as other members of the PGE2 pathway. In contrast to the export function of MRP4, the prostaglandin transporter PGT/SLCO2A1 imports PGE2 into the cell where 15-prostaglandin dehydrogenase (5-PGDH/HPGD) metabolizes PGE2 to terminate ligand-mediated activation of the prostaglandin E (EP) receptors. Results: We found that ABCC4, which encodes the MRP4 protein, is widely expressed in breast cancer but breast cancer subtypes have different expression profiles of PGE2 pathway members. ABCC4/MRP4 is elevated more frequently in basal breast tumors than in apocrine or luminal tumors. This is the first report that elevated expression of MRP4 is found in breast cancer subtypes (basal, HER2-enriched) with the worst prognoses. ABCC4 expression was also higher in breast cancer-associated stroma compared to healthy mammary tissue. Basal-type tumors also expressed the highest levels of PTGS2/COX-2 while the genes responsible for PGE2 metabolism (HPGD and SLCO2A1) are negatively correlated with TNBC. The same pattern was observed in breast cancer cell lines, where basal-type and HER2-enriched cell lines highly express MRP4 but luminal breast cancers do not. HPGD was decreased in most cancer cell lines compared to MCF10A immortalized cells, consistent with the known tumor suppressor role of HPGD/15PGDH. MRP4 exports PGE2 which was reduced in the presence of MRP4 small m.w. inhibitors or by MRP4 gene silencing. Lastly, expression of shMRP4 in highly metastatic, basal-type MDA-MB-231 human breast cancer cells reduced the ability of these cells to spontaneously metastasize to the lungs of NOD/SCID mice but did not affect the tumor growth properties of tumor cells implanted proximal to the mammary gland. Conclusions: Elevated MRP4 is a characteristic of aggressive breast cancer subtypes and is partially responsible for the high levels of PGE2 in the tumor microenvironment. Silencing of MRP4 reduces spontaneous metastasis in a xenograft model of TNBC. Further investigation of MRP4 as a potential therapeutic target is warranted.

#4133

Suppressed B7-H3 expression reduces glycolytic capacity and sensitizes tumor cells to anti-cancer agents.

Caroline E. Nunes-Xavier,1 Karine Flem Karlsen,1 Tove Øyjord,1 Ming Tan,2 Øystein Fodstad1. 1 _Institute for Cancer Research, Oslo, Norway;_ 2 _Center for Cell Death and Metabolism, Mitchell Cancer Institute, University of South Alabama, Mobile, AL_.

B7 family proteins are important immune response regulators, and can mediate oncogenicity-related signals and support cancer development. We have prevously shown that the B7-H3 protein promotes tumor cell invasion, proliferation and mestastasis, and increases resistance to chemotherapy. Now we have used human breast and melanoma cancer cell lines with different expression levels of B7-H3 to further evaluate the role of B7-H3 on the sensitivity to anticancer compounds. B7-H3 knockdown tumor cells showed enhanced chemosensitivity compared to their B7-H3 expressing counterparts, as well as increased inhibition to treatment with compounds that target proteins in the PI3K/AKT/mTOR- and MAPK-pathways (Nunes-Xavier et al. in revision). Treatment of control cells with an anti-B7-H3 monoclonal antibody resulted in similar sensitization, whereas B7-H3 overexpressing cells and xenografts were less sensitive. Of note, knockdown of B7-H3 decreased and B7-H3 overexpression increased the glycolytic capacity of the breast cancer and malignant melanoma cells. In conclusion, we have demonstrated a previously unknown relationship between B7-H3 expression and glycolysis in tumor cells, and found that B7-H3 confers resistance to also to pathway inhibiting agents. The results provide novel insights into the function of B7-H3 in cancer, and suggest that targeting of B7-H3 expression may be a promising alternative to improve current anticancer therapy. 

### Immune Cell Activity

#4134

Immune infiltration and PD-L1 expression in the tumor microenvironment are prognostic in osteosarcoma.

Pratistha Koirala,1 Michael Roth,2 Jonathan Gill,2 Jordan Chinai,1 Sajida Piperdi,1 David Geller,3 Bang Hoang,3 Vincent Poon,4 Xingxing Zang,1 Richard Gorlick1. 1 _Albert Einstein College of Medicine, Bronx, NY;_ 2 _Children's Hospital at Montefiore, Bronx, NY;_ 3 _Montefiore Medical Center, Bronx, NY;_ 4 _University of British Columbia Faculty of Medicine, Vancouver, British Columbia, Canada_.

Purpose: Over the past four decades, osteosarcoma (OS) survival rates have remained stagnant. There is a need to identify novel therapies to target OS. In this study we examined the expression of Programed Death Ligand 1 (PD-L1) and defined the tumor microenvironment in OS in order to assess the feasibility of utilizing immune checkpoint inhibitors as a treatment modality.

Experimental Design: PD-L1 expression in OS was examined in two patient cohorts using immunohistochemistry (IHC) (n=48, n=59) and expression was validated using quantitative real time PCR (qRT-PCR) (n= 21) and western blotting (n=9). IHC was used to determine presence of tumor infiltrating lymphocytes and antigen presenting cells (APCs) in the tumor. Expression of PD-L1 was correlated with the presence of immune cells and with event free survival in OS.

Results: PD-L1 is expressed in up to 25% of primary OS tumors. Of the PD-L1 positive tumors the majority were also PD-1 positive (92% vs 8%, p=0.002). In addition, the presence of immune cells in the tumor mass was significantly associated with PD-L1 expression. Although all immune cell types examined were present in OS, only infiltration by APCs, specifically CD1a positive dendritic cells (48.6% vs. 51.4%, p=0.0010) and CD68 positive macrophages (70.3% vs. 29.7%, p=0.0316), was associated with worse outcomes. PD-L1 expression was significantly associated with worsened survival (21.6% pos. vs. 78.4% neg., p=0.0146).

Conclusions: For the first time we have identified PD-L1 expression or presence of CD1a or CD68 positive antigen presenting cells as prognostic markers for worsened outcome in OS. Furthermore, we show that IHC is a valid detection method for PD-L1 in OS. With up to 25% of OS patients expressing PD-L1, this study provides rational for targeting the PD-L1:PD-1 axis for immunotherapy in OS.

#4135

A signature of rejection in colorectal cancer: immune markers and their epigenetic regulation.

Lena Sokol,1 Viktor H. Koelzer,2 Stefan P. Zahnd,1 Lucine Christe,1 Inti Zlobec,1 Alessandro Lugli1. 1 _University of Bern, Bern, Switzerland;_ 2 _Kantonspital Liestal, Liestal, Switzerland_.

Background

The immune system plays a pivotal role in the development and progression of colorectal cancer (CRC). Tumor immune rejection has been previously linked to activation of the interferon stimulated genes (ISG) pathway including STAT1, IRF5 and IRF1. Here, we tested the associations of ISG pathway genes with cytotoxic T cell (CTL) infiltration, granzymeB and perforin secretion, tumor expression of intercellular adhesion molecule 1 (ICAM-1), and clinicopathological factors. Further, we investigated the poorly understood regulation of STAT1 and IRF1 by miR-34a and miR-93 using sequential in situ hybridization (ISH) /immunohistochemistry and digital image analysis.

Methods

Resection specimens from 244 CRC patients with full clinicopathological data were included in this study. ngTMA (next generation tissue microarray) slides containing 8 punches of tumor and normal tissue from each case were immunostained for proteins of interest. The expression of the miRNAs was visualized on consecutive slides by ISH. Precise correlation analysis of miRNA expression and the target proteins was performed using digital image analysis.

Results

Active interferon signaling in the tumor microenvironment negatively correlated with the presence of distant metastasis (STAT1: p=0.013, OR=0.98, 95%CI=0.96-0.99; IRF1: p=0.014, OR=0.96, 95%CI=0.940.99; IRF5: p=0.009, OR=0.96, HR=0.93-0.99). High miR-34a and miR-93 expression corresponded to a 22.5 fold decrease of STAT1 and IRF1 (p=0.006, OR=0.39, 95%CI=0.20-0.77; p=0.058, OR: 0.48 95%CI=0.231.02). Finally, a combined score was generated from the three protein markers, representing an immune activated and a "quiescent" phenotype. The activated phenotype was associated with elevated CD8+ CTL infiltration (p=0.007, HR=2.07, 95%CI=1.22-3.52), an increase in granzymeB (p<0.001, HR=3.22, 95%CI=1.70-6.08), perforin (p=0.020, OR=1.90, 95%CI=1.10-3.26) and ICAM-1 expression (p=0.006, OR=0.2.17, 95%CI=1.263.85). Patients with immune activated CRC had a considerably reduced risk of developing distant metastases (p=0.001, OR=0.034, 95%CI=0.006-0.183).

Conclusion

Here, we describe a combined marker phenotype that can help to differentiate between patients with an activated and "quiescent" immune phenotype of CRC. The score is based on a quantitative assessment of STAT1, IRF1 and IRF5 expression by digital image analysis. Increased tumor infiltrating CTLs, granzymeB, perforin and tumor ICAM-1 expression, as well as a lower risk of distant metastases characterize the immune activated phenotype. Targeted inhibition of miR34a and miR-93 may be a promising strategy to activate the anti-tumoral immune response and will be validated in future experimental studies.

#4136

Properties of the immune microenvironment associated with clonal diversity in high-grade serous ovarian cancer.

Allen W. Zhang,1 Andrew McPherson,1 Andrew Roth,1 David R. Kroeger,2 Katy Milne,2 Wyeth W. Wasserman,3 Jessica N. McAlpine,1 Robert A. Holt,1 Brad H. Nelson,2 Sohrab P. Shah1. 1 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _BC Cancer Agency, Victoria, British Columbia, Canada;_ 3 _Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, Vancouver, British Columbia, Canada_.

High-grade serous ovarian cancer (HGSC) is a challenging disease characterized by poor survival and relapse. Our group has revealed extensive clonal diversity of malignant cells in HGSC, which is thought to facilitate the emergence of resistance and recurrence in response to chemotherapy. Moreover, we have shown that the presence of tumor infiltrating lymphocytes (TILs) is associated with longer patient survival in HGSC, suggesting that the immune system can contend with the clonal diversity of tumors in some patients. We sought to determine how TIL abundance and repertoire diversity relates to the degree of tumor clone diversity in HGSC.

We performed whole-genome sequencing, targeted amplicon deep sequencing, gene expression profiling, and T-cell receptor sequencing on 34 spatially separated HGSC tumor samples (8 patients) including ovarian masses and peritoneal foci. In addition, tissue sections were immunohistochemically stained for CD3, CD8, CD20, CD79a, and CD138 to detect major TIL subsets. Using PyClone, we decomposed each tumor sample into its constituent malignant clones and inferred clonal genotypes.

We discovered profound intra- and inter-patient variation in the degree of tumor clone diversity from phylogenetic analysis. Widespread inter-patient variation was also observed in TIL counts and T-cell receptor repertoires. Intraepithelial CD3+ cell counts ranged from 2.2-173.2 per HPF and the number of unique T-cell receptor clonotypes varied from 422 to 3164 per sample. Remarkably, quantitative metrics of tumor clone diversity—as defined by phylogenetic divergence and entropy in the malignant composition of each tumor—was associated with the 'proliferative' molecular subtype and, accordingly, low TIL density. Tumor clone diversity showed no correlation with the number of unique T-cell receptor species. Moreover, preliminary analyses reveal no association between tumor clone diversity and the expression of several ligands and receptors involved in inhibitory immune signaling (CTLA-4, PD-1, PD-L1, PD-L2).

Our results reveal an association between low TIL abundance and high diversity of malignant clones. This suggests that immune infiltration may inhibit tumor proliferation and clonal diversification, or vice versa. Collectively, our findings expose a relationship of the immune microenvironment with the clonal dynamics of malignant cells that could be exploited to overcome therapeutic resistance associated with clonal evolution in HGSC.

#4137

Loss of LKB1 mediates an immune inert phenotype in human lung adenocarcinoma.

Warren L. Denning,1 Ferdinandos Skoulidis,1 Li Shen,1 Vassiliki Papadimitrakopoulou,1 Lixia Diao,1 Yanyan Lou,2 Lauren A. Byers,1 Jing Wang,1 Jaime R. Canales,1 Ignacio I. Wistuba,1 John Weinstein,1 Don L. Gibbons,1 John V. Heymach1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Mayo Clinic, Jacksonville, FL_.

Background: We recently have reported that co-mutations within KRAS-mutated lung adenocarcinoma (LUAD) define a distinct biological phenotype and may offer insights into therapeutic targeting using this classification system. The analysis revealed striking differences in checkpoint ligand expression and T cell infiltration between TP53 and STK11 (LKB1) co-mutated KRAS LUAD. We, therefore, hypothesized that loss of LKB1promotes an immune-suppressed phenotype in KRAS LUAD. To test this, we analyzed 100 immune-related genes from The Cancer Genome Atlas datasets, as well as three other sets of resected tumors from NSLC patients from MD Anderson Cancer Center.

Results: Our results revealed that tumors lacking LKB1 (LKB1-loss), as defined by mutation or gene-expression signature, resulted in a down regulation of: tumor associated immunosuppressive ligands, PD-L1, PD-L2, B7-H3, B7-H4, HVEM; T cell associated immunosuppressive ligands PD-1, CTLA-4, BTLA; co-stimulation ligands such as CD80, -86, -40L, and -40. We further examined if this loss of immunosuppression and co-stimulation resulted in changes in markers of T cell phenotypes. Indeed, tumors lacking LKB1-loss tumors demonstrated a distinct lack absence of several T cell markers including CD3, -4, & -8, but also RORC, T-bet, GATA-3, and FoxP3. To determine if myeloid cell populations, known mediators of immune suppression, were also diminished in LKB1-loss tumors, we analyzed expression of CD11c, -103, -11b, -68, and CD33. Consistent with T cell makers and suppressive ligand markers, myeloid markers were down regulated in LKB1-loss tumors. Because common immune suppression markers were lost, we then examined the expression of antigen presentation (HLA-I, HLA-II) and amino acid transporters as possible alternative methods for immune suppression. In LKB1-loss tumors, both HLA-I and HLA-II markers were down regulated, compared to tumors with intact LKB1 expression. Furthermore, genes associated with the antigen presentation complex, LMP2, TAP1&2, and B2M were also down regulated. In contrast to the immune markers described above, cationic transporters (SLC7A2), anionic transporters (SLC7A11), and neutral transporters (SLC3A2) were universally upregulated in LKB1-loss tumors.

Conclusions: LKB1 loss is associated with an inert immune phenotype. Our data suggests that reduced antigen presentation and amino acid depletion, but not checkpoint factors or MDSCs, are likely mediator of this phenotype.

#4138

Immunological profile of metastatic or recurrent breast cancer patients.

Shigeru Imoto, Takayuki Ueno, Hirotsugu Isaka, Hiroki Ito, Kaisuke Miyamoto, Tomohiro Chiba, Hiroshi Kamma. _Kyorin Univ. School of Medicine, Mitaka, Japan_.

Background: We previously reported about immune suppression in breast cancer patients at this meeting. In brief, tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were analyzed in 50 early or advanced breast cancer (BC) patients. To compare between 38 early and 12 advanced BC cases, CD163-positive tumor cells and CCR4-positive tumor cells were detected more frequently in advanced cases than in early cases. Regulatory T (Treg) cells in PBMC significantly increased in percentage of the population in 37 BC patients than in 21 healthy volunteers (AACR 2011). In addition, several cytokines were examined in that cohort and plasma IL-17A had significantly higher levels in early BC than in advanced BC (AACR 2013). Then, we examined their prognosis and immunological profile.

Patients and methods: Treg cells were examined by counting CD4+CD25highCD127low/-cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, CD163 and CCR4. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/-, ++, +++). Human IL-2, IL-4, IL-6, IL-10, TNF, INFγ and IL-17A were measured using cytometric beads array system. Most patients received chemotherapy, hormonal therapy, and/or anti-HER2 therapy on the basis of intrinsic subtype and breast irradiation after breast-conserving surgery.

Results and discussions: At the median follow-up of 7 years after blood sample collection, only 2 operable patients relapsed and 3 patients including 2 cases of stage IV died of disease. Of 5 cases of stage IV or recurrent BC, CD163 and/or CCR4 were strongly stained positive in tumor cells. There were significant differences of staining intensity in CD163 and CCR4-positive tumor cells between those cases and the rest 45 cases (p<0.01 at Chi-square test). However, other immune cell profiles, Treg cells in PBMC and cytokines in plasma had no trend between them. Several reports demonstrated that cancer patients with CD163-positive tumor-infiltrating macrophages and CD163-positive tumor cells had poor prognosis due to tumor-associated macrophage. Phenotypic macrophage traits in cancer cells, like CD163 expression, may be explained by heterotypic cell fusion between monocytes/macrophages and cancer cells.

Conclusion: Targeted therapy against M2 macrophage or CCR4 is considered as a promising strategy of advanced breast cancer.

#4139

TfhX13 cells link breast cancer immune suppression and adaptive memory.

Chunyan Gu-Trantien, Edoardo Migliori, Denis Larsimont, Karen Willard-Gallo. _Institut Jules Bordet, Universite Libre de Bruxelles, Brussels, Belgium_.

Background:

Previously, we identified PD-1hiCD200hiCD4+ follicular helper T (Tfh) cells infiltrating human breast cancer (BC TIL) as principal producers of the B cell chemoattractant CXCL13 and correlated their presence with improved patient outcome. Our recent efforts have focused on understanding the relationship between Tfh, Th1 and Treg BC TIL, investigating the role(s) that CXCL13+CD4+ TIL play in tumor immunity and determining the condition(s) that favor their differentiation.

Methods:

Ten-color flow cytometry was used to characterize CD4+ TIL subpopulations in fresh tissues. Confocal microscopy was employed to study the in situ interaction between CXCL13+CD4+ T cells and B cells. CXCL13 was induced in normal PBMC in vitro.

Results:

Our data reveal that an exacerbated Treg response accompanies high CXCL13 expression within the activated CD4+ TIL compartment in BC. Th1 responses, using IFN-γ expression as a barometer, are limited. CXCL13+CD4+ TIL display both similar and distinct characteristics compared to their tonsillar counterparts. Tumor bed-localized CXCL13+ TIL, external to tertiary lymphoid structure (TLS) germinal centers (GC), are abundant in some tumors with extensive lymphoid infiltrates. Based on our observations, we designate the CXCL13+CD4+ TIL as TfhX13 cells.

A combination of PD-1 and ICOS can clearly segregate the CD4+ TIL into three distinct subpopulations: PD-1loICOSlo, PD-1hiICOSint and PD-1intICOShi. The PD-1hiICOSint TIL are enriched in TfhX13 and Th1 cells while the PD-1intICOShi TIL are principally FoxP3hi Treg. A linear correlation is observed between these two TIL subpopulations in 90% of BC while the remaining 10% contain an unbalanced, higher level of PD-1intICOShi Treg. qRT-PCR data confirmed the significant positive prognostic value of CXCL13 gene expression with ICOS expression signaling the adverse effects of Treg cell dominance observed in some BC. TfhX13 TIL abundance appears to parallel both a GC and post-TLS memory B cell presence. Confocal microscopy revealed TfhX13 TIL interact directly with B cells and plasma cells, potentially guiding B cell migration through the tumor and promoting TLS formation with functional GC. Finally, we show that IL-2 deprivation is critical for inducing CXCL13 while TGF-β1 treatment reduces IFN-γ and increases FoxP3 expression in normal CD4+ T cells.

Conclusion:

IL-2 consumption by Treg cells was shown to be essential for murine Tfh cell development and a subsequent GC response. Supported by this observation, our data indicate that TfhX13 cell differentiation is an important element of the Tfh cell program and suggests their propagation in BC occurs in response to Treg accumulation. CXCL13 expression may activate adaptive memory responses dependent upon in situ B cell maturation and thereby initiate a secondary attempt at protective anti-tumor immunity in the face of Treg-mediated immune suppression.

#4140

Secreted protein acidic and rich in cysteine (SPARC) promotes mammary tumor growth and dissemination through immune and non-immune mediated mechanisms.

Leandro N. Güttlein,1 Lorena G. Benedetti,1 Raúl G. Spallanzani,2 Cecilia Rotondaro,1 Ximena L. Raffo Iraolagoitia,2 Leonardo Sganga,1 Edgardo Salvatierra,1 Andrea S. Llera,1 Norberto W. Zwirner,2 Osvaldo L. Podhajcer1. 1 _Fundación Instituto Leloir, IIBBA-CONICET, Buenos Aires, Argentina;_ 2 _Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina_.

Secreted protein and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with breast cancer progression, both in human tumors and in experimental models. However, the molecular mechanisms responsible for this effect are still unclear in particular because most studies were performed in immunocompromised nude mice. We undertook a comprehensive study of SPARC role in this disease by using the highly metastatic and poorly immunogenic 4T1 and LM3 mammary tumor cells syngeneic in Balb/c mice. Knock down of SPARC expression using a targeted shRNA in both cell lines greatly reduced primary tumor growth and completely obliterated the establishment of metastatic foci in lung. We performed global gene expression analysis comparing primary tumors and metastatic foci of control 4T1 cells with those arisen from shRNA-treated, SPARC-deficient 4T1 cells. From these studies, a potential role of the inflammatory/immune response in controlling tumor evasion and metastasis, more specifically through genes associated with the prostaglandin pathway, was suggested. As we explored this hypothesis, we observed that knock down of SPARC expression in 4T1 3D spheroids, but not in 2D monolayers, downregulated COX-2 mRNA and protein levels. Moreover, exogenous addition of SPARC in SPARC-deficient spheroids of 4T1 and MDA-MB-231 human breast cancer cells increased COX-2 expression. Interestingly, the re-expression of COX-2 in SPARC-deficient 4T1 cells partially restored the in vivo primary tumor growth with no effect on the establishment of metastatic foci. COX-2 plays an important role in inducing myeloid-derived suppressor cells (MDSCs, CD11b+Gr-1+ cells), which promotes breast cancer outgrowth through immunosuppression. Flow cytometry analyses demonstrated that SPARC-deficient 4T1 tumor-bearing mice exhibited reduced levels of CD11b+Gr-1+ cells compared to control 4T1 tumor-bearing mice in the spleen (6,9±3,2 vs. 25,3±2,0 p<0,001), lungs (0,7±0,2 vs. 6,2±0,9 p<0,001) and bone marrow (48,0±6,4% vs. 62,1±5,3 p>0,05). COX-2 re-expression restored systemic MDSCs expansion in SPARC-deficient 4T1 tumor-bearing mice. The present data suggest that COX-2 expression is under SPARC control in breast cancer cells; moreover, COX-2 could mediate SPARC effects on tumor evasion from immune surveillance.

#4141

Role of interferon regulatory factor 5 in anti-tumor immunity: Orchestration of a functional tertiary lymphoid structure.

Erica Pimenta,1 Ryan Weiss,1 Dan Li,2 Betsy Barnes2. 1 _Rutgers, New Jersey Medical School, Newark, NJ;_ 2 _The Feinstein Institute for Medical Research, Manhasset, NY_.

The ability of breast cancer cells to metastasize is due to intrinsic intratumoral and extrinsic microenvironment changes. Many breast cancers are heavily infiltrated by immune cells and these infiltrating leukocytes are commonly observed around tumors in patients with invasive ductal carcinoma. Several recent studies show that the presence of tumor infiltrating leukocytes in human breast cancer tissue is associated with improved outcome, suggesting that the immune system is participating in the control and elimination of tumor cells. One way in which epithelial-derived cancers evade immunity is through dysregulation of cytokines and chemokines that "call in" specific immune cells to form a functional ectopic lymphoid-like structure termed a tertiary lymphoid structure (TLS) near the tumor. TLS are important for cell-mediated and humoral immunity and are extremely efficient immunological tools as the presentation of antigen occurs to both B and T cells. Although very little is known about the processes that drive TLS formation, data indicate that the presence of a TLS next to a solid cancer, including breast cancer, is a strong positive prognostic indicator. Recent data from our lab and others show that expression of the transcription factor interferon regulatory factor 5 (IRF5) in a human breast cancer molecular signature predicts increased survival and lower incidence of metastasis. We recently identified IRF5 as an important intrinsic regulator of mammary epithelial cell migration and metastasis. We also found that IRF5 within a breast tumor regulates the expression of cytokines/chemokines, such as CXCL13, that mediate immune cell trafficking. We previously reported that tumor-conditioned media (TCM) from IRF5-positive breast tumor cells contains CXCL13 and induces trafficking of CD19+CXCR5+ B cells and CD3+CD4+CXCR5\+ T follicular helper-like (Tfh) cells to the TCM. CXCL13 is a key player in the formation of a TLS as it recruits in the specific cells and activates the B cells to produce immunoglobulins. Further analysis of immune cell trafficking to TCM from IRF5+ and IRF5- breast tumor cells reveals specificity for the trafficking of anti-tumorigenic B and T cell subsets to IRF5-positive TCM, such as transitional, memory, and plasmablast/plasma B cells, along with Th1 and Tfh cells, rather than pro-tumorigenic, immunosuppressive Bregs, Th2, and Tregs that were found to specifically migrate to IRF5-negative TCM. Analysis of formalin-fixed paraffin-embedded breast tumors by immunofluorescence microscopy revealed a significant correlation between IRF5 intratumoral expression and the presence of a TLS. Furthermore, preliminary data from a murine model of invasive mammary tumorigenesis supports a critical role for IRF5 in anti-tumor immunity. Together, these data implicate IRF5 in the generation of a functional TLS and as a critical mediator of anti-tumor immunity.

#4142

Proteomics analysis of breast cancer cell-specific proteins that are transferred to immune cells via trogocytosis.

Eiji Suzuki,1 Masashi Inoue,1 Shinji Ito,2 Junko Satoh,2 Kosuke Kawaguchi,1 Ayane Yamaguchi,1 Moe Tsuda,1 Masakazu Toi1. 1 _Kyoto Univ. Hospital, Kyoto, Japan;_ 2 _Kyoto University, Kyoto, Japan_.

We previously reported that HER2 is transferred from HER2 overexpressing breast cancer cells to CD14+ monocytes via trastuzumab dependent trogocytosis (Suzuki et al. BMC cancer 2015) and EGFR of MDA-MB-231 (triple negative human breast cancer cell line) is also transferred to immune cells via trogocytosis and those immune cells express EGFR on the cell surface without therapeutic antibody administration (AACR 2015 #2349). There are several reports that suggest that trogocytosis might act to stimulate immunological tolerance or immune effector cell activation, however its immunological roles still remain unclear. Therefore, in order to clarify the role of trogocytosis, we study the profile of proteins that potentially are transferred from cancer cell to immune cell via trogocytosis.

Human triple negative breast cancer cell line, MDA-MB-231 that was cultured with the medium containing arginine labeled with ¹³C instead of normal ¹²C (SILAC method, ThermoFisher Scientific) was co-cultured with human peripheral blood mononuclear cell (PBMC) that was cultured with normal medium. The cell mixture was labeled with CD45+ magnetic beads and CD45+ cell (PBMC) and CD45- cell (MDA-MB-231) were separated by MACS cell separation kit (Miltenyi Biotec). Proteins of PBMC that were passed the trogocytosis were resolved on SDS-PAGE and LC-MS/MS was carried out to identify the cancer specific trogocytosed proteins in PBMC by SILAC method.

We found that proteins with higher H/L ratio were included in many of cytoskeletal proteins and interestingly mitochondrial metabolism related proteins of cancer cells were also detected in PBMC. Those up-regulated proteins in PBMC were reduced in MDA-MB-231. The findings suggested that the role of trogocytosis of PBMC on breast cancer cell might be involved in metabolisms of both cancer cell and immune cell. Thus, we are exploring the effect of trogocytosis on cancer cell metabolisms especially on mitochondrial related metabolism that could be reduced by immune cell trogocytosis and also on metabolism of immune cells themselves that capture the cancer cell proteins via trogocytosis.

#4143

Characterizing the immunogenicity of gastric cancer by transcriptomic expression based immune phenotyping.

Kai Wang,1 Hoicheong Siu,2 Shibing Deng,1 Jadwiga R. Bienkowska,1 Suet Yi Leung2. 1 _Pfizer, Inc., San Diego, CA;_ 2 _The University of Hong Kong, Hong Kong, Hong Kong_.

One of the main challenges in immuno-oncology is to quantify different types of immune cells in the tumor microenvironment, as such data can greatly facilitate our understanding of the mechanism of action of, and response to cancer immunotherapies. Traditionally immune profiling is performed by immunohistochemistry or flow cytometry experiments using surface markers specific to each immune cell type. However, both approaches suffer from practical limitations such as reagent availability and real world specimen conditions. Recently several computational deconvolution approaches using expression profiles of the bulk tumor tissue samples have been developed. This alternative approach utilizes existing database of purified immune cell gene signatures and relies on a rigorous mathematical framework of signal deconvolution. One of such algorithms, called CIBERSORT (Newman et al. 2015), has been demonstrated to perform robustly in deconvoluting the relative fractions of 22 human leukocyte subsets in solid tumor tissue samples, and benchmarked favorably against FACS analysis.

In this work, we performed immune phenotyping by the CIBERSORT-based expression deconvolution on a cohort of 100 gastric cancer (GC) patients. We found that the fractions of activated CD4 memory T cells, rather than CD8+ T cell, are most significantly correlated with tumor neo-antigen load, whereas the latter is most significantly associated with the expression of a "Cytolytic Activity" metagene and an interferon gamma signature. There is stronger pre-existing host immune response against the MSI and EBV subtypes of GCs mediated by either cytotoxic T cells or Natural Killer cells. Lastly, we found a high M2/M1 tumor associated Macrophages (TAMs) ratio is strongly associated with poor GC prognosis, corroborating recent reports on the role of a TAM and stromal response in gastric cancer angiogenesis.

Taken together, we demonstrated in this work that critical information about the tumor infiltrating immune cells can be gleaned from the computational deconvolution of bulk tumor gene expression profiling data. The comprehensiveness and cost-effectiveness of this approach can complement other immune profiling techniques in characterizing a wide variety of tumor specimens under various treatment conditions. We foresee its application in characterizing pharmacodynamics, understanding immunological mechanism of action and monitoring treatment response and disease progression for cancer immunotherapies and their combinations in both pre-clinical and clinical settings.

#4144

The prognostic value of tumor-infiltrating immune cells and clinical application of immunoscoring methods in advanced colorectal carcinoma.

Yoonjin Kwak,1 Duck Woo Kim,2 Sung Bum Kang,2 Woo Ho Kim,1 Hye Seung Lee3. 1 _Seoul National Univ. College of Medicine Department of Pathology, Seoul, Republic of Korea;_ 2 _Seoul National Univ. Bundang Hospital Department of Surgery, Seongnam, Republic of Korea;_ 3 _Seoul National Univ. Bundang Hospital Department of Pathology, Seongnam, Republic of Korea_.

Background Immunoscore(IS) which quantifies tumor-infiltrating lymphocyte (TIL) has been noteworthy for novel prognostic biomarker especially in colorectal cancer (CRC). Recent studies have been insisted that IS method is superior to current tumor-node-metastases staging system in various cancers. The prognostic significance of tumor-associate macrophage (TAM) also has been suggested. However, studies in the past included patients with stage I-III CRCs and the clinical implication of TIL and TAM has not been fully clarified in advance CRC patients who presented distant metastases. In this study, we investigated TIL and TAM in the primary site and the corresponding metastatic organ to identified the heterogeneity of each characteristics according to tumor location. We evaluated the prognostic significance of TIL and TAM in advanced CRCs. Also we designed new IS methods and conducted comparative analysis between existing and new methods for prognostic predictive value. Methods. Using immunohistochemistry and digital image analyzer, we measured the density of CD3, CD4, CD8 and FOXP3-positive lymphocyte of 196 advanced CRC patients. CD68 and CD163 immunohistochemistry was conducted to evaluate the density of tumor-associated macrophage. These properties were examined within tissue from four sites (the center (CT) and periphery of the primary cancer (IM), a distant metastasis (DM) and a lymph node metastasis (LM)). High density of lymphocyte marker and low density of macrophage marker in each region recorded as a score. IS method is based on the combination of two markers (CD3 and CD8) in two regions (CT and IM). Newly designed model, IS-metastatic and IS-macrophage included the additional scores of two markers (CD3 and CD8) in DM and macrophage markers (CD68 and CD163) in CT, respectively. Results. The each density of TIL and TAM showed significant heterogeneity according to the tumor location. Patients with high density of CD3-positive lymphocyte in CT presented favorable outcome (p=0.030). In contrast, patients showing high density of CD68 and CD163-positive macrophage in CT had significantly worse prognosis (p=0.011 and p<0.001, respectively). Higher IS (score 3-4) was significantly correlated with better survival rate (p=0.020). Patients with higher IS-metastatic as well as IS-macrophage (score 4-6) also showed significantly better outcome (p=<0.001 and p=0.005, respectively). In multivariate analysis, IS-Metastatic model was independent prognostic factor (p=0.012). Conclusions. TIL and TAM are distributed heterogeneously with respect to the tumor location in CRC patients and has a significant effect on patients outcome. IS is reproducible methods and applicable for predicting survival in advanced CRC. New IS model including CD3- and CD8-positive cells at distant metastasis could be an independent prognostic biomarker in advanced CRC patients.

#4145

Stromal heterogeneity and immune response in ovarian cancer.

Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. _UT MD Anderson Cancer Center, Houston, TX_.

Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer (HGSOC). Despite the increasing evidence on stromal involvement in tumor progression, the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) in ovarian cancer is not fully understood. How tumor heterogeneity contributes to a different in immune response remains a challenge question that needs to be addressed. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors.

By laser microdissection of tumor tissue samples from HGSOC patients, we generated cell type specific expression profiles and identified a CAF-specific gene signature for ovarian cancer. Genes expressed exclusively by CAFs that are associated with differential immune response were identified by comparing CAF expression profiles from HGSOC patients with high and low tumor-infiltrated CD8+ T cell densities. Among the differentially expressed genes identified, immunostaining results showed a significant inverse correlation between stromal MFAP5 expression and intratumoral CD8+ T cell density in HGSOC tissue samples (p=0.006). The results were further confirmed by correlating stromal MFAP5 protein expression and intratumoral CD8+ T cell density. Our recent studies showed that increased stromal MFAP5 expression is associated with poorer survival in HGSOC patients and MFAP5 modulates ovarian caner invasion and motility potential. Together with our preliminary data showing that MFAP5 modulates the expression of immune-related genes, we hypothesize that stromal MFAP5 may generate an immunosuppressive microenvironment through suppressing CD8+ T cell activation and trafficking in the ovarian tumor tissue. Cell culture experiment showed that recombinant MFAP5 protein treatment induced expression of CD47, an immune checkpoint protein, in cancer cells and downregulated CXCL13, a chemokine essential for immune cell adhesion, in endothelial cells. Using animal models, these findings were further validated. Based on our data, animal studies will be performed to further evaluate the efficacy of immune activation by targeting stromal MFAP5 in ovarian cancer treatment.

Delineating the molecular mechanism by which MFAP5 modulates the immune responses in ovarian cancer will help to design novel treatment modalities based on stromal MFAP5 blockade, which will promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates.

#4146

MultiOmyx multiplexed tumor infiltrating lymphocyte panel provides comprehensive immunophenotyping from a single FFPE slide.

Qingyan Au, Kathy Nguyen, Raghav Padmanabhan, Anne Kuller, Eddie Moler, Nicholas Hoe. _Clarient Diagnostics, GE Healthcare, Aliso Viejo, CA_.

Immune checkpoint therapies target immune regulatory pathways to enhance anti-tumor immune response. These therapies have contributed to important clinical advances, and are a promising approach to combat cancer. Development of effective immune checkpoint therapies requires an understanding of the host immune response within the tumor microenvironment. GE Healthcare, through its affiliate Clarient Diagnostics Service Inc., has developed a multiplexed Tumor Infiltrating Lymphocyte (TIL) Panel* consisting of 12 key cancer immune markers: CD3, CD4, CD8, CD20, CD45RO, CD56, CD68, CTLA4, FOXP3, PD1, PD-L1 and Pan-CK. This MultiOmyx TIL panel identifies individual Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), Tregulatory (CD3+CD4+FoxP3), memory T-cells (CD3+CD4+CD45RO), anergic T-cells (PD1+CD8+), natural killer cells (CD56+), macrophages (CD68+), and B-cells (CD20+) within the tumor and the stromal regions.

MultiOmyx multiplexed immunofluorescence technology enables qualitative and quantitative analysis of these 12 biomarkers' expression and co-localization from a single formalin-fixed, paraffin-embedded (FFPE) slide. MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining.

Herein, we report an analysis of immune response in the tumor microenvironment within solid tumors including breast cancer, lung cancer, colorectal cancer, esophageal cancer, prostate cancer, and melanoma utilizing the MultiOmyx TIL Panel. The results revealed two distinct immunologic phenotypes, high TIL tumors and Low TIL tumors. The high TIL tumors showed enhanced T cell population within the tumor and in the peritumoral stroma including CD8+ cytotoxic T cells, CD4+ helper T cells and CD45RO+ memory T cells. Increased expression of immune checkpoints markers such as CTLA4 and PD-1 were also observed. PD-1 was predominantly expressed in CD8+ cytotoxic T cells while CTLA4 was mostly found on CD4+ FOXP3+ regulatory T cells. PD-L1 expression was also induced, mainly on Pan-CK+ tumor cells and CD68+ macrophages. High density of PD-L1+ |CD68+ macrophages was localized in the stroma surrounding the tumor region. Conversely, many of the immune markers were not expressed in low-TIL tumors.

Immunophenotyping analysis offered by the MultiOmyx TIL panel may facilitate the identification of appropriate immunotherapeutic approach. Tumor with high TIL profiling may be effectively treated with single-agent immune checkpoint therapy, while tumor with low TIL profiling may require combination therapy with an agent that enhances endogenous antitumor response.

*MultiOmyx TIL panel is currently research use only tool.

#4147

T cell targeted peptides for monitoring immune response in melanoma.

Dustin Bauknight,1 Andrew Buckner,2 Lindsey Brinton,1 Timothy Bullock,2 Kimberly Kelly1. 1 _University of Virginia Department of Biomedical Engineering, Charlottesville, VA;_ 2 _University of Virginia Department of Pathology, Charlottesville, VA_.

Melanoma develops the ability to evade immune recognition through multiple mechanisms, despite being highly immunogenic. This is demonstrated by the effective and additive response checkpoint blockade therapies have had during combination clinical trials in melanoma [1]. While the results of these trials have been promising, large portions of patients do not respond to treatment [1,2]. Additionally, many responders take months to show a response using standard criteria. In order to monitor patient response and understand the limitations of immunotherapy, we performed phage display to discover peptides targeted to tumor infiltrating lymphocytes (TILs) in melanoma tumors.

To improve phage library characterization, we developed a method to deep sequence phage with the Ilumina MiSeq platform. Phage screens using PhD7 library (NEB) performed on HUVECS and on TILs, naïve and effector T cells, and B cells harvested from mice. The phage libraries generated from these screens were deep sequenced using the Illumina MiSeq system. The sequencing data was analyzed with an efficient, custom MATLAB script, which arranges sequences by frequency determining peptide frequencies for each library. Peptide frequency was normalized to a reference library generated by amplifying an unscreened phage library. T cell screens were compared with B cell and endothelial cell screens in sequence frequency matrices. Top TIL targeting sequences were cloned into phage, and then fluorescently labeled along with insert-less control phage. Labeled phage were incubated with TILs, or effector T cells and analyzed for specificity using flow cytometry. Four phage clones were identified with at least four fold increased binding for TILs over effector T cells isolated from the spleen. Interestingly, only subsets of the TILs were bound by the targeted phage.

We have identified and validated peptides that demonstrate specificity for CD8 TILs. These peptide sequences represent candidates for development into companion diagnostic imaging agents for use with checkpoint therapies. We are currently evaluating these peptides in an in vivo tumor model to further validate their specificity and to determine their biodistributions. Peptides that validate in vivo could be developed into useful imaging agents. After peptide sequences have been validated in vivo, we will determine the phage binding partner using a phage based pulldown. The peptide binding partners discovered through this approach will provide insight into the subset of TILs bound by the phage.

[1] Wolchok, J. D. et al., N Engl J Med 2013; 369:122-133.

[2] Drake, CG. et al., Clin Cancer Res November 15, 201117.

#4148

Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations.

Suvi Luoto, Juha Kesseli, Matti Nykter, Kirsi J. Granberg. _BioMediTech, University of Tampere, Tampere, Finland_.

Interactions between various components in the tumor microenvironment and dysregulated immune responses are thought to play important roles in cancer development. To better understand the role of immune cells in tumor pathogenesis and destruction, we computationally model the microenvironment of an aggressive brain tumor glioblastoma multiforme (GBM).

We downloaded GBM patient RNA-seq data from the Cancer Genome Atlas (TCGA). Using cluster analysis, we identified 16 clusters, each containing 10 to 933 genes that show a statistical enrichment of immune response related gene ontology terms. Utilizing a panel of RNA-seq data from normal cell types, we constructed global and cluster specific regression models to characterize the expression profiles of GBM samples in the clusters of interest as linear combinations of normal cell and reference GBM expression profiles. Simulated data was used to validate that the regression model coefficients accurately reflect the contributions of normal cell types to the expression profiles of tumor samples. Based on the regression analysis, we were able to uncover high variability in the composition of microenvironment across the TCGA cohort, suggesting diverse immune responses in tumors.

The results from global regression analysis were then associated with common genetic alterations in GBM and with GBM subclassification. Especially the estimated macrophage, granulocyte, and CD8+ T lymphocyte proportions show significant differences between different GBM subgroups. Accumulation of immune cells was increased in the mesenchymal and neural subtype compared to other subtypes. Furthermore, estimated immune cell proportions were associated with alterations in EGFR and NF1.

Taken together, our analysis provided a characterization of the immunomicroenvironment in GBM and linked immune cell responses to typical GBM alterations and GBM subtypes. More detailed characterization of diverse immune responses will facilitate patient stratification and might provide tools for personalized immunotherapy in the future.

#4149

Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1.

Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Masakazu Toi. _Kyoto Univ. Hospital, Kyoto, Japan_.

BACKGROUND:

We previously reported that direct co-culture of triple negative breast cancer cell line MDA-MB-231 and immune cells results in reduction of EGFR expression on cell surface of MDA-MB-231 (AACR 2015 #2349). However, a role of reduction of EGFR on MDA-MB-231 via co-culture with immune cells still remains unclear. Therefore, we evaluated a role of reduction of EGFR on MDA-MB-231 at the immunological point of view by testing expression of immune related genes including PD-L1.

METHODS:

In order to verify the importance of direct immune cell contact to breast cancer cell, MDA-MB-231 cells were co-cultured directly or indirectly with THP-1 cells (human monocytic cell line) at 1:50 cellular ratios. In order to study indirect co-culture assay, we used cell culture insert to avoid direct cancer cell-immune cell contact. We analyzed gene expression by quantitative real time PCR and membrane protein expression by flow cytometry of EGFR, also other HER family on MDA-MB-231 which is directly or indirectly co-cultured with THP-1. We also evaluated the expression of immune related genes including PD-L1.

RESULTS:

Both mRNA and protein level of EGFR on MDA-MB-231 cells directly co-cultured with THP-1 were significantly decreased as compared to those with indirectly co-cultured MDA-MB-231 cells. There are no significant differences in EGFR expression between indirectly co-cultured MDA-MB-231 cells and control MDA-MB-231 cells. Importantly, PD-L1 expression on MDA-MB-231 cells directly co-cultured with THP-1 was significantly decreased as compared to that with indirectly co-cultured MDA-MB-231 cells.

CONCLUSION:

It has been reported that PD-L1 expression in cancers is regulated by phosphatidylinositol 3-kinase (PI3K) and Akt signaling. Thus, our findings may give a novel insight on regulation of PD-L1 expression on cancer cells in tumor microenvironment that tumor infiltrated immune cell directly contact with cancer cells and EGFR down-regulation leads to reduction of PD-L1 expression on cancer cells.

Further investigation is needed to elucidate the mechanism of reduction of EGFR by direct immune cell contact to cancer cells and its interaction with modulation of PD-L1 expression. This will provide novel aspects for immune therapy of breast cancer patients.

#4150

Blockade of CXCR2 mediated granulocytic MDSC recruitment synergizes with CCR2 inhibition of inflammatory monocytes and restores anti-tumor immunity in pancreatic adenocarcinoma.

Timothy M. Nywening,1 Brian A. Belt,2 Roheena Z. Panni,1 Darren Cullinan,1 Dominic E. Sanford,1 Ryan C. Fields,1 William G. Hawkins,1 David G. DeNardo,1 William E. Gillanders,1 Peter Goedegebuure,1 David C. Linehan1. 1 _Washington University School of Medicine, St Louis, MO;_ 2 _University of Rochester Medical Center, Rochester, NY_.

Introduction: Pancreatic cancer (PC) is characterized by a dense tumor stroma with a heavy leukocytic infiltrate, comprised predominately of immunosuppressive bone marrow (BM) derived cells. We have previously demonstrated in a phase Ib clinical trial that CCR2 inhibition (CCR2i) prevents inflammatory monocyte (IM) recruitment from the BM and results in a significant reduction of tumor associated macrophages (TAM) and an increase in treatment efficacy. However, granulocytic myeloid derived suppressor cells (G-MDSC) remain in the tumor microenvironment (TME) following CCR2i. Herein, we explored the impact of targeting G-MDSC recruitment to PC tumors both alone and in combination with CCR2i.

Methods: Human BM, blood, and tumor was collected under an IRB approved protocol. A tissue microarray (TMA) from resected PC patients was analyzed for immune infiltrate. Mice were injected orthotopically with 2.5x106 syngeneic PC cells. CXCR2 and CCR2 inhibitors (Tocris) were given twice daily. Tumor growth was assessed and specimens obtained for analysis by flow cytometry, RNAseq, and IHC.

Results: Human PC overexpresses CXCL5 and CXCL8, corresponding with an abundance of tumor infiltrating CXCR2+ G-MDSC. Furthermore, the ratio of CD8 to G-MDSC correlates with survival in human PC patients. In an orthotopic murine model that recapitulates human disease, ƩCXCL ligands were also increased. Either Ly6G depletion or targeted blockade with a CXCR2 inhibitor decreased G-MDSC and reduced tumor burden. Intriguingly, blockade of IM from the BM did not reduce G-MDSC and paradoxically resulted in a modest increase in this population within the tumors from human patients following CCR2i. Thus, we explored the combination of CCR2/CXCR2 blockade both with and without FOLFIRINOX chemotherapy. This resulted in a synergistic impact when both BM derived populations were targeted and dual therapy was further enhanced by FOLFIRINOX. RNAseq analysis of tumors following monotherapy or dual inhibition revealed alterations in the TME favoring an anti-tumor immune response. To test the hypothesis that this effect was mediated by restoration of anti-tumor immunity we analyzed the tumor infiltrating lymphocyte (TIL) populations and found a significant increase in the relative and absolute numbers of CD8+ and C4+ TIL. Analysis of the activation status of these cells demonstrated an increase in effector CD8+ T-cell phenotype (IFNƴ+, CD69+, CD44+). Using Nur77GFP T-cell receptor reporter mice, we showed an increase in GFP expressing CD8+ TIL following dual blockade. CD8 depletion resulted in a loss of therapeutic efficacy of myeloid blockade, further confirming our hypothesis.

Conclusion: These findings suggest that combinatorial blockade strategies preventing tumor infiltration by myeloid cells may restore anti-tumor immunity in PC.

#4151

APC control of retinoic acid suppresses immune cell mediated intestinal proliferative response.

Amanda K. Templeton,1 Saher Hammoud,2 Brad Cairns,3 David Jones4. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _University of Michigan, Ann Arbor, MI;_ 3 _Huntsman Cancer Institute, Salt Lake City, UT;_ 4 _Oklahoma Medical Research Foundation, Oklahoma City, OK_.

Adenomatous polyposis coli (APC) tumor suppressor function is centered on its role in antagonizing Wnt/B-catenin mediated colonic proliferation. Our previous studies, however, established a second role wherein APC's control of retinoic acid (RA) results in intestinal epithelial cell differentiation. Surprisingly, the loss of APC or RA alone in zebrafish embryos was not sufficient to drive nuclear accumulation of B-catenin or intestinal cell proliferation. However, combining APC or RA deficiency with a KRAS mutation was sufficient to induce nuclear localization of B-catenin and the proliferative response. In contrast, the loss of RA alone in adult zebrafish results in both intestinal differentiation defects and proliferation. We were therefore interested in investigating these seemingly contradictory results. Using adult zebrafish, we show for the first time that following loss of retinoic acid adaptive immune cells (but not innate immune cells) are indirectly required to elicit the proliferative response independent of Kras activation. We demonstrate that IL-17 secretion from adaptive immune cells in response to loss of RA predisposes colonic epithelium to inflammation resulting in compromised barrier function, loss of cell polarity, reactivation of a progenitor intestinal program, and proliferation. Importantly, we find in both mouse and human intestinal organoid cultures an evolutionarily shared response with zebrafish of T cell dependent intestinal inflammation and proliferation following loss of RA. Our model demonstrates that loss of RA causes an increase in inflammatory mediators (Cox2 and Prostaglandins) that induces infiltration of IL-17 secreting T cells. In a feed-forward loop, the IL-17 acts on the primed epithelium to up-regulate proliferative signaling mediators (Raf and Rac1). Together, these studies provide an important new perspective on the ordering of molecular events that may underlie colon tumor progression.

#4152

Efferocytosis of prostate cancer cells induces a tumor-promoting inflammatory response in myeloid macrophages.

Hernan Roca, Marta Purica, Savannah Weidner, Amy J. Koh, Robert Kuo, Jacques E. Nör, Lonnie D. Shea, Laurie K. McCauley. _University of Michigan, Ann Arbor, MI_.

Tumor progression is characterized by persistent death of cancer cells, which are cleared by the innate immune system phagocytes. This process termed efferocytosis is enhanced with targeted therapies that induce apoptosis in tumor cells. Recent findings suggest that efferocytosis polarizes macrophages into M2-type and may induce tumor acceleration; however, this mechanism and its consequences in prostate cancer tumor fate remains largely unknown. Here the changes in cytokine expression (mRNA and protein) in primary bone marrow derived mouse macrophages (Mϕ) interacting with two apoptotic prostate cancer cells (human PC3 and mouse RM1) were analyzed. In response to efferocytosis, Mϕ produced known tumor-promoting inflammatory cytokines including IL-6, CXCL1 and CXCL5 that are chemoattractants of monocytes/macrophages and neutrophils. In vitro efferocytosis induced the activation of NF-κb signaling in Mϕ as analyzed by Western blot and functional luciferase reporter assays (TRACER). Inhibition of NF-κb with the chemical compound emetine (0.3 μM) blocked the efferocytosis of fluorescence-labeled apoptotic cancer cells and the expression of the inflammatory marker Ly-6B by Mϕ, suggesting a crucial role of NF-κb in the efferocytic function of Mϕ. An in vivo syngeneic tumor model was used in which apoptosis-inducible prostate cancer cells (RM1-iCasp9) were injected in vertebral bodies and implanted subcutaneously in immune competent mice. Seven days post-surgery mice were treated with vehicle (VEH) or the dimerizer drug AP20187 (AP) to induce apoptosis in RM1-iCasp9 cancer cells. Continuous analysis of tumor volume and the endpoint tumor weight (13d) revealed accelerated tumor growth in mice where apoptosis was induced (AP) as compared with controls (VEH) (p<0.01). Furthermore, the analysis of tumors by flow cytometry demonstrated a significant increase of tumor accelerating myeloid inflammatory cells in the AP-treated mice induced to undergo efferocytosis when compared to VEH-treated mice. These populations included total CD206+F4/80+ (M2 macrophages), Gr-1+CD11b(high)+ (myeloid granulocytes/monocytes associated with anti-tumor immunity), total Gr-1+ cells and CD68+ cells (phagocytes) that express high CD11b (p<0.05). In addition, Ly-6B, a marker associated with the activation of inflammatory myeloid (CD11b+) cells was significantly increased in the AP-treated tumors. In a similar experiment, the tumor protein analysis by ELISA showed a significant increase in CXCL5 in the AP-treated tumors relative to controls. Altogether these findings suggest that cancer cell apoptosis triggers an inflammatory response in myeloid efferocytic macrophages via activation of NF-κb and expression of cytokines that recruit and activate myeloid cells to accelerate tumor growth. This mechanism may be critical for metastatic bone colonization.

#4153

Abnormal expression of MUC1 mucin on colon epithelia stimulates production of pro-inflammatory cytokines promoting colitis-associated colon cancer in a murine model.

Sandra Cascio, Jacque Faylo, Jia Xue, Olivera Finn. _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

MUC1 is a transmembrane glycoprotein aberrantly expressed in human adenocarcinoma as well as chronic inflammatory conditions. We have previously demonstrated that the tumor form of MUC1, in association with p65, upregulates the expression of pro-inflammatory cytokines in the tumor microenvironment, including IL-6 and TNF-alpha, by modulating their transcriptional activity in epithelial cancer cells. Here, we explored the mechanism underlying MUC1/p65-induced transcription of IL-6 and TNF-alpha in colon cancer cells and its significance for the microenviroment of colitis that is known to be the precursor to cancer. We used a mouse model of colitis-associated colon cancer where wild type (WT) and human MUC1 transgenic (MUC1.Tg) mice are given carcinogen azoxymethane (AOM), followed by three cycles of 1.2% dextran sodium sulfate (DSS) in drinking water to induce colitis. MUC1.Tg showed higher tumor incidence, decreased survival, increased body weight loss and shorter colon length. These features accelerated the severity of inflammation-induced colon cancer. Consistent with our previous in vitro data, expression of NF-kB family members was higher in MUC1.Tg mice compared to WT. We previously showed that in tumors, MUC1/p65 complex modulated the expression of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) and interaction with pro-inflammatory cytokine promoters. In order to understand the significance of MUC1/p65-modulated cytokines in progressive colitis that gives rise to colon cancer, we analyzed infiltration of inflammatory cells into the inflamed colon tissues. The number of infiltrating macrophages was much higher in AOM/DSS-treated MUC1.Tg mice compared to WT. ELISA assay and gene expression analyses demonstrated that the treatment with AOM/DSS in the presence of human MUC1 on the colonic epithelia resulted in a significant increase in M1 type macrophage-associated genes, including IL-6, TNF-alpha and iNOS, whereas M2 type macrophage markers such as Arginase 1, Ym1, and IL-10 were drastically reduced when compared to colon tissues of treated WT mice. Taken together, our findings reveal a pro-inflammatory role for MUC1 in colitis-related carcinogenesis. Our study also provides a mechanism by which MUC1 accelerates tumor initiation and progression.

#4154

Neuropilin-1 expressing macrophages promote neuropilin-1 expression on lymphocytes by direct interaction and exert antitumor activity in HER2 positive breast cancer.

Kosuke Kawaguchi, Eiji SUzuki, Masashi Inoue, Isao Kii, Tatsuki R. Karaoke, Masahiro Hiram, Keiko Iwaisako, Pu Fengling, Mariko Niche, Ayane Yamaguchi, Hironori Haga, Masatoshi Hagiwara, Masakazu Toi. _Kyoto University, Kyoto, Japan_.

Tumor-infiltrating immune cells (TIIs) in HER2+ breast cancer (BC) play an important role in treatment with anti-HER2 therapy. However, the precise mechanisms of how TIIs exert anti-tumor activity remain unclear. Neuropilin-1 (NRP-1) on macrophages regulates immune functions in various cancers and thus we explored the role of NRP-1 expressing macrophages in anti-tumor activity in HER2+ BC. We show that NRP-1 on macrophages regulated the migration of and chemokine secretion from macrophages in vitro. Furthermore, in vivo studies using a humanized mouse model showed that NRP-1 knockdown of macrophages in adoptively transferring peripheral blood mononuclear cells (PBMCs) suppressed anti-tumor activity and infiltration of CD45+ immune cells into tumors. Interestingly, NRP-1 expressing TIIs were mainly CD4+ T cells, despite little expression of NRP-1 on CD4+ T cells in PBMCs. We found that NRP-1 expression on CD4+ T cells was induced by NRP-1 transfer from macrophages to T cells. In HER2+ BC patients, NRP-1 expressing TIIs correlated with better clinical outcomes. These results demonstrate that NRP-1 expressing macrophages are key subsets of immune cells in trastuzumab-mediated anti-tumor activity and may predict better outcomes for HER2+ BC patients. In conclusion, NRP-1 expression is required for differentiation and activation of macrophages. NRP-1 expressing macrophages promote NRP-1 expression on CD4+ T cells by direct interaction and contribute to anti-tumor immune responses in HER2+ BC.

#4155

Regulation of MDSC trafficking and function in RCC by CXCR4 in the presence of a VEGF-R antagonist.

David J. Panka,1 Robert D. Arbeit,2 James W. Mier1. 1 _Beth Israel Deaconess Med Ctr, Boston, MA;_ 2 _X4 Pharmaceuticals, Cambridge, MA_.

Using a murine model of 786-0 and A498 RCC xenografts, we have previously demonstrated that acquired resistance to sunitinib treatment was associated with a marked increase in the infiltration of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSC). These cells have also been implicated in the development of resistance to other anticancer therapies. Further, we observed that both the influx of MDSC and resistance to VEGF-targeted therapies could be prevented by concurrent administration of an HDM2 antagonist, a drug whose biological effects are mediated primarily through the up regulation of p53. MDSC trafficking into tumor tissue is regulated by chemokines, many of which (e.g. SDF-1/CXCL-12) are produced in response to HIF-2 expression. p53 is known to directly repress CXCL12 transcription, and we have shown that HDM2 blockade suppresses HIF-2 expression, suggesting that the drug has both direct and indirect effects on CXCL12 expression. Western blot analysis of tumor lysates confirmed that HDM2 antagonism mediates its effects on MDSC through the suppression of chemokine production, including CXCL12. These findings suggested that the ability of HDM2 antagonism to prevent sunitinib resistance might be due, at least in part, to the suppression of CXCL12 production and MDSC recruitment. Consequently we hypothesized that agents that block CXCL12/CXCR4 signaling directly would duplicate the effects of HDM2 blockade on MDSC trafficking and prevent resistance to VEGF-targeted therapies.

Mice were inoculated with 786-0 and A498 RCC xenografts, the tumors permitted to grow to ~300 mm3, and then treatment initiated with the CXCR4 inhibitor X4P-001 (AMD11070), axitinib, both agents in combination, or saline (control). Whereas either drug alone either had no (axitinib) or modest (X4P-001) effects on tumor growth, the combination of X4P-001 and axitinib had additive (synergistic) anti-tumor effects. Specifically, combination treatment resulted in massive tumor cell death, with the established implants actually regressing in size - an effect not previously seen with VEGFR-targeted drugs given as single agents. IHC staining demonstrated, as previously, that mice treated with axitinib alone had an increase in Ki-67 positive tumor cells. This effect was not observed in mice that received both X4P-001 plus axitinib, suggesting an anti-proliferative effect of the combination. Finally, the tumors from mice receiving axitinib alone had extensive MDSC infiltration, whereas the tumors from mice receiving either X4P-001 alone or the axitinib/X4P-001 combination had significantly less MDSC infiltration. These pre-clinical findings support both the role that MDSCs play in resistance to VEGF-R antagonists and the use of X4P-001 in combination with axitinib in upcoming clinical trials.

#4156

Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity.

Kazuya Kuwada, Shunsuke Kagawa, Megumi Watanabe, Shuichi Sakamoto, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Tetuya Kagawa, Toshiyoshi Fujiwara. _Department of Gastroenterological Surgery, Okayama, Japan_.

Objectives:

Postoperative recurrence occurs in approximately 80% of pancreatic cancer, and the peritoneum is the second most frequent metastatic site next to the liver. Recent evidence suggests that cancer microenvironment plays key roles in metastasis. To investigate mechanism of intraperitoneal metastasis in pancreatic cancer, we tried to detect cancer cell in the peritoneal wash from pancreatic cancer patients and analyze intraperitoneal cancer microenvironment. A genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-active cancer cells, was employed to detect cancer cells, and it enabled us to distinguish cancer cell from co-existence cells in the abdominal cavity. We explored the role of tumor-associated macrophages (TAMs) by using this virus-guided fluorescent imaging system.

Methods:

Peritoneal wash was obtained from 20 pancreatic cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Samples from cases with TelomeScan-expressing GFP-positive cells were further subjected to immunofluorescence assay for analysis of microenvironment. The antibodies against CD45, CD14, and CD204 were used in immunostaining as markers of leukocytes, monocytes and TAMs, respectively. To investigate the effect of TAMs on pancreatic cancer, Panc1 and BxPC-3 cell lines were co-cultured with PMA (phorbol 12-myristate 13-acetate)-treated monocytes and analyzed their changes.

Results:

The three out of 20 cases were positive in cytology, and GFP positive cells were detected after TelomeScan infection in those cases. In the wash, pancreatic cancer cells existed together with many leukocytes including macrophages. Further analysis demonstrated that those macrophages were TAMs. After Panc1 or BxPC-3 cells were co-cultured with TAMs, E-cadherin was decreased in Panc1, α-SMA and Vimentin was increased in BxPC-3. These results suggested that Epithelial to Mesenchymal Transition (EMT) was induced in pancreatic cancer cells by TAMs.

Conclusion:

Pancreatic cancer cells and TAMs were detected in the peritoneal wash using TelomeScan and immunostaining. The results suggested that EMT induced by TAMs may promote intraperitoneal metastasis of pancreatic cancer.

#4157

IL-6 glycoforms differentially modulate the polarization of tumor-associated macrophages in lung cancer.

Chun-Hua Hung,1 Hao-Chen Wang,2 Hsuan-Heng Yeh,3 Chien-Chung Lin,4 Wu-Chou Su1. 1 _Department of Internal Medicine, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan;_ 2 _Institute of Clinical Medicine, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan;_ 3 _Cancer Center, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan;_ 4 _Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan_.

Interleukin-6 (IL-6) is critical in modulating both tumor growth and anti-tumor immunity. Here, we describe the glycosylation pattern of lung cancer cell-secreted IL-6 and examine the function of the IL-6 glycoforms in polarization of tumor-associated macrophages (TAMs). IL-6 with different molecular weights were detected in various lung cancer cells. Because in NetNGlyc 1.0 Server, one possible N-glycosylation site has been predicted at N73 on IL-6, we used tunicamycin treatment and site-directed mutagenesis on N73 to demonstrate that lung cancer cell-secreted IL-6 is modified by N-glycosylation. To explore more detailed attached glycans, we measured the expression of glycosyltransferases by qPCR and found that the expression of fucosyltransferase 8 (FUT8), responsible for core fucosylation on N-glycosylated proteins, was higher in lung cancer cells than normal bronchial cells. Core fucosylation on IL-6 was reduced by silencing FUT8. We generated AS2-IL6, AS2-IL6-shFUT8, and AS2-IL6-N73Q cells for producing full-glycosylated IL-6 (G-IL6), core fucose-depleted IL-6 (DeCF-IL6), and Asn73-N-glycan-depleted IL-6 (N73Q-IL6). To examine the effect of the IL-6 glycoforms on differentiation of TAMs, THP-1 monocytic leukemic cells were incubated with phorbol-12-myristate 13-acetate for transforming to macrophages, followed by co-culture with the AS2 cell derivatives to mimic the education of TAMs. The co-cultured TAMs of each AS2 derivative showed distinct morphology. Intriguingly, the G-IL6-producing cells promote the expression of M2-TAM markers CD204, CCR1, arginase-1, and M2-specific cytokines that benefit anti-tumor activity, while DeCF-IL6 and N73Q-IL6 favored the formation of M1 macrophages. The phagocytosis capacity of cocultured macrophages is also altered by different IL-6 glycoform-secreting cells. On the other hand, the Stat3 activation status induced by IL-6 glycoforms is concordant to our previous results, in which the G-IL6 can induce a persistant Stat3 activation that is not observed in the DeCF-IL6- and N73Q-IL6-induced macrophages. We subsequently examined the distribution and polarization of TAMs in in vivo xenograft tumor model by both subcutaneous and intravenous injection of the IL-6 glycoform-overexpressing cells. Higher proportion of M2 TAM was attracted by the N73Q-IL6 tumor than other glycoform tumors. Together, we report the presence of specific IL-6 glycoforms secreted from lung cancer cells. Moreover, the glycosylation on IL-6 changes its activity on the polarization of TAMs, suggesting the pivotal role of soluble factor(s) in orchestrating the tumor microenvironment of lung cancer.

#4158

A distinct CD8+ tumor infiltrating lymphocyte subset is associated with high TIL density in patients with lung cancer.

Anusha-Preethi Ganesan,1 Eva M. Garrido-Martin,2 Oliver Wood,3 Toby Mellows,3 Serena Chee,3 Aiman Alzetani,4 Grégory Seumois,1 Tilman Sanchez-Elsner,2 Christian H. Ottensmeier,3 Pandurangan Vijayanand1. 1 _La Jolla Institute for Allergy & Immunology, La Jolla, CA; _2 _Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine University of Southampton, Southampton, United Kingdom;_ 3 _Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, United Kingdom;_ 4 _University Hospital Southampton, Southampton, United Kingdom_.

Purpose: High tumor infiltrating lymphocyte (TIL) density predicts for good prognosis in several cancers, however the degree of infiltration by TILs varies significantly even between individuals with the same cancer. An understanding of the molecular basis for this heterogeneity could lead to novel biomarkers and immunotherapeutic strategies. In this study, we performed transcriptomic profiling of the CD8+ TIL in a well-characterized cohort of patients with lung cancer.

Method: RNA sequencing was performed using CD8+ T cells flow-sorted from freshly resected lung tumors and matched normal lung tissue obtained from 36 untreated patients with stages I-III non-small cell lung cancer (NSCLC). CD8+ TIL density was quantified by immunohistochemistry using tissue microarrays.

Results: Principal component analysis (PCA) showed that CD8+ T cells isolated from each group (lung tumor vs normal lung) clustered together, forming spatially separated subsets reflecting their highly divergent transcriptional states. Pairwise comparison of RNA-Seq data from lung tumor vs normal lung tissue resulted in a total of 501 differentially expressed genes (DEGs; Benjamini-Hochberg adjusted P < 0.05 and greater than 2 fold change change in expression). Of these, 184 genes were up-regulated in CD8+ T cells from tumors while 317 genes were down-regulated, relative to normal tissue.. CD8+ TILs in lung tumor expressed a pattern of the canonical exhaustion molecules and this pattern was distinct from that observed in exhausted T cells from chronic viral infection models. CTLA4, PDCD1, 4-1BB, LAG3 and TIM3 were up-regulated while KLRG1 and IL7R were down-regulated. Exhausted CD8 T cells in LCMV model are known to up-regulate PTGER2 and CD160, but these molecules were down-regulated in CD8+ TIL. CD8+ TILs showed co-expression of the up-regulated exhaustion molecules, however there was marked heterogeneity in their expression in different patients. Unexpectedly, tumors that were characterized by high expression of PD1 in their CD8+ TILs also exhibited high TIL density. CD8+ TILs with 'PD1 Hi' phenotype appeared to have distinct properties including differences in activation, homing, chemokine production and transcription factors.

Conclusion: CD8+ TILs within lung tumors not only display transcriptional reprogramming but also are characterized by distinct TIL subsets that may, at least in part, contribute to the heterogeneity in TIL density. Targeted manipulation of this process may lead to new therapeutic opportunities.

#4159

Characterization of the T-cell receptor (TCR) repertoire in extensive disease small cell lung cancer (ED SCLC).

Maen Hussein,1 Sharon Wilks,2 Marc Monte,3 Donald A. Richards,4 Jerome H. Goldschmidt,5 David Waterhouse,6 Lisu Wang,7 Saumya Pant,7 Erik Yusko,8 Ryan O. Emerson,8 Darin M. Taverna,9 Kaushal Desai,7 Spyro Mousses,9 Zhenhao Qi,7 Jason D. Hipp,7 Harlan Robins,8 Kimary Kulig,7 Cory Batenchuk7. 1 _Florida Cancer Specialists, Lady Lake, FL;_ 2 _Cancer Care Center of South Texas, TX;_ 3 _Clopton Clinic of Jonesboro, Inc, AR;_ 4 _Texas Oncology – Tyler, TX;_ 5 _Blue Ridge Cancer Care, VA;_ 6 _Oncology Hematology Care, OH;_ 7 _Bristol-Myers Squibb, NJ;_ 8 _Adaptive Biotechnologies, WA;_ 9 _Systems Imagination, Inc, AZ_.

Background: The current study explores T-cell (TC) clonality and molecular factors associated with this metric. Tumors employ multiple mechanisms to evade antitumor immune responses. One process involves TC inhibition via upregulation of immune checkpoint (IC) ligands in the tumor microenvironment (TME). Gradual upregulation of inhibitory receptor at their cellular surface results in a decreased capacity to proliferate and activate cytotoxic pathways against tumor cells presenting antigenic peptides.¹ In melanoma, this subset of exhausted TCs has been described as highly clonal, where the majority of PD-1-expressing TC population shares the same TCR sequence specific against the same antigenic fragment.² Previous studies have demonstrated that a clonal TCR repertoire appears to be associated in part with therapeutic responses during IC blockade.³

Methods: We performed TCR sequencing on 82 blood and 73 archival tumor tissue samples collected from ED SCLC patients (pts) in an ongoing longitudinal cohort study in US community oncology practices. Of these, 82 blood and 48 tissue samples had sufficient material available to quantify a clonality metric. To quantify TC abundance as a fraction of total nucleated cells, 82 blood and 58 tissue samples had sufficient material available.

Results: Within the subset of 48 tumor samples, a more clonal (ie, less diverse) TCR repertoire was associated with less necrosis (P≤0.012) and lower levels of inflammatory cell infiltration in the local TME (P≤0.021). When pts were divided into 2 equal groups according to the median clonality level, pts with a less clonal TCR repertoire (n=24/48) who were treated with non-immune-targeted therapy trended toward a longer overall survival (OS; 446 vs 301 days; P≤0.039). In contrast, the percentage of TCs in the TME did not correlate with improved survival (P≤0.412), necrosis (P≤0.131), and inflammation (P≤0.615). This observation differs from results in melanoma describing the impact of IC blockade where pts responding to therapy were associated with a more clonal TME TC population and increased CD8 TCs in the tumor compartment and at the invasive margin.³ In blood, while a less clonal TCR repertoire was associated with a similar but non-significant trend toward longer survival (P≤0.148), pts with increased TC abundance had longer OS (P≤0.025). No association was observed between clonality in TME and clonality (P≤0.571) or TC abundance (P≤0.965) in blood.

Conclusion: We hypothesize that a diverse TCR repertoire in the TME and increased peripheral TC abundance are 2 predictors of longer OS in ED SCLC. To further explore factors that may influence TC responses in ED SCLC, the current TCR sequencing results will be integrated with transcriptome and whole genome sequencing analyses.

References

1. Wherry EJ. Nat Immunol. 2011;12:492-99.

2. Gros A, et al. J Clin Invest. 2014;124:2246-59.

3. Tumeh PC, et al. Nature. 2014;515:568-71.

#4160

Prognostic correlation of tumor-infiltrating lymphocytes (TILs) and cancer associated fibroblasts (CAFs) in patients with human esophageal carcinoma.

Takuya Kato, Kazuhiro Noma, Yuki Katsura, Hajime Kashima, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. _Okayama University, Okayama, Japan_.

Backgrounds: Cancer associated fibroblasts (CAFs) are thought to play an essential role in cancer invasion, migration, metastasis, and tumor immunosuppression. However, there has been still a little evidence of the correlation of tumor immunosuppression and CAFs in human esophageal carcinoma. The other hand, as like rising of PD-1 antibody targeting therapy to tumor immunosuppression have been shown dramatic cure responses in melanoma and now ongoing to other malignancies. Tumor-infiltrating lymphocytes (TILs) are considered typically to represent a host immune response against carcinoma. In many types of tumors TILs have been shown their strong correlation to patient's clinicopathological features. In this study, we evaluated the prognostic correlation of CAFs and TILs, which are classified respectively in tumor-associated CD8+ cytotoxic T lymphocytes (CTL) and FoxP3+ regulatory T cells (Treg) in surgically resected esophageal carcinoma.

Materials and methods: Total 58 cases with esophageal carcinoma in our institution were evaluated for the presence of CAFs and TILs by immunohistochemistry. TILs of CTL and Treg were calculated each in the intratumoral and the peripheral tissues, and the cutoff for subgroups was defined at the median value. CAFs were defined as fibroblasts expressing alpha smooth muscle actin (α-SMA), and evaluated with the α-SMA scoring by using "Area Index", which is calculated by imageJ. TILs and CAFs were assessed for the associations with pathological invasion depth (pT), lymph node metastases (pN), histological types, and disease-free survival (DFS) or overall survival (OS).

Result: In intratumoral tissues, Treg was significantly associated with advanced T stages, and Treg and a CTL/Treg ratio were associated with lymph node metastasis. Higher CTL, higher CTL/Treg ratio and lower Treg were significantly associated with improved DFS and OS in univariate analysis. Furthermore, multivariate analysis demonstrated selected higher CTL as an independent prognostic factor (P = 0.010). On the other hand, in peripheral tissues, CTL, Treg, and CTL/Treg ratio were not correlated with clinicopathological factors or the any prognosis. Additionally the overexpression of CAFs was strongly associated with poor prognosis (P=0.002) and the "Area Index" of α-SMA was inversely correlated with the CTL and CTL/Treg ratio in intratumoral tissues (P=0.029, 0.017). It suggests that CAFs accumulation in tumor tissue is correlated to status of intratumoral immunesuppression.

Conclusion: The CTL in intratumoral tissues are independent prognostic factors, and are considered to play an essential role in tumor immunity. Our results demonstrate that CAFs are significant correlation of immunosuppression in esophageal carcinoma. In the future, targeting CAFs therapy itself might improve tumor immunosuppression, and there is possibility to prolong a prognosis.

#4161

Bortezomib enhances expression of effector molecules in antitumor CD8+ T lymphocytes by modulating Notch-NF-kB-miR-155 crosstalk.

Ariana N. Renrick, Menaka C. Thounaojam, Duafalia F. Dudimah, Portia Thomas, Samuel T. Pellom, Roman V. Uzhachenko, Anil Shanker. _Meharry Medical College, Nashville, TN_.

The immunosuppressive tumor microenvironment usurps host antitumor immunity by multiple mechanisms including interference with the Notch system, which is important for various metazoan cell fate decisions and hematopoietic cell differentiation and function. We observed that treatment with the proteasome inhibitor bortezomib in mice bearing various solid tumors resulted in an upregulated expression of various Notch signaling components in lymphoid tissues, thereby increasing CD8+T-lymphocyte IFNγ secretion and expression of effector molecules, perforin and granzyme B, as well as the T-box transcription factor eomesodermin. Bortezomib also neutralized TGFβ-mediated suppression of IFNγ and granzyme B expression in activated CD8+T-cells. Of note, bortezomib reversed tumor-induced downregulation of Notch receptors, Notch1 and Notch2, as well as increased the levels of cleaved Notch intracellular domain (NICD) and downstream targets Hes1 and Hey1 in tumor-draining CD8+T-cells. Moreover, bortezomib promoted CD8+T-cell nuclear factor-κB (NF-κB) activity by increasing the total and phosphorylated levels of the IκB kinase and IκBα as well as the cytoplasmic and nuclear levels of phosphorylated p65. Even when we blocked NFκB activity by Bay-11-7082, or NICD cleavage by γ-secretase inhibitor, bortezomib significantly increased expression of Notch Hes1 and Hey1 genes as well as perforin, granzyme B and eomesodermin in activated CD8+T-cells. Data suggest that bortezomib can rescue tumor-induced dysfunction of CD8+T-cells by its intrinsic stimulatory effects promoting NICD-NFκB crosstalk. We are also elucidating components of microRNA regulation affecting NICD-NFκB crosstalk. Our preliminary data suggest that miR-155 plays a role in bortezomib-induced regulation of T cell function. These findings provide novel insights on using bortezomib not only as an agent to sensitize tumors to cell death, but also to provide lymphocyte-stimulatory effects, thereby overcoming immunosuppressive actions of tumor on anti-tumor T-cell functions.

#4162

Identifying T lymphocytes in IHC-stained tissues independently of CD3+ staining using morphometric features extracted by image analysis.

Nathan T. Martin, Joshua Black, Famke Aeffner, Logan Cerkovnik, Jasmeet Bajwa, Staci J. Kearney, Crystal Pulliam, A.J. Milici, Joseph Krueger. _Flagship Biosciences, LLC, Westminster, CO_.

Assessments of leukocyte populations in the context of cancer tissues are typically determined by staining for leukocyte subtype markers in formalin fixed tissues. This requires the identification, categorization, and localization of multiple leukocytes within tissue context utilizing multiple markers. Meeting these demands are challenging due to technical constraints on the number of individual markers which can be visualized or scored in a single slide, and the complexity of staining observed. The use of multispectral imaging with fluorescent multiplexed markers has better enabled the assessment of multiple markers in a single tissue section. However, these wet chemistry and image capture technologies are very complex, and can only be successfully implemented with significant laboratory infrastructure, specialty equipment, and experienced resources. For these reasons, this approach is not widely implementable in the clinical pathology laboratory setting, where such tests are oriented to a companion diagnostic utility. Approaches which rely on widely adopted chromogenic immunohistochemistry (IHC) staining are preferred in the clinical laboratory setting, but the number of assayable markers is limited to 1-3 unique markers in a single tissue. In order to create a useful approach that could be implemented in existing clinical laboratory workflow, Flagship Biosciences has developed an approach for deriving the complex endpoints often necessary in immuno-oncology studies which rely on 1-3 chromagenic stains and computer interpretation of the tissue using only hematoxylin counterstain to identify T-lymphocytes. In a proof-of-concept study, we utilized our Tissue Image Analysis (TIA) tools to identify morphometric parameters which could identify T-lymphocytes, independent of staining for the T-lymphocyte marker CD3. A cohort of non-small cell lung cancer (NSCLC) tissues was stained by CD3 IHC, and both CD3 and isotype-stained tissues were analyzed with Flagship's CellMap™ software to capture the morphometric and staining features of cells in the tissues. The morphometric features which characterized CD3+ cells were used to approximate the T-lymphocyte population frequency in the isotype-stained tissues. This T-lymphocyte classification scheme was defined based on hematoxylin staining alone, and accuracy of T-lymphocyte classification was verified by CD3 staining. Based on this study, the method described herein could be utilized to reasonably estimate the frequency of T-lymphocyte subsets (e.g. CD4+, CD8+, etc.) or different marker-positive leukocyte (e.g. macrophages) subsets relative to the total T-lymphocyte population without an additional T-lymphocyte marker such as CD3. The approach could, therefore, provide an added dimension of analysis for tissues stained by IHC without adding complexity to the wet assay by necessitating a marker for T-lymphocytes.

### Mechanisms of Tumorigenesis in Animal Models of Cancer 2

#4163

Molecular and physiological effects of splicing factor mutant U2AF1 in human lung cell lines and in mice.

Dennis Fei,1 Hayley Motowski,2 Sameer Prasad,2 Jovian Yu,2 Robert Bradley,3 Harold Varmus1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _National Human Genome Research Institute, MD;_ 3 _Fred Hutchinson Cancer Research Center, WA_.

The splicing factor gene, U2AF1, is commonly mutated in myeloid neoplasms and occasionally in solid tumors such as lung adenocarcinomas (LUADs), yet the molecular and physiological consequences of U2AF1 mutations are not well understood. Through genome editing of the endogenous locus, we created a common U2AF1 mutant allele (U2AF1-S34F) in an immortalized human bronchial epithelial cell line, and we inactivated the U2AF1-S34F allele in two LUAD cell lines that naturally harbor this mutation. Cells expressing U2AF1-S34F deployed altered splicing of many transcripts, and they displayed a S34F-characteristic sequence pattern at the proximal 3' splice site, as previously reported. Remarkably, S34F-associated alternative splicing patterns remained unaffected by changes in the amount of mutant U2AF1 protein, as long as the ratio of mutant and wild-type U2AF1 remained constant, as evidenced by RNA interference-mediated knockdown of the expression of both mutant and wild-type alleles. On the other hand, perturbing the ratio of mutant and wild-type U2AF1, either through overexpression or allelic-specific inactivation of wild-type U2AF1, greatly affected S34F-associated splicing. These results suggest that the ratio of mutant and wild-type U2AF1, rather than the absolute amount of either protein, is critical in controlling splicing outcome. Expression of U2AF1-S34F does not cause cell transformation and LUAD cells with endogenous U2AF1-S34F expression are not dependent on the U2AF1-S34F allele. Instead, wild-type U2AF1 is absolutely required for cell survival in the presence of the mutant allele. To study the effect of U2AF1-S34F in vivo, we created transgenic mice carrying a conditional mutant allele, MG-S34F, at the endogenous U2af1 locus. By crossing the MG-S34F mice with mice carrying a Mx1-Cre transgene and treating the progeny with poly IC, we activated MG-S34F by Cre-mediated recombination to express U2af1-S34F in bone marrow cells. This resulted in cytopenia in multiple blood cell lineages. Similar results were observed in recipient mice following non-competitive transplant. Bone marrow cells expressing U2af1-S34F were competitively disadvantaged in repopulating the hematopoietic system in irradiated mice, suggesting defective hematopoietic stem cell function, and we are currently assessing the long-term impact of U2af1-S34F expression on the incidence of myeloid neoplasms.

#4164

Synergism between NEDD9 overexpression and loss of PTEN and INK4A/ARF in melanoma tumorigenesis.

Kristen S. Hill, Xue Wang, Youngchul Kim, Jane L. Messina, Minjung Kim. _Moffitt Cancer Center, Tampa, FL_.

The fact that melanoma, the most aggressive form of skin cancer, is driven by multiple combinations of genetic lesions has supported the use of genetically engineered mouse (GEM) models in validating the etiological and biological roles of oncogenes and tumor suppressors. Most of the currently studied mouse melanoma models are driven by RAS/RAF/MAPK activation via activating RAS or RAF mutations in combination with loss of INK4A/ARF or PTEN; therefore, BRAF/NRAS wild-type melanoma remains less well characterized and the overall survival of patients with metastatic BRAF/NRAS wild-type melanoma remains poor.

In this study, we investigated the hypothesis that NEDD9 could synergize with the loss of PTEN and INK4A/ARF in melanoma tumorigenesis. This hypothesis is based on our observation of an inverse correlation between PTEN expression and levels of NEDD9 gene expression in human melanoma samples. NEDD9 (neural precursor derived, developmentally downregulated gene 9) was previously identified by us as a target of a recurrent focal amplification, associated with acquisition of metastatic potential in the RAS-Ink/Arf model. We have shown that NEDD9 is up-regulated in 35~50% of metastatic melanomas and enhances proliferation and invasion. First, we demonstrated that NEDD9 cooperated with PTEN loss in Ink4a/Arf-/- melanocytes to enhance cell proliferation, anchorage independent growth, and invasion in vitro. Additionally, when melanocytes were injected into nude mice either subcutaneously or intravenously, NEDD9 overexpression facilitated increased tumor growth and lung seeding, respectively. Further analysis showed that NEDD9 expression correlated with increased phosphorylation of ERK, SFKs (Src family kinases), AKT2, and STAT3. Of these pathways we have demonstrated through the use of small molecule inhibitors that both ERK and AKT2 signaling are required for the enhanced anchorage independent growth observed in NEDD9 overexpressing melanocytes. In addition, we generated a mouse model based on loss of PTEN and INK4A/ARF with or without NEDD9 upregulation. To do this, we generated a tet-inducible NEDD9 allele that carries a doxycycline-responsive, melanocyte-targeted NEDD9. These mice were crossed with a strain containing floxed PTEN and INK4A/ARF in combination with a melanocyte-targeted CreERT2. The development of cutaneous and ocular melanomas in these mice was enhanced by NEDD9 induction. This study will generate a body of knowledge for the in vivo roles of NEDD9 in melanoma tumorigenesis and identify signaling pathways that could be therapeutically targeted to treat patients with BRAF/NRAS wild-type melanoma

#4165

Myc vs. Akt therapy in RapidCap, a GEM model for metastatic prostate cancer.

Carlos E. Stahlhut,1 Alexandra J. Ambrico,1 Kaitlin E. Watrud,1 Hyejin Cho,2 Lily Wang,3 Jun Qi,2 Lewis C. Cantley,4 James Bradner,5 Lloyd Trotman1. 1 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Weill Cornell Medical Center, New York, NY;_ 4 _Weill Cornell Medical College, New York, NY;_ 5 _Dana-Farber Cancer Institute; Department of Medicine, Harvard Medical School, Boston, MA_.

We describe our efforts to determine the effect of MYC and PI3K inhibition in a genetically engineered mouse model of naïve and castration-resistant metastatic prostate cancer. Metastasis is a major driver of mortality and morbidity in prostate cancer, the most common cancer type in men and the second leading cause of cancer-related deaths in the western world. To recapitulate the metastatic process in genetically engineered mice, we have developed the RapidCaP system, which uses prostate-directed viral infection to trigger focal loss of Pten and Trp53. Using luminescence and fluorescence, the RapidCaP system allows us to track disease progression in real-time and isolate metastases in distant tissues. This system provides a powerful platform for the identification and validation of candidate driver genes and for the efficient testing of novel therapeutics. Using this system, we have recently identified Myc as a critical, spontaneously activated driver in Pten-negative metastatic prostate cancer. Preliminary trials indicate that the Myc-suppressing Brd4 inhibitor JQ1 is ineffective towards primary disease, but induces metastatic regression. This specificity correlates with a switch from AKT-driven primary disease to MYC-driven metastatic disease. Our current efforts seek to determine how, when and why targeting of Myc using JQ1 and the PI 3-Kinase pathway using NVP-BKM120 can be best used to treat naïve and castration-resistant metastatic prostate cancer. In addition, we are examining changes to the transcriptome and epigenetic alterations that characterize Pten-negative metastatic prostate cancer, with the goal of understanding the mechanisms of progression toward a metastatic state and establishing new therapeutic targets.

#4166

Role of calreticulin in the spontaneous development of lung adenocarcinoma: Evidence from a novel transgenic mouse model.

Nasrin Mesaeli, Hamid Massaeli, Divya Viswanathan, Dhanya Pillai, Mercy Anna Thomas. _Weill Cornell Medicine Qatar, Doha, Qatar_.

Lung cancer is the most common cause of cancer related deaths in both men and women worldiwide. The overall 5 year survival rate of lung cancer ranges between 5-15% (depending on the subtype). Based on histopathological criteria, lung cancer is subdivided into two main subtypes: small cell lung cancer (15%) and non-small cell lung cancer (NSCLC 85%). Adenocarcinoma a subtype of NSCLC is the most common type in both sexes, and it has a poor response to chemotherapy. We recently developed a transgenic mouse model of lung adenocarcinoma by overexpressing calreticulin (CRT) under the control of Tie2-promotor. The main phenotype of these mice is high incidence of metastatic lung adenocarcinoma similar to human patients. Immunohistological analysis showed these tumors to be SP-C+ve and CC10-ve phenotype. At the initial stages most of cells in the hyperplegic area and adenoma stained positive for exogenous CRT and HSCs markers, and as the tumor differentiated to carcinoma there were a significant loss of exogenous CRT and HSCs markers and increased expression of SP-C.

The aim of our current project was therefore to identify the mechanism of development of metastatic lung adenocarcinoma in this mouse model. To address this objective we carried out microarray and quantitative proteomic analysis of tissue and cell lines isolated from lungs of this transgenic mouse model.

Microarray analysis showed activation of cluster of genes involved in cellular development, growth and proliferation. Some of these pathways include genes invlved in VEGF signal transduction pathways and Epherin receptor signaling, at the same time there were decreased expression of cell-cell interaction and communication (needed for metastasis). At the protein level we observed a significant increase in proteins involved in tumor progression, invasion and metastasis. In addition we observed a significant change in the expression of genes invloved in lipid metabolism (both at protein and RNA level) in the lung of these mice. The change in the expression of lipid metabolizing genes suggest the metabolic shift needed during tumor progression.

In conclusion, our data suggests that CRT as an endoplasmic reticulum chaperone and a regulator of intracellular calcium homeostasis affects multiple genes invivo resulting in the development of tumor.

This research was funded by Qatar National Research Fund (NPRP4-043-3-016).

#4167

Ultraviolet radiation cooperates with individual oncogenes to drive melanomagenesis through distinct molecular mechanisms.

Amaya Viros,1 Malin Pedersen,2 Simon J. Furney,1 Maria Romina Girotti,1 Grazia Saturno,1 Elena Galvani,1 Berta Sanchez-Laorden,1 Kate Hogan,1 Charlotte Ng,3 Jorge S. Reis-Filho,3 Paul Lorigan,4 Martin Cook,1 Richard Marais1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _Institute of Cancer Research, London, United Kingdom;_ 3 _Memorial Sloan Kettering, New York, NY;_ 4 _The Christie NHS Foundation Trust, Manchester, United Kingdom_.

The contribution ultraviolet radiation (UVR) makes to genetically distinct melanoma subtypes is unclear. Melanoma driven by oncogenic BRAF or oncogenic NRAS differ in their epidemiological, clinicopathological and genetic features. NRAS melanoma in humans occurs in older individuals and presents at sites most exposed to UVR damage such as the head and neck.

In this study we show excessive sun exposure and NRAS-driven melanoma can co-occur by juxtaposing the epidemiological, clinical and genetic characteristics of a G12DNRAS-driven human melanoma case subject to extreme UVR exposure. To model the interaction of UVR with specific oncogenes and mimic human disease, we used mouse melanoma models driven by V600EBRAF or G12DNRAS. We previously reported that UVR cooperates with V600EBRAF by targeting the tumour suppressor TP53. In this study we confirm that UVR cooperates with G12DNRAS to drive melanoma and, as observed in humans, murine UVR-G12DNRAS melanoma occurs in older animals that accumulate higher lifetime exposure to UVR. Furthermore, we find murine UVR-G12DNRAS melanomas are histologically and genetically heterogeneous, and distinct to UVR-V600EBRAF murine and human melanomas. UVR-G12DNRAS melanomas present more UVR-induced mutations, a longer latency to tumour development and secondary driver mutations distinct from UVR-V600EBRAF melanoma. We investigated whether UVR cooperates equally with other RAS isoforms, and exposed G12DKRAS animals to UVR. These animals presented a different latency to melanoma development and specific cooperating secondary targets distinct from UVR-V600EBRAF and UVR- G12DNRAS. This implies that different RAS isoforms activate oncogenic pathways differently. Critically, UVR-V600EBRAF, UVR-G12DNRAS, UVR-G12DKRAS tumors present similar mutation rates when UVR exposure is held constant, which suggests that the differences in tumour latency might be explained if melanomas driven by different oncogenes require varying numbers of subsequent co-operators.

Finally, we present preclinical evidence showing how targeting the UVR-induced mutations found in the "long tail" of UVR-related mutations in NRAS melanoma can provide therapeutic options for NRAS mutant melanoma patients.

The insight gained begins to provide a molecular explanation for distinct associations between UVR and individual oncogenes in melanomagenesis, and we show how UVR-induced damage can be exploited to stratify patients for personalised therapy approaches.

#4168

Mig-6 ablation cooperates with oncogenic Kras in promoting mouse lung tumorigenesis.

Jian Liu,1 Sung-Nam Cho,2 Nili Jin,2 Seyed Javad Moghaddam,3 Jennifer Gilbert,4 Ignacio Wistuba,5 Francesco J. DeMayo1. 1 _NIEHS (National Institute of Environmental Health Sciences), Research Triangle Prk, NC;_ 2 _Baylor College of Medicine, TX;_ 3 _University of Texas M.D Anderson Cancer Center, TX;_ 4 _Maynooth University Department of Biology, Ireland;_ 5 _University of Texas, M.D. Anderson Cancer Center, TX_.

Lung tumorigenesis is a stochastic multistep process, during which different gene mutations accumulate, and Kras is an oncogene with the most frequent mutation in the Caucasian lung adenocarcinoma. Although Mig-6 (mitogen-inducible gene 6) has been shown to be a tumor suppressor gene in lung, skin and uterus, the interplay between Mig-6 and Kras in lung tumorigenesis remains elusive. Here, we found Mig-6 expression was down-regulated in oncogenic KrasG12D-induced lung tumors and Mig-6d/dKrasG12D mice showed an earlier onset of pulmonary tumor development and a significantly reduced life span as compared to KrasG12D mice though no obvious lung phenotype was observed in Mig-6d/d mice. Meanwhile, ablation of Mig-6 can change the lung epithelial cell fate in KrasG12D mice. Furthermore, lung tumor tissues of Mig-6d/dKrasG12D mice showed decreased cellular apoptosis, elevated EGF/Akt signaling and increased severity of inflammation as compared to those of KrasG12D mice. Thus, ablation of Mig-6 cooperated with oncogenic Kras in promoting lung tumorigenesis and this novel murine model of lung cancer can be applied for future study.

#4169

Systematic in vivo analysis of PI3K pathway aberrations in a mouse model of invasive lobular carcinoma.

Martine Van Miltenburg. _Netherlands Cancer Inst., Amsterdam, Netherlands_.

Invasive lobular carcinoma (ILC) is the second most prevalent breast cancer subtype, which develops in the milk-producing glands (lobules) and metastasizes to distant organs. A key feature of ILC is loss of the CDH1 gene, encoding the E-cadherin cell-cell adhesion receptor. Though E-cadherin is thought to be a tumour suppressor, it is interesting to note that mammary-gland specific deletion of E-cadherin does not lead to tumour development indicating that other factors are necessary for the induction of ILC. Recent data from the cancer genome atlas reveals that besides E-cadherin loss, mutations in PIK3CA, and to a lesser extent PTEN, TBX3, and FOXA1 were found as ILC enriched features. These data implicate PI3K signaling as a driving force in ILC development. Using our in-house established GEMM-ESC strategy, we have developed innovative mouse models for ILC to study novel aspects of the functional interplay between E-cadherin and PI3K in ILC development. We have discovered an intriguing new interplay between these components in which E-cadherin loss suppresses growth of normal mammary epithelium, which appears to involve senescence, whereas it effectively accelerates tumor growth in a PIK3CAmutant background. Therapeutically, PIK3CAE545K and AKTE17K ILCs were sensitive to AZD8055, an mTOR inhibitor, and BKM120, a PI3K 110α inhibitor in vivo, indicating that PI3K signaling is required for their tumor maintenance. Currently, we are dissecting the sensitivity of ILCs displaying PIK3CAE545K or AKTE17K mutations, and PTEN loss, to a large panel of inhibitors, which will likely provide new clinical options.

#4170

A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1.

Ya D. Gao,1 Maurice de Wit,1 Eduard A. Struys,2 Martine L.M. Lamfers,3 Gajja S. Salomons,2 Peter A.E. Sillevis Smitt,1 Pim J. French1. 1 _Department of Neurology, Erasmus Medical Center, Rotterdam, Netherlands;_ 2 _Department of Clinical Chemistry, VU University Medical Center, Amsterdam, Netherlands;_ 3 _Department of Neurosurgery, Erasmus Medical Center, Rotterdam, Netherlands_.

The gene encoding Isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas, chondrosarcomas, acute myeloid leukemia and intrahepatic cholangiocarcinomas. As there are few in-vivo model systems for IDH-mutated tumors we have created a transgenic zebrafish (Danio rerio) model expressing mutant IDH1. We have chosen the zebrafish as a model because they are transparent (allowing monitoring of the transgene in-vivo) and drug screening assays are straightforward (they are simply added to the aquarium).

IDH1R132H and IDH1R132C, mutations found in tumors that both produce D-2-hydroxyglutarate (D2HG) instead of alpha ketoglutarate, were cloned into an expression construct that is driven either by the Nestin or GFAP promoter. IDH1G70D (a loss of function mutation), IDH1wildtype and GFP were used as control. All IDH1 constructs were fused to GFP for visualization. These constructs were injected into fertilized zebrafish eggs at the one-cell stage.

All of our transgenic zebrafish lines remain healthy and produce offspring. Transgene expression was detected in the mid/hindbrain of the central nervous system by immunohistochemistry, Western blot and RT-QPCR. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing control constructs (IDH1wildtype, IDH1G70D or GFP). In contrast to reported, we failed to detect any differences in hydroxymethyl cytosine (the first step in DNA-demethylation) and mature collagen IV levels between wildtype and mutant IDH1 transgenic fish. We also performed microinjections on fertilized eggs to screen for early developmental effects of IDH1R132H and IDH1R132C. Despite of the high expression of the transgene, no developmental effects were found. Our observations therefore suggest that elevated levels of D2HG are insufficient to initiate tumorigenesis or other phenotypic effects in our fish. Treatment of the transgenic zebrafish with an IDH1 mutant inhibitor, AGI-5198, resulted in a reduction in the D2HG level in the mutant zebrafish. The L2HG level was not affected by AGI-5198. As no tumors were formed in our transgenic zebrafish lines, we backcrossed them with Tp53 mutant fish. Analysis of these lines is currently being performed.

In summary, we have generated a transgenic zebrafish model system that expresses mutated IDH1 that can be used to study effects of mutant IDH1 (or elevated levels of D2HG) in vivo and can be used for drug screening.

#4171

Lipid metabolism-independent role of apolipoprotein (E) levels in colon carcinogenesis through a regulating inflammation and active β-catenin.

Abdelmetalab F. Tarhuni,1 Ali H. El-Bahrawy,1 Hogyoung Kim,1 Youssef Errami,1 Mohamed A. Ghonim,1 Mohamed Alwan,2 Samar M. Hammad,2 Venkat N. Subramaniama,1 Ramadan A. Hemeida,3 Amarjit S. Naura,1 Hamid A. Boulares1. 1 _LSUHSC/NO, New Orleans, LA;_ 2 _MUSC, Charleston, SC;_ 3 _Al-Azhur University, Assuit, Egypt_.

Apolipoprotein E (ApoE) is critically important for cholesterol metabolism and lipid homeostasis. Polymorphisms in the ApoE gene have been implicated in the development of colon cancer and these polymorphisms may be used as risk indicators for the disease. Accruing evidence suggests new lipid metabolism-independent functions for ApoE both in inflammation and cardiovascular diseases. Ironically, ApoE was shown to up-regulate cyclooxygenase-2 in vascular smooth muscle cells and given the critical role of the COX-2 in colon carcinogenesis, these results suggest a pro-colon cancer function for the lipoprotein. In the present study, we show that a mere deletion of ApoE promotes systemic inflammation as assessed by TNF-α level in sera of mice under a regular diet. Concomitantly, TNF-α mRNA levels were significantly higher in the colon of ApoE-/- mice suggestive of localized inflammation. In contrast to smooth muscle cells, in vitro studies with primary colon epithelial cells (CECs) show that ApoE-deficiency substantially increased COX-2 expression in response to oxidized (ox)LDL both at the protein and mRNA levels. OxLDL-induced expression of COX-2 in WT CECs was highly sensitive to PP2 treatment, a Src inhibitor, and to LY294002, a pan-PI3K inhibitor. Surprisingly, while oxLDL-induced COX-2 expression was sensitive to PI3K inhibition, its sensitivity to Src inhibition was minimal suggesting that ApoE deficiency may promote COX-2 expression through a Src-independent mechanism. Similarly to COX-2, ApoE-deficiency also enhanced basal and ox-LDL-induced expression of MCP-1, IL-1β, ICAM-1, and VCAM-1. Using a cohort of colon cancer patients and healthy controls, we show that the overall levels of ApoE in sera of colon cancer patients were significantly lower than those of healthy controls (p=0.0028). We have utilized ApcMin mice, well-characterized mouse model of intestinal tumor. Using this genetic background, we have generated ApoE+/- and ApoE-/- lines which were utilized in this study. We found that inhibition of ApoE through gene heterozygosity led to ~50% reduction in the level of ApoE protein and a significant increased the number tumors in these mice compared to their wildtype counterparts. These changes in tumor burden were accompanied by slight changes in the lipid profiles of the ApoE+/- mice. Surprisingly however, when examining the results of ApoE-/-, we found that the tumor burden was not increased over that seen in the ApoE heterozygous mice, but the lipid profiles were elevated dramatically. This suggests that the mechanism through which ApoE affects lipid metabolism is separate from that which is involved in the formation of colon tumors. This aggravation of the tumor burden may be associated with a stimulus-independent increase in the levels of the active form of β-catenin. These results suggest that ApoE deficiency may be informative for the risk of developing colon cancer.

#4172

A role for RAD52 as a lung cancer susceptibility gene in the 12p13.33 locus.

Rachel Lieberman, Donghai Xiong, Ming You. _Medical College of Wisconsin, Milwaukee, WI_.

Although 80-90% of lung cancer patients smoke, only about 10% of heavy smokers develop lung cancer. This suggests that although tobacco smoke undoubtedly increases one's risk for lung cancer, genetic risk factors most likely predispose certain individuals. Recent genome-wide association studies identified variation in the recombination repair gene, RAD52, associated with increased lung cancer risk, and particularly with squamous cell lung carcinoma (SCC). SCC development is strongly associated with smoking and individuals who smoke have increased DNA repair, presumably to address ongoing DNA damage from exposure to tobacco smoke. Hence, it fits that the genomic region of 12p13.33 containing RAD52 shows both copy number amplification and an increase in gene expression in SCC tumors compared to normal. Functionally, we have previously demonstrated that overexpression of Rad52 in mouse bronchial epithelial and lung tumor cells leads to an increase in growth rate and that depletion leads to decreased cell growth, senescence, and an accumulation of cells in G2/M.

Our current study focuses on genetic instability within lung tumor progression and how the DNA repair gene RAD52 allows primary tumor cells to become genetically stable enough to be able to invade and avoid apoptosis. C57BL6 wildtype or Rad52-/- mice at 8 weeks of age were randomized into two groups and treated topically with 0.04 M NTCU in 100-microliter drop, twice a week for 40 weeks. Unlike mouse lung adenocarcinomas, mouse SCC does not form visible solid nodules on the surface of the lung. Lungs were fixed and stained with H&E and examined histologically under a light microscope to establish tumor multiplicity and the types of lesions (invasive SCC, SCC in situ, or bronchial hyperplasia/metaplasia). Lung tissue was also stained for TUNEL analysis as well as markers of SCC and proliferation. Our novel findings demonstrate that depletion of Rad52 in vitro generates increased DNA damage and sensitivity to cell death, while knockout of the Rad52 gene in an in vivo mouse model of carcinogenesis decreases murine lung hyperplasia, in situ carcinoma and lung SCC. In addition, knockout appears to increase apoptosis. Thus, our functional studies continue to support genetic findings by revealing that loss of RAD52 can enhance cell death and DNA damage in lung tumor cells. This complementary evidence further strengthens the notion of RAD52 as a potential oncogene and implicates a major role for the process of recombinational repair in determining risk for SCC.

#4173

The oncopig cancer model as a validated model for human hepatocellular carcinoma.

Kwame A. Darfour-Oduro,1 Arun K. De,1 Laurie A. Rund,1 Ron C. Gaba,2 Charles E. Ray,2 Regina M. Schwind,3 Kyle M. Schachtschneider,1 Kuldeep B. Singh,4 Lawrence B. Schook1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _University of Illinois Hospital and Health Sciences Center, Chicago, IL;_ 3 _University of Illinois Cancer Center, Chicago, IL;_ 4 _Department of Veterinary Pathobiology, University of Illinois, Urbana, IL_.

Human hepatocellular carcinoma (HCC) is the fifth most common cancer globally and is the third most common cause of cancer-related deaths worldwide, accounting for more than 600,000 deaths each year. While the information gained from human clinical trials may enhance the efficacy of HCC treatment, such trials provide limited capability for investigation into the processes contributing to procedural effectiveness and disease progression. Immunological, genetic and physiological differences between rodents and humans represent major limitations for the use of rodent based cancer models in HCC studies. Pigs represent an attractive model given that they share many genetic, anatomical, and physiological similarities with humans. In a previous study, we demonstrated the functionality of a porcine inducible model of cancer (Oncopig; Schook et. al., 2015). In order to develop and validate the Oncopig HCC model, we established primary (control) hepatocyte cultures from 3 individual Oncopigs and transformed them with Cre recombinase (HepCre). Within a week the activated cells began to proliferate, taking on transformed morphologic characteristics. Transcriptional analysis confirmed both the primary and transformed cells expressed hepatocyte specific marker genes (albumin, HNF4 and G6P). The Cre treated cell lines also expressed oncogenic P53R167H and KrasG12D. Each of the three transformed cell lines were then injected into the livers of SCID mice to verify tumorigenicity. SCID mice (9/9) developed tumors that recapitulated several morphological similarities with human HCC including cellular arrangement and marked pleomorphism. The primary hepatocytes expressed cytokeratin and the HepCre cell lines expressed both cytokeratin and vimentin, indicative of an epithelial-mesenchymal transition, which is a common tumor progression signal observed in human HCC and other highly proliferative carcinomas. Alpha-fetoprotein is often found in the serum of HCC patients and was measurable in the medium of porcine HepCre cells but not primary hepatocytes. Oncopig HCC cells were polygonal with eosinophilic granular cytoplasm as observed in human HCC. Principal component analysis of gene expression data showed clear separation between the primary hepatocytes and HepCre cells. Differential expression analysis revealed upregulation of proto-oncogenes and downregulation of tumor supressor genes in the HepCre cells, as well as enrichment of several pathways that are concordantly dysregulated in human HCC, including the TP53 pathway, fatty acid metabolism pathway and complement and coagulation cascade pathway. These data demonstrate the utility of the Oncopig model in terms of relevance to human HCC and therefore aid in improved detection, treatment, biomarker discovery for liver tumors and other unmet clinical needs.

Reference

Schook et al. A Genetic Porcine Model of Cancer. PLoS One. 2015;10: e0128864

#4174

SPOP mutation drives tumorigenesis in mouse prostate - a novel model of ETS negative prostate cancer.

Mirjam Blattner,1 Deli Liu,1 Dennis Huang,1 Dong Gao,2 Andrea Sboner,1 Yu Chen,2 Mark A. Rubin,3 Christopher Barbieri1. 1 _Weill Cornell Medicine, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 3 _Englander Institute for Precision Medicine, Weill Cornell Medicine, New York, NY_.

Background: Recurrent mutations in SPOP are the most common point mutations in prostate cancer, occurring in about 10% of cases across multiple independent cohorts. F133V is the most frequently (50%) mutated residue. SPOP mutation defines a distinct molecular subclass of prostate cancer, universally negative for ETS rearrangements. No available prostate cancer cell lines harbor endogenous SPOP mutations, making model systems a critical need. Here, we describe a new mouse model with conditional transgenic expression of SPOP-F133V in the mouse prostate.

Experimental Design: We generated a conditional mouse with the SPOP-F133V transgene knocked in to the Rosa26 locus (R26F133V); these mice were crossed with Pb-Cre4 mice to express SPOP-F133V specifically in the prostate. Since PTEN deletion is known to drive prostate cancer progression, we crossed Pb-Cre4; R26F133V mice with Ptenf/f mice to study the impact of SPOP mutation in the background of conditional heterozygous (f/+) and homozygous (f/f) loss of Pten. Organoid lines were derived from transgenic mouse prostates and infected with inducible Cre-ERT2 to serve as in vitro platforms, 2D as well as 3D, for additional studies. RNA-Seq was performed on independently induced samples.

Results: Pb-Cre4;R26F133V mouse prostates showed a minimal histological phenotype, with rare cytological changes of atypical nuclei (p<0.005) in one year old mice. In combination with Ptenf/+, SPOP-F133V expression resulted HG-PIN in 80% (n=6) of mice, age 6 month, compared to only 20% (n=8) in control mice. In addition, the HG-PIN in R26F133V;Ptenf/+ mice showed a distinct phenotype with strong nuclear atypia absent in controls. In the background of Ptenf/f, SPOP-F133V leads to poorly differentiated, invasive cancer (n= 8 out of 9) compared to control mice (Ptenf/f), which displayed only HG-PIN (n=6). Organoids with expression of SPOP-F133V showed increased proliferation and increased ki67 staining. To define signaling pathways controlled by mutant SPOP in the prostate, we performed RNA-seq on mouse organoids expressing SPOP-F133V and controls. We interrogated the gene space nominated by the mouse F133V mutation to the human prostate cancer TCGA transcriptome. Unsupervised clustering demonstrated a highly significant enrichment of ETS negative human prostate cancer (p<2.2x10-16), supporting the relevance of our transgenic models to human prostate cancer.

Summary: Mutation in SPOP causes an early onset of HG-PIN in the prostate of Ptenf/+ mice, and progression to poorly differentiated invasive cancer in Ptenf/f mice. HG-PIN shows a very distinct histological phenotype with strong nuclear atypia. Gene expression in murine prostate organoids expressing SPOP-F133V strongly correlates with human tumors, providing relevance for this novel mouse model in defining the biology and therapeutic vulnerability of this subclass of prostate cancer.

#4175

ERG oncogenic activation leads to the endoplasmic reticulum stress and cell survival mechanisms.

Taduru L. Sreenath,1 Shiela Macalindong,1 Natallia Mikhalkevich,1 Shashwat Sharad,1 Parameet Kumar,1 Denise Young,1 Rishita Gupta,1 Shilpa Katta,1 Ahmed Mohamed,1 Shyh-Han Tan,1 Albert Dobi,1 Gyorgy Petrovics,1 Isabell A. Sesterhenn,2 Charles J. Bieberich,3 Peter Nelson,4 David G. McLeod,5 Valeri Vasioukhin,4 Shiv Srivastava1. 1 _Uniformed Services Univ. of the Health Sci., Rockville, MD;_ 2 _Joint Pathology Center, Silver Spring, MD;_ 3 _University of Maryland Baltimore County, Baltimore, MD;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _Walter Reed National Military Medical Center, Bethesda, MD_.

Introduction: High levels of the ERG expression due to gene fusions (predominantly TMPRSS2-ERG) is frequently detected in prostate cancer (CaP) in Western countries. Better understanding of the roles of the ERG oncogenic functions in CaP initiation and/or progression will enhance its utility as the therapeutic target. Transgenic mice generated to understand role of ERG in the CaP development have shown focal proliferative and dysplastic PIN lesions and adenocarcinoma in older mice. The present study was aimed towards understanding the molecular mechanism underlying the role of ERG in early stages of oncogenic transformation of prostate epithelium.

Methods: Morphological analyses of mouse prostate glands were performed by H&E staining and by electron microscopy. Luminal cell surface markers were evaluated by FACS analysis. Luminal cells were assessed for their potential to proliferate and form spheres by prostate sphere formation assay. Effects of ERG on ER stress and UPR marker proteins were analyzed in transgenic mice prostates and ERG expressing cell culture models. Representative ER stress pathway in ERG-positive human prostate cancer was analyzed by CPDR-Affymetrix GeneChip data from well and poorly differentiated tumors.

Results: Histological examination of ERG-Tg mouse prostates revealed increased luminal cell death due to apoptosis. Subsequently, TEM analysis revealed significant morphological differences such as increased numbers of lysosomes, autophagosomes, concentric membrane bodies with ribosomes, and lipid droplets in the cytoplasm of transgenic secretory luminal epithelial cells. Epithelial cells of ERG-Tg mouse prostates showed increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to induced cell death. ERG-Tg mouse prostate tissues and LNCaP-ERG transfectants showed increased expression of ER stress sensors and UPR proteins. Further, in human prostate tumors, a strong correlation was also observed between expression of the ERG and P4HB/PDI, an ER stress response protein.

Conclusions: A critical of function of ERG in early prostate tumorigenesis may involve ER stress resulting into the activation of UPR, autophagy and cell survival through clonal selection. These observation also define potential new therapeutic targets in CaP-ERG network.

Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and USU-CPDR funds.

#4176

Leptin mediates the anti-breast cancer effects of environmental enrichment.

Grant Foglesong, Wei Haung, Lei Cao. _The Ohio State University, Columbus, OH_.

Obesity affects 35% of U.S. adults and is a leading risk factor for breast cancer in postmenopausal women, but worsens prognosis regardless of menopausal status. Importantly, progression to obesity can largely be slowed or reversed with aggressive lifestyle changes, thus also having the potential to mitigate cancer onset. A mouse model of environmental enrichment (EE) to improve motosensory, cognitive, and social stimulation by increasing physical engagement and social interaction triggers vast improvements in overall health. These include reducing adiposity, promoting the white to brown fat transition, mitigating diet-induced obesity (DIO), and decreasing progression of multiple cancer types including colon cancer and melanoma. We have elucidated the primary mechanism of the EE-induced phenotype to be the activation of the hypothalamic-sympathoneural-adipocyte (HSA) axis, a specific neuroendocrine route in which the brain communicates with adipose tissue. These benefits may also be partially attributable to the sharp drop in serum leptin levels following EE which has been implicated in diminishing tumorigenesis, invasiveness, and metastasis. Here we investigated the effects of EE on breast tumorigenesis in all body mass states and menopause-associated hormone conditions. Our preliminary studies showed that EE delayed the cancer onset in the MMTV-PyMT spontaneous mouse model of breast cancer. In addition the role of leptin signaling in the EE-induced effects on breast tumorigenesis was investigated by utilizing obese models with varied leptin signaling including leptin receptor-defective db/db mice that express extremely high levels of leptin, leptin-defective ob/ob mice that do not express leptin but are leptin-sensitive, and a diet-induced obesity (DIO) model with elevated leptin but intact leptin signaling. These obese mice were housed in EE or control housing until significantly attenuated weight gain was observed followed by implantation of primary PyMT-derived mammary tumor cells. EE was highly effective at reducing adiposity in each of these obesity models, regardless of leptin signaling. However, the effects of EE on mammary tumor progression were dependent on leptin signaling. EE decreased leptin level and inhibited mammary tumor growth in DIO mice. In contrast EE failed to attenuate tumor progression in ob/ob mice in the absence of leptin, suggesting that leptin is a key mediator of the EE anti-cancer effects on breast cancer. The elucidation of mechanisms of EE and the HSA axis-induced improvement in overall health will provide effective prevention as well as therapeutic strategies for metabolic syndromes and associated cancer types.

#4177

Dynamic visualization of cancer cell engraftment into immune compromised zebrafish.

John C. Moore,1 Qin Tang,1 Nora Torres Yordan,2 Timothy Mulligan,3 Finola E. Moore,1 Riadh Lobbardi,1 Ashwin Ramakrishnan,1 Anthony Anselmo,1 Ruslan Sadreyev,1 Jason Berman,4 Robert Liwski,4 Brant Weinstein,3 John Rawls,5 David M. Langenau1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Harvard University, Cambridge, MA;_ 3 _NIH, Bethesda, MD;_ 4 _Dalhousie University, Halifax, Nova Scotia, Canada;_ 5 _Duke University, Durham, NC_.

Cell transplantation into immune compromised mice has transformed our understanding of cancer and is now the gold standard for assessing therapeutic responses in vivo. However, mouse models are expensive and engraftment is often difficult to visualize directly. To overcome these challenges, we have developed immune compromised zebrafish (ICZ) in the transparent casper background using genome editing techniques. We have successfully targeted genes required for immune cell function and are well known to cause immune deficiency in human and mice. To date, we have developed homozygous viable mutants for recombination-activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), janus kinase 3 (jak3), interleukin 2 receptor gamma (Il2rg), zeta-chain (TCR) associated protein kinase 70 (zap70), and forkhead box N1 (foxn1/nude). Gene expression analysis of marrow cells using RNAseq has identified novel transcript changes correlated with loss of specific cell types, and in conjunction with large-scale single cell transcriptional profiling, has identified specific cellular defects associated with T, B, and NK cell loss. For example, homozygous prkdc (SCID) mutant fish lack mature T and B cells, but have intact NK cell signaling. By contrast, il2rg-deficient zebrafish lack T and NK cells. Importantly, these ICZ models accurately recapitulate known human severe combined immune deficiencies and established mouse models that are commonly used for cell transplantation. Thus, it is not unexpected that a subset of zebrafish mutants have reduced immune cell function, permitting engraftment of normal hematopoietic and muscle satellite cells from allogeneic donors. Additionally, we have demonstrated robust and persistent engraftment of fluorescently labeled leukemia, rhabdomyosarcoma, neuroblastoma, and melanoma from a wide range of zebrafish strains. Because mutations have been created in optically-clear, casper-strain zebrafish and cancers are fluorescently labeled, we now have unprecedented access to directly visualize tumor cells at single cell resolution in live animals. To date, we have optimized our models to visualize neovascularization, intratumoral cell heterogeneity, clonal evolution and metastisis. The ability to transplant non-immune matched cell types will likely revolutionize the types and scale of cell transplantation experiments performed in the zebrafish and will likely permit engraftment of mouse and human cells into compound mutant ICZ models in the near future.

#4178

Pigs as a new weapon against cancer: Modeling solid tumors in porcine.

Andrew Diaz,1 Daniel Principe,2 Brian DeCant,1 Paul J. Grippo,1 Laurie Rund,2 Lawrence Schook2. 1 _University of Illinois-Chicago, Chicago, IL;_ 2 _University of Illinois, Urbana-Champaign, IL_.

Widely-used genetically modified mice have revolutionized the field of cancer research by providing reliable in vivo systems with similar, if not identical, molecular alterations. Furthermore, Cre/Lox technology has afforded greater tissue specificity, with many of the developing cancer phenotypes recapitulating those observed in humans. However, despite these strengths, there are a number of notable limitations when comparing mice to humans including small sample sizes, dramatic differences in physiology, and often dissimilar drug responses. Pig models would alleviate these shortcomings if they were to have a repertoire of gene signatures observed in human cancer, like those engineered in mice. Therefore, to achieve this goal, we have engineered a pig model with the Cre-responsive transgene encoding KRASG12D and TP53R167H. Hence, tissue-specific targeted Cre would generate simultaneous expression of mutant KRAS and p53 alleles to induce cellular changes leading to cancer. Pig pancreases were injected with adenovirus containing a cre expression vector (adenoCre) in an attempt to generate a model of pancreatic cancer. Initial injections of adenoCre into the parenchymal body of the pancreas led to the development of acinar-ductal metaplasia (ADM), though the clear predominant histotype was leiomyosarcoma immediately adjacent to the pancreas. Pancreatic ADM was characterized by concomitant localization of amylase (acinar cell marker) and CK19 (ductal cell marker) in the same pancreatic acinus, with an occasional few cells expressing both cell markers. These lesions were further characterized and exhibited increased proliferation via PCNA staining , as well the development of surrounding desmoplastic stroma (enhanced trichrome, vimentin, and aSMA staining; a marker of pancreatic stellate cells) with a robust inflammatory component (strong CD11b and CAE staining). Based on the lack of pancreas specificity, the experiment was repeated by injecting adenoCre into the main pancreatic duct. This 10-month-old pig appears relatively healthy after, which was expected considering this was a fully adult pig prior to manipulation. We will fully access this pig in 2 months to determine if strict pancreatic duct injection of adenoCre induces ADM, neoplastic disease, and/or ductal adenocarcinoma. Based on these encouraging findings, we anticipate having a robust model of pancreatic cancer in the pig, and plan to extend this study to generate models of other solid tumors with high rates of KRAS and p53 mutations. These porcine models may significantly impact preclinical studies, as diseases induced in pigs are likely more physiologically relevant to those in humans, with a potentially similar pharmacokinetic profile following drug treatment. Indeed, due to the size of the organs and volume of blood, samples can be more readily shared among multiple groups of investigators facilitating greater collaboration and expanding future research.

#4179

Expression profiling of zebrafish rb1- neuroectodermal-like brain tumors: A new model of CNS PNETs.

Maura A. McGrail,1 Staci L. Solin,1 Jeffrey A. Haltom,1 Laura E. Schultz,1 Jeffrey J. Essner,1 Heather R. Shive2. 1 _Iowa State University, Ames, IA;_ 2 _North Carolina State University, Raleigh, NC_.

We examined the gene expression signature of zebrafish brain tumors induced by somatic inactivation of the retinoblastoma tumor suppressor rb1 to determine the similarity with human CNS primitive neuroectodermal tumors (PNETs). Childhood CNS PNETs are aggressive tumors characterized by undifferentiated cells with features of the embryonic neuroepithelium. Previously our histological analysis showed the majority of zebrafish rb1- lesions were proliferative neuroectodermal-like tumors composed of neoplastic cells with small hyperchromatic nuclei that form rosettes. To obtain the transcription profile of the zebrafish rb1- tumors, RNA-Seq was performed on 10 tumors from fish ranging in age from 3.5 to 10 months of age. Duplicate RNA-Seq libraries were synthesized for each tumor and multiplex sequenced on an Illumina HiSeq NextGen Sequencer at the Genome Sequencing Core, Kansas University. Our initial gene expression profiling of the zebrafish rb1- brain tumors revealed a molecular signature consistent with an undifferentiated tumor phenotype. In each zebrafish tumor sample, standard markers of neuronal differentiation (sypa, sypb, neurod1/2, neurog1/2) and glial differentiation (GFAP, S100β) were not significantly expressed. In contrast, the neural stem/progenitor marker sox2 was highly expressed in all tumors, consistent with the primitive neuroectodermal-like tumor histology. Comparison with human childhood CNS-PNET gene expression analysis revealed a similar profile. Four of the nine genes that define the human oligoneural (group 2) molecular subgroup (olig2, bcan, sox8b, sox10 and ascl1) described by Picard et al, 2012 were highly expressed in the zebrafish tumors. Together, the data indicate the zebrafish brain tumors induced by somatic inactivation of rb1 model human CNS PNETs. Additional gene expression, pathway, and cross-species comparative analyses are in progress. The zebrafish rb1- tumor model will be useful for investigating the molecular pathways driving the aggressive and undifferentiated features of CNS PNETs.

#4180

Rewiring the cytokine network in melanoma.

Raghavendar Nagaraju,1 Christopher Dee,1 Helen Young,1 Jorge Barriuso,1 Christopher Secombes,2 Adam Hurlstone1. 1 _University of Manchester, Manchester, United Kingdom;_ 2 _Scottish Fish Immunology Research Centre, University of Aberdeen, Aberdeen, United Kingdom_.

Malignant melanoma is one of the most aggressive types of malignancies in humans resulting in 50,000 deaths worldwide annually. Fortuitously, malignant melanoma is highly immunogenic and it is now well established that the host immune system can detect and kill melanoma. T helper type-1 lymphocytes (TH1) play a pivotal role in inducing and maintaining anti-tumour immune responses. In melanoma patients, it has been observed that higher number of TH1 cells in the tumour micro-environment lead to better prognosis. In this study, using an autochthonous zebrafish melanoma model, when we forced the expression of interferon gamma (IFNg) locally in the tumour microenvironment, thereby potentially enhancing TH1 differentiation, we observed an inflammatory response against melanoma that eventually lead to complete tumour regression.

Using transposon mediated BAC transgenesis, we have generated a Tg(cd4:mcherry) reporter line labelling CD4+ cells in zebrafish. By driving Human NRASQ61L in zebrafish melanocytes we have modelled melanoma in zebrafish and later when these fish developed tumours we forced the expression of IFNg in melanoma cells using a tamoxifen inducible LSL/CreERT2 system driven by mitfa minimal promoter. Experiments were performed using animals with and without CreERT2. Following tamoxifen administration, we observed a loss of pigmentation in all the IFNg induced tumours (n=12) by 2 weeks post induction which gradually increased over the study period. Whereas no loss of pigmentation was observed in control tumours (n=12) throughout the study period. At 9 weeks post induction, the study was terminated and tumours were analysed using histology. In tumours were IFNg was induced, we observed that the tumour tissue was now replaced with fibrotic tissue accompanied by a marked lymphocyte infiltration and patches of necrosis. However, new tumour nodules were also observed to develop in the vicinity of regressing nodules which are unpigmented and potentially hypo-immunogenic. We observed no signs of tumour regression in control tumours.

Although preliminary, our data is very exciting and promising as it will open new avenues for developing combinatorial therapies in melanoma. Using a high throughput in-vivo model system such as zebrafish, it is an exciting proposition to test the impact of combining anti-PD1/PDL1 inhibitors with various cytokines/chemokines in inducing effective and sustained tumour regression in melanoma.

#4182

Statins induce apoptosis in osteosarcoma cells by activation of Ampk and p38-MAPK via suppression of mevalonate pathway.

Walied Abd-Elghani Kamel,1 Eiji Sugihara,1 Sayaka Iwai Yamaguchi,1 Hiroyuki Nobusue,1 Kenta Maki,2 Akihiro Muto,2 Hideyuki Saya,1 Takatsune Shimizu2. 1 _Keio University School of Medicine, Tokyo, Japan;_ 2 _Hoshi University, Tokyo, Japan_.

Osteosarcoma (OS) is the most common, non-hematopoietic, primary malignant bone tumor. OS is characterized by its aggressive local growth and systemic dissemination. Although combination of surgical operation and adjuvant chemotherapy greatly improved the prognosis, more than 20% of patients still cannot get long-term survival. Therefore, novel therapeutic approaches should be expected to be developed. Previously, we developed an OS mouse model by overexpressing c-MYC in bone marrow stromal cells derived from Ink4a/Arf knockout mice. We isolated highly tumorigenic cells (designated AXT cells) from tumors after serial transplantation. Inoculation of AXT cells into syngeneic C57BL/6 mice results in the development of lethal OS with metastatic lesions including lung, which pathologically and clinically mimics human osteoblastic osteosarcoma. To obtain the novel therapeutic agents for OS, we performed drug screening using existing drug collections and found that statins strongly suppressed AXT cell growth and migration in vitro. Simvastatin induces caspase dependent apoptosis, which can be completely rescued by the supplement of mevalonate and geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate, suggesting that OS cells are susceptible to the inhibition of the mevalonate pathway. As the responsible effectors, we found treatment of simvastatin drives RhoA-GTP translocation from cell membrane to cytosol as well as the disruption of interaction between RhoA and Rho-GDI, indicating that, in spite of the accumulation of RhoA-GTP in OS cells, RhoA cannot work properly, which appears to affect cell motility and anchorage dependent survival. As downstream signaling of RhoA, p38-MAPK and AMPK are strongly activated by simvastatin treatment. Activation of p38-MAPK is not accompanied by the ROS production. The specific inhibitor for p38-MAPK or AMPK can rescue this activation as well as the simvastatin-induced cell death, respectively, suggesting that activation of both kinases is responsible for the anti-tumor effect of simvastatin. A single treatment of statins attenuated OS tumor growth in vivo. These findings suggest that the activation of p38-MAPK and AMPK by statins become a potential therapeutic option for OS.

#4183

Targeting mTOR signaling during gliomagenesis.

Daniela Torres, Angelina Mela, Canoll D. Peter. _Columbia University, New York, NY_.

Diffusely infiltrating gliomas are the most common type of primary brain tumor seen in adults. In a subset of patients, these tumors present as low grade lesions that progress to high grade glioma (HGG). Conventional treatment for gliomas includes surgical resection followed by radiation and temozolomide. Glioma cells invade peripheral brain tissue making complete surgical resection impossible and despite aggressive treatment tumor recurrence is inevitable. Current research has focused on identifying new therapeutic targets, mainly in HGG. However, investigating the therapeutic targets at early stages of gliomagenesis could provide a novel approach for the treatment of low grade gliomas (LGG). Gliomas have been characterized by gene expression profiles and classified into distinct subtypes including proneural, classical and mesenchymal. Proneural gliomas, which account for the majority of LGG, show high expression of PDGFRα and frequently harbor p53 mutations. The resulting alterations in PDGFRα signaling and inactivation of p53 both induce an upregulation in mTOR signaling, which contributes to gliomagenesis via its regulation of cell growth and proliferation. Thus, inhibiting mTOR signaling should have a robust effect on glioma growth. However, inhibiting mTOR signaling in HGG has had limited success. This may be due to the accumulation of genetic alterations that occur during glioma progression resulting in loss of sensitivity to the anti-tumor effects of mTOR inhibitors. We hypothesize that inhibiting mTOR signaling at early stages of gliomagenesis will be more effective. To test this hypothesis we are characterizing the effects of mTOR inhibition at different stages of gliomagenesis in our proneural glioma mouse model. We assessed the effects of inhibiting mTOR with the ATP-competitive mTOR inhibitor (AZD8055) in vitro and in vivo. Our preliminary data show that AZD8055 inhibits mTOR activity and reduces glioma cell viability in vitro. Inhibition of mTOR in our proneural glioma model showed that AZD8055 crosses the blood brain barrier and effectively inhibits mTOR in glioma cells within the tumor, as well as in the surrounding brain tissue. In addition, we have characterized mTOR activity at different time points in glioma formation and show upregulation of mTOR activity during early stages of gliomagenesis. Our findings support the role of mTOR in early stages of gliomagenesis and motivate our ongoing studies to test the effects of AZD8055 for the treatment of LGG.

#4184

Pre-clinical study using KRAS mutant mouse models for lung cancer immunotherapy together with MEK inhibition.

Jiehui Deng,1 Shuai Li,1 Grit Herter-Sprie,2 Paul D. Smith,3 Gordon J. Freeman,1 Jeffrey A. Engelman,4 Peter Hammerman,1 Kwok-Kin Wong1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _University of Cologne, Cologne, Germany;_ 3 _AstraZeneca, Cambridge, United Kingdom;_ 4 _Massachusetts General Hospital, Boston, MA_.

Activating KRAS mutations are identified as the most common driver oncogene for lung adenocarcinomas, accounts for 20~30% of lung cancer patients. The development of therapeutic strategy for KRAS mutant lung cancer patients remains to be an unmet need, due to poor prognosis and therapeutic resistance to conventional therapy. The purpose of our study is through utilizing different types of genetically engineered mouse models (GEMMs), to understand the molecular heterogeneity of KRAS mutant lung cancer, to define the immune microenvironment, and to determine the feasibility and strategy of using MEK inhibition in combination with immune checkpoint inhibitors for the treatment of KRAS mutant lung cancer.

We generated two different types of GEMMs with KRAS mutations that develop tumor in the lung: Kras with G12D mutation or KRAS with G12C mutation. Continuous treatment of either KrasG12D or KRASG12C mice with MEK inhibitor Selumetinib (AZD6244, ARRY-142886) shows short-term response followed by drug resistance to single regimen treatment. The immuno-profiling shows that Selumetinib can increase cytotoxic T cells percentage and decrease PD-L1 expression on both myeloid cells and lymphocytes. Prolonged treatment of Selumetinib on KRAS mutant mice will lead to down-regulation of the expression of immune-checkpoint inhibitory factors Ctla-4 and Pd-1 on both CD4+ and CD8+ T cells. When KRAS mutant mice are treated with Selumetinib intermittently, they show improved response comparing with continuous treatment. When combining MEK inhibition with immune checkpoint blockade by using Selumetinib together with Pd-1 antibody treatment, Tim3 and Ctla-4 expression on T cells is increased potentially leading to T cell exhaustion and immune suppression in lung cancer. These pre-clinical results provide molecular insight for the immune response to MEK inhibition in KRAS driven lung cancer. Furthermore, these data support our hypothesis that MEK inhibition combination with immune checkpoint blockade treatment will have better outcome for lung cancer patients that have KRAS mutation through activating T cell response.

#4185

Randomized double-blind clinical study of F14512, a new polyamine-vectorized anticancer drug and Etoposide in naturally occurring canine lymphoma.

Bruno Gomes,1 François Serres,2 Juliette Hordeaux,2 Zacharie Segaoula,2 Emmanuel Bouchaert,2 Séverine Wadoux,2 Laurent Marescaux,3 Franck Floch,3 Pierre Boyé,3 Kevin Geeraert,3 Ingrid Bemelmans,2 Thierry Marchal,4 Corinne Fournel,4 Grégoire Zorza,1 Pierre Ferré,1 Aurélie Pétain,1 Nicolas Guilbaud,1 Dominique Tierny2. 1 _Institut de Recherche Pierre Fabre, Toulouse, France;_ 2 _Oncovet Clinical Research, France;_ 3 _Oncovet, France;_ 4 _VetAgro-Sup, France_.

F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in Phase I/II clinical trials in patients with acute myeloid leukemia. The aim of this study is to investigate the potential of F14512 in a new clinical indication and to compare its clinical efficacy with the reference topoisomerase II inhibitor Etoposide. Because of the many similarities between human and dog lymphomas, we are seeking to determine the tolerance, efficacy, PK/PD relationship of F14512 in this indication and potential biomarkers that could be translated into human trials.

Firstly, we successfully initiated two Phase 1 dose-escalation trials with 23 and 27 dogs with naturally occurring lymphomas using F14512 and Etoposide, respectively, with endpoints including safety, therapeutic efficacy and biomarker studies. Secondly, we initiated a randomized double blind study with two groups of 24 dogs with naturally occurring lymphomas in order to compare the clinical efficacy of F14512 and Etoposide in both newly diagnosed and relapsing cases.

The Phase 1 trial demonstrated that F14512 could be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy with a response rate of 91% (21/23) with 10 complete responses, 11 partial responses, one stable disease and one progressive disease. Phosphorylation of histone H2AX was studied as a potential pharmacodynamic biomarker of F14512. Etoposide displayed modest therapeutic efficacy with a response rate of 19% (5/27) with 1 complete response, 4 partial responses, and 6 stable diseases. The comparative clinical study was then initiated with both compounds administered at the recommended dose identified in the Phase 1 trial. Inclusion of all the dogs from the randomized double blind comparative study will be finalized at the beginning of 2016 and the results of the study will be disclosed at the AACR 2016 meeting.

This work shows that naturally occurring cancers in dogs can be of great interest in translational research in order to support preclinical and clinical development of new compounds.

#4186

Syngenomic fingerprint: the biomic characterization of the mouse syngeneic tumor models.

John E. Prime,1 Suzanne Mosely,1 Jens-Oliver Koopmann,1 Dennis YQ Wang,2 Danielle Greenawalt,3 James Harper,1 Miika J. Ahdesmaki,2 Rebecca Leyland,1 Olivia Harris,1 Ross Stewart,1 Philip Brohawn,4 Brandon Higgs,4 Bryony Langford,2 Athula Herath,1 Robert Kozarski,1 Jane Coates-Ulrichsen,1 Judith Anderton,1 Michelle Morrow,1 Richard C. A Sainson,1 Robert W. Wilkinson1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _AstraZeneca, Cambridge, United Kingdom;_ 3 _AstraZeneca, Waltham, MA;_ 4 _MedImmune, Gaithersburg, MD_.

The pre-clinical assessment of immuno-oncology (IO) therapies can be enabled by the use of murine syngeneic tumors established in immuno-competent mice. With the aims of selecting relevant models and of minimizing animal experimentation by reducing the number of models tested, the full characterisation of syngeneic models at the transcriptomic and genomic level is a key objective for pre-clinical scientists.

Model characterisation includes global aCGH, exon array analysis and FACS profiling alongside exome sequencing. The model data is undergoing hypothesis free and driven analyses which are already generating valuable insights. Comparison of in vivo tumor samples with their in vitro equivalents has highlighted enrichment for a number of immune pathways; as has the comparison of different tumor lines. The genomic, transcriptomic and 'proteomic' model data are being integrated to give a functional output which will act as a 'Syngenomic Fingerprint' for each model.

The resulting Syngenomic fingerprints will help pre-clinical scientists to refine their in vivo plans through an improved understanding of the limits and advantages as well as the clinical relevance of some of our preclinical models. It is also supporting the targeted modification of models to better match specific human cancer types.

#4187

Loss of SLFN11 or gain of TWIST1 promote chemotherapy resistance in small cell lung cancer.

Eric E. Gardner,1 Benjamin H. Lok,2 Valentina E. Schneeberger,2 Elisa de Stanchina,2 John T. Poirier,2 Charles M. Rudin2. 1 _Johns Hopkins University School of Medicine, Baltimore, MD;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

The majority of patients with small cell lung cancer (SCLC) who are treated with first-line platinum/etoposide chemotherapy develop therapeutic resistance, but little is mechanistically understood how resistance develops in patients. This is partly due to limited access to historic paired tissue samples, few investigational re-biopsy programs and the aggressive nature of this disease.

In this study we used a collection of 10 treatment-naïve, patient-derived xenograft (PDX) models of SCLC to determine how resistance to chemotherapy can be acquired in vivo, by cycling weekly cisplatin and etoposide, followed by serial passaging of tumor cells capable of surviving and proliferating through treatment. Applying whole transcriptome (RNAseq), exome and high-depth targeted sequencing approaches to paired parental and resistant populations revealed minimal recurrent mutations; however, two consistent gene expression changes were observed, implicating either a decrease in SLFN11 or an increase in TWIST1 transcript levels may promote resistance. We validated both mechanisms in vitro using conditional gain and loss of function experiments in SCLC cell lines of human and mouse origin. Where suppression of SLFN11 was associated with resistance to select classes of DNA damaging agents, gaining TWIST1 produced a broad resistance phenotype, consistent with an epithelial-to-mesenchymal transition.

Increased sensitivity to chemotherapy through overexpression of SLFN11 was observed only in cell lines with low levels of TWIST1. Importantly, TWIST1 knockdown or knockout did not dramatically change the sensitivity to chemotherapy in multiple TWIST1-high cell lines tested, suggesting that directly targeting TWIST1 may be of limited utility in SCLC. We extended these results by modeling acquired resistance to cisplatin and etoposide in a triple knockout (p53/Rb/p130) mouse allograft model of SCLC, finding that increased expression of TWIST1 was associated with chemotherapy resistance, but did not confer a greater metastatic potential in a tumor type with an already high predilection for metastasis. Understanding the relative expression of SLFN11 and TWIST1 at baseline and upon clinical relapse may be informative in determining therapeutic options for SCLC patients that no longer respond to platinum and etoposide.

#4188

**RNAi and CRISPR/Cas9 based** in vivo **models for drug discovery.**

Prem K. Premsrirut,1 Gregory Martin,2 Lukas Dow,3 Sang Yong Kim,4 Johannes Zuber,5 Scott Lowe,6 Greg Hannon7. 1 _Mirimus Inc., Woodbury, NY;_ 2 _Charles River Laboratories, Wilmington, ME;_ 3 _Weill Cornell Medical College, New York, NY;_ 4 _New York University, New York, NY;_ 5 _Research Institute of Molecular Pathology, Vienna, Austria;_ 6 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 7 _Cancer Research UK, Cambridge, United Kingdom_.

With the advent of CRISPR-Cas9 technology, the speed and precision in which genetically engineered mouse models can be created is unprecedented. We now have at our disposal a genetic toolbox that will enable the rapid generation of sophisticated mouse models of cancer. Recently, an inducible CRISPR-Cas9 (iCRISPR) system was described that enables doxycycline-regulated Cas9 induction of widespread gene mutagenesis in multiple tissues. Previously, we also demonstrated how inducible RNA interference (RNAi) can be exploited experimentally to effectively and reversibly silence nearly any gene target not only in vitro but also in live mice. Here, we take advantage of these powerful technologies and combine both tet-inducible CRISPR-Cas9 and inducible RNAi-mediated gene silencing to develop animal models in which both de novo tumorigenesis can be induced by Cas9-mediated genome editing and therapeutic strategies assessed downstream via RNA interference-mediated gene silencing. By using this combination of CRISPR/Cas9 and RNAi technologies, we are able to not only model disease pathogenesis, but also mimic drug therapy in the same mice, giving us exceptional capabilities to perform preclinical studies in vivo. Using our robust flexible system, we have created a cost-effective and scalable platform for the production of complex genetically engineered mouse models of cancer with RNAi silencing of nearly any gene - mice with enormous predictive power that will shape our development of better tolerated therapies.

#4189

Cancer drugs on the fly: whole-animal chemical screening in Drosophila identifies unexpected role for EGFR in RAF intestinal stem cell tumors.

Michele Markstein, Hannah Dayton, Samantha Dettorre. _University of Massachusetts Amherst, Amherst, MA_.

A major challenge in cancer therapeutics is to identify drugs that target cancer stem cells. This presents a problem for traditional cell-based screening approaches because the complex cellular interactions underlying stem cell biology are difficult to recapitulate in vitro. Conversely, mice, which are arguably among the best animal models for cancer—are not amenable to high-throughput screening. However, many aspects of stem cell initiated tumorigenesis can be modeled in the fruit fly Drosophila melanogaster, which due to its small size is well suited for large-scale screens. The Drosophila intestine, in particular, presents a compelling model to study stem-cell derived intestinal tumors because Drosophila intestinal stem cells share many features with their mammalian counterparts including a propensity to form tumors in response to mutations in WNT, NOTCH, or EGFR signaling. We therefore developed an in vivo screening pipeline using adult Drosophila, to identify compounds that can suppress the growth of stem cell initiated tumors. In previous work we showed that ectopically expressing a constitutively active human c-RAF transgene, RAF(gof) in Drosophila intestinal stem cells results in fast growing tumors that are sensitive to several FDA approved chemotherapeutics. These results establish the clinical relevance of using Drosophila to model stem cell cancers.

Here we present results from a screen of known bioactives, that reveal an unexpected role of the EGF receptor, EGFR, in RAF(gof) intestinal stem cell tumors. Based on the linear order of genes in the canonical RAF pathway—EGFR→RAS→RAF→MEK→ERK—we expected that drugs that inhibit genes acting downstream of RAF would score as hits in the screen, whereas genes that act upstream of RAF would not. Indeed, that same logic is applied when assessing whether to give human patients certain pathway specific drugs: patients with activating mutations in RAS or RAF are typically not given drugs that target the upstream EGFR. Consistent with our expectation, we found that drugs that inhibit genes downstream of RAF, effectively stopped Raf(gof) tumor growth. However, to our surprise, we also found that inhibitors of EGFR blocked the growth of RAF(gof) tumors. Using RNAi against EGFR, we genetically validated the chemical results. Our preliminary evidence suggests that EGFR is required in a parallel pathway, upstream of AKT. These findings are significant because they suggest that in some cases, human patients with RAF activated tumors may benefit from drug therapies that target EGFR. Importantly, our results demonstrate that taking an unbiased approach in assessing which drug therapies to apply to a given cancer, may yield unexpectedly good results.

#4190

Expression of a human CIC-DUX4 fusion is sufficient to induce malignant small round blue cell tumors in zebrafish: a new model of CIC-DUX4 sarcoma for translational research.

Sarah Watson,1 Genevieve Kendall,2 Olivier Delattre,1 Franck Tirode,1 James F. Amatruda2. 1 _Inserm U830, Laboratory of Genetics and Biology of Cancer, Institut Curie, Paris, France;_ 2 _Departments of Pediatrics, Molecular Biology and Internal Medicine, UT Southwestern Medical Center, Dallas, TX_.

CIC-DUX4 sarcomas are aggressive soft-tissue malignancies characterized by frequent metastases and uniformly poor survival. Histologically, CIC-DUX4 sarcomas resemble Ewing sarcomas but do not express EWS-FLI1 or other FET-ETS fusion genes typical of Ewing sarcoma. The molecular origins of these tumors began to be clarified with the discovery that they harbor characteristic t(4; 19) or t(10; 19) chromosomal translocations that fuse the transcriptional repressor CIC with the homeodomain-containing protein DUX4. The resulting CIC-DUX4 fusion protein acts as an aberrant transcription factor. However, the critical targets of CIC-DUX4 have been only partly described, the cell of origin and mechanism of transformation are unknown and animal models of the disease are lacking. Owing to this lack of biologic understanding, patients with CIC-DUX4 sarcomas are treated with the same regimens used for classical Ewing sarcoma, despite generally poor outcomes.

To comprehensively address these problems we are taking a cross-species comparative oncology approach via analysis of human CIC-DUX4 sarcomas and genetically engineered in vivo zebrafish models. To study in vivo effects of CIC-DUX4, we used highly efficient Tol2 transposon-based transgenesis techniques to induce tumors in the zebrafish. In the absence of a known cell of origin for CIC-DUX4 sarcoma, we used a variety of ubiquitous promoters to induce mosaic expression of human CIC-DUX4 in zebrafish, along with GFP or mCherry fluorophores to mark transgenic cells. CIC-DUX4 exerted stong effects on zebrafish embryonic development. In particular, expression of CIC-DUX4 was prominent in the developing vasculature including hemogenic endothelium, indicating that the endothelial environment is favorable to the expression of CIC-DUX4. Beginning at 5 weeks of age, almost 40% of injected animals developed invasive, rapidly-growing tumors. Similar to human CIC-DUX4 sarcomas, tumors in the zebrafish occurred in soft tissues including the trunk musculature, the ventral abdominal tissue and the head and neck. The tumors exhibited histologic features of small round blue cells, identical to the appearance of human CIC-DUX4 sarcomas. Zebrafish CIC-DUX4 sarcomas were readily transplantable as allografts into recipient zebrafish. To further understand the molecular mechanisms of tumorigenesis, and to establish aspects of CIC-DUX4 function common to human and zebrafish tumors, we are carrying out detailed molecular analysis of the zebrafish tumors including RNASeq and whole-exome sequencing. Taken together these findings demonstrate that CIC-DUX4 expression is sufficient to promote the development of SRBCT, give insight into the cellular origins of CIC-DUX4 sarcomas and provide a valuable new animal model for translational research in this disease.

#4191

Alterations in endothelin axis during pancreatic acinar to ductal metaplasia.

Suprit Gupta,1 Satyanarayana Rachagani,1 Sushil Kumar,1 Kavita Mallya,1 Surinder Kumar Batra,2 Maneesh Jain2. 1 _Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE;_ 2 _Department of Biochemistry and Molecular Biology, Fred and Pamela Buffet Cancer Center University of Nebraska Medical Center, Omaha, NE_.

Background: Acinar-ductal-metaplasia (ADM) is one the earliest recognizable alterations in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). Inflammation-induced ADM, which is typically reversible upon removal of inflammatory insult, becomes irreversible in the presence of oncogenic K-Ras and progresses to Pancreatic Intraepithelial neoplastic lesions (PanINs) and subsequently PDAC. Endothelin (ET) axis comprising of endothelin isoforms (ET-1, ET-2 and ET-3) and two receptors A (ETAR) and B (ETBR), has been demonstrated to contribute to the pathobiology of pancreatitis and its components exhibit aberrant overexpression in pancreatic cancer PDAC. However, the expression patterns of ET axis in oncogene-associated early lesions remain unknown. We hypothesize that alterations in ET axis contribute to oncogene associated irreversible ADM. Thus, we studied expression pattern of ET axis in the pancreatic inflammation in the presence and absence of oncogenic KRas and in preneoplastic lesions.

Methods: Expression of ET-1, ETAR and ETBR was analyzed in murine models of preneoplastic lesions [KC model: (Pdx1-Cre, KrasG12D)) by IHC. To determine the changes in ET axis during ADM, wild type (WT) and KC mutant mice were treated with cerulein to induce pancreatitis and tissues collected at day 0, 2, 7 and 21 days post treatment were analyzed for mRNA and Immunofluorescence analysis. Expression pattern of ET axis components was also determined in the pancreas of KC and WT animals following exposure to cigarette smoke.

Results: A progressive increase in the expression of ET-1, ETAR and ETBR was observed in the PanIN lesions in KC mice. In mice with WT Kras, cerulein treatment resulted in a notable increase in the expression of ET-1 and ETAR at day 2, while the levels of ETBR increased marginally; however a recovery to basal levels was observed for all three molecules by day 7. In contrast, significant increase (p<0.005) in ET-1, ETAR and ETBR transcripts was observed following cerulein treatment and these levels continued to remain high even at 21 days post-trauma in KC mice. The changes in the expression ETAR and ETBR were corroborated by confocal microscopy. Cerulein treatment resulted in increased expression of ETAR and ETBR in acinar compartment in both WT and KC mice as indicated by their co-localization with amylase. In KC mice, cerulein induced acinar-to-ductal metaplasia and the resulting CK19 positive ductal components continued to express increased levels of both receptors with distinct magnitude and kinetics. Similarly, in the smoking models, ET-axis components exhibited a more robust and sustained overexpression in KC mice as compared to the WT.

Conclusions: The expression of oncogenic KrasG12D results in an enhanced and sustained activation of ET-axis following inflammatory insults in the pancreatic tissues, suggesting its possible role in tumor initiation and progression.

#4192

The association between gastric Mucin 5AC (MUC5AC) expressions, transforming growth factor-β (TGF-β) signal, and histopathological phenotypes of lung cancer in xenograft models.

Ryo Sato,1 Takashi Semba,1 Hirotsugu Kohrogi,2 Hideyuki Saya,1 Yoshimi Arima1. 1 _Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan;_ 2 _Department of Respiratory Medicine, Kumamoto University, Kumamoto, Japan_.

According to the 2015 World Health Organization (WHO) classification of lung tumors, invasive lung adenocarcinomas are pathologically classified as lepidic, acinar, papillary, micropapillary, or solid types. Adding the cribriform pattern as an additional subtype to the original WHO classification has recently been proposed, because patients with cribriform predominant tumors have a poorer prognosis than that for patients with acinar or papillary predominant tumors. Understanding the molecular mechanisms contributing to the pathological subtypes may provide a basis for the development of new therapeutic strategies and an improved prognosis for lung cancer patients.

We found that the human lung adenocarcinoma A549 cells developed distinct histological types of tumors; acinar types and mucinous cribriform types, when cells were inoculated into immunodeficient mice. Acinar type tumors showed a gland-like pattern containing stromal cells, and cribriform type tumors contained abundant acidic mucins stained with Alcian blue. We also found that the cribriform type tumors, but not acinar type tumors, expressed Mucin 5AC (MUC5AC), which is one of the common mucin core proteins. In contrast, acinar type tumors upregulated the phosphorylation of Smad3, which is the downstream of the transforming growth factor-β (TGF-β) signal, indicating that the TGF-β/Smad pathway is activated in acinar type tumors. We went on to culture A549 cells in several conditions and found that MUC5AC is upregulated in serum-free floating culture conditions. To determine the relationship between TGF-β and MUC5AC expressions, TGF-β1 was added to the culture medium, and we found that TGF-β1 completely inhibited MUC5AC induction.

These data imply that cancer cells and/or cancer associated fibroblasts (CAFs) release TGF-β1 and inhibit MUC5AC expressions, leading to acinar type tumor formations. 

### Molecular and Cellular Imaging of Cancer 1

#4193

Reporter systems to measure dynamics and specificity of targeted cancer therapy.

Rebecca T. Marquez, Liang Xu, Experimental and Preclinical Imaging Core. _University of Kansas, Lawrence, KS_.

The Precision Medicine Initiative has driven the need for targeted therapy development to increase cancer patient survival. Due to this initiative, developing enhanced methods for measuring the accuracy and specificity of lead compound delivery is critical for moving these compounds through preclinical studies at a more efficient rate. The Experimental and Preclinical Imaging Core at the University of Kansas (epicore.ku.edu) is developing reporter constructs to assist in validating lead compound inhibition of oncogenic targets. Our lentiviral reporter constructs are used to establish stable cell lines that can be used either in vitro or in vivo to ensure optimal target inhibition. Our constructs include promoter reporters as well as 3' untranslated-region (UTR) reporters to measure either changes in mRNA stabilization or translation by RNA-binding proteins or microRNA target validation. Our goal is to provide reporter constructs that will enable industry and academic institutions to ensure optimal inhibition of signaling pathways targeted by their lead compounds or microRNA-based therapies. Currently, we are constructing stable cancer cell lines that express near-infrared proteins, such as iRFP670, for measuring primary tumor and metastatic growth in vivo. In comparison to luciferase-expressing cell lines, we have found that cells expressing iRFP670 provided a more robust signal and were easier to image without the need for injecting a substrate. New promoter and 3'UTR reporters are being developed using iRFP670 and will be tested in vivo.

#4194

Assessment of novel benzosuberene-based vascular disrupting agents (VDA) on diverse tumor lines.

Li Liu,1 Mary Lynn Trawick,2 Kevin Pinney,2 Ralph Peter Mason1. 1 _UT Southwestern Medical Ctr., Dallas, TX;_ 2 _Baylor University, Waco, TX_.

Introduction: In developing any new drug, non-invasive assays of efficacy provide vital insight into activity with respect to dosing and pharmacodynamics of action. We have previously demonstrated the utility of dynamic bioluminescence imaging (dBLI) to evaluate VDA activity [1,2]. The current study, which evaluated the vascular-disrupting activity upon treatment with the promising novel benzosuberene-based agents KGP18 and KGP265 [structurally inspired by the natural product, combretastatin A-4 (CA4)] led to significantly decreased and delayed light emission indicative of vascular disruption.

Methods: Human breast cancer cells (MDA-MB-231), prostate cancer cells (PC3), and lung cancer cells (A549) along with mouse breast tumor 4T1 cells were implanted in male SCID/Balb/c mice. The dose of KGP265 ranged from 5 to 15 mg/kg and the well studied VDA combretastatin A-4P (CA4P; 120 mg/kg) was used as positive control. Dynamic BLI was performed at baseline, 4, 24, 48 and 72 hours after IP administration of each VDA. Fresh D-luciferin was administered at each time point. As a control, additional tumor bearing mice were injected with the same volume of saline.

Results: Dose escalation indicated that higher doses produced more effective vascular shutdown, and doses of 3 mg/kg KGP18 and 7.5 mg/kg KGP265 appeared optimal, being effective while causing little observable toxicity with the MBA-MD-231 tumor model. The optimal dose of KGP265 was 5 mg/kg for 4T1, and 10 mg/kg for A549 and PC3. Treatment of the 4T1, PC3 and A549 tumors with the above dose of KGP265 greatly reduced signal output in BLI images. By 4 hours, about 90% of signal was reduced and the signal stayed down for the next 48 hours post-injection. In vivo observations were confirmed by post mortem histology. Treatment with KGP265 led to significantly decreased and delayed light emission indicative of vascular disruption in different tumor types. The BLI signal was reduced by 90% at 4 hours compared to baseline. Light emission remained significantly depressed up to 48 hours and 72 hours, whereas CA4P showed substantial recovery by 24 hours and further recovery by 48 hours. Meanwhile light emission was highly consistent following administration of saline as control with reproducible kinetics and intensities.

Conclusion: The results indicate that KGP18 and KGP265 cause significant vascular disruption. KGP265 is a phosphorylated prodrug of KGP18; it has enhanced water solubility, and maintains substantial vascular disrupting efficacy. Compared to CA4P (optimal dose 120 mg/kg), the new VDAs are effective at lower doses and achieve similar imaging results, suggesting enhanced efficacy. We are continuing to explore efficacy and therapeutic response through combination studies.

Acknowledgment: CA4P was kindly provided by Dr. David Chaplin of OXiGENE. Supported by NIH R01CA140674

References: 1.Zhao D et al, FASEB J. 22(7): 2445-2451, 2008

2.Mason RP et al, Integrat. Biol., 3: 375-387, 2011

#4195

Targeting the immune microenvironment to improve colorectal cancer anti-angiogenic therapy.

Keehoon Jung, Takahiro Heishi, Andy Yun, Timothy P. Padera, Rakesh K. Jain, Dai Fukumura. _Harvard Medical School / MGH, Boston, MA_.

Colorectal cancer (CRC) is the leading cause of cancer-related death worldwide. Anti-angiogenic therapy with bevacizumab has been proven efficacious in metastatic CRC. However, the improvement in overall survival has been modest and rather disappointing. Thus, there is an urgent need to identify the underlying mechanisms responsible for the resistance to anti-angiogenic therapy and develop strategies to overcome it. Clinical and preclinical interest in tumor immunotherapy has been renewed in a significant manner recently. However, the role of the immune microenvironment in colorectal cancer resistance to anti-angiogenic therapy is not well understood.

We have developed miniaturized confocal endomicroscopy technique for spontaneous CRC models in mice. We also established a novel cecum window recently. These unique systems enable us to monitor the CRC and its immune microenvironment longitudinally with a video-rate intravital multi-photon microscope. Using these CRC models and in vivo imaging methods, we found that anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody increases infiltration of Gr1+ myeloid-derived suppressor cells (MDSCs) and they alter the tumor microenvironment toward an immune-suppressive condition to avoid attack of the host immune system. We also found that the Gr1+ MDSCs express CXCR4, which has important roles in cell-autonomous survival and chemotaxis, through its downstream signaling pathways. When we treat the VEGF signaling blocker together with a CXCR4-specific inhibitor, the level of the Gr1+ MDSC infiltration is dramatically decreased nearly to that of control group, and subsequently, tumor growth is significantly delayed compared to separate treatment of each inhibitor.

Taken together, we identified Gr1+ MDSCs as key players in tumor resistance to anti-angiogenic therapy in CRCs, and the blockade of CXCR4 signaling has a synergistic effect with anti-VEGF therapy, proven by state-of-the-art in vivo imaging modalities.

#4196

Real-time single-walled nanotube (SWNT)-based imaging system improves tumor detection and survival in ovarian cancer animal model.

Lorenzo Ceppi,1 YoungJeong Na,1 Neelkanth M. Bardhan,2 Andrew Siegel,3 Nandini Rajan,3 Angela M. Belcher,4 Michael J. Birrer1. 1 _Center for Cancer Research, The Gillette Center for Gynecologic Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA;_ 2 _Department of Biological Engineering, Massachusetts Institute of Technology; The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA;_ 3 _Lincoln Laboratory, Massachusetts Institute of Technology, Lexington, MA;_ 4 _Department of Materials Science and Engineering, Department of Biological Engineering, Massachusetts Institute of Technology; The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA_.

Residual tumor detection in advanced stage ovarian cancer (OC) represents a critical challenge in the treatment of this disease. Optimal abdominal cytoreduction to reduce tumor to no evidence of macroscopic disease is the main endpoint of tumor debulking surgery, with demonstrated survival advantages, but the current clinically available imaging modalities have shown low tumor detection sensitivity of tumors smaller than 10 mm diameter. Instead, targeted molecule-based imaging systems offer higher detection rate and are likely to extend the aim of complete resection to the microscopic scale. We applied a custom designed imaging system to a preclinical ovarian cancer tumor model and observed higher microscopic tumor detection accuracy and survival advantage compared to non-image guided debulking surgery.

A two component imaging system was developed. The contrast agent is an intra-peritoneal injectable nanomolecular probe, composed of a single walled carbon nanotube (SWNT) coupled with an engineered M13-bacteriophage carrying a modified peptide targeting secreted protein, acidic and rich in cysteine (SPARC), extracellular protein overexpressed in OC. The imaging device system is capable of detecting SWNT inherent fluorescence in near-infrared second window (NIR2) and of displaying real-time images to guide the intraoperative tumor debulking.

In an orthotopic ovarian cancer animal model, the imaging system detected microscopic tumors in the millimeter and sub-millimeter scale resolution with a mean tumor diameter of 2.1 vs 3.6 mm (p< 0.01) compared to eye detected tumors. The imaging system demonstrated sensitivity of 97.2% and specificity of 69.7%, and a Receiver Operator Area under the Curve of 0.85 + 0.05. In a survival surgery animal model, the imaging guided resection surgery compared to non guided surgery showed a survival advantage for the experimental arm, with median survival time of 41.0 days vs 28.5 days, respectively (HR 0.22, 95% CI of ratio 0.04 to 1.08 Log Rank test: p: 0.063).

The accuracy of the imaging system and its survival advantage are promising. The survival outcome improvement in this preclinical model supports the translation to first-in-human investigation. Moreover, the imaging system accuracy suggests further exploitation in targeted delivery therapies.

#4197

Combined contrast enhanced ultrasound and photoacoustic imaging reveals both functional flow patterns and dysfunctional vascular pooling in tumor models.

Melissa Yin,1 Avinoam Bar-Zion,2 Dan Adam,2 Stuart Foster1. 1 _Sunnybrook Research Institute, Toronto, Ontario, Canada;_ 2 _Technion - Israel Institute of Technology, Haifa, Israel_.

The tumor vasculature and its hypoxic microenvironment are constantly undergoing changes. These alterations are key attributes associated with aggressive cancer phenotypes, raising the need for non-invasive methods to track these changes. Similarly, in many cases, various cancer treatments also affect tumor vasculature, and preferably – should be monitored. Dynamic contrast-enhance ultrasound (DCEUS) and photoacoustic (PA) imaging are two promising candidates. DCEUS has the ability to measure functional tissue perfusion, whereas multispectral PA imaging can be used to evaluate tissue oxygenation related parameters. This study investigates the relationship between blood perfusion, oxygen saturation levels and hemoglobin concentration in two hind-limb tumor models, and evaluates the ability of these two modalities to image vascular structures and functions.

Xenograft tumors were induced in SHO mice using either LS174T human colorectal cancer cells (n=6), or PC3 human prostate cancer cells (n=6). Tumors were grown to a depth of 4-6 mm before imaging was performed using a laser integrated high-frequency ultrasound system (Vevo®LAZR, VisualSonics Inc.). Contrast enhanced images were collected after a 50μL bolus injection of MicroMarker ultrasound contrast agents (VisualSonics Inc.) using non-linear contrast imaging. Perfusion parameters were quantified after applying wavelet denoising to the DCEUS clips. PA images were acquired using a 21MHz linear array transducer with fiber optical bundles integrated to each side, used to deliver light from a 680-970 nm tunable laser. Oxygen saturation levels and hemoglobin concentration were estimated from the PA measurements using spectral un-mixing. Tumor vascularity and hypoxia were confirmed with immunohistochemistry staining for CD31 and CA9.

Reasonable correlations were found between corresponding pixels in the DCEUS perfusion maps and oxygen saturation maps (R=0.63 and R=0.5 for LS174T and PC3 respectively). In contrast, the correlation between blood perfusion and hemoglobin concentration was nil for LS174T tumors (R=-0.1), and low for PC3 tumors (R=0.34). This discrepancy was explained by the presence of blood pools in LS174T tumors, observed in tumor histology. The presence of hemoglobin inside regions of hemorrhage together with the limited capability to separate hypoxic and necrotic regions, impeded the ability of PA imaging to detect blood vessels inside tumors. Compared to PA imaging, DECUS provides better detection of functional vasculature and enables the visualization of single blood vessels around the tumor core, without including blood pools.

This study demonstrates that a multi-modality imaging scheme combining DCEUS and PA imaging can provide both distinctive and complementary information on tumor microenvironment in experimental animal studies.

#4198

Optoacoustic imaging of blood vasculature and study of angiogenesis in orthotopic breast cancer models.

Isabel Quiros-Gonzalez, Michal Tomaszewski, James Joseph, Sarah E. Bohndiek. _University of Cambridge, Cambridge, United Kingdom_.

The outcomes of anti-angiogenic drugs in breast cancer have been disappointing. There is an urgent clinical need to better understand the existing and emerging anti-angiogenic therapies in order to: select appropriate patients therapy; define 'windows' for combination therapy; and reduce healthcare costs of 'precision medicine'. MultiSpectral Optoacoustic Tomography (MSOT) is emerging as a new imaging modality, cheaper and less toxic than existing functional imaging methods. It is based on the absorption of laser energy in tissues, which produces pressure waves detectable by ultrasound. MSOT can detect binding of O2 to haemoglobin (Hb and HbO2) based on changes in the optical absorption spectrum, making it a very useful tool to image blood vasculature and measure tissue oxygenation.

We have used MSOT to study blood vessel formation in a breast cancer xenograft model (MCF7, Estrogen Receptor+, n=10). MSOT images were acquired at 3 and 6 weeks after innoculation and at 6 weeks, tumours were collected for histopathological study. The endothelial protein CD31 was use to identify blood vessel density. Serum levels of vascular endothelial growth factor (VEGF) were measured at 3 and 6 weeks. Presence/absence of VEGF receptor levels were assessed in MCF7 cell line by WB and IF.

The MSOT parameters mean intensity (MI) and maximum intensity (MaI) for total Hb (THb) and O2 saturation (SO2, HbO2/(HbO2+Hb)) did not change significantly during tumour development (p-values: Hb=0.952, 0.716; HbO2=0.102, 0.19; mean/max respectively, SO2=0.12), indicating that there is no substantial change in blood vessel density in this tumour model despite a size increase (mean, cm3 3w=0.139 and 6w=0.458, p-value=0.04). The serum levels of the pro-angiogenic cytokine VEGF were significantly decreased (mean, pg/ml 3w=116.97 and 6w=86.24). The decrease in VEGF could explain the apparent lack of further blood vessel formation by 6 weeks. Unexpectedly, although the mean intensity for total Hb is lower in the tumour than the reference (cava artery-vein), there is no difference in SO2. Finally, comparing measurements from MSOT to histopathology, HbO2 MI correlates with CD31 staining intensity (CD31si) (Spearman correlation r=0.57 p-value=0.041) indicating that both measurements mark presence of blood vessel in the tumour. Microvessel density and CD31si do trend towards correlation with THb MI but this is not statistically significant at present.

In conclusion, MSOT is a direct method to image blood vasculature in our tumour model non-invasively and indirectly to determine blood vessel density. The xenograft mouse model from MCF-7 cell line shows good vascularization, oxygenation and stability during tumour growth. In the next steps, we will investigate in this xenograft model the utility of MSOT biomarkers to monitor response to anti-angiogenic therapies, hence establishing the potential of the technique as a companion diagnostic.

#4199

Acidic pH-targeted nanorods facilitate detection of orthotopic pancreatic tumors via multispectral optoacoustic tomography.

Matthew Zeiderman, Kelly M. McMasters, Phillip Chuong, Lacey R. McNally. _University of Louisville Brown Cancer Center, Louisville, KY_.

We present a precision cancer nanomedicine based on acidic pH targeted chitosan coated mesoporous silica coated gold nanorods (CMGs) designed for multispectral optoacoustic tomography (MSOT). We have conjugated pH-sensitive variant 7 pHLIP peptide to the CMGs (V7-CMG) to provide targeting specificity to the acidic tumor microenvironment. For treatment and detection of resistant malignancies such as pancreatic cancer, such targeting modalities may help to improve treatment and enhance tumor detection. The V7-CMGs provide the ideal contrast and targeting specificity necessary for MSOT imaging of retroperitoneal orthotopic pancreatic tumors. The pH-sensitivity of the targeting pHLIP peptide and chitosan coating makes the particles suitable for simultaneous in vivo tumor imaging and drug delivery. In vitro studies confirm the pH-sensitivity of CMG drug release and tumor cell specific nano-cargo delivery.

#4200

Novel imaging strategy to assess the antitumor efficacy of treatments in an orthotopic mouse lung cancer model using ultrasound and photoacoustic imaging.

Florian Raes,1 Julien Sobilo,1 Sharuja Natkunarajah,1 Philippe Trochet,2 Dieter Fuchs,2 Stephanie Lerondel,1 Alain Le Pape3. 1 _Phenomin-TAAM CIPA CNRS UPS44 French National Center for Scientific Research, Orléans, France;_ 2 _FUJIFILM Visualsonics Inc., Amsterdam, Netherlands;_ 3 _Phenomin-TAAM CIPA CNRS UPS44 French National Center for Scientific Research / CEPR INSERM U1100, Orléans / Tours, France_.

Introduction

The aim of this study is to develop new strategies for the exploration of relevant preclinical lung tumor models dedicated to onco-pharmacology studies in mice. Here we refine a translational approach while overcoming physical ultrasound (US) imaging limitations allowing lung tumors exploration. Our data were compared to Computed Tomography (CT) and Bioluminescence (BLI) aiming to validate this promising approach.

Methods

Human lung cancer cells NCI-H460-luc2 were orthotopically xenografted in Balb/c nude mice. BLI was performed 7 days after implantation then once a week until the end of the study (Day 28) using an IVIS-Lumina II (Perkin Elmer). Tumors were imaged with the Vevo®LAZR System (FUJIFILM VisualSonics Inc.), 3D scans of US, Contrast Enhanced Ultrasound (CEUS) (Vevo MicroMarkerTM) and photoacoustic (PA) image being recorded digitally. A comparison between in vivo US and CT was achieved with additional ex vivo weight and volumetric measurements.

Results

The control of the tumor cell implantation was determined by BLI allowing the early quantification of tumor burden. 3D US measurements made possible longitudinal tumor volumes assessment. Compared to the other in vivo measurements, results clearly indicated the gross correlation of volumes processed with US and CT (R²= 0.75). Such a correlation is also observed with ex vivo US weight and volumetric measurements (R²= 0.72, R²=0.75 and R²=0.65 respectively). PA imaging evidenced the hemoglobin saturation with oxygen inside tumors, and targeted CEUS imaging allowed assessing the relative VEGFR2 expression in lung tumors.

Conclusions

This study proved the ability of US imaging to detect and control the in vivo orthotopic lung tumor growth. This real-time approach is fast while being completely inert regarding the in situ proliferation, as compared to dosimetry related to repeated CT examinations. Furthermore it allows overcoming the limitation of BLI at the time tumors become hypoxic since the luciferase/luciferin reaction is strictly dependent upon O2 and ATP. We described here a PA and US imaging strategy to follow the orthotopic lung tumors growth and consider biomarkers such as VEGFR2 expression or hypoxia in vivo for the first time. This procedure would be of great interest to longitudinally investigate tumor proliferation in animals when evaluating new anticancer therapies efficacy, avoiding any perturbation of the tumor progression compared to other available imaging modalities.

#4201

Targeting tumor-associated neovasculature for delivery of optical enhancers detects ovarian cancer micrometastasis.

Ayesha B. Alvero, Eydis Lima, Dongin Kim, Sean Orton, Natalia Sumi, Mary Pitruzzello, Yang Yang-Hartwich, Dan-Arin Silasi, Tarek Fahmy, Gil Mor. _Yale University School of Medicine, New Haven, CT_.

Background: Patients with epithelial ovarian cancer have the best overall survival when maximal surgical effort is accomplished. While identification and removal of large metastases do not pose a challenge, micrometastases are impossible to distinguish intra-operatively and contribute to the high mortality. Our objective is to develop specific tumor-targeting optical enhancers that can aid surgeons in the performance of microscopic tumor debulking with the goal of minimizing microscopic residual disease. We hypothesize that we can utilize overexpressed αVβ3 integrins in the tumor-associated neovasculature. Specific targeting is achieved by encapsulating fluorescent probes in FDA-approved PLGA-based nanoparticle (NP) coated with the peptide sequence arginine-glycine-aspartate (RGD), which binds with high affinity to these integrins.

Materials and methods:

Ovarian cancer xenograft is established intra- peritoneally (i.p.) in nude mice using cancer cells that stably express mCherry fluorescent protein. The following formulations were tested: soluble deep infrared dye (DIR), DIR encapsulated in naked NP (DIR-NP), and DIR encapsulated in RGD-coated NP (DIR-RGD-NP). Formulations were delivered i.p. and colocalization of fluorescent signals were determined ex vivo. Staining of micrometastasis was visualized using fluorescent dissection microscope.

Results: The best colocalization was observed in mice administered DIR-RGD-NP. We observed 75% colocalization in this group compared to 26% and 0% in DIR-NP and DIR groups, respectively. Ex vivo analysis of DIR intensity in tumors less than 2 mm showed mean fluorescent intensity (MFI) of 1209 in DIR-RGD-NP group versus 155 MFI DIR-NP group. Based on DIR staining, we can locate 81% of mCherry+ micrometastasis in animals administered DIR-RGD-NP. In these animals, tumors less than 1 mm were detected due to a halo of DIR staining around each micrometastatic lesion. In these foci, DIR signal was observed to stain the vasculature surrounding the small tumor implants especially those in the mesentery and diaphragm. In contrast, we were able to detect only 18% of the mCherry+ micrometastasis in animals administered DIR-NP.

Conclusion:

We demonstrate that we can utilize the specific phenotype of tumor-associated neovasculature to target optical enhancers to locate and delineate micrometastasis. RGD-coated nanoparticles are able to carry probes to the tumor microenvironment leading to optimal staining of micrometastasis. Our results highlight the use of this nanotechnology platform in microscopic surgical debulking to assure maximal surgical effort, minimize residual disease, and improve patient survival.

#4202

Peptide probes binding to radiation-inducible cell surface GRP78 as a novel targeting strategy for various tumors.

Vaishali Kapoor, David Dadey, Kim Nguyen, Hua Li, Buck Rogers, Dinesh Thotala, Dennis Hallahan. _Washington University in St. Louis School of Medicine, St. Louis, MO_.

Traditional radiation therapy is often associated with significant toxicity to normal cells that could limit the success of cancer therapy. The greater understanding in the molecular differences between cancer cells and normal cells has lead to targeted therapies in cancer treatment. We have discovered novel radiation-inducible antigens in cancer using phage-display peptide libraries. One such inducible-antigen is Glucose regulated protein 78kDa (GRP78) that was shown to bind the hexapeptide GIRLRG. GRP78 is known to regulate cellular stresses, including hypoglycemia, hypoxia and the ER stress response. It is over-expressed in different cancer subtypes and has been correlated to their poor prognosis.

In this study, we evaluated the cell surface induction of GRP78 post irradiation (IR) on lung cancer (A549 and LLC), glioma (D54 and GL261) and endothelial (HUVEC and 3B11) cell lines using flow cytometry. Significantly higher surface expression of GRP78 was observed in all cell lines. We determined the specificity of GIRLRG to bind tumors in vivo using nano SPECT technology. GIRLRG was conjugated to a 40KDa PEG (for longer circulation time) and radiolabelled with 111Indium (111In) using diethylene triamine pentaacetic acid (DTPA) as the chelator. The A549 tumor bearing nude mice were irradiated (3Gy x 3 times) or sham irradiated prior to tail vein injections of 111In labeled GIRLRG. The mice were imaged 48h and 96h post injection using nano CT-SPECT imager. The SPECT images revealed that GIRLRG specifically bound to the irradiated A549 tumors while little or no binding was seen in the sham irradiated tumors. The post-SPECT imaging bio-distrubution also revealed maximum uptake in irradiated tumors. We further evaluated tumor binding of GIRLRG in Glioma (D54), esophageal cancer (OE33), cervical cancer (HT3), and pancreatic cancer (BxPC3). The nano-SPECT imaging showed that GIRLRG specifically bound to all the irradiated tumors tested. Phosphor images of the tumor sections showed that GIRLRG specifically bound to the tumors and not to normal tissues.

In conclusion, GIRLRG peptide has high affinity to GRP78 in vitro and in vivo. Radiolabeled GIRLRG is a novel tool for imaging tumors and may be developed further as a therapeutic agent to deliver cancer specific drugs or therapeutic radioisotopes.

#4203

Non-invasive imaging of tumor cell death induced by a TRAILR2 agonist.

Bangwen Xie,1 Andre Neves,1 Stefanie R. Mullins,2 David Tice,3 Danielle Carroll,2 Robert W. Wilkinson,2 Kevin M. Brindle1. 1 _University of Cambridge, Cambridge, United Kingdom;_ 2 _MedImmune Limited, Cambridge, United Kingdom;_ 3 _MedImmune LLC, Gaithersburg, MD_.

Imaging tumour cell death can give an early indication of treatment efficacy. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor2 (TRAILR2) is a member of the TNF receptor superfamily, which interacts with death receptors (DRs) and induces apoptosis in a broad range of cancer cells but not normal cells[1]. MEDI3039, a newly described agonist of TRAILR2, was used to induce tumour cell death. A NIRF fluorophore-labelled phosphatidylserine (PS)-binding protein (~15kDa), the C2A domain of Synaptotagmin-I (C2Am-750), which binds to the PS exposed by apoptotic and necrotic cells, was used to image MEDI3039-induced cell death[2]. Non-specific binding was assessed using a site-directed mutant that is inactive in PS binding, which was conjugated to a different fluorophore (iC2Am-680).

Binding of C2Am-750 to MEDI3039-treated human colon and breast adenocarcinoma cells in vitro (Colo205 and MDA-Dual respectively) was assessed by flow cytometry and confocal microscopy. Both methods showed that C2Am-750 labelled MEDI3039-treated Colo205 cells, while the inactive iC2Am-680 showed only low non-specific binding. C2Am-750 and iC2Am-680 were also used to monitor the effect of treatment in a Colo205 xenograft model. One cohort of mice (n=5) received a single dose of MEDI3039 (0.4 mg/kg), while another untreated cohort (n=5) served as a control group. All mice received a single i.v. injection of a 1:1 mixture of 0.1 µmole/kg C2Am-750 and iC2Am-680 at 16 h after drug treatment, followed by whole body fluorescence imaging (FLI) measurements using an IVIS200 camera at 0 and 3 h post probe injection. FLI measurements in MEDI3039-treated Colo205 tumours showed significant increases in the uptake of C2Am-750 relative to iC2Am-680, both in vivo (4-9 fold, P value <0.0001) and ex vivo (7-11 fold, P value <0.0001). Uptake of iC2Am-680 was low in both treated and untreated groups. Subsequent histological analyses showed that the C2Am-750 signal was strongly correlated with both cleaved caspase-3 and TUNEL staining of dead cells.

NIRF fluorophore-labelled C2Am showed a favourable biodistribution profile, with good tumour penetration and quick clearance of unbound material in vivo. The probe can be used to investigate the efficacy of targeted therapy, or other anti-tumour therapies, at an early stage following treatment.

1. Prasad, S., Kim, J.H., Gupta, S.C. & Aggarwal, B.B. Targeting death receptors for TRAIL by agents designed by Mother Nature. Trends in pharmacological sciences 35, 520-536 (2014).

2. Alam, I.S., Neves, A.A., Witney, T.H., Boren, J. & Brindle, K.M. Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death. Bioconjugate chemistry 21, 884-891 (2010).

#4204

Intraarterial delivery of doxorubicin-loaded hollow gold nanospheres for photothermal ablation chemotherapy of hepatocellular carcinoma in the liver of rats.

Junjie Li,1 Min Zhou,1 Chiyi Xiong,1 Wanqin Wang,1 Qizhen Cao,1 Xiaoxia Wen,1 Xin Ji,2 Andrew Wang,2 Sanjay Gupta,1 Chun Li1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Ocean Nanotech, San Diego, CA_.

Purpose: We sought to use combretastatin A-4 phosphate disodium (CA4P) to enhance the tumor uptake of doxorubicin (Dox)-loaded, polyethylene glycol (PEG)-coated hollow gold nanospheres (HAuNS; Dox@PEG-HAuNS) for improved photothermal ablation (PTA)-chemotherapy of hepatocellular carcinoma (HCC) in rats under image guidance. Materials and Methods: Two groups of 10 HCC-bearing rats each received intrahepatic arterial (IA) injection of either PEG-HAuNS/Lipiodol alone or CA4P followed by PEG-HAuNS/Lipiodol 5 min later. Each group was subdivided, and the Au content of tumors and tissues excised at 1 h or at 24 h was quantified using neutron activation analysis (n=5/time point). Five rats received CA4P plus PEG-[64Cu]-HAuNS/Lipiodol and underwent µPET/CT. Therapeutic effects were compared among 3 groups of 6 rats each that received IA injection of saline (control group), CA4P plus Dox@PEG-HAuNS/Lipiodol (chemotherapy group), or CA4P plus Dox@PEG-HAuNS/Lipiodol plus near-infrared irradiation (PTA-chemotherapy group). Findings were verified with postmortem histopathology and/or autoradiography. Results: PEG-HAuNS uptake in CA4P-pretreated tumors was significantly higher than that in non-CA4P-pretreated tumors at both 1 h (P<0.05) and 24 h (P<0.01). The tumor-to-liver PEG-HAuNS uptake ratios at 1 h and 24 h were 5.63±3.09 and 1.68±0.77, respectively, in the CA4P-treated group and 1.29±2.40 and 0.14±0.11, respectively, in the non-CA4P-treated group. µPET/CT clearly delineated the tumors, enabling quantitative imaging guidance. Tumor volumes 10 d after therapy were 1.68±1.01, 3.96±1.75, and 6.13±2.27 cm3 in the PTA-chemotherapy, chemotherapy, and control groups, respectively, with significant differences between the PTA-chemotherapy group and other groups (P<0.05). Conclusion: CA4P pretreatment causes a higher concentration of Dox@PEG-HAuNS to be trapped inside the tumor, thereby enhancing the anti-HCC efficacy of µPET/CT-guided PTA-chemotherapy in rats. (Supported in part by the John S. Dunn Foundation)

#4205

**Targeting a variety of cancers with NOTA-derivatized pH (low) insertion peptide (pHLIP) complexes with** 64 **Cu and** 18 **F: What cancers are targetable.**

Dustin W. Demoin,1 Kimberly J. Edwards,1 Linden C. Wyatt,2 Mirkka Sarparanta,1 Jacob Pourat,1 Oleg A. Andreev,2 Yana K. Reshetnyak,2 Nerissa Viola-Villegas,3 Jason S. Lewis1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _University of Rhode Island, Kingston, RI;_ 3 _Karmanos Cancer Institute, Detroit, MI_.

Introduction: Although heterogeneous, the extracellular tumor environment is generally marked by decreased pH relative to nontumor tissues due to cancer cell metabolism. Using a pH-sensitive peptide to deliver diagnostic PET isotopes can provide clinicians with improved diagnostic tools for cancer-specific imaging and an alternative to receptor-based cancer targeting. pH (low) Insertion Peptides (pHLIPs) have previously been shown to target cancerous tissues and are able to deliver PET metals to the cancer site utilizing a NOTA-based chelator.

Methods: The thiol-containing cysteine moiety on the pHLIP peptide sequence was conjugated to the NOTA chelator. The NOTA-derivatized peptides were labeled with 64CuCl2 in 100 mM NH4OAc (pH 5) solution; the [18F]-fluoride was reacted with AlCl3 before forming the [18F]-AlF-NOTA-pHLIP complexes in NH4OAc buffered reaction mixtures (pH ~ 4.1). After purification and formulation, the radiolabeled peptides were injected intravenously into 4T1 breast, PC-3 prostate, LNCaP prostate, or B16-F10 melanoma tumor-bearing mice.

Results: The 4T1 tumor-bearing mice showed significant uptake > 10 %ID/g by 4 h p.i. and continued to show increased uptake of the 64Cu-NOTA-cysVar3 over 20 h. Tumors were visible in the 4T1 tumor-bearing mice as early as 2 h, but had the greatest tumor-to-background ratios at 24 h. Initial studies with B16-F10 tumor-bearing mice showed > 9 %ID/g uptake in the tumor by 4 h p.i. with slight increase of 64Cu-NOTA-cysVar3 uptake over 24 h. Due to the placement of the melanoma tumors away from internal organs, tumors were visible by 1 h p.i. Additionally, the size of the melanoma tumors did not affect the %ID/g of radiotracer uptake.

Conclusions: Our studies indicate that the NOTA-cys(pHLIP), specifically NOTA-cysVar3, radiolabeled with 64Cu or 18F is a viable imaging tool for detecting highly metabolic tumors in preclinical mouse models.

Funding: NIH F32 CA186721 (D.W.D.), NIH R01 CA138468 (J.S.L.), NIH MSKCC Center Grant (P30-CA08748).

#4206

A dual-motif CAIX inhibitor, [64Cu]XYIMSR-06, for PET imaging of clear cell renal cell carcinoma.

IL MINN, Soo Min Koo, Hye Soo Lee, Mary Brummet, Steven P. Rowe, Michael A. Gorin, Polina Sysa-Shah, William D. Lewis, Hye-Hyun Ahn, Yuchuan Wang, Sangeeta Ray, Ronnie C. Mease, Sridhar Nimmagadda, Mohamad E. Allaf, Martin G. Pomper, Xing Yang. _Johns Hopkins University, Baltimore, MD_.

Carbonic anhydrase IX (CAIX) is a cell surface enzyme that is over-expressed in approximately 95% of clear cell renal cell carcinomas (ccRCCs), the most common renal cancer. CAIX is also a surrogate marker of hypoxic tumors for various human cancers; however, a clinically viable molecular imaging agent targeting CAIX is not available. We have synthesized and performed in vitro and in vivo evaluation of a dual-motif, low-molecular-weight inhibitor of CAIX, [64Cu] XYIMSR-06, for imaging CAIX expression on ccRCC tumors using positron emission tomography (PET). [64Cu] XYIMSR-06 was synthesized in two steps from reported dual-motif precursor 1. Upon radiolabeling, [64Cu] XYIMSR-06 was evaluated in immunocompromised mice bearing CAIX-expressing SK-RC-52 tumors for in vivo PET imaging and biodistribution. [64Cu] XYIMSR-06 was generated in radiochemical yields of 51.0 ± 4.5% (n=5) and specific radioactivity of 6.0 GBq/μmol (170Ci/mmol ± 70, n = 5). Tumor could be visualized on PET images by 1 h post-injection with high tumor-to-background levels achieved within 24 h. Biodistribution studies of [64Cu] XYIMSR-06 demonstrated a maximum tumor uptake of 19.3 ± 4.51% injected dose per gram of radioactivity at 4 h. Tumor-to- blood, muscle and kidney ratios were 129.6 ± 18.8, 84.3 ± 21.0 and 2.1 ± 0.26, respectively, at 8 h post-injection. At 24 h, a tumor-to-kidney ratio of 7.1±2.5 was achieved. These findings represent increased tumor-to-background ratios at the indicated times relative to previously reported CAIX-targeted imaging agents. [64Cu] XYIMSR-06 is a promising candidate for PET imaging of CAIX expressing tumors, especially ccRCC.

#4207

microPET/CT imaging of the mobilization of CD11b+ cells.

Qizhen Cao, Qian Huang, Chun Li. _UT MD Anderson Cancer Center, Houston, TX_.

Objectives: Mobilization of immune cells is often associated with prognosis and treatment response for many diseases. The objective of this study was to test whether mobilization of CD11b+ myeloid cells could be noninvasively imaged by PET/CT. Methods: Anti-mouse CD11b antibody was labeled with 64Cu. Immunocompetent mice with 12-o-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in the ears, lipopolysaccharide (LPS)-induced inflammation in the lung, mice bearing 3 different syngeneic murine tumors (4T1 mammary carcinoma, CMS4 sarcoma, and CT26 colon carcinoma), and mice bearing ID8 ovarian cancer undergoing photothermal therapy were imaged with [64Cu]-labeled anti-CD11b. microPET-CT imaging and biodistribution were obtained at 24 h after intravenous injection of the radiotracer. Subpopulations of bone marrow cells, cells from spleen, and cells from the disease sites were analyzed by flow cytometry and/or immunohistochemistry (IHC) staining. Results: 64Cu-labeled anti-CD11b bound specifically to CD11b+ myeloid cells. microPET-CT images showed significantly higher uptake of 64Cu-DOTA-anti-CD11b antibody at the inflammatory sites and tumors undergoing photothermal therapy. In addition, microPET-CT images also revealed mobilization of CD11b+ cells from bone marrow to secondary lymphoid organs (spleen and/or lymph nodes). Interestingly, mice bearing 4T1 carcinoma displayed a markedly different imaging/distribution pattern compared to mice bearing CMS4 and CT26 tumors, characterized by a significant increase in blood pool and liver activities with corresponding reduction in the uptake of the radiotracer in the bone marrow and the spleen in different stages of tumor development. These findings were evaluated in the context of flow cytometry and IHC analyses. Conclusions: The differential distribution pattern of 64Cu-labeled anti-CD11b in different disease models could be attributed to mobilization of CD11b+ cells. PET/CT may be used to image systems response of immune cells to disease development and treatments. (Supported in part by the John S. Dunn Foundation)

#4208

**Development of** 18 **F-IL2: a PET radiotracer for imaging activated T-cells.**

Elly L. van der Veen, Petra Maarsingh, Anton G.T. Terwisscha van Scheltinga, Marjolijn N. Lub-de Hooge, Geke A.P. Hospers, Erik F.J. de Vries, Elisabeth G.E. de Vries. _University Medical Center Groningen, Groningen, Netherlands_.

Introduction: Activation of T-cells is accompanied by a strong up-regulation of interleukin-2 (IL2) receptor (CD25). Therefore PET imaging of IL2 receptors might be a suitable imaging biomarker for T-cell activation. 18F-IL2 PET could detect CD25-positive T-cells and the migration of these T-cells to distant sites of inflammation in SCID mice subcutaneously injected with human peripheral blood mononuclear cells1 and NOD mice with insulitis. Also a strong correlation was found between the accumulation of 18F-IL2 and the number of injected activated T-cells in immune-competent rats.2 In tumor bearing mice, 18F-IL2 PET could detect treatment-induced accumulation of activated T-cells in the tumor following local radiotherapy and/or vaccination.

Cancer immunotherapy is increasingly obtaining a place in clinical practice. However not all patients benefit. A biomarker for upfront or early response prediction for these immunotherapies might support patient selection before and during therapy. Potentially 18F-IL2 PET might serve this purpose. Therefore we aimed to accommodate the production of 18F-IL2 for use in clinical imaging studies.

Material and methods: In order to produce a GMP-compliant tracer the production is being implemented on the Eckert & Ziegler PharmTracer synthesis module. In this synthesis module, disposable cassettes, reactors and vials are used to avoid cross-contamination between productions. First the prosthetic group N-succinimidyl 4-fluorobenzoate (18F-SFB) is produced in 3 steps from cyclotron-produced 18F-fluoride. Subsequently, 18F-SFB is conjugated to human recombinant IL2 (Proleukin®). Various methods for synthesis and purification of 18F-SFB have been evaluated. Also purification of 18F-IL2 has been optimized. Quality control has been performed using ultra performance liquid chromatography (UPLC) and Thin Layer Chromatography (TLC).

Results: 18F-SFB was successfully synthesized with the Eckert & Ziegler PharmTracer synthesis module with decay-corrected radiochemical yields comparable to literature (range 28-64%). Major challenges have been encountered, most importantly regarding the purification of the 18F-SFB and 18F-IL2, stability of the IL2 and specific activity. The activated ester 18F-SFB was purified by high performance liquid chromatography (HPLC) to remove any impurities that could interfere with the conjugation. 18F-IL2 has been purified using PD-10 columns with PBS containing 0.05% SDS as mobile phase.

Conclusions: Several challenges for the GMP-compliant production of 18F-IL2 have been overcome. In the near future this tracer will be used in preclinical and clinical studies to non-invasively image activated T-cells before and during cancer immunotherapy. This can provide insight in the effects of cancer immunotherapy on the immune response.

References:

1. Di Gialleonardo V, et al. J Nucl Med.2012;53(5):679-86.

2. Di Gialleonardo V, et al. Eur J Nucl Med Mol Imaging.2012;39(10):1551-60.

#4209

Targeting GPNMB with 89Zr-CR011 for PET imaging of triple negative breast cancer.

Bernadette V. Marquez,1 Supum Lee,1 Tibor Keler,2 Thomas Hawthorne,2 Jeremy Hoog,1 Shunqiang Li,1 Cynthia Ma,1 Suzanne E. Lapi3. 1 _Washington University School of Medicine, Saint Louis, MO;_ 2 _Celldex Therapeutics, Hampton, NJ;_ 3 _University of Alabama Birmingham, Birmingham, AL_.

Glycoprotein non-metastatic melanoma B (GPNMB) is a transmembrane protein overexpressed in 30 - 40% of triple negative breast cancer (TNBC) and has shown to be associated with metastasis and disease recurrence. An anti-GPNMB antibody drug conjugate, Glembatumumab Vedotin (CDX-011), is currently in Phase II clinical trials for the treatment of metastatic TNBC patients, with promising outcomes. Positron Emission Tomography using radiolabeled antibodies could be advantageous in stratifying patients who may benefit from CDX-011, tracking the biodistribution of CDX-011, and assessing GPNMB expression in vivo. To this end, we radiolabeled the naked antibody, Glembatumumab (CR011), with the positron-emitting 89Zr (half life = 3.3 days). We characterized the stability, affinity, rate of cellular internalization, and specificity of 89Zr-CR011 using various cell-binding assays in human TNBC cell lines. We determined that 89Zr-CR011 is stable in serum solution for up to 5 days, binds specifically to GPNMB+ TNBC cells with high affinity (KD = 16 nM), and internalizes rapidly (50% within 30 - 60 min). We conducted a biodistribution study from 1 - 12 days post administration via tail vein in GPNMB+ MDA-MB-468 xenografts to determine the time point at which we achieve the optimal tumor-to-nontarget ratios. A subset of mice was administered a blocking dose of unlabeled CR011 (100-fold excess, 1 mg/mouse), where we observed a 2.5-fold reduction in 89Zr-CR011 tumor uptake, confirming the specificity of 89Zr-CR011 for GPNMB+ TNBC tumors. PET imaging studies and dosimetry calculations are currently in progress. This preliminary study demonstrates that 89Zr-CR011 may be an excellent companion diagnostic agent for CDX-011 therapy and an essential tool to assess the function of GPNMB in vivo.

#4210

Non-invasive molecular imaging of SIRT1-mediated epigenetic regulation in cancer using PET/CT with a novel substrate-type radiotracer 2-[18F]PhAHA.

Robin E. Bonomi,1 Maxwell Laws,1 Aleksandr Shavrin,1 Thomas Mangner,2 Juri G. Gelovani2. 1 _Karmanos Cancer Institute, Wayne State University, Detroit, MI;_ 2 _Karmanos Cancer Institute, Detroit, MI_.

SIRT1 is involved in a wide variety of cellular processes and functions and is known to mediate cleavage of the acetyl moiety from the acetylated lysine residues of several proteins, including p53, PPARƔ, members of the FOXO family and NF-KB. Therefore, SIRT1 has emerged as an important target for therapy of cancer. However, currently there are no methods for non-invasive monitoring of pharmacodynamics of novel SIRT1 isoform-selective inhibitors in norm and disease. Therefore, there is a need for development of agents for non-invasive imaging of expression and activity of various SIRT1 in vivo.

Previous studies demonstrated that SIRT1 can dephenylacetylate a lysine residue at a rate of ~56% of its natural deacetylation rate, while other SIRT isoforms cannot cleave an aromatic leaving group. This provided an opportunity to develop a SIRT1-selective imaging agent for PET imaging, which would allow for quantitative measurement of the SIRT1 expression-activity product in vivo. We developed a focused library of compounds to elucidate the structure-activity relationship of SIRT1 and to identify the most selective and efficient imaging substrate for SIRT1. This preliminary screening utilized high-throughput fluorogenic assay with the Carbazol-L-Lysine-Aminomethylcoumarin as a backbone [1]. Subsequently, this backbone was exchanged for 6-aminohexanoicanilide, AHA [2] to develop PET radiotracer. The lead compound, 2-fluorophenylaminohexanoicanilide, was developed in both F-19 (2-FPhAHA) and F-18 (2-[18F]PhAHA) versions. 2-[18F]PhAHA was injected i.v. into Sprague-Dawley (SD) rats followed by dynamic imaging using MicroPET R4 (Siemens, TN), followed by CT imaging on INVEON microCT (Siemens, TN). The kinetics of radiotracer uptake was quantified using Logan graphical analysis [3], which demonstrated differential accumulation of 2-[18F]PhAHA-derived radioactivity in specific areas of the rat brain with high expression of SIRT1 (i.e., the dentate gyrus, CA1, nucleus accumbens, and caudate putamen). Following characterization of 2-[18F]PhAHA-derived radioactivity in the normal rat brain, 2-[18F]PhAHA was administered to SD rats bearing intracerebral 9L gliomas. PET/CT demonstrated significantly increased uptake of 2-[18F]PhAHA-derived radioactivity in the 9L tumors vs. normal brain tissue (P<0.01) and matched with tumor localization as observed with MRI and histological staining.

In summary, SIRT1 activity is upregulated in intracerebral 9L gliomas, as evidenced by increased accumulation of 2-[18F]PhAHA on PET/CT images, which demonstrates the feasibility of this method for visualization and quantification of SIRT1 activity in brain tumors. Also, 2-[18F]PhAHA on PET/CT should allow for future monitoring of pharmacodynamics of SIRT1 inhibitors during anti-cancer therapy.

#4211

Discovery of a novel peptide for improvement of radiotherapeutic efficacy and development of prognotic diagnosis.

Kyoung Jin Lee,1 Jae Hee Lee,1 Si Yeol Song,2 Seong-Yun Jeong,1 Eun Kyung Choi2. 1 _Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Radiation Oncology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea_.

Cancer can be treated by the various modalities including surgery, chemotherapy, radiotherapy, among which the radiotherapy is the most important therapeutics in the cancer treatment. Despite the progression of modalities, cancer therapy has been constrained by impasse of prediction of treatment outcome and direction of cancer therapeutics because of diagnosis date, metastasis and genetic diversity of the individual patients.

In this study, we discovered specific tumor targeting peptides and radiotherapy-responding peptides suitable to apply to cancer therapy and monitoring of radiotherapy response. For a non-invasive imaging for potential cancer diagnosis system, we screened peptides using the phage display technique. Phage display is used in various pharmaceutical biotechnologies, and the one of the most powerful technologies to identify novel peptides regulating carcinogenesis. To emphasize the function of peptide ligands in a living body and consider the tumoral microenvironment, we used the patient-derived xenograft (PDX) tumor models for in vivo peptide displayed phage screening. It is warranted that the peptides specifically targeting in vivo human cancer xenografts with radiation treatment or not in colorectal cancer PDXs. We performed in vivo peptide screening in the two different cases of colorectal cancer PDX mouse models, and selected peptides were confirmed to radiation responding peptides and radiation resistant peptides. The results suggested that selected peptides were a promising diagnostic lead molecule for rapid and accurate detection for the radiotherapy response.

Taken together, we described a strategy to identify the peptides by in vivo phage display, and peptides possessed the specific targeting ability which would be potentially feasible for the development of personalized precision treatment. Its might be potential IR-guided probe and prognostic diagnosis in radiotherapy for colorectal cancer.

Keywords: Peptide, Colorectal cancer, Patient-derived xenograft (PDX), Radiation, In vivo peptide screening

#4212

Molecular imaging of EGFR-expressing tumors with novel targeted protein scaffold, anti-EGFR repebody.

Misun Yun, Dong-Yeon Kim, Hyeon-Sik Kim, Jin Hai Zheng, AYoung Pyo, Jung-Joon Min. _Chonnam National University Medical School, Gwangju, Republic of Korea_.

Repebody is a binding scaffold based on variable lymphocyte receptors which are nonimmunoglobulin antibodies composed of leucine-rich repeat modules in jawless vertebrates. Repebody can be developed against variety of epitopes by module engineering. In this study, EGFR-specific repebody (RBEGFR) was developed to visualize the status of receptor expression and to prevent ligand binding that may inhibit autophosphorylation and downstream intracellular signaling.

We developed RBEGFR by phage display. H1650, HCC827, A549 (human non-small cell lung cancer) and HT29 (human colon cancer) were selected as EGFR expressing cell lines. MDA-MB-435 (human melanoma) and SW620 (human colon cancer) was selected as a negative control. Specific binding of RBEGFR to cells and cancer tissue was determined by immunofluorescence (IF) staining and/or FACS analysis. In vivo imaging was done by i.v. injection of FNR-675 labeled RBEGFR (30 μg/mouse) or 64Cu-NOTA- RBEGFR (7.4 MBq/mouse) in H1650, HCC827 and HT29-bearing mouse models using cooled CCD camera or microPET, respectively. Orthotopic colon cancer mice were generated by i.p. injection of Azoxymethane (AOM; 10 mg/kg) and oral administration of 2% dextran sulfate sodium (DSS). In vivo imaging was done by i.v. injection FNR-675 labeled RBEGFR (30 μg/mouse) in AOM/DSS mouse models using cooled CCD camera or fluorescence microendoscopy.

In vitro and in vivo IF staining demonstrated that strong binding of RBEGFR to H1650, HCC827 and HT29, but not to MDA-MB-435 and SW620. In vivo near infrared (NIR) imaging demonstrated specific targeting of FNR-675-RBEGFR to grafted H1650, HCC827 and HT29 tumor in mice. The 64Cu-NOTA- RBEGFR was detected at the implanted tumor from 1 h (SUVmax: 1.34±0.12) after the injection, peaked at 6 h (1.75±0.18), maintained to 24 h (1.33±0.17). The radioactivity significantly decreased by blocking with cold form of 50 μM naïve RBEGFR 1 day before injection of 64Cu-NOTA- RBEGFR, indicating specific binding of RBEGFR to EGFR in vivo. Optical NIR imaging after i.v. injection of FNR-675-RBEGFR showed specific signal in the abdomen of AOM/DSS mice, but not in control mice. Correlation with surgical/necropsy imaging, fluorescence endoscopy and pathology revealed strong accumulation of FNR-675-RBEGFR in malignant dysplasia, but weak or no accumulation in low grade tumor or benign lesion.

In conclusion, the RBEGFR could be developed for specific targeting of cancer overexpressing EGFR. The fluorescence-labeled RBEGFR could be developed for imaging agent for detecting colonic dysplasia and assessing EGFR status. In particular, this agent may have a potential as an imaging companion diagnostics to predict therapeutic outcome of targeted therapy with monoclonal antibody for EGFR through pre-therapeutic visualization of EGFR status. Our work provides a basis to develop potential strategy of targeted immune-detection of cancers which may replace monoclonal antibodies.

#4213

Gold nanostar as theranostic probe for brain tumor sensitive PET-optical imaging and image-guided specific photothermal therapy.

Yang Liu,1 Austin Carpenter,2 Hsiangkuo Yuan,3 Zhengyuan Zhou,4 Michael Zalutsky,5 Ganesan Vaidyanathan,4 Hai Yan,2 Tuan Vo-Dinh6. 1 _Department of Chemistry, Department of Biomedical Engineering, Fitzpatrick Institute for Photonics, Duke University, Durham, NC;_ 2 _Department of Pathology, Duke University Medical Center, Durham, NC;_ 3 _Department of Biomedical Engineering, Duke University, Durham, NC;_ 4 _Department of Radiology, Duke University Medical Center, Durham, NC;_ 5 _Department of Radiology, Department of Biomedical Engineering, Department of Radiation Oncology, Duke University Medical Center, Durham, NC;_ 6 _Department of Biomedical Engineering, Department of Chemistry, Fitzpatrick Institute for Photonics, Duke University, Durham, NC_.

Malignant brain tumors are one of the most lethal cancer forms causing over 14,000 deaths each year. In spite of extensive research efforts over many decades, the median overall survival (OS) still remains at just 15 months for the common malignant giloma, glioblastoma multiforme (GBM). We have developed a multifunctional gold nanostar (GNS) probe that shows potential to improve cancer management by providing personalized cancer theranostics, a combination of diagnostic and therapeutic functions. The GNS nanoprobe has a tunable plasmon absorption peak in the near infrared (NIR) "tissue optical window" for deep-tissue penetration and tip-enhanced surface plasmon resonance, which makes the GNS as a superior nanoprobe for cancer detection using surface-enhanced Raman spectroscopy (SERS), two-photon photoluminescence (TPL) imaging and photothermal therapy with NIR light. We have also functionalized the GNS with iodine-124 radioisotopes for sensitive 3D positron emission tomography (PET) and achieved a 10-picomolar limit of detection (LOD). The GNS nanoprobe accumulates selectively in brain tumor due to the enhanced permeability and retention (EPR) effect, which is caused by defective endothelial tight junctions in blood vessels surrounding the tumor. On the contrary, the GNS nanoprobe has minimum uptake in normal brain tissue due to the intact blood-brain-barrier (BBB). The GNS uptake ratio of tumor-to-normal (T/N) brain tissue is measured to be up to 30:1 by using inductively coupled plasma mass spectrometry (ICP-MS). By combining the high detection sensitivity of PET imaging and specific accumulation of GNS in brain tumor, we have demonstrated the feasibility of 1-mm brain tumor detection with the I-124 labeled GNS nano radiotracer. The obtained T/N ratio is up to 10:1 with our GNS nanoprobe labeled with I-124, which is much better than that (typically less than 1.5:1) for traditional F-18 fluorodeoxyglucose (FDG) PET contrast agent due to high glucose uptake in normal brain tissue. Furthermore, we have utilized GNS's high TPL intensity to delineate brain tumor margin with excellent sensitivity and high spatial resolution (µm level). In addition to brain tumor imaging, we have successfully demonstrated photothermal ablation with a NIR laser taking advantage of GNS's superior photothermal conversion efficiency. We have also performed a 6-month toxicity study for the GNS nanoprobe and experiment results, including body weight monitoring, blood chemistry test, and H&E histopathology examination, show no deleterious effect after infusion of GNS nanoprobe up to 100 mg/kg dose. In summary, the GNS nanoprobe, with limited toxicity and multifunctional capabilities, exhibits great promise to fulfill the unmet needs in brain tumor management in future translational medicine investigations.

#4214

Assessment of early antimitotic treatment response in ovarian cancer using temporal diffusion spectroscopy.

Xiaoyu Jiang, Hua Li, Ping Zhao, Jingping Xie, Dineo Khabele, Junzhong Xu, John C. Gore. _Vanderbilt University, Nashville, TN_.

A quantitative MRI method, namely, temporal diffusion spectroscopy (TDS), has been used to measure tumor microstructural changes in response to a well-established antimitotic drug treatment (protein-bound paclitaxel) and evaluated as an early indicator of tumor responsiveness. Current radiological methods of monitoring treatment response in solid tumors rely on measuring changes in tumor size as measured by CT or MRI, and it is well recognized this approach suffers from major limitations. In recent years, tumor size measurements have been supplemented by a number of imaging techniques that aim for in vivo characterization and measurement of biologic processes at the cellular and molecular level, but to date none has proven robustly successful in practice. For example, diffusion MRI has shown a high sensitivity to tumor response, but a relatively lower specificity e.g. sometimes unable to distinguish real response of responder tumors from the progression of some non-responders.

Temporal diffusion spectroscopy measures a series of apparent diffusion coefficients (ADC) over a wide range of effective diffusion times, corresponding to diffusion distances ranging from subcellular to cellular levels (~ 3-20 μm). By fitting the measured ADC spectra to a simplified tissue model, parameters reflecting structural properties (such as cell size) in tissues can be extracted. Abraxane, one of the most successful microtubule-targeted chemotherapeutic drugs, has been reported to induce aberrant mitosis in tumor cells leads to mitotic arrest, the consequence of which is a cell size increase. In the current study, we implemented TDS in Abraxane/PBS-treated two types of human ovarian xenografts and cultured cells (OVCAR-8 as a responder to Abraxane, and NCI/ADR-RES as a resistant type). Abraxane-induced cell size increases were confirmed by flow cytometry and light microscopy in cell culture. The efficacy of Abraxane in treating tumors in vivo was examined histologically by calculating percentages of Caspase-3-activated area within Caspase-3 stained slides. Most excitingly, the fitted restricted size, one of the spectral parameters obtained from TDS, was able to quantify cell size increases which were not detected by conventional diffusion-MRI. All the MR results had a high degree of consistency with other flow, microscopy, and histological data. Moreover, with an appropriate analysis, the Abraxane-responsive tumors in vivo could be easily distinguished from all the other drug-vehicle-treated tumors and Abraxane-resistant tumors. In conclusion, temporal diffusion spectroscopy detects antimitotic-therapy-induced microstructural variations (notably, increases in cell sizes) in tumors in vivo which occur before changes in tumor volume or conventional diffusion MRI metrics. A combination of restricted size and zero-frequency diffusion coefficient can potentially be used as an early indicator of tumor responsiveness.

#4215

Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts.

Prasanta Dutta, Mayrim Rios Perez, Travis Cole Salzillo, Michael Pratt, Yaan Kang, Niki Zacharias, Anirban Maitra, Jason B. Fleming, Pratip Bhattacharya. _MD Anderson Cancer Center, HOUSTON, TX_.

Pancreatic cancer, the most lethal of solid tumors is an aggressive disease that develops relatively symptom-free. The absence of early symptoms has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic intervention to improve the survival rate of pancreatic cancer patients. Our goal is to identify metabolic biomarkers for early detection with an understanding of pancreatic cancer progression from benign lesions to a malignant state. Hyperpolarized magnetic resonance imaging (HP-MRI) provides a >10,000-fold signal enhancement for detecting of endogenous metabolic substrates to monitor metabolic fluxes through the multiple key biochemical pathways including glycolysis and citric acid cycle. In particular, conversion between hyperpolarized 13C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase (LDH), has been shown to have a number of potential applications such as diagnosis, staging tumor grade and monitoring therapy response. Genetic mutation is evident in causing tumorigenesis and often time these mutations trigger the signaling pathways that are associated with "metabolic transformations. There is currently tremendous research interest in dissecting the mechanisms of metabolic transformation as the cancers progress. In this effort, we are applying high-resolution NMR (nuclear magnetic resonance) metabolomics and the real-time hyperpolarized metabolic imaging of patient-derived xenograft tumors for probing the underlying mechanism of altered-metabolism and correlate with pancreatic cancer progression and its aggressiveness. The protocol detailing heterotopic engraftment of pancreatic cancer patient tumors into immunodeficient mice and expansion of direct xenograft tumors was recently reported elsewhere. These patient-derived xenografts (PDX) were well annotated with original patient tumors. For the NMR metabolomics study, the fresh-frozen tumor samples were homogenized, lyophilized, and resuspended in D2O and 1-D proton NMR spectroscopy was employed to characterize the water-soluble portion of the metabolome. These data suggest that elevated glycolysis (production of high lactate) and alterations in choline metabolism may arise in pancreatic cancer development. Also, the metabolite concentrations were significantly higher in aggressive tumors compare to non-aggressive one. The real-time metabolic imaging shows the immediate conversion of lactate after injection (i.v.) of hyperpolarized 13C pyruvate and probe the upregulated LDH activity as the cancer progress. The basic genomic profiling of these tumors is in progress. This model may provide an excellent platform where mutational status of these patient tumors can be correlated with both high resolution and hyperpolarized dynamical metabolomics data for characterizing individual tumor phenotypes.

#4216

**Experimental imaging in orthotopic xenograft models of renal cell carcinoma: comparative evaluation of high-resolution ultrasonography,** in vivo **micro-CT, and 9.4T MRI.**

Johannes Linxweiler,1 Christina Körbel,2 Andreas Müller,3 Volker Jung,1 Eva Jüngel,4 Stefan Siemer,1 Michael Stöckle,1 Kerstin Junker,1 Michael D. Menger,2 Matthias Saar1. 1 _Saarland University Medical School, Department of Urology, Homburg/Saar, Germany;_ 2 _Saarland University Medical School, Department of experimental surgery, Homburg/Saar, Germany;_ 3 _Saarland University Medical School, Department of diagnostic and interventional Radiology, Homburg/Saar, Germany;_ 4 _Frankfurt University Medical School, Department of Urology, Homburg/Saar, Germany_.

Introductory Sentence

Orthotopic xenografts are increasingly used as preclinical in-vivo models for the study of renal cell carcinoma (RCC). However, monitoring of these tumors requires sophisticated imaging techniques. In this study, we comparatively evaluated modern small animal imaging tools like high-resolution 3D-ultrasonography (3D-US), contrast-enhanced in-vivo micro-CT (CE-CT) and 9,4T MRI (MRI) to non-invasively monitor tumor growth and progression in an orthotopic RCC xenograft model.

Material and Methods

106 CAKI-1 cells were injected under the renal capsule of 18 Balb/c-nude mice. Every 14 days from week 4 imaging was performed by CE-CT and 3D-US. 10 weeks after tumor cell inoculation, all animals additionally underwent multiparametric MRI (T1, T2, T2*, DWI). At autopsy, tumor volumes were determined using a caliper and compared to in-vivo imaging results. CE-CT, MRI and 3D-US were evaluated regarding tumor detection, analysis of tumor volume, radiographic imaging properties and examination time. Finally, similar experiments were performed with the RCC cell lines KTCTL30 (n=2) and 786-0 (n=1) to test their applicability for the orthotopic xenograft model.

Results

Tumors were detected in 16/18 animals (89%) 4 weeks after orthotopic inoculation. All methods enabled to adequately visualize orthotopic tumors and to display their growth over time. While the tumors had a homogenously radiolucent signal in CT scans, sonography and MRI were better able to visualized intratumoral structures (e.g. solid areas or intratumoral bleedings) and surrounding soft tissue. CT had the best spatial resolution, followed by 3D-US and MRI. The median examination time was 4.8min per 3D-US, 12.5min per CT and 37.9min per MRI, respectively. Of interest, tumor volumes determined by KM-CT and ultrasonography showed strong correlation with each other (n=85, R=0.985) as well as with caliper measurements at autopsy (CT: n=16. R=0.922; sonography: n=16, R=0.934). Similarly, tumor volumes measured with T2-weighted MRI correlated well with those determined by CT, sonography and caliper. No side effects due to radiation exposure were observed. Mice inoculated with KTCTL30 cells developed partly cystic, partly solid tumors. 786-O cells yielded purely solid, exponentially growing tumors.

Conclusion

All three imaging modalities are feasible tools for the non-invasive monitoring of orthotopic tumor growth and show excellent correlation with each other. While sonography allows for a fast analysis of tumor volume with excellent resolution and no radiation exposure, MRI provides the best visualization of soft tissue and can give information about tumor biology when acquiring additional sequences. CT has the best spatial resolution of all methods and enables simultaneous screening for bone and lung metastases but relies on the use of contrast agent and ionizing radiation.

#4217

First pre-clinical validation of radiogenomics in glioblastoma.

Pascal Zinn,1 Sanjay Singh,2 Markus M. Luedi,3 Faramak Zandi,2 Aikaterini Kotrotsou,2 Masumeh Hatami,2 Ginu Thomas,2 Ahmed Elakkad,2 Joy Gumin,2 Erik P. Sulman,2 Frederick Lang,2 David Piwnica-Worms,2 Rivka R. Colen2. 1 _Baylor College of Medicine, Houston, TX;_ 2 _UT MD Anderson Cancer Center, Houston, TX;_ 3 _Berne University, Bern, Switzerland_.

A plethora of Magnetic Resonance Imaging (MRI) features have been correlated to cancer genomics to date, however, none have established causality. Here, we present an in vivo xenograft RNA interference validated, potentially clinically applicable test method termed "Magnetic Resonance Radiomic Sequencing" (MRRS) for the noninvasive detection of cancer genomics in Glioblastoma. MRRS comprehensively assesses the entire tumor mass using imaging texture-based algorithms that generate thousands of variables (features) inherent to the tumor. Two independent glioblastoma stem cells (GSC1 and GSC3) harboring doxycycline inducible short hairpin RNA against Periostin (POSTN), a gene previously identified in our radiogenomic screen, were implanted at orthotopic location in nude mouse brain. In vivo knockdown of >90% and ~40% POSTN gene was achieved in GSC3 and GSC1 respectively. The T2 and T1 post MRI texture features, in edema and contrast enhancement phenotype features were compared between doxycycline (POSTN knockdown) and sucrose (control) group of mice using T test statistics. The significant features were included in a Stepwise Forward Logistic Regression analysis to build the final predictive model. The accuracy of the model was tested using ROC cure analysis. Among 3600 features in GSC3 mice cohort, 117 features were significantly (p value<0.05) different between the two groups. The significant features were included in a Stepwise Forward Logistic Regression analysis, 2 textures features (feature 234 of edema T1 and feature 251 of edema T2) were selected to be included in the final predictive model. The AUC of the model with leave one out cross validation method was 100%. The similar analyses were done in the GSC1 mice. The final predictive model in the GSC1 group was statistically insignificant (p value=0.15) with AUC (95% CI) = 73% (46%-98%), suggesting that MRRS is reflective of underlying gene expression levels. Our results therefore describe the `first mouse model derived MRRS signature to describe a causal link of gene alteration to MRRS. This novel test method may open an avenue for human-mouse matched co-clinical trials and noninvasive Radiogenomic diagnostics.

#4218

**Diffusion-weighted MRI can predict response to aflibercept in** in vivo **models.**

Karianne G. Fleten,1 Kine M. Bakke,2 Andreas Abildgaard,1 Gunhild M. Mælandsmo,1 Katrine Røe Redalen,2 Kjersti Flatmark1. 1 _Oslo University Hospital, Oslo, Norway;_ 2 _Akershus University Hospital, Lørenskog, Norway_.

BACKGROUND: The liver is the most frequent metastatic site in colorectal cancer (CRC), and relevant in vivo models are needed to study the efficacy of anticancer drugs in this setting. Aflibercept, targeting vascular endothelial growth factors A and B and placental growth factor, is one of the most recently approved antiangiogenic agents for treatment of metastatic CRC. In the present work, the efficacy of aflibercept was investigated in experimental models of liver metastases in nude mice and growth progression was monitored using non-invasive imaging.

METHODS: Liver metastases were established in mice by intrasplenic injection of CRC cell lines HCT116 and HT29 transfected with luciferase. Magnetic resonance imaging (MRI) (7T) and bioluminescent imaging (IVIS spectrum) were used to monitor tumor growth. Aflibercept was delivered intraperitoneally. To further characterize treatment response in metastatic tumors, diffusion-weighted (DW)-MR images were obtained. At termination tumors were sampled for histopathologic analysis.

RESULTS: Mice bearing HCT116 xenografts responded well to treatment and a significant increase in survival compared to vehicle treated animals was observed (p< 0.001), in addition to decreased tumor burden. No increase in survival was observed upon aflibercept treatment in HT29 xenografts (p=0.155), whereas in one of two experiments performed a significant reduction in tumor volume was observed, suggesting that there was a slight response to treatment.

T2-weighted MRI was used to quantify tumor volume by manual delineation using the OsiriX software. MRI-assessed tumor volumes correlated well with tumor weight at the time of termination, while for bioluminescence measurements no such correlation was observed. In contrast, bioluminescence was a sensitive detection method early in the experiments when MRI did not show liver tumor.

DW-MRI was obtained and apparent diffusion coefficient (ADC) tumor maps were calculated using the nordicIce software. A slight increase in ADC values was observed for HT29 tumors while there was a large increase in ADC values for HCT116 tumors after treatment with aflibercept. This corresponded well with histologic evaluation of tumors, showing increased necrosis in HCT116 tumors, but not in HT29 tumors.

CONCLUSION: Our results demonstrate efficacy of aflibercept in an orthotopic model of liver metastases in CRC. MRI could be used to monitor treatment efficacy with high precision, while bioluminescence measurement could detect small-volume disease with high sensitivity, but was less specific in high-volume disease. Interestingly, ADC values obtained from DW-MRI were highly predictive of treatment response by clearly visualizing and quantifying tumor necrosis.

#4219

Surface-displayed RGD enhanced the targeting and therapeutic efficacy of attenuated Salmonella typhimurium.

Seung-Hwan Park, Jin Hai Zheng, Hee-Seung Yun, In-Kyu Park, Yeongjin Hong, Hyun E. Choy, Jung-Joon Min. _Chonnam National University Medical School, Hwasun, Republic of Korea_.

Anticancer therapy using engineered bacteria aims to overcome limitations of current cancer therapy by actively targeting and efficiently removing cancer. In order to achieve this goal, new approaches are essential to target therapeutically resistant regions of tumors, to maintain enough number of bacteria in tumor and to deliver drugs at sufficient concentrations during the period of therapeutic process. Therefore, it is necessary to enhance the bacterial targeting efficiency through bacterial surface engineering. Here, we demonstrate that Salmonella tumor tropism can be strengthened significantly via surface displaying of RGD peptide sequence (ACDCRGDCFCG; RGD sequence) in the external loop of outer membrane protein A (OmpA) of attenuated Salmonella typhimurium. RGD-displaying Salmonella strongly bound to αvβ3 over-expressing cancer cells while showed weak binding to non-expressing cancer cells, indicating the feasibility of surface display with preferential homing peptide. In vivo bioluminescence imaging showed strong targeting efficiency of RGD-displayed Salmonella in αvβ3 over-expressing cancer xenografts (MDA-MB-231, MDA-MB-435, M21, and U87MG). The surface engineered bacteria significantly suppressed both human breast tumor (MDA-MB-231) and human melanoma (MDA-MB-435), and prolonged survival in mice. In conclusion, engineered bacteria displaying RGD peptides on the surface could advance both targeting efficiency and therapeutic effects.

#4220

Tumor-targeting Salmonella typhimurium A1-R efficacy on colon cancer liver metastasis alone and in combination with fluorescence-guided surgery.

Takashi Murakami,1 Yukihiko Hiroshima,1 Yong Zhang,1 Ming Zhao,1 Ryusei Matsuyama,2 Takashi Chishima,2 Kuniya Tanaka,2 MIchael Bouvet,3 Itaru Endo,2 Robert M. Hoffman1. 1 _AntiCancer, Inc., San Diego, CA;_ 2 _Department of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University, Yokohama, Japan;_ 3 _Department of Surgery, University of California San Diego, San Diego, CA_.

Liver metastasis is the most frequent cause of death from colon and other cancers. Surgical treatment of liver metastasis has had limited success. The aim of this study is to determine the efficacy of tumor-targeting Salmonella typhimurium A1-R alone and in combination with fluorescence-guided surgery on liver metastasis in orthotopic mouse models. HT-29 human colon cancer cells expressing red fluorescent protein (RFP) developed liver metastasis after orthotopic implantation. In the orthotopic nude-mouse models, S. typhimurium A1-R targeted liver metastases and significantly reduced their growth (p < 0.01) after i.v. administration. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase overall survival (p = 0.01) and disease-free survival (p < 0.01) after bright-light surgery (BLS) of liver metastasis. The results of this study demonstrate the future clinical potential of S. typhimurium A1-R treatment of liver metastasis.

#4221

Imaging formation of the tumor microenvironment in orthotopic liver tumors in red fluorescent protein (RFP) transgenic nude mice.

Atsushi Suetsugu,1 Yukihiko Hiroshima,2 Takuro Matsumoto,1 Kosuke Hasegawa,1 Miki Nakamura,1 Masahito Shimizu,1 Shigetoyo Saji,1 Hisataka Moriwaki,1 Michael Bouvet,3 Robert M. Hoffman2. 1 _Gifu University Graduate School of Medicine, Gifu, Japan;_ 2 _AntiCancer, Inc., San Diego, CA;_ 3 _University of California, San Diego, CA_.

We report here dual-color imaging of the formation of the TME in orthotopic liver cancer in transgenic red fluorescent protein (RFP) nude mice, which express RFP in all organs. Non-colored HUH-7 human hepatoma cells were injected in the spleen of RFP nude mice to establish an orthotopic liver cancer model. TME formation in the liver tumor was observed using the Olympus OV100 small animal fluorescence imaging system. Non-colored liver cancer cells formed colonies in the liver 28 days after cell transplantation to the spleen. In the liver, RFP host-mouse cells accumulated around and in the liver tumor as visualized by fluorescence imaging. A desmin and sirus-red positive area increased around and within the liver tumor over time. These results indicate cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis suggesting that CAFs, along with the angiogenic tumor blood vessels, could serve as visible therapeutic targets.

#4222

Usefulness of VE1 immunohistochemical detection of BRAFV600E in aggressive thyroid cancers (PDCs and UCs).

Noa Feás-Rodríguez,1 Angela M. Costa,2 Tomás Alvarez Gago,3 Juan José Mateos Otero,3 Jose Manuel Cameselle Teijeiro,4 Raquel Muñoz,1 Ginesa Maria Garcia-Rostan1. 1 _Institute of Molecular Biology and Genetics (IBGM), Valladolid, Spain;_ 2 _Universidade do Minho, Braga, Portugal;_ 3 _Department of Pathology - School of Medicine - Valladolid University, Valladolid, Spain;_ 4 _Department of Pathology, Hospital Clinico Universitario de Santiago de Compostela, Santiago de Compostela, Spain_.

Activating BRAF mutations are frequent in thyroid follicular cell carcinogenesis. The BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency the BRAFV600E mutation is also present among less differentiated, more aggressive, I131 resistant forms of thyroid cancer as poorly differentiated carcinomas (PDCs) and undifferentiated carcinomas (UCs). Small molecule inhibitors targeting either the BRAF V600E protein or upstream or downstream kinases involved in MAPK signalling are currently under pre-clinical or clinical investigation in advanced, metastatic, I131 resistant thyroid cancers. Recently, immunohistochemical (IHC) studies on PTCs, using the VE1 mouse anti-human BRAF V600E antibody have shown to be a reliable means for detecting the BRAFV600E mutation in a clinical setting without molecular genotyping units. In this study we sought to determine the usefulness of the VE1 antibody for detecting the BRAF V600E mutant protein in a series of 103 aggressive thyroid cancers (59 PDCs and 44 UCs) previously characterized by PCR-SSCP for the presence of BRAF mutations in exons 11 and 15. The BRAFV600E mutation was present in 10/59 PDCs (17%) and 11/44 UCs (25%). Immunohistochemistry revealed 9 mutated PDCs (sensitivity 90%) and 9 mutated UCs (82% sensitivity). The staining intensity in BRAF V600E mutated samples ranged from weak to strong. Non valuable results were found in 3 PDCs and 4 UCs. Overall the results indicate that immunohistochemistry with VE1 antibody may be an alternative to molecular biology approaches for the routine detection of BRAFV600E point mutations in clinical settings without molecular genotyping facilities. It may help clinicians in targeted therapy decision making. 

### Molecular and Cellular Imaging of Cancer 2

#4223

Novel strategy for diagnosis of solid tumors by visualization of fusion transcripts.

Fatu Badiane Markey, Mona Batish. _Rutgers University, Newark, NJ_.

The discovery of chromosomal translocations as one major cause of cancers has led to the introduction of numerous diagnostic techniques to detect these chromosomal abnormalities. Chromosomal banding, DNA fluorescence in situ hybridization (DNA FISH), and RT PCR are all techniques used for the diagnosis of cancers. Despite the advancement these techniques have brought to the field, they are primarily optimized for the detection of blood cancers and are not very efficient for the detection of solid tumors which are very heterogeneous. One such cancer, Ewing's sarcoma (ES), is a prevalent pediatric, bone and soft tissue tumor that is caused by a chromosomal translocation between Ewing's sarcoma breakpoint region 1 (EWSR1) on chromosome 22 and various members of the ETS protein family. The most common fusion partner, in 85% of cases, is Friend leukemia integration 1 (FLI1) on chromosome 11 which leads to the formation of the fused EWS/FLI1 gene that codes for an aberrant transcription factor. However, even within this single translocation there are multiple breakpoint-fusion scenarios that can occur which results in heterogeneity and increases the chances of a faulty diagnosis with standard techniques. Considering the diversity that exists within the molecular abnormalities that result in Ewing's sarcoma and other solid tumors, there is a need for an alternative and more robust method of detection. The technique of single molecule fluorescence in situ hybridization (smFISH) has been modified by our group to target the EWS/FLI1 transcript that results from the chromosomal fusion; this new technique is referred to as "Fusion FISH". In Fusion FISH, two differently labeled probe sets are designed to target each half of the EWS/FLI1 fusion transcript. A co-localization of the two probe sets indicates the presence of the EWS/FLI1 translocation and a positive test for Ewing's sarcoma. Preliminary data has established the success of Fusion FISH in detecting both type 1 and type 2 translocations in several Ewing's sarcoma cell lines. The fusion transcript is expressed at similar levels in all cell lines and clearly distinct from the signal of wild type EWS and FLI1 mRNA. Additional studies will examine this new technique in other solid cancers, patient tumor samples, and for validation of additional relevant mRNA biomarkers.

#4224

Multiplexing single cell time lapse imaging of intra-cellular oxygen in cancer cells.

Heiko Düssmann,1 Sergio Perez,1 Ujval Anil Kumar,1 Dmitri B. Papkovsky,2 Jochen HM Prehn1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Cork, Cork, Ireland_.

Oxygen plays an important role in aerobic energy metabolism and signal transduction. The detection of intracellular or subcellular molecular oxygen (O2) levels is important for the understanding of cell physiology and mitochondrial energy metabolism, and the alterations correlated with cancer. Detection of O2 has recently been improved significantly with the development of live cell time lapse microscopy compatible oxygen sensitive probes.

We here utilized nanoparticle probe MitoImageTM-MM2 with embedded poly(9,9-dioctylfluorene) (PFO) as an O2-independent component and Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) to perform time lapse ratiometric measurements of intra-cellular oxygen levels. Loading of HeLa cervical cancer cells with MM2 enables us to image kinetics of intracellular O2 concentration in response to the mitochondrial respiratory chain inhibitors Antimycin A (complex III) or sodium azide (complex IV). These changes were detected in respiration-supporting medium equilibrated with 5% (53 μM) and 20% (225 μM)O2. Also exposing cells to varying extracellular oxygen concentrations (21 - 225 μM) showed strong dependence of the intracellular oxygen levels. The mitochondrial membrane potential was monitored by simultaneous imaging of Tetra-Methyl-Rhodamine-Methyl ester (TMRM) in the same sample. Multiplexing with GFP finally allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilisation (MOMP) in HeLa expressing cytochrome-c-eGFP and stained with TMRM.

In conclusion, the MitoImageTM-MM2 nanoparticle probe enables single-cell, time-lapse imaging of alterations in intracellular oxygen levels in cancer cells, and can be employed in multiplex assays to investigate alterations in intracellular oxygen levels relative to protein dynamics and mitochondrial function.

Keywords: Intracellular oxygen; phosphorescent oxygen-sensitive nanoparticles; HeLa cells; MitoImage ratiometric probes; confocal microscopy.

#4225

Dynamic control of tumor vasculature with direct observation using intravital microscopy.

Emmanuel M. Gabriel, Daniel Fisher, Minhyung Kim, Colin Powers, Anthony Visioni, Jason Muhitch, Joseph Skitzki. _Roswell Park Cancer Institute, Buffalo, NY_.

Introduction

Intravital microscopy (IVM) provides in vivo real-time imaging of tumor-associated vasculature. Our group was the first to use IVM successfully in human subjects to image melanoma associated vessels. In this preclinical study, we hypothesized that intravenous (IV) nicardipine increases observable blood flow to tumor through vasodilatation and therefore may facilitate drug delivery.

Methods

Standard window chambers for IVM were implanted into female BALB/c mice. The murine colon cancer cell line CT26HA was inoculated in the skin within the chamber. Fluorescein isothiocyanate dextran (FITC-dex) was injected IV to enhance vessel observation prior to nicardipine (8 µg/mouse). Observations were performed at 100x magnification using a modified Olympus microscope for 10 minutes. Naïve mice were used for control observations. Mice bearing CT26HA were observed on post-inoculation day 5, 12 and 20. Vessel architecture and vessel diameter in response to nicardipine were compared.

Results

In non-CT26HA bearing mice, nicardipine resulted in a greater than 10% increase in skin vessel diameters directly measured using IVM (pre-nicardipine diameter 82.4 ± 3.6 µm versus post-nicardipine diameter 93.0 ± 3.9 µm. In CT26HA bearing mice, abnormal vessels were observed as early as day 7 even in the absence of a solid tumor mass. Vessel abnormalities included haphazard areas of increased vessel density, aberrant vessel branching patterns, nonfunctional vessels, and altered flow rates. Following nicardipine injection, the diameter of larger tumor-associated vessels did not increase and remained constant (pre: 111.2 ± 2.7 µm vs post: 109.1 ± 3.3 µm). Paradoxically, for smaller vessels (< 20 µm), tumor-associated vessels vasoconstricted following nicardipine injection and returned to baseline diameter after approximately five minutes. Blood flow in these small vessels decreased or stopped during this observation.

Conclusions

Observation of dynamic vessel changes from vasoactive agents is feasible using IVM. For tumor-associated vessels, nicardipine was not shown to increase vessel diameter and counterintuitively decreased diameters of smaller vessels. Implication on drug delivery from vasoactive agents will lead to further pre-clinical study of applications for human IVM.

#4226

The rodent eye as a non-invasive window for understanding cancer nanotherapeutics.

Xinlei Wang,1 Wenwu Xiao,2 Yuanpei Li,2 Pengfei Zhang,1 Mayank Goswami,1 Robert J. Zawadzki,1 Edward N. Pugh,1 Kit S. Lam2. 1 _UC Davis, Cell Biology & Human Anatomy, Davis, CA; _2 _UC Davis Cancer Ctr., Sacramento, CA_.

We have successfully used the mouse eye as a non-surgical window for highly efficient, optical investigation of xenograft models, using a state-of-the-art ocular imaging facility, the UC Davis "EyePod". The EyePod employs single-cell resolution intravital confocal microscopy and optical coherence tomography, performed completely non-invasively through the natural optics of the eye (1). This technology enables repeatable in vivo imaging over days, weeks and even months, quantitative tracking of tumor development and delivery of theranostic nanoparticles, and the measurement of tumor and tumor microenvironment responses. Moreover, the retina is a highly "approachable part of the brain", so that non-invasive study of ocular tumors provides a platform for examining such critical issues as drug delivery across the blood retinal barrier (BRB) and blood brain barrier (BBB). Clinical metastasis of solid tumor to the uvea is not uncommon. Therefore, uvea/subretinal xenograft implant, even though not orthotopic, is clinically relevant.

To visualize and photo-manipulate nanoparticle delivery to intraocular xenograft, we have used our recently reported nanoporphyrin (2) as the model system. This novel multifunctional porphyrin-based micellar nanoplatform allows (i) efficient encapsulation of hydrophobic chemotherapeutic drugs or fluorescent dyes, (ii) near-infra red fluorescent (NIRF) detection of the tumor via the intrinsic fluorescence of porphyrins, (iii) photodynamic therapy (PDT) and photothermal therapy (PTT) via efficient free radical and heat generation at the tumor site, respectively. Thorough understanding of how this nanocarrier distributes within the tumor microenvironment, and how it responds to controlled optical stimulation will enable us to maximize its therapeutic potential as a nano-theranostic agent. Preliminary FRET study with doxorubicin-loaded nanoporphyrin has allowed us to visualize in vivo and in real time the release of doxorubicin at the tumor site upon illumination with 680nm laser. In addition, the EyePod has enabled us to follow the tumor response over a period of 35 days. Work is currently underway to (i) develop patient-derived xenograft (PDX) model in the eye of NSG mice, and (ii) use EyePod to study the effect of tumor targeting and brain endothelial cell targeting peptides, recently developed in our laboratory, for nanodelivery into the tumor cells and across the BRB.

#4227

Influence of anti-angiogenic therapy on the prevalence of breast cancer stem cells.

Connor Holloway,1 Huisi Ai,2 Mario Dzemidzic,2 Marc Mendonca,2 Harikrishna Nakshatri,2 Keith Stantz1. 1 _Purdue University, West Lafayette, IN;_ 2 _Indiana University School of Medicine, Indianapolis, IN_.

Purpose: The objectives of this study are to investigate the effects of anti-angiogenic therapy on breast cancer stem cell (BCSC) prevalence and hemodynamic imaging factors associated with these biomarkers.

Introduction: Angiogenic inhibitors in breast cancer patients have shown to increase progression-free survival but not overall survival. Targeting the tumor vasculature can be used to alter the microenvironment (hypoxic)-niche, either enhancing BCSC subpopulations or causing these cells to differentiate. In this study, mice bearing triple-negative inflammatory breast tumors were treated with different dose regimens of VEGFR2 inhibitors and changes in ALDH1, EpCAM, and DLL1 measured.

Material and Methods: Four cohorts of athymic nude mice bearing SUM149 breast tumors were treated with a low-(LD) (10mg/kg), moderate-(MD) (40mg/kg), and a high-dose (HD) (120mg/kg) of DC101, an anti-VEGFR2 monoclonal antibody, 3-times every three days. One group was not treated. All groups were imaged prior to and after treatment using DCE-CT and PCT-S to assess vascular physiology (perfusion, fractional plasma volume, permeability) and hemoglobin status (SaO2), after which the tumors were excised, fixed, sectioned at 4-6 different planes (1-2mm apart) and stained for ALDH1, EpCAM, and DLL1. Expression was calculated by determining the percent of the tissue exceeding a threshold determined from positive controls.

Results: A reduction in ALDH1 and DLL1 expression was observed between untreated and MD group (3.6 to 1.0%, P=0.03; 18.3 to 1.5%, P=0.1), with a subsequent increase from MD to HD groups (1.0 to 1.9%, P=0.08; 1.5 to 3.6%, P=0.02). Changes in EpCAM were not observed (P>0.25). In vivo imaging results (DCE-CT and PCT-S) indicate, on average, favorable changes in tumor hemodynamic parameters may have occurred within the MD treated group, where perfusion (+26%) and Fp (-30%) on average resulted in a shorter mean-transit-time and the fraction of the vasculature with low SaO2 levels decreased (from 23% to 10%) not observed in LD or HD groups (but not reaching significance).

Conclusions: Anti-angiogenic drugs targeting VEGFR2 in inflammatory TNB cancer has been shown to modulate ALDH1 and DLL1, but not EpCAM. Preliminary data demonstrate that vascular physiologic parameters obtained from DCE-CT and PCT-S may provide potential diagnostic factors. Given the overlap of ALDH1 and DLL1 with Notch and subsequent VEGF pathways, both a biomechanistic and oxygen microenvironment influence may be involved in modulating BCSC maintenance.

#4228

Imaging and targeting of hypoxic microenvironments in prostate cancer.

Balaji Krishnamachary,1 Louis Dore-Savard,1 Santosh Kumar Bharti,1 Flonne Wildes,1 Yelena Mironchik,1 Margaret E. Black,2 Zaver M. Bhujwalla1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Washington State University,, Pullman, WA_.

Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to hypoxia response elements (HRE) in the promoter region of target gene that results in their increased transcription [1]. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation [1]. Bacterial or yeast cytosine deaminase (yCD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU) that is widely used in cancer treatment [2]. Using a lentivirus approach, we established controlled expression of yCD by HRE in prostate cancer cells (PC-3). These cells also report on HIF-1α expression with regulated luciferase (Luc) expression, allowing detection of hypoxia, and the generation of 5-FU from 5-FC by yCD in the presence of hypoxia. Transduction efficiency and reporter activity in response to hypoxia was evaluated by performing luciferase assays, and bioluminescence imaging (BLI) of cells in vitro or in vivo using a Xenogen IVIS Spectrum system. Cell viability in vitro in response to hypoxia in the presence of 5-FC was assessed by MTS assay. In vivo studies were performed by inoculating 2x10^6 PC-3-HRE-Luc cells and PC-3-HRE-yCD+Luc cells on either flank of 5-week-old male severely combined immune deficient (SCID) mice. BLI was performed once tumors reached ~200mm3 followed by 5-FC injection through the tail vein (200mg/kg) and intraperitoneally (250mg/kg). BLI was performed 3 days after the first 5-FC injection and continued through the treatment protocol. At the end of the treatment protocol, tumors were excised, and a part of the tumor was processed for immunohistochemistry. Bioluminescence was detected in both PC3-HRE-Luc and PC-3-HRE-yCD+Luc cells only in response to the hypoxia mimetic cobalt chloride or hypoxia (1% O2) confirming the regulation of luciferase by hypoxia and activation of CD. Expression of yCD and its ability to convert the prodrug 5-FC to 5-FU, with increased cell kill was evident under hypoxia. In vivo, engineered PC-3-HRE-yCD+Luc cells reported hypoxia, and showed significant reduction of hypoxic regions and tumor volume. Morphologically, PC-3-HRE-yCD+Luc tumors exhibited extensive necrosis. We are currently evaluating the effects of eliminating hypoxic cancer cells on distant metastasis as well as on aggressive subpopulations such as cancer stem cells in the primary tumor.

References: [1] Philip, B., et al., Carcinogenesis, 2013. 34(8):1699-707.,[2] Longley DB, et al., Nat Rev Cancer, 2003. 3: 330-38.

Acknowledgements: This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for technical assistance

#4229

Physical expansion of tissue microarrays for high-resolution imaging of normal and cancer samples with conventional microscopy.

Octavian Bucur,1 Yongxin Zhao,2 Edward Boyden,2 Andrew H. Beck1. 1 _Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, MA;_ 2 _Massachussetts Institute of Technology, Boston, MA_.

BACKGROUND

Archival formalin fixed paraffin embedded (FFPE) tissues are an excellent and abundant source of human cancer and normal tissues, useful for cancer research and diagnosis. Moreover, FFPE tissue microarrays enable analysis of hundreds of samples from many different patients on the same slide. Super resolution microscopes have been used for cancer tissue imaging; however, these methods are expensive and require long recording times, limiting their usefulness for the cancer research community. Recently, a new approach (Expansion Microscopy) was developed to enable physical magnification and high resolution imaging of cell lines and fresh-frozen (and fixed) mouse brain with conventional microscopes (Chen F, Tillberg PW, Boyden ES, 2015, Science 347:543-548). In the current study, we developed expansion microscopy for expanding and imaging formalin fixed paraffin embedded normal and cancer human tissues, both in tissue microarrays and whole tissue slides.

METHODS

Expansion microscopy (ExM) physically magnifies tissue samples by embedding them in a dense swellable polymer, anchoring key biomolecules to the polymer mesh, and adding water to swell the polymer. ExM physically magnifies the brain tissue with nanoscale isotropy and a post-expansion measurement error of 2-4%. Proteins can also be visualized by a modified immunofluorescence assay (Chen F et al., Science, 2015).

In the present study, we optimized the ExM chemistry, labeling, and imaging methodologies to enable ExM to be used in cancer research and diagnosis, for morphological and protein imaging and analysis of both tissue microarrays and whole tissue slides on a wide variety of human tissues. Nuclei were detected by DAPI, and protein markers, including those of stroma (vimentin) and epithelium (keratins), were detected using immunofluorescence.

RESULTS and CONCLUSIONS

We developed ExM protocols to enable expansion of human normal and cancer tissues ~4.5x in linear dimension, with a post-expansion measurement error of 5% or less. We successfully expanded normal and cancer human FFPE tissue microarrays containing over eight different tissue origins, including breast, prostate, lung, colon, pancreas and ovary. We expanded fresh frozen tissue samples of normal and cancer tissues. Our method makes possible the use of ExM on routinely collected FFPE pathology samples, enabling super resolution optical investigation of morphology and protein expression/localization in tissue microarrays with conventional fluorescent microscopy. Future applications include both large-scale retrospective and prospective studies of carcinogenesis in clinical tissue samples. * Yongxin Zhao and Octavian Bucur contributed equally; # Edward Boyden (esb@media.mit.edu) and Andrew H. Beck are corresponding authors (abeck2@bidmc.harvard.edu);

#4230

Visualizing the distribution of metabolites and the efficacy of BIBF1120 on metabolic status of lung cancer derived tumors by imaging mass-spectrometry (MS).

Daisuke Arai,1 Hiroyuki Yasuda,1 Kenzo Soejima,1 Shizuko Kagawa,1 Junko Hamamoto,1 Shigenari Nukaga,1 Katsuhiko Naoki,1 Katsura Emoto,2 Yuki Sugiura,3 Makoto Suematsu,3 Tomoko Betsuyaku4. 1Division of Pulmonary Medicine, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan; 2Department of Pathology, Keio University School of Medicine, Tokyo, Japan; 3Department of Biochemistry & Integrative Medical Biology, Keio University School of Medicine, Tokyo, Japan; 4Division of Pulmonary Medicine, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.

Background: Imaging MS (Shimadzu CO., LTD.) is a novel technology that can quantitatively visualize the distribution of hundreds of metabolites in the selected areas of tissues. BIBF1120 (gift from Boehrlinger Ingelheim Seiyaku CO., LTD.) is a multiple tyrosine kinase inhibitor, targeting VEGFR, FGFR and PDGFR, and is known as an anti-angiogenic agent. Anti-angiogenic drugs are supposed to induce under-nutrition in tumors. However, no study has visualized the regional differences in the metabolic status of tumors induced by anti-angiogenic agents.

Aim: The aims of this study are to visualize the distribution of metabolites in tumors and metabolic status induced by BIBF1120 and to obtain the direct proof that BIB1120 induce under-nutrition status in tumors by Imaging MS.

Method: We used lung cancer derived cell lines, namely, PC9, H1975, H3122, A549, H69, and H520. We evaluated distribution of metabolites in tumors derived from xenograft models by Imaging MS. The efficacy of BIBF1120 was evaluated by MTS assay and mouse xenograft model. We treated the engrafted mice with BIBF1120 or vehicle for 4 weeks and harvested xenograft tumors with isotope labeled glucose injection. Micro-vessel density and percentage of apoptotic cells in the tumors were evaluated by immunohistochemistry using anti-CD34 antibody and anti-cleaved caspase 3 antibody, respectively. The effect of BIBF1120 on metabolic status in xenograft tumors was evaluated by imaging MS.

Result: Imaging MS revealed heterogeneity of metabolites distribution in the tumors before BIBF1120 treatment. In areas with rich ATP, many viable cells were seen with abundant glutathione, an anti-oxidant metabolite and glutamate, a metabolite derived from oxidative phosphorylation. Otherwise, other areas with rich lactate, a metabolite derived from glycolysis pathway, have less ATP and glutathione level compared with the areas with rich ATP. BIBF1120 inhibited growth of all of the xenograft tumors tested, although it did not directly show inhibitory effect for proliferation rate of those cells in vitro. BIBF1120-treated tumors exhibited significantly lower micro-vessel density compared to the vehicle-treated tumors (p<0.05) and reduced 2-3 DPG (a maker of blood flow), glucose, and ATP levels. Lactate level was increased in BIBF1120-treated tumors. Imaging MS with isotope labeled glucose injection revealed that BIBF1120 treatment induced inactivation of oxidative phosphorylation pathway and activation of glycolysis pathway, especially in the peripheral areas of tumors.

Conclusion: We successfully visualized the heterogeneous distribution of metabolites in the tumors, and the effect of BIBF1120 on human lung cancer derived tumors. This is the first report to visualize the distribution of metabolites in engrafted tumors and the efficacy of BIBF1120 on the nutrition status of tumors.

#4231

Analysis of FFPE treated clinical tissue sections obtained from human intraocular malignancy, uveal melanoma by mass spectrometry imaging (MSI).

Laura M. Cole,1 Hardeep S. Mudhar,2 Karen Sisley,2 Andrew Peck,3 Mike Batey,4 Emmanuelle Claude,4 Malcolm Clench1. 1 _Sheffield Hallam University, Sheffield, United Kingdom;_ 2 _Royal Hallamshire Hospital, Sheffield, United Kingdom;_ 3 _Waters Corporation, Milford, MA;_ 4 _Waters Corporation, Wilmslow, United Kingdom_.

Introduction

Clinical research into human intraocular disease UM FFPE tissue sections is described. UM remains the most common intraocular malignancy in adults, with poor prognosis for UM within the choroid region and distant sites UM metastasis. Two imaging MS techniques Matrix Assisted Laser Desorption/Ionisation (MALDI) and Desorption Electrospray Ionisation (DESI) MSI were applied. Molecular profiles were obtained and analysed by multivariate statistical approaches, providing insight into the biochemical and biological differences/similarities within the patient sample cohort (n=15).

Methods

Enucleations were collected over 6 years and subjected to the standard fixation and paraffin embedding protocols. In preparation for MS, 5 µm sections were produced with removal of the paraffin followed by heat induced antigen retrieval.

MALDI MSI tissue sections were prepared by applying a matrix solution onto the tissue sections to help ionization of molecules directly from the tissue. All MSI experiments (MALDI and DESI) were carried out using a SYNAPT mass spectrometer (Waters Corporation, Manchester, UK). Multivariate analyses were performed using MATLAB (MathWorks, Inc., Natick, MA, US) and the Eigenvector PLS_Toolbox.

Results

Initial MALDI MSI images acquired with a spatial resolution of 100 µm x 100 µm in positive ion mode showed distinct spatial distribution of many molecular species throughout the choroid, cornea, retina, lens and UM tumor regions. Further MALDI MSI experiments using consecutive tissue sections at 50 µm x 50 µm spatial resolution show substantial variation in the spatial distribution of species within the low molecular mass range. DESI MSI experiments were carried out at 200 µm x 200 µm in negative ionization mode. Deprotonated molecular ions were detected from a variety of lipid related species, localized to specific regions within the eye including the tumor region.

Multivariate analysis classified UM samples (good vs. poor prognosis). Using unsupervised PCA, an unbiased representation of the data was generated, with clear sample grouping and differentiation observed based upon tumor status. Use of the supervised PLS-DA technique provided even clearer separation between the selected samples, with tumor profiles displaying dominant discriminatory peaks. These peaks could be identified as sphingolipids and Lyso-phosphocholine, a phosphatidylcholine degradation product.

Conclusions

It was possible to analyze clinical research FFPE tissue sections by MALDI and DESI MSI, illustrating that specific small molecular species remained localized to certain tissue types including the UM tumor. Initial correlation with tumor status was determined from the statistical analysis of the MALDI MSI datasets.

#4232

PET imaging of galectin-3 expression with [18F]FPDTG for detection of early breast carcinoma lesions in dense breast tissue.

Robin E. Bonomi, Vadim Popov, Thomas Mangner, Avraham Raz, Anthony F. Shields, Juri G. Gelovani. _Karmanos Cancer Institute, Wayne State University, Detroit, MI_.

Mammography is the gold standard for breast cancer detection with sensitivity of 85%, which decreases to 68% in women with dense breasts and to 48% in women with extremely dense breasts, making cancer detection challenging. Additional methods for breast cancer screening have been developed, including: contrast-enhanced mammography, tomosynthesis, automated whole breast ultrasound, advanced MRI and molecular imaging (99mTc-MIBI gamma camera; 18F-FDG). However, early detection and differential diagnosis of malignant vs benign breast lesions remains a major challenge in patients with dense breasts.

We have been developing novel approaches to early detection and differential diagnosis of malignant vs benign breast lesions using PET imaging of galectin-3 (Gal-3), that is significantly overexpressed in breast carcinomas, as compared to pre-malignant lesions. We developed a novel radiotracer 3-[18F]fluorophenylthio-digalactoside (18F-FPTDG) with high binding affinity (Kd 14 nM) and specificity to Gal-3. In vitro radiotracer binding studies in a panel of MCF10-derived cell lines with different degrees of malignancy and Gal-3 expression, demonstrated a highly significant linear relationship between the level of Gal-3 expression and the magnitude of 18F-FPTDG binding to cell membranes (R2=0.99).

Preliminary in vivo PET imaging studies with 18F-FPTDG in mice bearing MCF10DCIS tumor xenografts demonstrated a rapid bi-exponential clearance of 18F-FPTDG from the blood (fast and slow clearance half-times: 0.5 and 10.5 min, respectively). 18F-FPTDG cleared predominantly via the hepatobiliary route, as evidenced by high levels of radioactivity in the liver, increasing levels in duodenum and upper intestinal tract. The liver and kidney clearance followed mono-exponential kinetics with half-times of ~120 min and 37 min, respectively. Renal clearance was more active in mice, as evidenced by higher amounts of radioactivity in the bladder. The magnitude of catabolism of [18F]FPDTG was negligible, as evidenced by very low levels of [18F]F2 accumulation in the bone at 60-75 min post injection. The level of accumulation of [18F]FPDTG-derived radioactivity in the lungs, muscles, and other organs and tissues was extremely low. Also, there was no measurable levels of [18F]FPDTG-derived radioactivity in the brain. In contrast, there were distinct regions in the breast carcinoma xenograft with higher levels of [18F]FPDTG, especially in the central regions (hypoxia). The half-time clearance of [18F]FPDTG from tumors was ~8 min, which was about twice longer than clearance half-time from lungs and muscles - 4.5 and 4 min, respectively.

Additional studies are being conducted in various breast tumor xenograft models, as well as in PyVmT transgenic mice, to assess the utility of PET/CT with [18F]FPDTG for differential diagnosis of benign vs malignant breast lesions and for biopsy guidance.

#4233

Early detection of pemetrexed-induced inhibition of thymidylate synthase in non-small lung cancer with FLT-PET imaging.

Xiao Chen, Ian Berger, Urooj Khalid, Jenny Cai, Akash Patel, Sharyn I. Katz. _Hospital of the University of Pennsylvania, Philadelphia, PA_.

Introduction: Successful inhibition of thymidylate synthase (TS) can be imaged with FLT ([18F]thymidine)-positron emission tomography (PET) thereby providing a measure of therapy response on day 1 of therapy. Here we characterize this imaging strategy for implementation into 1st line therapy with pemetrexed and cisplatin for NSCLC.

Materials and Methods: The kinetics of pemetrexed-mediated TS inhibition mediated DNA salvage pathway "flare" in DNA was first characterized in vitro using 3H-thymidine DNA incorporation assays, analysis of TK1 protein expression and mobilization of the equilibrative nucleoside transporter 1 to the cell surface membrane. Kinetics of the pemetrexed-mediated "flare" in the salvage pathway was then confirmed in an in vivo mouse model of NSCLC. Finally, a proof-of-principle clinical trial was performed to demonstrate feasibility of imaging of pemetrexed-mediated TS inhibition in human subjects with NSLC.

Results: In vitro examination of pemetrexed-induced changes in the salvage pathway revealed a burst in DNA activity that peaked at 2 hrs following the administration of pemetrexed. The addition of cisplatin did not impact the amplitude or timing of the pemetrexed-induced "flare" in the salvage pathway. In vivo imaging of TS inhibition with FLT-PET confirmed a peak in salvage pathway activity at 2 hrs. Imaging of pemetrexed-induced TS inhibition in a human clinical trial demonstrated feasibility with imaging at the 2 hr time point.

Conclusion: FLT-PET measured efficacy of pemetrexed-induced TS inhibition is optimal at 2 hrs from the start of therapy. This timing of FLT-PET imaging is feasible in human clinical trials.

#4234

Elucidating structure-activity relationships of SIRT2 and fluoroalkyl chain length to identify the lead candidate substrate radiotracer for PET imaging.

Robin E. Bonomi, Aleksandr Shavrin, Vadim Popov, Thomas Mangner, Juri G. Gelovani. _Karmanos Cancer Institute, Wayne State University, Detroit, MI_.

Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation through post translational modifications of histone core and other critical proteins in cellular pathways. The HDACs class III, sirtuins (SIRTs) 1-7, possess limited deacetylase activity. Each of these nicotinamide dinucleotide (NAD+) dependent enzymes have unique structures of catalytic sites that prefer larger lysine-modifying groups as substrates (i.e., glutaryl, succinyl, lipoyl, crotanyl, myristoyl).

In particular, SIRT2 is a key regulator of nuclear H4K16 and cytoplasmic alpha-tubulin proteins, thereby acting as a mitotic exit regulator. From the literature, it is clear that SIRT2 plays a pivotal role in the regulation of cancers, so much so that it may switch between tumor suppressor and regulator at different stages of tumor progression. For this reason, SIRT2 has been identified as a possible target for cancer therapy but due to the pleiotropic roles of SIRT2 under normal and pathologic conditions, it is important to assess its activity during pharmacomodulation (i.e. activation or inhibition). Therefore, non-invasive PET imaging using SIRT2-specific substrate-type radiotracers may aid in the development of new SIRT2 inhibitors for therapy of cancer.

Herein, we report novel synthesis of a focused library of compounds to assess the structure activity relationship (SAR) of SIRT2 with fluoroalkyl chain length varying between 3 and 16 carbons. The preliminary screening was performed with the tert-butyloxycarbonyl-lysine-aminomethylcoumarin (Boc-lys-AMC) backbone and Fluor-de-lys assay [1] to determine the chain length for optimal SIRT2 catalytic efficiency. Based on the analysis of Michealis-Menten kinetics of each substrate, myristoyl (14-carbon carboxylic acid) was cleaved most efficiently, followed by 12-fluorododecanoic and 10-fluorodecanoic acids (kcat/km = 4E-3, 1E-4, 8.9E-5 s-1uM-1, respectively). For in vivo PET imaging radiotracer development, we linked the 12-fluorododecanoic acid with the aminohexanoicanilide (AHA) backbone [2]. This compound was developed with a terminal bromine (precursor), iodine, fluorine, and F-18 (12-[18F]DDAHA). In vitro cellular uptake studies demonstrated a three-fold increase in accumulation of 12-[18F]DDAHA in U87 glioma cells as compared to MiaPaCa, MDA-MB-231, and MCF10A cell lines. The increased accumulation in the U87 glioma cells indicates that this compound may prove useful for detection of brain gliomas and for monitoring therapies with novel SIRT2 specific inhibitors. This insight into the catalytic activity of SIRT2 provides a basis for the development of a SIRT2-specific PET imaging agent to further probe the role of SIRT2-mediated epigenetic regulation in normal physiology and cancer.

#4235

Choline metabolism is an early predictor of EGFR-mediated survival in NSCLC.

Rosy Favicchio,1 Nicos Angelopoulos,1 Diana Brickute,1 Robin Fortt,2 Frazer Twyman,3 Georgios Giamas,4 Juan Carlos Lacal,5 Eric O. Aboagye1. 1 _Imperial College London, London, United Kingdom;_ 2 _Kings College, London, United Kingdom;_ 3 _Kings College London, London, United Kingdom;_ 4 _University of Sussex, London, United Kingdom;_ 5 _, Instituto de Investigación Sanitaria del Hospital Universitario La Paz, Madrid, Spain_.

Oncogenic signalling and metabolic reprograming are hallmarks of tumour progression, yet little is known about the regulatory elements that coordinate their interface. Aberrant choline and phospholipid metabolism are strongly correlated to malignant progression in NSCLC and provide the essential components required by both hallmarks and yet mechanistic links to either remain scarce. Choline kinase alpha (ChoKα) regulates the conversion of choline to phosphocholine and although its regulatory cascade has not been described, it is thought to act in conjuction with EGFR. We used an integrated systems approach and queried whether pharmacoproteomic pathway mapping could identify regulators of the cholinic phenotype. Proteomic and phosphoproteomic Stable isotope labelling by amino acids in cell culture (SILAC) analysis was used to describe the interactome following ChoKα or EGFR inhibition. Bioinformatic analysis was used to identify the significant (Significance-B test) subset of targets for each condition. These subsets were clustered according to GeneOntology, Reactome and KEGG databases and the resulting maps identifed the potential regulators of choline metabolism. Choline uptake, phosphorylation and efflux were further evaluated in vitro in response to erlotinib, cisplatin, pemetrexed and paclitaxel using radio-labelled Choline analogues. Derived metabolites were characterised using radio-HPLC. Uptake was further characterised under hypoxic and nutrient deficient conditions. In vivo, [18F]-D4-Choline PET dynamic imaging was performed following treatment. Pharmacoproteomic analysis revealed a 40% overlap between ChoKα and EGFR inhibition providing direct evidence of the pathways and targets involved in, mostly, biosynthesis. Rapid modulation of the cholinic phenotype was directly dependent on ChoKα activity. Intracellular uptake was induced by nutrient deprivation, hypoxia and reversed through second messenger signalling or growth factor stimulation. Choline uptake within 3 hours of treatment correlated to survival at 72 hours. In vivo, [18F]-D4-Choline tracer kinetics were diagnostic of choline kinase expression and sensitive to treatment. Significant correspondence between ChoKα and EGFR inhibition provided mechanistic evidence that ChoKα and lipid metabolism are effectors of the EGFR signalling cascade in NSCLC. Choline can act as a sensor by synchronizing the survival response via metabolic and signalling reprograming and is thus an early predictor of therapeutic efficiency in vitro and in vivo.

#4236

SUPR-PET: Nuclear imaging of Her2-positieve breast cancer with SUPR peptides.

Lindsay E. Kelderhouse,1 Amanda Hardy,2 Federica Pisaneschi,1 Joshua P. Gray,1 Seth Gammon,1 Richard W. Roberts,3 Terry T. Takahashi,3 Steve Fiacco,2 Steven W. Millward1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _EvoRx Technologies, Pasadena, CA;_ 3 _University of Southern California, Los Angeles, CA_.

In recent years, patients with Her2-positive breast cancer have benefitted greatly from targeted therapies such as Trastuzumab and Pertuzumab. However, there are currently no FDA-approved Her2-targeted imaging agents to diagnose and monitor Her2-positive breast cancer leaving physicians to rely on biopsies to determine Her2 status. Whole-body visualization of Her2 would allow the noninvasive identification of all Her2 primary and metastatic lesions as well as serve as a powerful tool to monitor the effectiveness of Her2-targeted therapeutics. We recently described the generation of Her2-targeted SUPR (Scanning Unnatural Protease Resistant) peptides that selectively bind the Her2 receptor in vitro with low nanomolar affinity. Cy5-labeled SUPR peptides showed rapid and Her2-specific tumor uptake and minimal retention in non-tumor tissues after 24 hours of washout. In this study, we describe 18F radiolabeling of Her2-targeted SUPR peptides and their evaluation as PET radiotracers to visualize Her2 expression in vivo. The lead compound, SUPR4, was labeled with 18F-fluoroethylazide in high radiochemical yield and specific activity on an automated radiochemical synthesis platform. The resulting radiotracer (SUPR-18F) showed rapid and Her2-selective tumor uptake between 30-60 minutes post-injection with minimal liver uptake. The majority of the tracer was cleared by the kidneys at 2 hours post-injection although some activity was observed in the GI tract suggesting hepatobiliary excretion. No significant uptake was observed in the brain. 90 minute dynamic PET scans were performed to estimate the rate of tumor uptake and clearance in major organ systems and the biodistribution quantified by autoradiography post-mortem. Pre-injection of unlabeled SUPR peptide, Trastuzumab, and Pertuzumab followed by PET imaging with SUPR-18F was used to confirm tumor uptake as a function of specific binding to the Her2 receptor. Having established the utility of SUPR-18F in pre-clinical mouse models, we anticipate that this class of PET tracers could be employed in same-day imaging procedures throughout the administration of Her2-targeted therapy.

#4237

**Decreased hypoxia in a HER2** + **breast cancer model following trastuzumab therapy.**

Anna G. Sorace,1 C. Chad Quarles,2 Violeta Sanchez,1 Thomas E. Yankeelov1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Barrow Neurological Institute, Phoenix, AZ_.

Introduction: Hypoxic tumors demonstrate treatment resistance with an increased risk of metastasis. Therefore, alleviating hypoxia has potential to improve efficiency of combination therapies. The primary goal of this study is to quantify alterations in hypoxia in response to trastuzumab in a murine model of HER2+ breast cancer through imaging and histology.

Experimental Design: Mice were implanted subcutaneously with BT474 breast cancer cells (107) and randomly assigned into treated (10 mg/kg trastuzumab) or control (saline) groups. After tumors reached ~250 mm3, animals (n=32) were utilized to identify longitudinal changes in functional vascular through an intravenous injection of Hoechst 33342 nuclear stain (immediately prior to sacrifice) and hypoxia through pimonidazole intravenous injection (one hour prior to sacrifice). Tumors were extracted for immunofluorescence between days 0 through 7. Tumor sections were flash frozen and stained with anti-pimonidazole and propidium iodide (nuclear counterstain). Additionally, a set of tumors (n=36) were sacrificed for immunohistochemistry (days 0 through 7), formalin fixed and stained for CA-IX. All stained sections were scanned in high resolution (20×) and quantitatively analyzed with Leica SCN400 software. Another cohort of animals (n=10) were imaged with 18F-FMISO (fluoromisonidazole) PET between days 0 through 7 and quantified via mean tracer concentration (%ID/g).

Results: Immunohistochemistry revealed significantly increased hypoxia in the control group compared to treated on days 3 (p=0.03) and 7 (p=0.002), as measured through CA-IX staining. Additionally, on day 4, functional vascular delivery was increased while hypoxia (pimonidazole) decreased in treated tumors compared to control. 18F-FMISO PET imaging corroborated histology findings with significantly decreased hypoxia in treated tumors compared to control tumors on day 7 (-47%; p<0.0001).

Conclusion: Trastuzumab has been shown to decrease hypoxia, as measured through gold-standard immunofluorescence. Additionally, this trastuzumab-induced improvement in tumor hypoxia can be measured via clinically relevant, noninvasive imaging (18F-FMISO PET). Temporarily improving the tumor's functional vasculature and decreasing hypoxia during trastuzumab treatment has potential to enhance the effectiveness of combination therapies. Identifying windows of improved vascular and cellular normalization with noninvasive medical imaging provide opportunity to decrease resistance to standard-of-care treatments and optimize therapeutic timing and regimens.

#4238

**Multispectral optoacoustic tomography detects orthotopic pancreatic tumors** in vivo **using a nano-contrast agent.**

Phillip Chuong,1 Matthew Zeiderman,1 William E. Grizzle,2 Lacey R. McNally1. 1 _University of Louisville Brown Cancer Center, Louisville, KY;_ 2 _University of Alabama Birmingham, Birmingham, AL_.

There have been very modest developments in pancreatic cancer detection and treatment with mortality essentially remaining unchanged in the last four decades. The major challenge for both early detection and treatment of pancreatic tumors lies in overcoming various biological barriers, especially poor perfusion, insufficient contrast agents/drugs uptake by the tumor, and limitations of traditional imaging modalities. The combination of accurate real-time imaging with tumor-specific nano-contrast agents could mitigate these impediments. We constructed highly stable nano-contrast agents by encapsulating GNRs having aspect ratio 3:1 in PAA (1.5 ± 0.5 nm), and MS (6.25 ± 0.25 nm) shell, respectively and targeted to insulin growth factor 1 receptor (IGF1-R) positive pancreatic tumor cells via Syndecan-1 ligand. IGF1-R specific binding of Syndecan-MS GNRs and Syndecan-PAA-GNR was determined using in an IGF1-R negative cell line (MiaPaca-2) or a blocking antibody to IGF1-R in S2VP10L pancreatic cancer cells determined via flow cytometry. In vivo, mice bearing orthotopic pancreatic tumors were iv injected with 100 nM of Syndecan-MS GNRs and accumulation was determined four hours post injection via Multispectral Optoacoustic Tomography. Biodistribution was determined using a Region of Interest method. The cellular uptake of Syndecan-MS-GNRs significantly enhanced in the S2VP10L cells (503.4 counts) compared to MiaPaca-2 cells (64.1 counts), blocking agent (323.7counts) and untargeted MS GNRs (average 47 counts) revealing that Syndecan-MS-GNRs enhanced both tumor specificity and OA signals by binding with IGF1-R in S2VP10L cells. In vivo, Syndecan-MS-GNRs significantly accumulated in S2VP10L orthotopic pancreatic tumors (233.1 MSOT a.u.) with minimal accumulation in kidney(29.7 MSOT a.u.) and liver (31.4 MSOT a.u.). The Syndecan-MS-GNRs did not accumulate within the orthotopic MiaPaca-2 (IGF1-R negative) mouse model with the biodistribution of pancreas tumor (12.4 MSOT a.u.), kidney (114.3 MSOT a.u.), and liver (35.2 MSOT a.u.).

#4239

**Monitoring** in vivo **biodistribution of superparamagnetic nanoparticles using superparamagnetic relaxometry (SPMR).**

Andrew Gomez,1 Kayla E. Minser,1 Caroline L. Weldon,1 Bill Anderson,1 Todor Karaulanov,1 Helen J. Hathaway,2 Dale L. Huber,3 Edward R. Flynn,1 Erika Vreeland1. 1 _Senior Scientific LLC, Albuquerque, NM;_ 2 _University of New Mexico Health Sciences Center and University of New Mexico Comprehensive Cancer Center, Albuquerque, NM;_ 3 _Center for Integrated Nanotechnologies, Sandia National Laboratories, Albuquerque, NM_.

We report on the use of Superparamagnetic Relaxometry (SPMR) as a technique for monitoring the distribution of superparamagnetic magnetite (Fe3O4) nanoparticles in vivo. SPMR uses superconducting quantum interference device (SQUID) sensors to measure the remnant magnetization of nanoparticles bound to cells following a brief magnetizing pulse. Unbound nanoparticles are not detected, making SPMR uniquely capable of discriminating between nanoparticles that are circulating freely in the bloodstream from those that have been immobilized in organs and tissues throughout the body.

The rate of clearance of magnetic nanoparticles from circulation is largely dependent on their hydrodynamic diameter and surface chemistry. In this study, biodistribution of PrecisionMRX™ nanoparticles with 25 nm cores and two different surface coatings were followed using the MRX™ instrument. In independent experiments, negatively charged carboxylate nanoparticles and methoxy-PEG (mPEG) functionalized nanoparticles with a near neutral charge were suspended in 100 µL of saline. Nanoparticle suspensions were injected intravenously via tail vein into Balb/C mice at a dose of 5 mg/kg of body mass, while control mice were injected with 100 µL of saline solution. Mice were measured individually on the MRX instrument at successive time points over the course of 24 hours. At selected intervals during the 24 hour period, mice were euthanized, exsanguinated, and organs harvested for ex vivo measurements on the MRX. Organs and blood were subsequently assayed for iron content.

MRX measurements of mice injected with carboxylate nanoparticles showed a strong magnetic signal localized in the region of liver that reached a maximum several minutes after injection. The negative surface charge of the carboxylate nanoparticles likely resulted in their rapid opsonization in the blood and removal from circulation by phagocytic cells in the liver. Conversely, MRX measurements of mice injected with mPEG coated nanoparticles indicated an extended circulation time, with a gradually increasing magnetic signal in the liver that had not yet reached a maximum after four hours. Together, these experiments demonstrate the utility in using SPMR to measure the clearance of nanoparticles from the bloodstream following injection, an important parameter in the design of optimal coatings for in vivo diagnostic and therapeutic use.

This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

#4240

Optical imaging and detection of distal site micrometastatic lesions in vivo: Design of rare-earth albumin nanocomposites.

Margot A. Zevon,1 Harini Kantamneni,1 Laura Higgins,1 Derek Adler,1 Xinyu Zhao,2 Sheng Yang,2 Mei Chee Tan,2 Mark Pierce,1 Richard Riman,1 Vidya Ganapathy,1 Charles Roth,1 Prabhas Moghe1. 1 _Rutgers University, Piscataway, NJ;_ 2 _Singapore University of Technology and Design, Singapore, Singapore_.

The presence of micro-metastatic lesions in the bone marrow is an accurate indicator of poor prognosis in breast cancer patients. Detection of these lesions has been a challenge due to lack of sensitive clinical imaging modalities, leading to poor prognosis. Optical imaging technologies are challenged by tissue scattering and absorption of visible light, limiting resolution of deeper tissue and micro-lesions. While optical imaging modalities have the potential for real-time in vivo monitoring, poor resolution and penetration through biological media limit their application to large and subcutaneous lesions. Our approach utilizes rare earth (RE) nanoprobes that absorb near infrared (NIR) radiation and emit in the shortwave infrared (SWIR) spectrum (1000-3000 nm), allowing for greater depth of detection and improved spatial resolution.

In this study we demonstrate the ability of RE Albumin NanoComposites (ReANCs) to preferentially accumulate in distant lesions in long bones, prior to detection by clinically relevant imaging modalities in an experimental model of metastatic breast cancer. Athymic nude mice were inoculated directly into their tibias with MDA-MB-231-derived bone tropic cells. ReANCs were injected via the tail vein once a week and SWIR imaging was performed to determine particle accumulation. Animals were imaged via MRI and BLI to assess tumor burden. Longitudinal imaging revealed significantly improved accumulation of nanoprobes in the tibia of tumor bearing animals compared to healthy controls, leading to SWIR tumor detection prior to identification via MRI. Tumor presence was confirmed via ex vivo SWIR imaging and immunohistological analysis.

The ability of ReANCs to seek distant metastases was validated in an experimental metastatic model of luminal breast cancer that phenocopies the human disease. Athymic nude mice were inoculated with luciferase labeled MCF7 subclone cells via their left ventricle. Cell- seeding and tumor progression in the adrenals, mandibles and long bones were assessed via bioluminescence imaging. In parallel, animals were treated with either ReANCs or AMD3100 (small molecule inhibitor of CXCR4) functionalized ReANCs and imaged to detect micro-metastatic lesions prior to their detection via conventional imaging modalities such as MRI and CT. Ex-vivo validation at the study end-point confirmed association of REANCs to tumors. Enhanced resolution of lesions by SWIR emitting RE nanoprobes renders them as potential novel optical imaging agents to detect micro-metastasis of breast cancer to bone, and highlights an innovative paradigm in optical imaging. Given the multifunctional nature of albumin nanocarriers as drug delivery vehicles, the imaging probes designed here are capable of simultaneous detection and treatment of difficult to detect lesions, suggesting future clinical relevance for cancer nanomedicine.

#4241

Predicting clinical response in breast cancer using cellular-resolution optical metabolic imaging.

Joe T. Sharick,1 Alex J. Walsh,2 Melinda E. Sanders,1 Ingrid Meszoely,1 Mary A. Hooks,1 Mark C. Kelley,3 Melissa C. Skala1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Air Force Research Laboratory, Fort Sam Houston, TX;_ 3 _CHI Memorial Medical Group, Chattanooga, TN_.

While over 50 drugs have been approved by the FDA to treat breast cancer, there are no reliable methods for optimizing treatment regimens for individual patients. Currently, oncologists choose drug treatments based on expression levels of tumor cell signaling receptors (i.e. HER2, ER) and assess whether the treatment is effective after weeks or months of precious time have passed. Unfortunately, over one third of patients exhibit resistance to their initial treatment. The toxic side effects and morbidities resulting from suboptimal drug regimens could be eradicated by applying a personalized medicine approach to breast cancer treatment. This approach would allow clinicians to determine the optimal treatment plan for individual patients early on, at the time of diagnosis.

While current methods track therapy response via changes in tumor size (i.e. MRI, mammography, ultrasound), changes in cell metabolism precede changes in tumor size and thus present an earlier marker of treatment response. Optical metabolic imaging (OMI) is sensitive to these early changes in metabolism by exploiting the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), as well as the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox state, and the fluorescence lifetimes of NAD(P)H and FAD report on the binding activity of these coenzymes. Additionally, OMI has the unique ability to measure these endpoints in individual cells, which allows for the detection of heterogeneous subpopulations of responsive or resistant cells within a tumor. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These samples are grown as "organoids" in a 3D matrix that mimics the natural tumor environment.

We have demonstrated that OMI accurately predicts treatment response in organoids derived from breast cancer xenografts compared with gold standard tumor growth curves in vivo. We have also shown that OMI can measure drug response and detect heterogeneous cell populations in organoids derived from triple negative, ER+, and HER2+ human breast tumors. The ability of OMI to predict treatment response has also been demonstrated in the polyoma middle-T mouse model of breast cancer, which exhibits more cellular heterogeneity than cell line xenografts and also incorporates the influence of the immune system on cancer drug response.

Preliminary data shows that OMI of organoids generated from biopsies of newly diagnosed breast cancer patients can accurately predict how the patient clinically responds to neoadjuvant treatment. This methodology could allow oncologists to determine the ideal treatment regimen for their patients at the time of diagnosis.

#4242

Rhodamine esters as fluorescent tumor painting agents for glioblastoma.

Yu-Hsi Lin,1 Steven Millward,1 Seth Gammon,1 Nikunj Satani,1 Naima Hammoudi,1 Joshua Philip Gray,1 Lindsay E. Kelderhouse,1 Argentina Ornelas,1 Haley Schroeder,2 Florian L. Muller1. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Colorado College, Colorado Springs, CO_.

GBM remains one of the most difficult cancers to treat. Despite surgery, radiation and chemotherapy, tumors invariably recur. There is a strong correlation between extend of resection and subsequent time to recurrence and ultimate patient survival. A key challenge for the neurosurgeon is to minimize removal normal brain tissue whilst being as aggressive as possible with regards of removal of tumor tissue. Defining tumor boundary during surgery remains challenging. Fluorescent dyes such as 5- Aminolevulinic acid, have been studied as aids for tumor delineation but have failed to gain widespread acceptance in clinical practice. It has long been known that a substantial number of cancers, including Glioblastoma, exhibit a hyperpolarization of the plasma and mitochondrial membrane potential. This hyperpolarization is manifest by increased retention of lipophilic cationic dyes (Nernstian probes), such as the SPECT-CT probes 99Tc-Sestamibi and 99Tc-Tetrofosmin in both glioma cells in culture and primary tumors. We synthesized a series of ester derivatives of Rhodamine B with high fluorescence quantum yield in the red spectrum and a very favorable biological safety profile. Utilizing a series of intracranial xenografted glioblastoma pre-clinical mouse models, we show that a single I.V. injection of 1 mg/kg fluoroethylrhodamine ester (RhoFe) results in dramatic selective fluorescence in tumor but not surrounding normal brain tissue. This was observed even in tumors with minimal breakdown of the blood brain barrier, as determined by T1-contrast enhancing agents. RhoFe-tumor-selective fluorescence can remain visible up to 72 hours after injection. Similar results were obtained with Tetramethylrhodamine ester (TMRE) and rhodamine 123 (Rho123), but unlike RhoFe, TMRE proved highly toxic, while Rho123 fluorescence was both weaker and in the green spectrum, accompanied by higher endogenous auto-fluorescence. While well tolerated in vivo, RhoFe shows strong photoxicity in cell culture, suggesting potential as a photodynamic therapy agent. Taken together, these data suggest that RhoFe may be a promising tumor painting agent, with potential utility in fluorescence guided surgery as well as photodynamic therapy.

#4243

ChAcNLS-A14-B105, a novel fluorescence imaging agent for enhanced detection of muscle invasive bladder cancer.

Laurent Fafard-Couture, Simon Beaudoin, Samira Osati, Véronique Dumoulon-Perreault, Réjean Lebel, Brigitte Guérin, Johan E. van Lier, Jeffrey Leyton. _Université de Sherbrooke, Sherbrooke, Quebec, Canada_.

Fluorescence imaging (FI) provides increased sensitivity for the detection of bladder tumors compared to white light cystoscopy. However, most advanced dyes used in FI suffer from nonspecific cellular accumulation resulting in increased false-positives. In addition, these dyes have not made significant progress in detecting the most aggressive bladder tumors such as muscle invasive bladder cancer (MIBC). Our objective is to develop an antibody-conjugate (AC) PDI agent against interleukin-5 receptor α-subunit (IL-5Rα)-positive MIBC cells. IL-5Rα is overexpressed on MIBC cells relative to superficial bladder cancer or healthy bladder cells. We selected a class of fluorescent dyes termed BODIPY for its availability, facile coupling to proteins, and easy chemical modifications to accommodate required absorption and fluorescence properties. BODIPY was attached to an AC composed of the antibody A14 functionalized with a moiety termed ChAcNLS, which we have shown enables it to escape endosome entrapment and redirect its trafficking to the nucleus. This novel approach results in increased intracellular accumulation of attached molecular payloads in MIBC cells. We aim to determine if ChAcNLS-A14 will increase intracellular accumulation of BODIPY to enhance fluorescence detection of MIBC by probe-based confocal laser endomicroscopy (pCLE). In addition, we aim to develop in an improved orthotopic rat bladder tumor model of human MIBC for testing.

Methods

We have prepared a carboxyl BODIPY derivative (B105) that allows for facile coupling to A14-ChAcNLS using routine procedures and reagents. The number of B105 and ChAcNLS per A14 was determined by fluorescence spectroscopy and SDS-PAGE, respectively. Intracellular accumulation of B105 was determined by treating HT1376 MIBC cells with A14-B105 and ChAcNLS-A14-B105. Cells were then imaged by fluorescence confocal microscopy. For the in vivo model of MIBC, RNU rats were anesthetized and their bladder washed in mild acid to disrupt the endothelium. HT1376 cells marked with the fluorescent marker DiL and instilled in the rat bladder. Intravesical pCLE was performed 2 weeks later.

Results

ChAcNLS-A14-B105 conjugate contained 7 molecules of B105 and 3 ChAcNLS peptides per A14. Confocal microscopy revealed the level of B105 intracellular accumulation in IL-5Rα-positive MIBC cells was increase ≥10-fold with ChAcNLS-A14-B105 relative to A14-B105. pCLE revealed HT1376 cells survived and colonized the rat bladder urothelium.

Conclusion

ChAcNLS-A14-B105 was successfully constructed and shown to significantly enhance B105 intracellular accumulation in target IL-5Rα-positive MIBC cells. A novel orthotopic rat bladder tumor model of human MIBC was also generated. Future testing of ChAcNLS-A14-B105 administered intravesically in RNU rat bladders bearing orthotopic tumors of human MIBC will be performed to determine the potential clinical translation of this new FI agent.

#4244

Optical imaging of ovarian cancer metastasis in mouse models of obesity.

W. Matthew Leevy, Yueying Liu, Matthew Metzinger, Kyle Lewellen, M. Sharon Stack. _University of Notre Dame, Notre Dame, IN_.

Epithelial ovarian cancer (EOC) is a deadly gynecologic malignancy, owing to its propensity for intraperitoneal (IP) metastasis. Indeed, almost 75% of women with EOC are diagnosed during the later stages of the disease in which metastasis is already underway. Pathogenesis of EOC is marked by diffuse and widely disseminated metastatic sites within the IP compartment, often with peritoneal ascites. Mouse models of EOC have been developed to facilitate the pre-clinical research of this deadly disease, however the quantification of metastatic tumor burden in these animals has remained challenging due to the broad distribution of lesions in the IP space. Here we describe a combined In Vivo/Post-Mortem method to monitor and measure EOC metastasis in mouse models of obesity. RFP-labeled ovarian cancer cells were injected IP in obese and control cohorts of mice, followed by weekly in vivo imaging until time of sacrifice. Subsequent post-mortem imaging was conducted after surgical exposure of the peritoneal cavity to reveal organs that were then imaged in situ. Ex vivo imaging was performed last on dissected organs from each animal. Imaging signal was enhanced via multispectral imaging methods to remove tissue auto-fluorescence. Semi-quantitative methods to measure tumor burden are demonstrated in their application to the study of metastasis in both obese and control animals. Ultimately these results demonstrated that obesity dramatically increased ovarian cancer metastatic success, and likely contributes to the reduced EOC survival rates noted in clinical reports of obese patients.

#4245

BLZ-100 provides optical contrast in animal models of breast cancer.

Stacey Hansen,1 Janean Fidel,2 Katie Kennedy,2 William Dernell,2 Boel Fransson,2 Josh Ramsay,2 Valorie Wiss,2 Mark Stroud,1 Julia E. Parrish-Novak1. 1 _Blaze Bioscience, Inc., Seattle, WA;_ 2 _Washington State University, Pullman, WA_.

Over 230,000 new breast cancer cases occur in the US annually. Complete surgical removal of tumor tissue is critical to reduce the risk of local recurrence. About 24% of patients who undergo breast conservation surgery require a second surgery due to positive margins. Second surgeries can be psychologically and financially costly to patients. BLZ-100, a peptide-fluorophore conjugate, is a Tumor Paint product candidate being developed to provide real-time, high resolution, intraoperative optical contrast between tumor and normal tissue, potentially helping surgeons accomplish more complete and accurate tumor resections. Intraoperative imaging with BLZ-100 may potentially improve the percentage of breast cancer patients achieving a complete resection and negative margins at their first surgical procedure. To better define the relationship between tumor pathology and BLZ-100 fluorescence, data from mouse models and canine mammary cancer cases were evaluated. Human breast cancer was modeled in mice using cell lines and xenograft models using patient-derived tumors. Review of histopathology was also conducted for each dog case. Animals received a single IV dose of BLZ-100, and tumors were excised one day after dosing. Excised tumor and normal tissues were imaged using the Odyssey Clx (LI-COR) to determine the ratio of signal in tumor compared to normal (background) tissue (TBR). Detailed histopathology reports for the dog cases were also analyzed to determine factors potentially associated with fluorescence intensity and TBR. A significant BLZ-100 TBR was observed. Histopathologic features potentially associated with TBR in dogs included cellularity and the degree of infiltration and demarcation. The studies supported the concept that BLZ-100 might potentially serve as an intraoperative optical imaging agent for breast cancer surgery.

#4246

Multispectral open-air fluorescence-guided imaging and detection of tumors using a hands-free translational platform with liquid crystal tunable filters (LCTF).

Ali Behrooz, Kristine Vasquez, Peter Waterman, Jeff Meganck, Jeffrey Peterson, Peter Miller, Joshua Kempner, Wael Yared. _PerkinElmer, Hopkinton, MA_.

Intraoperative identification and resection of tumors currently relies on the ability of the surgeon to visually detect or palpate the tumors and residual malignant tissue. As such, minuscule tumor nodules can go undetected or be inadequately removed, with such cases often resulting in the need for secondary treatment or additional surgical intervention. The Solaris™ platform is an open-air fluorescent imaging instrument designed for large animal fluorescence-guided surgery, with the advantage of real-time acquisition of fluorescence images/video under surgical light conditions. Solaris supports four fixed fluorescent channels ranging from visible to near infrared (NIR), and a multispectral channel where a liquid crystal tunable filter (LCTF) is used to acquire spectral data by sweeping across the green-to-red portion of the visible spectrum. This range of imaging channels allows for single-wavelength and multispectral imaging of widely used reagents (e.g. indocyanine green [ICG] and Fluorescein isothiocyanate [FITC]) and unique NIR fluorescent dyes used for detecting and labeling tumors.

While fluorescent imaging using NIR imaging agents (680, 750, 800 nm) offered effective tumor detection, identification of tumors implanted in nude mice or rats using visible (400-650 nm) reagents such as FITC presented challenges considering the presence of auto-fluorescence originating from tissue and food (alfalfa). For these reagents, Solaris acquired multispectral images using the LCTF under ambient light conditions, and an automated spectral unmixing algorithm was applied to the multispectral data, after background correction and ambient light removal, to separate tissue and food auto-fluorescence from the reagent fluorescent signal. The algorithm used vertex component analysis to automatically extract the primary pure spectra present in the multispectral images and unmix the reagent fluorescent signal by non-negative least squares fitting. To test the spectral unmixing capabilities of Solaris, in vivo experiments were performed using small amounts of locally injected FITC in mice and rats. In the absence of unmixing, it was not possible to accurately detect sites of FITC signal, but with unmixing the labeled regions were well defined. Additional studies in tumor-bearing mice and rats substantiated the ability to spectrally unmix FITC agent signal in deep tumor masses imaged under ambient light, enhancing the ability to surgically resect them. To further validate this concept, bioluminescent tumor cell lines were implanted in mice. After image-guided tumor resection, both the residual tumor bed and the resected tumors were imaged to confirm complete removal. These data demonstrate that intraoperative image-guided resection of fluorescent-labeled tumors can be achieved using LCTF-based open-air multispectral imaging on the Solaris.

#4247

TOLD MRI validation of reversal of tumor hypoxia in glioblastoma with a novel oxygen therapeutic.

Jason Lickliter,1 Jeremy Ruben,2 David B. Wilson,3 Heling Zhou,4 Ralph Mason,4 Evan C. Unger5. 1 _Nucleus Network, Melbourne, Australia;_ 2 _William Buckland Radiotherapy Centre, The Alfred Hospital and Monash University, Melbourne, Austria;_ 3 _NuvOx Pharma, Tucson, AZ;_ 4 _UT Southwestern, Dallas, TX;_ 5 _University of Arizona, Tucson, AZ_.

Background: Glioblastoma multiforme (GBM) is known to be a hypoxic tumor and hypoxia adversely affects response to radiation therapy. Dodecafluoropentane emulsion (DDFPe) on a weight basis, delivers over 100x as much oxygen as other higher molecular weight fluorocarbons and is under study as an oxygen therapeutic in GBM patients treated with chemo-irradiation. Tissue oxygen level dependent (TOLD) MRI is an oxygen sensitive imaging technique that is being used to study tumor re-oxygenation in these patients.

Methods: With informed consent and IRB approval adult patients with GBM with residual tumor post-surgery were enrolled into the dose escalation phase of the study. Patients received DDFPe (2%w/vol DDFPe) at doses of 0.05 cc/kg, 0.1 cc/kg or 0.17 cc/kg I.V. 30-60 minutes prior to each fraction of radiation (2-Gy each, 30 fractions over 6 weeks). Temozolomide chemotherapy was given concurrently. Radiation was performed while the patients were breathing carbogen via a non-rebreathing circuit; the post-radiation MRI was performed while the patients were breathing oxygen. All patients underwent standard MR imaging at 1.5 Tesla. TOLD MRI was performed on patients at the higher two dose levels pre and post DDFPe on two separate occasions, days #1 and days #5 or 10 after initiation of radiation. Steady state free precession (SSFP) Look-Locker was used to obtain T1 maps. T1 maps were calculated from 3 axial slices obtained through tumor from either 3 or 4 repetitions.

Results: TOLD MRI showed substantial decreases in T1 of tumor tissue after dosing with DDFPe and breathing oxygen (mean T1 decreased from 1501±454 ms to 1371±35 ms on day one and 1459±326 ms to 1183±271 ms on day 5 for the patient receiving 0.1 cc/kg DDFPe; from 1511±93 ms to 1250±85 ms on day 1 and from 1454±75 ms to 1281±77 ms on day 10 for the patient receiving 0.17cc/kg DDFPe), while inconsistent changes were found in contralateral normal brain (ranging from 863 to 1144 ms for both patients at all time points). The two different doses of DDFPe showed similar effect. Patients had second surgeries following completion of RT showing radiation necrosis consistent with enhanced effect of RT due to tumor re-oxygenation.

Discussion and conclusion: The change in T1 (TOLD) with oxygen breathing after DDFPe administration indicated improved tumor oxygenation. The utility of TOLD MRI as a potential non-invasive prognostic imaging biomarker holds promise for precision medicine in terms of assessing tissue reoxygenation of hypoxic tumors. The ability to alter tumor oxygenation in GBM patients pre-treated with DDFPe could have important therapeutic implications. Dose expansion is currently underway with the dose of 0.1-cc/kg DDFPe.

Figure: Representative T1 maps of two patients pre (air breathing) and post (oxygen breathing) DDFPe injection and radiation treatments. Arrows indicate tumors.

#4248

Functional imaging markers for blockade of breast cancer metastasis by IGF1R and insulin receptor targeted drugs using novel MRI and targeted iron oxide nanoparticles.

Deepali Sachdev,1 Huy Donguyen,1 Naoharu Kobayashi,2 Sidath C. Kumarapperuma,2 Joeseph C. Weber1. 1 _Masonic Cancer Center, Minneapolis, MN;_ 2 _University of Minnesota, Minneapolis, MN_.

IGF1R and insulin receptor (IR) regulate biology of estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. The results of initial clinical trials with anti-IGF1R drugs have been disappointing due to feedback upregulation of insulin receptor (IR) signaling and absence of biomarkers for these drugs.

We evaluated the effects of BMS-754807, a dual tyrosine kinase inhibitor of IGF1R/IR on ER+ and TNBC. In ER+ MCF-7 cells, it inhibited xenograft growth of MCF-7 tumors (n=10 per treatment) compared to vehicle. Interestingly, while tumor growth was suppressed over a period of five weeks, eventually the tumors displayed resistance to BMS-754807. In TNBC, it inhibited motility in vitro. In contrast to ER+ cells, BMS-754807 did not inhibit primary tumor growth of TNBC cells injected into the mammary fat pad of mice. But at a dose of 50 mg/kg daily inhibited metastasis of TNBC cells, MDA-231-LM2 and MDA-MB435A/LCC6 cells, in the orthotopic and tail vein models of metastasis compared to vehicle (n=10/group). Our data indicate that regulation of metastasis and tumor growth by IGF1R can be discrete events and functional imaging to identify biological properties of metastatic breast cancer regulated by IGF1R/IR are needed to better define treatments. While MRI is a powerful tool for detecting and imaging cancer, its utility in imaging metastasis to the lung is limited due to the challenges of lung MRI with conventional 3D gradient echo (GRE). MRI does not visualize lung well, mainly due to the abundance of air-tissue interfaces, which cause the MR signal to decay too rapidly for conventional MRI pulse sequences to capture. Clinically metastases are monitored by CT or PET but exposure of patients to ionizing radiation is a concern and problematic in longitudinal studies monitoring response to a targeted drug. Therefore, we recently reported the utility of a novel MR sequence called sweep imaging with Fourier transformation (SWIFT), where the data is acquired quasi-simultaneously with the radiofrequency pulse, to image lung metastasis of breast cancer. Here, we monitored response to IGF1R and IGF1R/IR targeted drugs in preclinical models of lung metastasis of breast cancer. We used MDA-231-LM2 cells with the tail vein injection model of metastasis. Mice with breast cancer metastases in the lungs were treated with either huEM164, an antibody against IGF1R, or BMS-754807. Metastasis was monitored by BLI and SWIFT MRI weekly. SWIFT was more sensitive in detecting inhibition of metastasis by these drugs.

Thus, dual inhibition of IGF1R and IR is effective in blocking growth of ER+ and metastasis of TNBC. However, combination of this therapeutic strategy with other agents may be necessary to prevent or delay onset of resistance. Further, noninvasive biomarkers of response to IGF1R/IR targeted drugs can be developed with SWIFT imaging.

#4249

**Quantification of murine lung tumor pH** in vivo **by acidoCEST MRI.**

Leila R. Lindeman, Edward A. Randtke, Christine M. Howison, Kyle M. Jones, Mark D. Pagel. _University of Arizona, Tucson, AZ_.

Introduction: Our objective was to accurately measure the extracellular pH (pHe) of lung tumors in vivo using an innovative, non-invasive imaging method known as acidoCEST MRI, to potentially improve the early detection of lung tumors via molecular imaging.

Methods: A spontaneous murine lung tumor model was initiated through orthotopic injections of urethane in seven A/J mice to induce formation of lung adenocarcinomas. Starting at 8 weeks post injection, we performed coronal anatomical scans to monitor tumor growth with Bruker BioSpin 7T small animal MRI instrument. AcidoCEST MRI was then performed when tumors reached a diameter of 1 mm in one dimension at approximately 17 weeks post injection, followed by additional scans each month. For all MRI scans, mice were anesthetized with 2.0% isofluorane, the respiration rate was monitored, and body temperature was maintained at 37 °C. Respiration-triggering (gating) was used in all imaging sequences to compensate for motion artifacts in the lung. For optimal gating, the mouse's respiration rate was maintained at < 40 breaths per minute. Each mouse was scanned with acidoCEST MRI using 370 mg/mL iopamidol (200 μL IV bolus, 400 μL/hr IV infusion), using a 6 sec saturation period at 3.5 μT power, 300 ms acquisition time, with 468x468 μm spatial resolution, updated with improved respiration gating. Parametric maps of pixel-wise pHe values of the tumor were produced by fitting the Bloch-McConnell equations in Matlab 2014a. The average tumor pHe and concentration of iopamidol agent in the tumor tissue were recorded.

Results: AcidoCEST MRI was successfully applied to the in vivo imaging of murine lung tumors. Our innovative, respiration-gated pulse sequence and Bloch-McConnell fitting method were essential to overcome the noise created by motion artifacts inherent in lung imaging. Lung tumors demonstrated successful uptake of the iopamidol contrast agent, with an average concentration of approximately 40 mM. The pH values in the tumor ranged from 6.5 to 7.0 with an average value of 6.64, demonstrating tumor acidosis. Anatomical MRI was used to monitor tumor growth from 1 mm diameter at 17 weeks post injection, to 4 mm diameter at 36 weeks post-injection.

Conclusion: Our study has established that acidoCEST MRI can be used to quantify the pH of murine lung tumors in vivo, showing that this method is promising for improved early detection of lung tumors. These pHe measurements can be compared with tumor growth rates to determine how tumor acidosis correlates with aggressive phenotype. Tumor pHe measurements can also be related to concentration of the agent as a biomarker of vascular perfusion. Furthermore, the spatial heterogeneity of lung tumor pHe can be assessed with this noninvasive imaging method. Further pre-clinical studies are also warranted to determine if the acidoCEST MRI method is suitable for early therapy response monitoring.

#4250

pHLIP® technology for imaging acidic tumors.

Anna Moshnikova,1 Michael Anderson,1 Ramona-Cosmina Adochite,1 Donald M. Engelman,2 Oleg A. Andreev,1 Yana K. Reshetnyak1. 1 _University of Rhode Island, Kingston, RI;_ 2 _Yale University, New Haven, CT_.

Introduction:

Extracellular acidosis promotes tumor development, progression and invasiveness. A combination of effects acidifies tumor cell interiors, so to maintain intracellular pH, cells pump out lactic acid and protons, acidifying the extracellular space. In addition, overexpression of carbonic anhydrases on the surfaces of cancer cells further contributes to an acidification of the environment especially near the cell surface. Thus, the pH near tumor cell surfaces is expected to be the lowest, and it increases with distance from the membrane. pH (Low) Insertion Peptides (pHLIP® peptides) pertain to the class of pH-sensitive agents able of sensing pH at the cellular surface and delivery of imaging and therapeutic agents to the cancer cells in tumors.

Experimental Procedures:

We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, SNARF, conjugated to the pH Low Insertion Peptide (pHLIP® peptide). The measurement tool was validated using cancer cells grown in spheroids, in mice and in excised tumors. Also, we performed imaging of primary tumors and metastatic lesions testing various fluorescent pHLIP® constructs on animal tumor models and human tissue.

Results:

pHLIP® is a platform technology using pH-triggered, membrane-associated folding for targeting acidic tumors. In contrast to other approaches developed to measure extracellular pH and target acidic environments (mostly in endosomes, where the pH is around 5.0-5.5), the pHLIP® technology has the capability to "sense" and target pH at the surfaces of tumor cells. We established that the pH at the surface of cancer cells is 0.2-0.4 pH units lower (in some parts of tumor it reaches values of pH6.0-6.1) than bulk extracellular pH measured up to now and demonstrated that the surface pH is sensitive to cell glycolytic activity. The approach was sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by injection of glucose. In response to low pH at the surface of cancer cells, pHLIP® peptides insert across the cell membrane and undergo coil-helix transition, which gives a high cooperativity for the transition compared with any pH-dependent diffusion-limited process, and allow targeting imaging agents to cancer cells in tumors. Even small differences in pH between normal and cancerous cells can be distinguished, and since both the pK of insertion and the cooperativity can be tuned, the system can be refined to target specific pH ranges. We have demonstrated targeting of acidic tumors in mice tumor models and in human tissue.

Conclusions:

Even individual cancer cell maintain acidity on its surface and could be detected in a well perfused areas. pHLIP® technology can be used in vivo and ex vivo on tissue specimens and opens both a path to medical utility and an exploration of other functions of acidity on cell surfaces. The leading fluorescent pHLIP® construct was selected for translation to clinics for diagnostic and surgical resection of primary tumors and submillimeter metastatic lesions.

### New Cell Lines and 3D Models

#4251

Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting.

Hubert Tseng,1 Jacob A. Gage,1 Pujan K. Desai,1 Reynolds Brobey,2 Sheri Skinner,2 Mehdi Dehghani,3 Kevin P. Rosenblatt,3 Wenliang Li,2 Robert J. Amato,2 Glauco R. Souza1. 1 _Nano3D Biosciences, Houston, TX;_ 2 _University of Texas Health Science Center - Houston, Houston, TX;_ 3 _CompanionDx, Houston, TX_.

Precision medicine holds the promise of designing patient-specific therapies to improve therapeutic efficiency. However, the scarcity of tumor and biopsy tissue is a limiting factor in the development of diagnostic assays. Cells isolated from these tissues could be used to overcome these issues, while serving as the basis for assays to diagnose and guide treatment. It is critical that the in vitro culture of these cells be performed in three-dimensional (3D) environments that can better replicate the native tumor microenvironment. However, currently available 3D cell culture platforms, like Matrigel, suffer from technical limitations in reproducibility and handling that make the development of such assays difficult.

Towards that end, this study isolates cells from human prostate cancer (PC) and renal cell carcinoma (RCC) tumor biopsies and patient-derived xenografts (PDX) and prints them into spheroids using magnetic 3D bioprinting. The core principle of magnetic 3D bioprinting is the magnetization of cells and their aggregation using mild magnetic forces. Once aggregated, these cells form spheroids that mimic native tumor environments in extracellular matrix and cell-cell and cell-ECM interactions. This technique can be used to actively magnetize cells and generate spheroids from a scarce cell source, while overcoming the limitations of other 3D cell culture platforms.

In this study, we demonstrated our ability to print spheroids from cells isolated from human tumor biopsies and PDX. Isolation techniques ranging from simple mincing and filtration to enzymatic digestion were employed. Next, these cells were magnetized

by incubation with a biocompatible magnetic nanoparticle assembly, NanoShuttle. Once magnetized, these cells were printed into spheroids of varying sizes, from 1,000-20,000 cells, in 384-well plates. These cells were cultured for days, after which viability was measured using CellTiter-Glo.

Our preliminary studies demonstrated our ability to isolate cells and print them into spheroids. Isolation was best with either mincing and filtration alone or collagenase II (400 U/mL) digestion for 1 h. These cells were then successfully magnetized and printed into spheroids, which remained viable after 72 h. Spheroids of 10,000-20,000 cells were the most successful, and further optimization is needed to reduce the size needed for viable spheroids to take full advantage of scarce resources such as tumor biopsies. We also demonstrated the ability to assay compound toxicity, showing a dose-dependent toxicity on spheroids derived from PDX tumors.

In all, we demonstrated our ability to isolate cells from human tumor biopsies and PDX models and print them into spheroids with high throughput. These preliminary results will serve as a platform for the further development of precision medicine assays to optimize PC and RCC treatment.

#4252

Establishment of a multicellular 3D cell culture model for tumor - endothelial cell interaction.

Arno Amann,1 Marit Zwierzina,1 Johann Kern,2 Gabriele Gamerith,1 Stefan Koeck,1 Edith Lorenz,2 Johannes Rainer,3 Heinz Zwierzina1. 1 _Medical University Innsbruck, Innsbruck, Austria;_ 2 _Oncotyrol - Center for Personalized Cancer Medicine GmbH, Innsbruck, Austria;_ 3 _European Academy of Bolzano/Bozen (EURAC), Bolzano, Italy_.

Introduction: Due to the increasing understanding of the mechanisms relevant to the genesis of cancer, we are experiencing a transition from disease to target-oriented therapy. One major hurdle for the development of these targeted therapeutic regimens, however, is the limited availability of predictive in vitro models. The critical challenge is to develop culture models better reflecting in vivo conditions. We present data that highlights the differences of RNA expression of in vivo like 3D microtissues consisting of tumour cells, fibroblasts and two different endothelial cell lines compared to normal 2D cell culture conditions.

Methods: 96-well hanging drop microtiter plates (InSphero AG, Zürich, Switzerland) were applied for the production of 3D mono-, co- and tri-cultures including the human lung cancer cell lines A549 or Colo699 alone or in combination with a human lung fibroblast cell line (SV-80) and either a human umbilical vein endothelial cell line (HUVEC) or the primary human lung microvascular endothelial cell line (HMVEC-L).

In addition, to conventional histology (H&E), tumour endothelial spheroid aggregation was displayed immunohistochemically (IHC) by protein expression of e-cadherin, CD31, von Willebrand factor (vWF) and α-muscle actin (α-SMA).

RNA expression profiling by Affymetrix chip analysis was performed for multicellular 3D microtissues and 2D cultured cell lines.

Results: Endothelial cells aggregated in coherent tube like structures preferentially in the fibroblast consisting core of all microtissues. Furthermore, endothelial cells expressed α-SMA only in microtissues that consisted both of fibroblasts and tumour cells indicating an interaction between these two cell types.

RNA expression profiles revealed a high number of regulated genes in tri-cultures when compared to microtissues only consisting of mono- or co-cultures or to traditional 2D cultivated cells. Regulated genes played an important role either in cell cycle, organelle fission, wound healing and DNA packing.

Interestingly, no difference in the RNA expression was displayed in microtissues containing either immortalized or primary endothelial cells,

Finally, a relation of RNA expression between our cell culture model and patient data was identified.

Conclusion: We demonstrate that cultivation of cells as multicellular microtissues in a 3D environment led not only to a difference in RNA but also in protein expression due to cell - cell interactions. Our data support the importance of performing complex co-culture for investigating tumour stroma interactions.

#4253

Modeling the effects of inflammatory stress on human intestinal epithelial cells in 3D enteroid co-culture system.

Fang Wang, Dorottya Laczkó, Mary Ann Crissey, Gary Falk, Bradley Johnson, Anil Rustgi, John Lynch. _University of Pennsylvania, Philadelphia, PA_.

The molecular pathogenesis of colitis-associated colorectal cancer (CAC) has been suggested to involve oxidative stress-induced DNA damage, resulting in mutations of tumor-suppressor genes and activation of pro-oncogenic pathways. However, the exact relationship between inflammation and colorectal cancer is not well understood due to the lack of a human model system that not only recapitulates in vivo growth/differentiation patterns, but also can be easily manipulated. In order to gain insight into the mechanism of colitis-associated colorectal cancer, we explored human intestinal enteroids as an ex vivo model to study the interaction of intestinal epithelial cells with immune cells under inflammatory conditions. Human intestinal crypts were isolated from adult intestinal tissue and grown in Matrigel culture to form enteroids. To model inflammation, we incorporated MHC-mismatched human peripheral blood mononuclear cells (PBMCs) into the Matrigel. TH1 immune response was induced by a cocktail of cytokines including IL-2, IL-7 and IL-15. A TH1 response was confirmed by the production of pro-inflammatory cytokines INFγ and TNFα over the course of 6-day treatment with the cytokine cocktail. Several cytokines related to TH1 and TH2 responses were induced. Interestingly, co-culture with human intestinal enteroids significantly enhanced the level of pro-inflammatory cytokines INFγ, IL-6 and TNFα, anti-inflammatory cytokine IL-10, as well as TH2 cytokine IL-13. Immunophenotyping of PBMCs shows that both CD4+ T cells (10-30%) and CD8+ T cells (1-10%) were activated upon TH1 induction. In addition, the percentage of activated CD8+ T cells was much higher in co-culture than in PBMC alone controls. Moreover, the cytokine cocktail dramatically induced the production of INFγ by purified CD4+ or CD8+ T cells but only when in co-culture with enteroids and not when cultured alone. In the enteroids, the TH1 response significantly inhibited cell proliferation and increased the apoptosis of epithelial cells when in co-culture only. Increased γH2AX (DNA damage marker) and impaired integrity of intestinal enteroids were also observed when in co-culture with the TH1 response. Cell-cell contact between enteroids and immune cells is required for these effects. Moreover, the TH1 response increases the mRNA levels of GRP78 (master regulator of ER stress) and Nrf2 (regulator of oxidative stress); decreases the mRNA level of Lgr5 [marker of active intestinal stem cells (ISCs)], but has no obvious effect on Bmi1 (marker of quiescent ISCs) in co-culture. Taken together, our data provide insight into the interaction of intestinal epithelial cells with immune cells at the molecular and cellular levels and establish this approach as a viable model for exploring the mechanism of CAC. Future studies will examine specifically for oxidative DNA damage and the effects of specific tumor suppressor inactivation on epithelial cell responses.

#4254

Dynamic change of differentiation in spheroids derived from mixed small cell carcinoma/adenocarcinoma of the uterine cervix.

Satoshi Kubota. _Osaka university, Osaka-fu Suita-shi, Japan_.

Small cell carcinoma of the uterine cervix (SCCC) occurs mainly in young women with poor prognosis. Clear treatment algorithm remains difficult to develop for its rarity. Herein, we report a case of SCCC with adenocarcinoma component, and analyzed the mechanism of differentiation using a spheroid culture method.

CTOS (cancer tissue-originated spheroid) is a primary culture system by which highly purified and viable cancer cells are cultivated. The principle of this method is maintaining cell-cell contact throughout the preparation and culture process. CTOS preserves characters of original tumors, especially their differentiation status.

We generated a panel of CTOSs from 9 SCCCs. The success rate of CTOS preparation in SCCC was 100%, indicating SCCC is one of the most suitable cancers for CTOS method.

We focused on SCCCs with adenocarcinoma component. These admixed tumors have been reported in other organs, such as lung, ovary, stomach, and so on. However, the mechanism underlying mixed tumor remains unclear. One of the CTOSs retained the characteristics of mixed tumor in original tumor.

Immunostaining revealed that CD99 was positive in the region of small cell carcinoma region, and CK7 in that of adenocarcinoma. CTOSs were sorted by CD99 and EpCAM (a possible stem cell marker) expression levels, and transplanted into NOD-Scid mice. Xenotumors derived from both CD99lowEpCAMlow cells and CD99lowEpCAMhigh cells showed mixed cell type in H&E staining. Immunohistochemistry revealed that CD99 was positive in the region of small cell carcinoma, and CK7 in that of adenocarcinoma. On the other hand, CD99highEpCAMhigh cells-derived xenotumor consisted of only small cell carcinoma. The tumors were positive for CD99, but negative for CK7.

Next, we searched for the key factor of differentiation. It has been reported that hypoxia induced adenocarcinoma in lung adenosquamous cell carcinoma. When CTOSs were incubated in hypoxia (3%O2), FACS analysis revealed that proportion of CD99low cells increased (20.6% in normoxia, 40.2% in hypoxia). Immunostaining of CTOSs showed similar results. Thus differentiation status of a mixed type SCCC showed dynamic change of differentiation in spheroids depending on culture conditions.

The research can provide insight into the origin of mixed tumor of SCCC, and may be useful for developing treatment strategy.

#4255

EpCAM positive lung cells are involved in organoid formation.

Takashi Semba,1 Ryo Sato,1 Makoto Suzuki,2 Hideyuki Saya,1 Yoshimi Arima1. 1 _Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio Unive, Tokyo, Japan;_ 2 _Department of Thoracic Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan_.

A combination of fluorescence activated cell sorting (FACS) and 3D culture techniques enable us to isolate specific lung cells and to form organoids, which recapitulate organ architectures in vitro. To clarify the cells of origin in the formation of lung organoids and to apply for oncogene-mediated transformation, we conducted spheroid culture and 3D culture experiments using mice lung cells.

We isolated the CD31-/CD45- cells from the lungs of wild-type (wt) or Ink4a/Arf-knockout (Ink4a/Arf-/-) C57BL/6 mice. The single cells were then cultured in serum-free medium in a low-attachment dish for 7 days to form spheres. The spheres were dissociated into single cells again, and were then infected with retroviruses expressing KRASG12V, EML4-ALK, EZR-ROS1 (Arai et al., PLoS ONE 2013), or KIF5B-RET (Saito et al., Carcinogenesis 2014) genes, which were identified as oncogenes in human lung adenocarcinomas. Those oncogene-expressing sphere-derived cells from wt and Ink4a/Arf-/- lung cells neither expressed EpCAM (the epithelial marker) nor TTF-1 (the lung lineage marker). We found that those cells formed undifferentiated invasive lethal tumors in lung lobes when the cells were inoculated into the lungs of mice via the trachea, after bleomycin treatment.

We then isolated the EpCAM+/CD45-/CD31- lung cells from wt or the Ink4a/Arf-/- C57BL/6 mice and these cells were subjected to a 3D culture. Those cells were suspended in a serum-free medium with keratinocyte growth factor, and the 1x104 cells were seeded on a 24 well 0.4μm insert pre-coated with growth factor-reduced Matrigel. After 2 weeks, the cells formed morphologically distinct phenotypes of colonies, such as cystic colonies, dense sphere colonies, and branched colonies. A histopathological study revealed that the EpCAM-positive colonies were composed of TTF-1-positive cells, and included differentiated lung cell markers, SP-C, Aquaporin 5, and CC-10. Interestingly, the EpCAM-/CD45-/CD31- cells from the lungs of the Ink4a/Arf-/- mice did not form organoids under 3D culture conditions.

Our data indicates that the EpCAM-positive normal mice lung cells, but not the EpCAM-negative cells, formed lung organoids including TTF-1-positive cells in a 3D culture. Inoculation of EpCAM+/CD45-/CD31- lung cells expressed oncogenes into the lungs of mice might allow us to develop a well-differentiated lung adenocarcinoma model.

#4256

Functional analysis for cancer precision medicine using patient-derived 2D and 3D cell models.

Xuefeng Liu, Ewa Krawczyk, Ogla Timofeeva, Nancy Palechor-Ceron, Aleksandra Dakic, Vera Simic, Bhaskar Kallakury, Anatoly Dritschilo, Richard Schlegel. _Georgetown University, Washington, DC_.

Background: Preclinical models provide an essential tool to study both cancer biology with particular relevance to identifying appropriate therapies and new targets. Both Patient-derived xenografts and 3D methods require significant time for the necessary amplification of the tumor cells, which limits clinical utility. Even classical 2D cultures suffer from their long time requirements for establishing a cell line with a low efficiency (1-30%). Conventional cancer cell lines usually fail to reflect the complex genotypes and phenotypes of the corresponding primary tumor. Recently, we described the use feeder cells and a ROCK inhibitor to induce the conditional reprogramming (CR) of adult epithelial cells into a basal or stem-like state. The induction of these CR cells is reversible, and the removal of feeders and ROCK inhibitor, coupled with their placement in environments that mimick their native environment (Matrigel, air-liquid interface (ALI), and the renal capsule in mice) allows cells to differentiate normally. Importantly, the CR technology can generate 2×106 cells in a week from small biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. A recent study utilized the CR method to initiate cultures from CT-guided lung biopsies and identify combination of therapies (Science 2014). Three recent reviews in Nature series highlighted CR method as one of the next-generation patient-derived cancer models (Nat Rev Clin Oncol, 2014, Nat Rev Genetics and Nat Rev Cancer 2015). Primary goal: The clinical utility and standard protocols for generating patient CR cultures. We therefore initiated study to examine whether CR cultures reflected the biology and genotype of the original tumor and whether cultures might be used to predict clinical responses. Procedures and Results: We first worked out standard protocols for clinical sampling, storage, shipping, freezing and the preparation of conditioned medium. CR methods were then used to generate matched cultures from both tumor cells and adjacent normal cells from patients with prostate cancer or lung cancer. CR cultures were established efficiently from these tumors (>95% for prostate and lung cancer CRCs). Following the rapid establishment of CR cultures, they were then transferred 3D cultures. The data with Matrigel 3D and (ALI) demonstrated that CR cells reexpressed cell specific markers and were well-differentiated. We also characterized them for their growth properties, induction of tumors in immunodeficient mice, karyotype, and their exome and transcriptome profile. Conclusion: CR cells, coupled with sequential 3D culture conditions, appears to provide optimal conditions for inducing the differentiation of normal cells and for differentiating normal from tumor cells. Finally, this cell-based approach should be useful for defining the functional heterogeneity of the respective primary tumors and for evaluating appropriate therapies.

#4257

Gene expression and genomic drift comparative analysis between patient-derived conditionally reprogrammed cells and original tumors.

Jessica Li, Bonnie Liu, Rin Nakamura, Heidi Savage, Shoji Ikeda, Timothy Wilson, Teiko Sumiyoshi, Garret Hampton, Lukas Amler, Mark Lackner, Shih-Min A. Huang, Walter C. Darbonne. _Genentech Inc., South San Francisco, CA_.

First two authors contributed equally

Last two authors contributed equally

Recent studies have shown that ex vivo propagation of normal tissues or patient-derived tumor cells in presence of irradiated fibroblast feeder cells and ROCK inhibitor can rapidly establish conditionally reprogramed cells (CRCs). In case of normal tissues, the induction of CRCs was reversible when the ROCK inhibitor and the feeder cells were removed, resulting in CRCs differentiating to its tissue origin (Liu et al.2012). Previous publications suggested that the establishment of such cell models provides new strategies to understand acquired resistance during treatment (Crystal et al 2015) and to predict treatment response (Liu et al. 2014).

However, gene expression modulations and genomic drifting during the establishment of CRC propagation have not been thoroughly studied. The primary goal of this study is to molecularly characterize alterations between the original tumor tissues and the derived models growing with or without ROCK inhibitor. Understanding in-depth molecular fluctuations in this patient-derived ex vivo system will facilitate its appropriate use for tumor biology experimentation.

Herein, tumors from prostate cancer and breast cancer patients were surgical removed and cryopreserved at the clinical site then processed and cultured as previously described (Liu et al. 2012). Gene expression profiling and next-generation sequencing were carried out on the original tumor tissues and cellular models passaged during the CRC propagation in the presence or absence of ROCK inhibitor. Gene expression analysis of the prostate cancer cells and the breast cancer cells were carried out using a 93-gene prostate cancer-focused Fluidigm panel and a 800 gene NanoString breast cancer-focused panel, respectively. Cancer hotspot mutations were analyzed using the Ion Torrent Cancer Hotspot v2 NGS assay.

Through aforementioned genomic and transcriptomic interrogations, we demonstrated the extent of indication-relevant gene expression modulation during establishment and propagation of these cells. We also characterize cancer hotspot mutations in the primary tumor cells and the stability of those mutations during ex vivo propagation. These results should begin to inform the appropriate use of the CRC model for tumor biology experimentation.

#4258

Multi-tumor cell culture medium supports a high take rate and improves culture growth rate in five tumor types.

Elin S. Agoston,1 Marian Novak,2 Naghmeh Salimi,1 Alex Chao,3 Jeffrey Kent,3 Agoston T. Agoston,4 Ronny Drapkin2. 1 _Cellaria Biosciences, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA;_ 3 _Northeastern University, Boston, MA;_ 4 _Brigham and Women's Hospital and Harvard Medical School, Boston, MA_.

The efficiency of primary cell line derivation is inadequate, which presents a substantial hurdle in the study of many solid tumor types. We examined whether substitution of a novel multi-tumor cell culture medium into existing cell culture protocols may improve the efficiency of primary cell culture and cell line derivation.

The multi-tumor medium RETM (Renaissance Essential Tumor Medium) was initially screened for the ability to achieve primary, extended, and continuous cultures from solid tumors of the breast, lung, colon, prostate and ovary (serous and endometrioid). A pan-cytokeratin antibody was then used to confirm epithelial origin of the expanded cell lines. Performance of RETM was compared to two established media types (RPMI, and Fallopian Tube Medium) in extended primary culture of five high-grade serous carcinoma (HGSC) tumor samples isolated from patients' ascites. Immunofluorescent staining of Pax8, an important Mullerian lineage marker, was used to confirm the purity and the expected lineage identity of the resulting cell lines. To examine genetic drift, we performed SNP genotyping on the original tumor and the cultured cells in our extended primary cultures of HGSC and our continuous cultures from breast, lung, and colon. The resulting genotype was then compared to the original tumor and the percent matching genotype was determined.

Of the six tumor types screened, five grew as long term cultures. Without additional optimization of methodology in HGSC, RETM supported a higher take rate (extended culture in 4 of 5 isolates versus 1 of 5 for established media types) as well as a 10-fold higher average growth rate in the same four cases (RETM doublings/day: 0.309 versus 0.030 for RPMI [p=0.004] and 0.023 for FTM [p=0.001]). In all cases, fibroblast overgrowth was ruled out by positive cytokeratin staining. The genetic analysis demonstrated a high degree of fidelity in genotypes between the patients' tumors and the corresponding cell lines as follows: DF30 (HGSC at 23 doublings) 99.7%; DF68 (HGSC at 25 doublings) 95.5%; Wood (breast ductal and lobular carcinoma at 150 doublings) 98.3%; Jacket (lung adenocarcinoma at 150 doublings) 87.2%; and Ferry (colon adenocarcinoma at 30 doublings) 99.6%.

We have shown for the first time that a single cell culture medium can support establishment of long-term cell lines established from a number of different tumor types including breast, lung, colon, endometrioid ovarian, and HGSC. Our results demonstrate that, compared to the current standard, RETM significantly improves both culture take rate and growth rate of the extended HGSC primary cultures. Together with the absence of cell culture crisis, the maintenance of genotype across a large number of population doublings indicate these cell lines are genetically stable and may be cultured to higher passages with reduced concern for genetic drift.

#4259

Long term cell culture models to identify biomarkers of response.

Usha Warrior, Jonathan Crane, Karen Bernards, Brian Nelson, Katie Snead, Alison Angione, Amber Sherman, Shawn DeVault, Jim Hnilo, jameel Shah. _Eurofins Pharma Discovery Services, St. Charles, MO_.

In order to test the efficacy of many cancer therapeutics, culturing cells in the presence of compounds for longer than the conventional 72-hr cell growth assay may be necessary. It is shown that the inhibition of many epigenetic targets results in a significantly delayed drug response not revealed by conventional shorter term cell growth assays. OncoPanel™ from Eurofins Pharma Discovery Services is a collection of 300 cell lines with broad cancer type and subtype representation and extensive genomic characterization. We have developed a 10-day assay to test inhibitors of epigenetic targets. Using the EZH2-selective inhibitor, GSK343, we have demonstrated the advantages of cell line profiling under long-term assay conditions when drug response reflects a protracted mechanism of action. We also tested Olaparib, a PARP inhibitor, in this system and showed substancial increased sensitivity against several cell lines versus the 72 hr assay. The significant biomarkers identified from the genomic analysis of the data from both GSK343 and Olaparib substantiate the relevance of long term cell profiling.

#4260

3D colorectal cancer organoids as preclinical models for assessment of activity of RAF inhibitor BGB-283.

Xiaoran Wu,1 Chunyan Fu,1 Xi Yuan,1 Lianhai Zhang,2 Xiaohong Wang,2 Jiafu Ji,2 Lusong Luo1. 1 _BeiGene (Beijing) Co., Ltd., Beijing, China;_ 2 _Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Surgery, Peking University Cancer Hospital and Institute, Beijing, China_.

Tumor organoids are three-dimensional (3D) cultures of cancer cells. They can be derived from the tumor cell lines, patient-derived xenografts and tumor tissues from cancer patients, thereby providing an attractive in vitro assay to optimize anticancer treatment. Oncogenic B-RAF, which drives cell transformation and proliferation, has been detected in 5-15% of colorectal cancers (CRC). Despite the remarkable clinical activities achieved by vemurafenib in treating B-RAF V600E metastatic melanoma, their clinical efficacy in B-RAF V600E CRC is far less impressive. BGB-283, currently under clinical development for RAF and RAS mutated solid tumors, is a novel inhibitor of the RAF dimer with inhibitory activities against RAF family kinases. In this study, we evaluated the anti-proliferative effect of BGB-283 and compared it to BRAF inhibitor vemurafenib in CRC patient tumor derived organoids models. BGB-283 exhibited therapeutic potentials in tumors with mutations in the MAPK pathway. Herein, we reported the establishment of tumor organoid models from 19 CRC patients' tumor tissues with a success rate of 83%. Organoids maintained many characteristics of their original tumors based on their histology. Our studies using primary CRC tumor organoids showed that in contrast to vemurafenib, which only inhibited B-RAF V600E CRC tumors, BGB-283 had inhibition effect on both B-RAF V600E and KRAS mutated tumors. These findings support BGB-283 as a promising clinical stage antitumor agent in treating CRC harboring B-RAF V600E and KRAS mutations. In addition, we showed that patient-derived organoids can be a valuable tool to test drug sensitivity in a personalized treatment and help to fill the gap between tumor genetics and patient trials.

#4261

Novel tumor microenvironment (TME) restored 3D cell culture model and humanized patient-derived xenograft model for cancer research and drug evaluation.

Jing Zhang, Dabing Yang, Huilin Wang, Qiong Song, Hui Li, JP Shaw, Tianjing Deng. _BioDuro, Shanghai, China_.

New anticancer drug development is an expensive and time consuming process, which typically can take more than 10 years and cost over a billion dollars. Although a majority of late discovery drug candidates demonstrate potency in preclinical models, most will ultimately fail to achieve efficacy in clinical trials. While recent strides have been made to improve preclinical models, the continued widespread use of traditional models creates translational challenges during clinical development. An intrinsic challenge for drug developers is to close the gap between preclinical modeling and translational efficiency for patients. Accurately modeling core attributes of the human tumor microenvironment (TME) in preclinical models is key for identifying novel targets, lowering toxicity and improving translational success. Even for advanced patient-derived xenograft (PDX) in vivo models, the cytokines generated by murine stromal cells fail to replicate many important paracrine networks, which can mislead preclinical pharmacology outcome data. To address this gap, we have begun to establish and characterize nearly 1,000 novel patient tumor samples, spanning over a dozen cancer indications, into robust human TME-restored 3D cell culture models and humanized patient derived xenograft models. Our goal is to improve overall patient-derived tumor model initiation success rates and subsequent translational pharmacology.

#4262

Combined 3D quantitative imaging and 3D cell culture for cancer drug discovery.

Guillaume Vidal,1 Valérie Lobjois,2 Zied Souguir,1 Pauline Pannetier,1 Elise Demange,1 Jean-Michel Lagarde3. 1 _Celenys, Rouen, France;_ 2 _ITAV USR3505 CNRS, Toulouse, France;_ 3 _Imactiv 3D, Toulouse, France_.

Monitoring drug activity in multicellular tumor spheroid in vitro models is an important challenge as these 3D models have demonstrated their major relevance in pharmacological screening and anticancer drugs evaluation (1). Optical microscopy coupled to the selective plane illumination microscopy (SPIM)3D microscopy, and, and automatic automated images processing and quantitative analysis are promising approaches to process optimizes the evaluation of drug response in a 3D cell culture models with, in a high/mediumhigh throughput screening waycapabilities.

BIOMIMESYS® used as a 3D cell model is based on hyaluronic to provide an in vivo like environment to the cells.

The In this study the effects of the topoisomerase I inhibitor Topotecan were investigated on colon adenocarcinoma HCT-116 3D cell culture. Spheroids were prepared and grown in BIOMIMESYS® used as a 3D cell model isa matrix based on hyaluronic acid tothat provides an in vivo like environment to the cells.

was monitored and coupled with mMorphometric parameters and with nuclei fluorescence markers were used to monitor analysis for drug activity . Data provideand to characterization characterize of both cytostatic and cytotoxic effects of this drug withon the basis of both quantitative 3D and imaging of the mechanisms.

These data highlight the relevance of combining a biomimetic microenvironment for 3D cell culture recreating the the in vivo features of a tumor cell population biology, together with and high resolution content microscopy-based quantification for drug screening.

This biomimetic model provides an innovative platform for the in-depth analysis of tumor development and for the discovery or the characterization of novel therapeutic targets.

Ref:

1) Breslin S, O'Driscoll L. Drug Discov Today.18: 240-249 (2013)

#4263

Establishment and initial characterization of human hepatocellular carcinoma organoid culture.

Sebastian Schölch, Lahiri K. Nanduri, Daniel Kühn, Jürgen Weitz, Moritz Koch, Nuh N. Rahbari. _University Hospital Carl Gustav Carus, Dresden, Germany_.

Background:

Hepatocellular carcinoma (HCC) is the 3rd most leading cause of cancer death in the world. Despite extensive research, HCC has a strikingly bad prognosis due to the limited therapeutic options: Other than surgical resection, there is no curative treatment. No cytotoxic agent has shown efficacy so far, and the only currently approved systemic agent, sorafenib, is only used in a palliative context.

This dilemma is due to the therapy-resistant biology as well as its variable pathogenesis and biological diversity, which requires excellent preclinical models to allow differential and innovative studies.

Innovative 3D culture methods such as organoid culture have been increasingly implemented in routine in vitro research for many tumor entities. However, due to its distinct biology, organoid culture of HCC has been a challenge. We here describe the initial establishment and characterization of HCC organoid culture.

Materials/Methods:

Surgical HCC tissue specimens were obtained in the operating rooms of the University Hospital Dresden; biopsy specimens were obtained via CT-/ultrasound-guided biopsy of HCC lesions. All samples were processed according to a novel protocol and cultured in Matrigel supplemented with a highly specialized culture medium. Organoids were propagated, passaged and frozen at regular intervals. Proliferation was measured using a modified WST proliferation assay protocol. Next-generation sequencing (NGS) was performed on corresponding triplets of healthy tissue, tumor tissue and organoid samples using the TruSight One Sequencing Panel (Illumina) on an Illumina MiSeq Sequencer. Histology was performed on formalin-fixed and paraffin-embedded organoids using standard protocols.

Results:

We were able to establish a method to culture HCC samples as organoids in vitro. Proliferation assays revealed distinct differences in organoid growth of HCCs from different patients and correlated well with both clinical behavior and histological grading of the tumors. Amplicon sequencing (targeting 4,813 cancer-associated genes) of corresponding sets of tumor specimens and HCC organoids demonstrated the HCC origin of the organoids and their genetic and genomic stability in culture. Treatment of HCC organoids with sorafenib showed the expected reduction in proliferation and viability. Interestingly, treatment of the HCC organoids with hepatocyte growth factor (HGF) resulted in a marked reduction in proliferation as opposed to healthy liver organoids, which had increased proliferation activity upon stimulation with HGF. Histology of the HCC organoids showed distinct features of HCC and corresponded well with the corresponding HCC tissue samples.

Conclusion:

We successfully established a protocol reliably allowing 3D organoid culture of HCC. The organoids faithfully recapitulate the biology of the corresponding tumor and can be used for multiple purposes including in vitro therapeutic testing.

#4264

Engineered in vitro models of cancer metastasis using decellularized biomatrix.

XI TIAN, Michael E. Werner, Henry P. Foote, Ariel D. Hanson, Lola M. Reid, Andrew Z. Wang. _University of North Carolina-Chapel Hill, Chapel Hill, NC_.

Background: Metastasis contributes to majority of death in cancer patients. However, cancer metastasis is difficult to study due to the lack of experimental models that can fully recapitulate the biological process, especially the organ specificity of cancer metastasis. Recent advances in tissue engineering demonstrated that decellularized tissue, organs that are denuded of cells, are excellent scaffolds for tissue engineering of complex organs, such as liver and lung. These decellularized organs preserve the organ microenvironment, which is critical in cancer metastasis. We hypothesized that we can utilize decellularized organ and engineer in vitro models of cancer metastasis that can recapitulate the organ specificity of cancer metastasis. Here we report the proof-of-principle of this approach by employing colorectal cancer as a disease model. Since the primary cause of morbidity and mortality in colorectal patients is liver and lung metastasis, we aimed to engineer in vitro models of colorectal cancer lung and liver metastasis.

Methods: Decellularized biomatrix was prepared by sodium deoxycholate based perfusion decellularization of rat liver or lung. We compared the growth factors on the biomatrix scaffold to that of normal rat liver and lung using semi-quantitative ELISA. We cultured colorectal cancer cell lines: HT-29, SW480 and Caco2, on tissue culture dishes coated with liver and lung decellularized biomatrix scaffolds. Cell colony morphology and structure was examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and hematoxylin and eosin stain. Therapeutic response (chemotherapy and radiation) for cells grown on biomatrix was also examined.

Results: Decellularized organs preserved 92% growth factors bound to biomatrix scaffold at near physiological levels. HT-29, SW480 and Caco2 were able to proliferate on dishes coated with biomatrix scaffold and spontaneously formed three-dimensional (3D) colonies when maintained at higher levels. These tumor colonies are millimeter in size and are spherical in shape, similar to in vivo metastases. SEM showed cells on the tumor surface appeared to form tight junctions with each other. TEM images further confirmed that these 3D tumor colonies contain areas of necrosis, consistent existing literature 3D colonies and in vivo metastasis. Importantly, we identified signet ring cell formation in the engineered 3D metastases. We also found the cells in engineered liver and lung metastasis responded to chemotherapy treatment differently than the cells cultured under standard conditions or on collagen or on matrigel. Interestingly, cells in both engineered liver and lung metastasis are more sensitive to radiotherapy than standard condition.

Conclusions: Our engineered in vitro tumor metastasis models closely mimic the in vivo metastasis phenotypically, histologically, and biologically.

#4265

Automation-enabled 3D tumor chemosensitivity screening by imaging and flow cytometry analysis.

Michael Kowalski,1 Vipat Raksakulthai,2 Kristin Prasauckas,2 Tara Jones-Roe1. 1 _Beckman Coulter, Indianapolis, IN;_ 2 _Molecular Devices, Sunnyvale, CA_.

Three-dimensional (3D) cancer cell cultures are being utilized for drug screening with increasing frequency since they provide a more physiologically relevant solid tumor model than two-dimensional cell cultures. Spheroids are a well characterized, in vitro tumor model system that has been utilized with multiple cancer cell lines. Growth and manipulation of these cultures in hanging drop plates (HDPs) can be challenging to manipulate and these challenges are amplified as sample throughput increases. We automated the culture and compound treatment of cancer spheroids in HDPs, as well as the preparation of these spheroids for analysis by high content imaging and flow cytometry. Automated cancer spheroid cultures showed excellent consistency in size and shape, providing higher confidence in the screening results. Creating single cell suspensions from the cancer spheroids for flow cytometry analysis was another significant challenge overcome through automation. Imaging provided information such as the location of drug-responding cells within the cancer spheroid while flow cytometry removed the potential for spheroid exclusion of staining reagents leading to false negatives. Automation enabled the use of these complementary analyses, thereby achieving a more complete understanding of compound sensitivity in solid tumors and illustrating how spheroids can be utilized to screen for novel cancer therapeutics.

#4266

Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds.

Pauline Pannetier,1 Fiona Louis,1 Zied Souguir,1 Agathe Devaux,1 Didier Le Cerf,2 Jean-Pierre Vannier,1 Elise Demange,1 Guillaume Vidal1. 1 _Celenys, Rouen, France;_ 2 _Laboratoire PBS UMR6270, Rouen, France_.

Extracellular matrix (ECM) characteristics, including stiffness, porosity, composition and spatial interaction with the surrounding cells and soluble factors are key components for cell growth in a tissue microenvironment. However, poor performance of 2D in vitro systems and animal models demands physiologically relevant well controlled 3D platforms for mechanistic assays, drug resistant phenotypes, new drug efficacy, toxicity assessment.

Technical limitations in the current use of multicellular spheroids prevent their widespread use in cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange, cell retrieval and microscopy analysis challenging. An ideal 3D cell culture system would form spheroids in an in vivo like microenvironment, while being easy to handle and compatible with all analytical methods.

To address this unmet needs, we use a controlled hyaluronic acid-based scaffolds for spheroid formation. We have enriched 2 hyaluronan scaffolds with other components of the ECM such as collagen I, collagen IV, collagen VI, RGDs motif or galactosamine. Functionalised scaffolds overpass the 2D flat culture limitations by recreating cell/cell interactions and cell/matrix interaction to recreate a more physiologically authentic 3D architecture. The 2 functionalized scaffolds recapitulate the microenvironment for Hepatocyte and Adipocyte growth.

Once formed, the spheroids can be cultured long-term, the scaffold is transparent allowing reproducible High Content Screening, the spheroids and the cells can be retrieve, avoiding the technical issues of other 3D systems to retain samples. Moreover, the scaffold is compatible with fluorescence/luminescent kit and immunofluorescent microscopy.

We demonstrated this technology using cell lines, primary cells (adipocytes, hepatocytes). We assayed the spheroids over time using various endpoint spheroid morphology, growth and viability, resistance to anti-cancer drug, relevant cell organization formation and toxicity endpoint.

Thus, this study introduces functionalized HA scaffold for the use of in vitro culture model as that represent native cell environments is ready to ready to use and compatible with HTS and all analytical methods for drug development and compound screening.

#4267

Establishment and biological characterization of a Chilean ascites-derived gallbladder cancer cell line.

Javier Retamal,1 Carolina Bizama,1 Jaime Espinoza,1 Lorena Rosa,1 Francisca Alfaro,1 Diego Romero,1 María José Apud,1 Bruno Nervi,1 Pamela Leal,2 Helga Weber,2 Juan Carlos Roa,1 Patricia García1. 1 _Pontificia Universidad Católica de Chile, Santiago, Chile;_ 2 _Universidad de La Frontera, Temuco, Chile_.

Gallbladder cancer is the most common biliary tract cancer. Globally, it is considered as a rare malignancy but shows a high incidence in certain geographic areas, such as Eastern and South Asia, Eastern Europe and Latin America. In Chile, this aggressive neoplasia is the second leading cause of cancer death among women, with a mortality rate of 15 deaths per 100.000 women, only slightly lower than breast cancer. Most patients with gallbladder cancer are diagnosed at advanced stages and the prognosis still remains low, even using the most current diagnostic techniques. Tumor biology of gallbladder cancer is still poorly understood and there are no therapeutic options to improve the prognosis of patients with advanced gallbladder cancer. The establishment of cell lines for their use as in vitro models is essential for the study of tumor biology and drug susceptibility. The aim of this study was to characterize and compare the malignant properties of three gallbladder cancer clones isolated from ascites-derived primary culture cells. Tumor cells were isolated from the ascites of an advanced gallbladder cancer patient using a previously established protocol. The primary culture cells were characterized to determine their epithelial origin by using immunohistochemical markers. Due to the heterogeneous nature of these cells, individual clones were isolated from them and maintained in culture until their establishment as immortal cell lines (less than 20 passages). Finally, three clones were obtained and evaluated in order to characterize and compare their malignant properties, determining their growth rate, chemosensitivity to gemcitabine, cisplatin and 5-fluoracil, migration capability and the in vivo tumorigenesis induction. The ascites-derived primary culture cells showed high expression of epithelial and tumor markers (Cytokeratin 7 and 19, CA 19-9, CA 125, CA 15-3) and negative expression of mesothelial markers (calretinin and mesothelin). Individual clones (clone 1, 2 and 3) derived from the primary culture showed differences from each other. The calculated doubling time was 60h for Clone 1, 35h for Clone 2, and 28h for Clone 3. All three clones were equally sensible to gemcitabine, cisplatin and 5-Fluoracil, compared to other established gallbladder cancer cell lines. Clone 5 exhibited the greater migration potential and Clone 6 resulted to have the most tumorigenic capability, although all were able to form xenograft tumors before 2 weeks. Conclusions: To our knowledge, these are the first gallbladder cancer cell lines established from a Chilean patient and they may provide a useful tool for the study of gallbladder cancer biology and for in vitro and in vivo analysis aimed at identification of new potential therapeutic targets. Research supported by FONDECYT 11130515, 1151008, 1130204, 3140426 and 3140308.

#4268

Examining the 3D tumor microenvironment via microbioreactors.

Matthew Rogers, Tammy Sobolik, David Schaffer, Philip Samson, John Wikswo, Ann Richmond. _Vanderbilt University, Nashville, TN_.

Microfluidic devices can offer a unique opportunity to more fully examine key factors of the tumor microenvironment that mediate metastasis. A microfluidic bioreactor was fabricated and cast in (poly)dimethylsiloxane (PDMS). Highly metastatic MDA MB 231-GFP+ tagged cells were allowed to aggregate into spheroids over a 24 hour period via a hanging drop method. The cancer cells were pipetted in droplets of media onto the inverted lid of a 100 mm well dish. The lid was then re-inverted onto its base and the dish was incubated overnight, where the cells in each drop would aggregate into a single spheroid. Afterward, the center channel of the microfluidic device was coated with an extra-cellular matrix (ECM), which consisted of 56% HEPES buffer, 24% Type 1 rat tail collagen, and 20% Matrigel. A single spheroid was then introduced into the center of the device by lowering the lid of the well dish onto the device such that the hanging drop was allowed to fall into an inlet hole, taking with it the spheroid. After a brief incubation, the device was submerged in media in a petri dish and monitored for 14 days. The device was used to study the metastatic potential of the cancer cells as a function of the microenvironment. In these experiments, NIH3T3-mcherry+-tagged fibroblasts were loaded into the channel along with the ECM. The spheroid remained fairly tightly aggregated during the first week, while the fibroblasts on either side of the channel grew an array of tubules within the ECM, migrating toward the spheroid. As the fibroblasts tubules approached the spheroid in the center of the device, the spheroid began branching out toward the incoming fibroblast tubules. The tumor cells used the fibroblast tubules as a scaffold to migrate outward in a single-file pattern. The cancer cells migrated a distance of roughly 0.55 mm/day. In other experiments, when a device containing a spheroid and fibroblasts was constantly exposed to 10 ng/mL CXCL12, there was an inhibition of fibroblast growth and tubule formation in the ECM, while the cancer cells migrated extensively even in the absence of fibroblast/matrix tubules, at a rate of roughly 0.37 mm/day. This contrasted with the control devices, where the cancer cells did not migrate until the fibroblast tubules reached the spheroid. In devices that consisted of a spheroid alone, the cells remained tightly aggregated and did not migrate into the surrounding environment. These data indicate that the microbioreactor utilized herein will be useful to dissect the interaction between cancer cell migration and the microenvironmental factors that facilitate this migration, leading to metastasis. When exposed to a pro-tumor chemokine environment or tubular fibrils laid down by fibroblasts, cancer cells can migrate freely. However, in a neutral microenvironment, the cancer cells may remain tightly aggregated until appropriate stimuli are provided, even though they have an intrinsic capability to metastasize.

#4269

Interstitial fluid pressure alters cell motility and collective invasion via EMT marker expression in an engineered model of a human breast tumor.

Alexandra S. Piotrowski-Daspit,1 Joe Tien,2 Celeste M. Nelson1. 1 _Princeton University, Princeton, NJ;_ 2 _Boston University, Boston, MA_.

We developed a three-dimensional (3D) engineered model of a solid human breast tumor to study the effects of interstitial fluid pressure (IFP) on collective invasion and the expression levels of epithelial-mesenchymal transition (EMT) markers. Many solid tumors exhibit elevated IFP; as these tumors grow, intra-tumoral vascular and lymphatic vessels collapse. The non-functioning lymphatic system impairs drainage, and immature hyperpermeable blood vessels cause fluid to accumulate within the interstitial space. As a result, IFP rises steeply beyond the tumor periphery and plateaus at pressures as high as 50 mm Hg above normal at the tumor core. This pressure profile, in turn, leads to outward fluid flow from the core of the tumor. IFP has been shown to affect the migratory behavior of individual cells in 3D cell culture models, though its role in collective cancer invasion remains unknown. Moreover, the underlying molecular mechanisms linking IFP to changes in cell motility remain unclear. We sought to address these questions using our engineered model.

Our 3D culture model consists of an aggregate of MDA-MB-231 breast cancer cells (mimicking a solid tumor) embedded within a 3D collagen gel that is flanked by two media reservoirs. The IFP profile experienced by the cancer cells is established by altering the heights of the media reservoirs on either side of the collagen, creating a hydrostatic pressure gradient. Transcript levels of EMT markers in the aggregates subjected to a variety of pressure profiles were determined using quantitative real-time PCR. Expression of these markers was also manipulated ectopically through the creation of stable cell lines. Time-lapse imaging and cell tracking were used to determine the persistence and motility of individual cells within the aggregates.

We found that the direction of IFP-induced flow determines the invasive phenotype of tumor cells. Additionally, high expression levels of both mesenchymal (Snail1, vimentin) and epithelial (E-cadherin, keratin-8) markers were characteristic of collectively invading aggregates, suggesting that partial EMT is important for collective invasion. Ectopic expression and knockdown of EMT markers revealed that they are necessary and sufficient for collective invasion in response to IFP. Time-lapse imaging analysis demonstrated that IFP and EMT marker expression also affect the motility and persistence of individual cells within the aggregates, further confirming that IFP is an important regulator of collective invasion. In conclusion, we used a robust culture model of a human breast tumor to gain insight into the mechanisms guiding collective invasion from primary tumors in response to IFP; IFP alters expression levels of EMT markers, thereby regulating collective invasion.

#4270

UMR957: a new osteogenic osteosarcoma cell-line derived from an osteoprotegerin null mutant mouse.

Benjamin Navet, Jérome Amiaud, Céline Charrier, Thibaut Quillard, Bénédicte Brounais-Le Royer, Dominique Heymann, Frederic P.R. Lezot. _INSERM, Nantes, France_.

Background: Mouse osteosarcoma cell-lines enabling in vivo studies in syngeneic mouse strains are rare but important for preclinical studies. Existing cell-lines when injected are mainly osteolytic with a moderate tumor osteoid tissue formation. There is a crucial need for new cell-lines with higher osteogenic characteristic.

Methods: A female osteoprotegerin null mutant mouse, breed in the animal house of the Faculty of Medicine from the University of Nantes (France), spontaneously developed an osteosarcoma on the left tibia. The mouse was sacrificed, a biopsy was place in culture for further in vitro and in vivo (injections) studies and the tumor was analyzed using μ-CT, μ-radiography, classical histology and immunohistochemistry.

Results: μ-CT and μ-radiography analyses evidenced that the tumor developed in the Opg-/- mouse was highly osteogenic with an important tumor osteoid tissue that colonized most of the surrounding soft tissues. A total destruction of the tibia was observed. Histology and immunohistochemistry confirmed the presence of an osteosarcoma with an important expression of most bone markers as Osterix. An important vascularization was evidenced in the tumor as the presence of numerous TRAP positive cells. Monocyte/macrophage and T-lymphocyte cells were also present in this tumor. From the biopsy a cell-line was obtained and named UMR957. This cell-line evidenced all markers of the osteoblast cell. When injected in the tibia vicinity of either naïve C57BL/6J, Nude or Opg-/- mouse a highly osteogenic osteosarcoma was observed with similar features to the initial tumor.

Conclusions: A new mouse osteosarcoma cell-line was obtained from a spontaneous tumor developed in an Opg-/- mouse. This cell-line when injected in syngeneic mouse recapitulates all features of the initial tumor more particularly its massive osteoid tissue formation.

#4271

Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM).

Teresa W-M Fan,1 Andrew N. Lane,1 Jun Yan,2 Richard M. Higashi,1 Jeremiah T. Martin,1 Michael Bousamra2. 1 _University of Kentucky, Lexington, KY;_ 2 _University of Louisville, Louisville, KY_.

Introduction: We use SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1,2). We have now extended the range of models to fresh human tissue slices, which retain the original tissue architecture and heterogeneity with a paired benign versus cancer tissue design under defined cell culture conditions. β-glucan is a polysaccharide that repolarizes tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype in mice (3). Here we report the activation of TAMs in human NSCLC tissue slices.

Experimental: Freshly resected paired tissue slices from individual patients (approx. 1 mm or less thick and 5-40 mg wet weight) were incubated ± particulate β-glucan in standard cell culture conditions, with gentle rocking to enable efficient gas, nutrient, and waste product exchange. Tissue slices could be maintained metabolically viable for at least 48 h of incubation. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched common nutrient tracers such as glucose and glutamine, using high resolution mass spectrometry, GC-MS, and NMR after a period of incubation (2).

Findings: Time-course analysis of the slices by NMR, MS, and histology revealed that NSCLC tissue slices, both benign and tumorous, retained their architecture and a broad spectrum of metabolic activities. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues ex vivo, as the in vivo case. The paired tissues from different patients showed significantly different metabolic responses to β-glucan, as expected

Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer and stromal cells in their tissue context, and response to drug treatment (4). As the microenvironment of the target human tissue is maintained, including the resident immune cells, individualized response to immune-active agents can be determined in a clinically relevant setting.

Supported by NCI P01CA163223-01 and NIEHS 1R01ES022191-01

1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., et al. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182.

2. Sellers, K., Fox, M.P., Bousamra, M., II, et al. (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. Journal of Clinical Investigation, 125, 687-698.

3. Liu, M., Luo, F., Ding, C., et al. (2015) Particulate β-Glucan Converts Immunosuppressive Macrophages into M1 Phenotype Through Dectin-1-induced Syk-Card9-Erk Pathway and Raf-1-c-Maf Pathway. J. Immunol., 195, 5055-5065.

4. Xie, H., Hanai, J., Ren, et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809

#4272

Organoids and patient-derived tumor xenograft of pancreatic adenocarcinoma share morphological and genetic features with the primary tumor.

Isabel Romero Calvo,1 Ashwin Akki,2 Andrey Ugolkov,3 Mary M. Buschmann,4 Samantha M. Sparrow,1 Teresa Barry,4 Margaret Eber,4 Tongjun Gu,1 Shuang Qin Zhang,4 Hedy Kindler,4 William Dale,4 Kevin Roggin,5 Andrew P. Mazar,3 Kevin P. White,1 Christopher R. Weber2. 1 _The University of Chicago, Institute for Genomics & Systems Biology, Chicago, IL; _2 _The University of Chicago Medicine, Department of Pathology, Chicago, IL;_ 3 _Department of Pharmacology, Feinberg School of Medicine, Center for Developmental Therapeutics, Northwestern University, Evanston, IL;_ 4 _The University of Chicago Medicine, Department of Medicine, Chicago, IL;_ 5 _The University of Chicago Medicine, Department of Surgery, Chicago, IL_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death, with a 5-year overall survival rate <7%. Several active systemic therapies are now available for PDAC. Personalizing therapy may be improved with the development of realistic tumor models from a patient's explanted tumoral tissue. Human pancreatic tumor patient-derived xenografts (PDX) implanted into immunodeficient mice and tumor organoids grown in vitro 3D culture are two promising models. However, it is uncertain if these models are histopathologically and genetically similar to the primary PDAC.

Histopathological comparison of sections from PDX tumor, organoids, and primary PDAC from a 63-year-old female patient were performed on paraffin embedded tissue. H&E and immunohistochemical staining for cytokeratins (CK7, CK20), p53, Claudin-4, and CEA were performed. DNA and mRNA sequencing was performed. Both PDX and organoids exhibited histopathological features remarkably consistent with the original patient tumor including the histologic grade (moderately differentiated), cytological appearance (irregular nuclear membranes, open chromatin and prominent nucleoli), mitotic activity (5 -7/10 HPF), and immunohistochemical profile. The PDX and organoids demonstrated diffuse moderate-to-strong positivity for CK7, CEA, p53, and Claudin-4 and focal weak positivity for CK20 - all similar to the primary tumor. The immunohistochemical staining pattern was consistent with mRNA sequencing of the primary tumor which showed that CEA, Claudin-4 and p53 expression were ∼960-fold, ∼27-fold and ∼3 fold higher respectively (vs. benign pancreatic tissue). DNA sequencing revealed somatic mutations in KRAS and TP53 genes seen in >90% and ∼70% of PDACs respectively, and a few rare somatic mutations, such as a sodium leak channel (NALCN) mutation, seen in ∼3% of PDAC.

Both PDX and organoid models of PDAC maintain key histological features, immunohistochemical profile and basic gene expression pattern akin to the primary tumor. These findings suggest that PDXs and organoids have the potential to serve as reliable pathophysiological models for optimizing individual therapy for patients with PDAC.

#4273

Oncogenic BRAFV600E drives stem cell niche factors-independent growth and tumorigenic transformation in colon organoids.

Byunghak Kang,1 Julie In,1 Nicholas Zachos,1 David Huso,1 Shinji Maegawa,2 Jean-Pierre Issa,2 Hariharan Easwaran,1 Stephen B. Baylin1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _Temple University, Philadelphia, PA_.

Herein, we report a model for how BRAF mutations, in contrast to KRAS mutations, specifically associate with and induce, in mouse colon organoids without a microenvironment, the phenotype of human sporadic right-sided colon adenocarcinomas (COAD). The human BRAF mutant COAD are particularly distinguished by having an increased incidence of promoter CpG island methylation, termed CpG island methylator phenotype (CIMP)-High. This latter phenotype contrasts with the majority of the COAD which are classified in the CIMP-low to intermediate groups and have mostly KRAS mutations. To understand how these mutations influence tumor evolution and the methylation landscape, we modeled the early carcinogenesis of colorectal cancer by inducing BRAFV600E and KRASG12D mutations individually in 3D organoids prepared from mouse proximal colon. The induction of BRAFV600E mutation, but not KRASG12D mutation, showed various features of progressive transformation. In vitro induction of BRAFV600E drove the organoids to derive cystic changes in morphology and promoted adoption of a stem cell niche independency characterized by growth in the absence of added stem cell niche factors including Wnt3a, R-Spondin, and Noggin. This independency is not due to increased secretion of niche factors by the Paneth-like cells in BRAFV600E mutant organoids. In addition, BRAFV600E organoids showed dysplastic changes such as high nuclear to cytoplasmic ratio and abnormal budding. The most exciting finding is that induction of BRAFV600E mutation, but not KRASG12D mutation, induced complete transformation of the organoids forming xenograft tumors in NOD/SCID mice. The tumors exhibit histological characteristics of human mucinous adenocarcinoma, which is the tumor type highly associated with BRAFV600E mutation in human COAD. Gene expression analyses revealed up-regulation of intestinal stem cell signature genes and down-regulation of genes related to intestinal differentiation in BRAFV600E organoids. In addition, BRAFV600E organoids showed increased expression levels of Wnt pathway target genes indicating an enhanced and sustained Wnt-signaling. Analyses of CpG-island methylation in a panel of genes showed increased CpG-island methylation only in the BRAFV600E mutant organoids. In conclusion, in mouse colon organoids, BRAFV600E drives a human right-sided COAD phenotype with adoption of a stem cell niche independency, activation of the Wnt pathway, and induction of CpG island methylation.

#4274

Porcine ovarian matrix supports maintenance of human intestinal organoid cultures.

Jay George, Sol De Gese, Gabriel Benton. _Trevigen, Inc., Gaithersburg, MD_.

Currently, extracellular matrix hydrogels derived from the Engelbreth-Holmes-Swarm murine tumor (Cultrex BME, Matrigel) are used for stem cell maintenance and differentiation of both normal and tumor organoid culture models. While this matrix is suitable for many in vitro applications, there is increasing demand for a 3D organoid culture platform that is more suitable for clinical applications. Here, we have developed a soluble extracellular matrix derived from normal porcine ovaries that forms a hydrogel polymer at physiological temperature and that supports maintenance of human small intestine organoids in culture. This matrix supports expansion of LGR5+ human stem cell populations using Wnt, EGF, Noggin, and R-Spondin 1 cell culture medium. The matrix also supports differentiation of stem cells into mini organs by removing Wnt which is characterized by the formation of intestinal crypts which exhibit immunostaining for lineage specific markers. This new extracellular matrix hydrogel represents a non-tumor sourced, normal extracellular matrix for evaluating both development and cancer progression.

#4275

Exploring the metastatic potential of exosomal NM23 signaling using a triple negative breast cancer model in mice.

Suzann Duan, Senny Nordmeier, Iain L.O. Buxton. _University of Nevada School of Medicine, Reno, Reno, NV_.

Decades of research has established the idea that cancer cells of the primary tumor prime the metastatic microenvironment prior to establishing metastases at distant sites in the body. While this process is thought to facilitate neocolonization of dislodged primary tumor cells, the mechanisms by which cancer cells communicate to their remote targets remain largely unknown. Our lab has previously shown that triple negative breast cancer cells release exosomes carrying NM23, a nucleoside diphosphate kinase (eNDPK) implicated in promoting angiogenesis and pro-metastatic events extracellularly. To further elucidate the role of eNDPK in breast cancer signaling, we are developing a novel murine model that will allow us to examine its effects in vivo, as well as establish a timeline for the occurrence of metastases.

To establish our metastasis model in mice, we first showed that injecting human MDA-MB-231 cells into the mammary fat pad results in the formation of a primary tumor and subsequent development of metastases in the lung. Treating mice with inhibitors of NDPK activity reduces the size of the primary tumor and prevents secondary tumor formation in the lung. To mimic the pulmonary tumor microenvironment, we have isolated endothelial cells from the lungs of 6-8 day old mouse pups using magnetic beads conjugated to the endothelial cell markers CD54, CD102, or CD106. Purity of endothelial cells was confirmed by immunofluorescence staining. In preparation for in vivo experiments that demonstrate eNDPK targeting to lung endothelial cells, we have 3D printed biocompatible polylactic acid (PLA) tubular inserts that are capable of supporting lung endothelial cells encompassed in a gelatinous cell scaffold. Inserts containing lung endothelial cells are surgically introduced into the subcutaneous tissue of adult SCID mice. Human MDA-MB-231 cells expressing GFP-tagged tetraspanin are then injected orthotopically into the mammary fat pads of mice. After a period of 7-21 days, the inserts are excised and analyzed for the presence of GFP-labeled exosomes containing eNDPK and reorganization of the insert microenvironment.

Using this model for metastasis, we will be able to confirm the involvement of eNDPK in cell-cell communication between primary tumor cells and their targets in vivo. The significance of uncovering the mechanism(s) by which cancer cells metastasize is emphasized by the fact that recurrence of cancer at distant sites is associated with the most negative outcome in women diagnosed with breast cancer. Implication of eNDPK signaling in metastasis will lead to future research in developing novel small molecule inhibitors. Further, eNDPK can be used as a biomarker for the beginning stages of metastatic breast cancer, replacing current unreliable early detection methods and improving long-term survival outcomes.

#4276

Cystathionine-β-synthase overexpression increases cell proliferation, migration, bioenergetics and tumorigenesis in a non-tumorigenic colorectal cancer (CRC) cell line.

John R. Zatarain, Ches'Nique M. Phillips, Michael E. Nicholls, Paul Johnson, Steven G. Widen, Thomas G. Wood, Nadiya Druzhyna, Bartosz Szczesny, Craig Porter, Katalin Modis, Csaba Szabo, Celia Chao, Mark R. Hellmich. _UT Medical Branch, Galveston, TX_.

Introduction: We recently described overexpression of the enzyme, cystathionine-β-synthase (CBS) in human CRC (but not normal colonic mucosa) produces endogenous hydrogen sulfide (H2S) increasing tumor bioenergetics, cell proliferation, invasion, migration and promotes tumor angiogenesis. Its role in the progression of a colorectal adenoma to carcinoma remains elusive. The purpose of this study was to determine whether CBS overexpression in a non-tumorigenic human CRC cell line (NCM356) is sufficient to increase cell proliferation, migration, tumorigenesis, and metastasis. Methods: NCM356-p (parental) are non-tumorigenic when xenografted into athymic nude mice and express low levels of endogenous CBS, similar to normal colonic mucosa. RNASeq analysis determined mutation status. NCM-p were transduced with a lentiviral vector containing a CBS cDNA (NCM-C) or vector (NCM-v). H2S production was visualized with a fluorescent probe, 7-azido-4-methylcoumarine (AzMC). Cell proliferation rates where determined with a Coulter Counter. Cell migration and invasion assay were performed in Boyden chambers with NIH3T3 conditioned media (CM) as a chemoattractant. Anchorage-independent growth was assessed by soft-agar assay. Cellular bioenergetics was assessed using Oxygraph-O2K respirometer chamber. Tumorigenesis and metastasis were assessed injecting cells subcutaneously and orthotopically into nude mice, respectively. Aminooxyacetic acid (AOAA) was used to inhibit CBS activity. Statistical significance (p≤0.05) set using ANOVA or non-parametric Student t-test. Results: CBS overepression and H2S production was verified by Western blot and AzMC fluorescence in the NCM-C cells. CBS overexpression demonstrated significantly increased proliferation rate (p<0.03 NCM-C vs. NCM-v or NCM-p) over 4 days in culture, invasion through matrigel (p<0.01), migration (p<0.001) toward NIH3T3 CM and increased colony formation in soft agar (p<0.01). CBS inhibition with AOAA (1mM) decreased invasion and migration (p<0.01) in the NCM-C cells compared to NCM-v. In high-resolution respirometry, respiratory rate was significantly higher in the NCM-C compared to -p and -v. (141.8 ± 1.5 vs. 33.7 ± 0.8 vs. 72.2 ± 1.77 pmol·s−1·mg−1, -p v -C p < 0.0001, -v v -C p < 0.0005, -p v -v p < 0.001). Exome-wide sequence analyses of the NCM-p identified inactivating mutations in the APC and TP53 tumor suppressor genes, and an activating mutation in KRAS. Although the NCM-C cells produced significantly larger tumors in mice compared with NCM-v or NCM-p (p<0.001 2-w ANOVA), none of the cell lines caused liver metastasis in vivo. Conclusion: A non-tumorigenic cell line with some known mutations can progress to a tumorigenic, but not metastatic, phenotype with CBS overexpression and H2S production. Our data supports the importance of CBS/H2S axis in the adenoma to carcinoma sequence.

#4277

Development and validation of a novel IDH1-mutant astrocyte cell line as a model for high-grade gliomas.

Nathaniel D. Robinson,1 Karin R. Purshouse,2 Nathan R. Fons,2 Gregory A. Breuer,2 Stefan Pusch,3 Andreas von Deimling,3 Ranjini K. Sundaram,4 Ranjit S. Bindra4. 1 _Yale School of Medicine, Yale University, New Haven, CT;_ 2 _Department of Experimental Pathology, Yale University, New Haven, CT;_ 3 _Department of Neuropathology, Institute of Pathology, Ruprecht-Karls-Universitaet Heidelberg, Heidelberg, Germany, Germany;_ 4 _Department of Therapeutic Radiology, Yale University, New Haven, CT_.

High-grade gliomas (HGGs) are devastating malignancies of the central nervous system, and few treatment options are available for these tumors. In the most malignant form of the disease, glioblastoma multiforme (GBM), over 90% of patients will succumb to their tumor within 5 years after standard of care treatment, consisting of surgery, radiation therapy, and temozolomide chemotherapy. It is now clear that gliomas are molecularly heterogeneous entities, with mutations in tumor suppressors and oncogenes defining many distinct sub-types with important therapy implications. However, almost all HGGs are treated with a limited array of initial therapies, regardless of these molecular differences. Isocitrate dehydrogenase-1 (IDH1), a gene recently found to be mutated in many gliomas, is involved in the conversion of isocitrate to 2-oxoglutarate in cells. The IDH1 R132H mutant enzyme converts 2-oxoglutarate to the oncometabolite (R)-2-hydroxyglutarate (D2HG), which leads to profound metabolic alterations in tumor cells. In addition, recent studies indicate that mutations in IDH1 may also induce altered DSB repair, differential sensitivities to chemo-radiotherapy, and substantial changes in chromatin modifications. Here, we present the creation of a novel astrocyte cell line harboring an engineered heterozygous IDH1 R132H mutation at the endogenous gene locus using CRISPR/Cas9 gene editing. We confirmed expression of the engineered mutation at the protein level, and we have characterized this cell line in a comprehensive panel of functional assays. In particular, we demonstrated that our mutant cell clones secrete high levels of D2HG, and we confirmed that the levels of this oncometabolite can be suppressed with small molecule inhibitors of mutant IDH1. We also characterized the DNA damage response network in IDH1-mutant cells using high-content DNA damage foci assays recently developed by our group, and also in clonogenic survival assays. To our knowledge, this is the first report of an astrocyte cell line harboring an engineered, heterozygous R132H mutation at the endogenous locus. This novel cell line represents a new model system for studying gliomas and has tremendous applications for further cell characterization, mechanistic studies, and drug screening.

#4278

Unique cell-based transposon mutagenesis screen for studying EMT process of tumor in the liver.

takahiro kodama,1 Justin Newberg,1 Michiko Kodama,1 Robert Rangel,1 Milton J. Finegold,2 Nancy Jenkins,1 Neal G. Copeland1. 1 _Houston Methodist Research Institute, Houston, TX;_ 2 _Texas children's hospital, Houston, TX_.

Background and Aims: Epithelial mesenchymal transition (EMT) is a complex differentiation process that epithelial cells lose its own characteristic and acquire mesenchymal property. EMT is reported to contribute vascular invasion, metastasis and poorer prognosis of hepatocellular carcinoma (HCC). However, how and what types of genes are involved in EMT process of HCC is still not fully understood. Here we develop cell-based transposon mutagenesis system to screen genes involved in EMT process in the liver, Methods: Mouse hepatoblasts were isolated from embryos of hepatocyte-specific T2Onc2 transposon transgenic and SB11 transposase knock-in mice (Alb-Cre/T2Onc2/SBase) or their control littermates. They were cultured in vitro and waited for spontaneous immortalization. Multiple immortalized hepatoblast cell lines (IHBCs) were then injected into the flank or the liver of nude mice and monitored for tumor growth. Results: IHBCs from Alb-Cre/T2Onc2/SBase mice showed hepatoblast characteristics with active T2Onc2 transposition and maintained differentiation capacity into mature hepatocytes in vitro. Five of 7 IHBCs developed tumors at flank and 3 of 5 inside the liver, while injection of 8 IHBCs from the control littermates never developed any tumors, indicating that transposon conferred tumorigenic potential on IHBCs. Histological analysis revealed tumors were positive for transposase in addition to Sox9 and EpCAM, indicating that they were originated from hepatoblasts with albumin expression. However, they were spindle-shaped mesenchymal tumors with positive staining for Vimentin, suggesting that transposon induced EMT of hepatoblasts during malignant transformation. qPCR array also showed the strong activation of EMT-related genes in these tumors. We further confirmed these phenotypes by additional injections of IHBCs and collected 52 xenografted tumors from 5 IHBCs. Sequence of transposon insertion sites in these tumors identified 803 candidate cancer genes (CCGs). Trunk driver analysis identified oncogenic activation of Met/Gab1 signaling as the driving force of tumor development. Pathway analysis revealed that CCGs were enriched in known signaling pathways involved in EMT process including Wnt, TGF-beta, MAPK and Notch signaling as well as adherence junction, focal adhesion and regulation of actin cytoskeleton, indicating that transposon targeted these pathways, inducing EMT in these tumors. Furthermore, analysis of RNA-seq data from TCGA human HCC samples showed that CCGs were enriched in genes that show significant correlation between their mRNA levels and those of EMT-related genes, suggesting their involvement in EMT process. Conclusion: Our unique transposon tumor model mimics EMT process of the tumor in the liver. Therefore, transposon-identified CCGs may be a good resource to discover genes involved in EMT process in the liver.

#4279

Human HER2-overexpressing mouse breast cancer cell lines derived from MMTV.f.HuHER2 mice: characterization and use in a model of metastatic breast cancer.

Sunju Park, George Sgouros. _Johns Hopkins University, School of Medicine, Baltimore, MD_.

Preclinical evaluation of therapeutic agents against metastatic breast cancer require cell lines and animal models that recapitulate clinical metastatic breast cancer as much as possible. We have previously used cell lines derived from the neu-N transgenic model to investigate anti-neu targeting of metastatic breast cancer using an alpha-emitter labeled antibody reactive with the rat variant of HER2/neu expressed by the neu-N model. To investigate alpha-emitter targeting of metastatic breast cancer using clinically relevant, commercially available anti-HER2/neu antibodies, we have developed cell lines derived from HuHER2 mice (MMTV.f.HuHER2 obtained from Genetech). HuHER2 mice develop breast cancer spontaneously and with a higher frequency than normal mice.

We extracted primary mammary gland tumors, purified the epithelial breast cancer cells, and established 7 different HuHER2 cell lines. We also established 2 different cell lines from spontaneous lung metastases (HuHER2-L1 and -L2). We evaluated HuHER2 protein expression in the cell lines by western blot analysis. Cell surface receptor expression was evaluated by immunofluorescence (IF) staining. We performed qRT-PCR to assess phenotypic measures of aggressiveness and metastatic propensity. The following were evaluated: ER, PR, Twist1, Vimentin and E-Cadherin. Sensitivity to trastuzumab antibody, in vitro, was also assessed by evaluating changes in cellular metabolism via the MTT assay.

HuHER2 protein was overexpressed in all of the isolated cell lines. One of the cell lines (denoted HuHER2-6) had approximately 1.5-8 times more protein expression compared to the other 6 cell lines developed. The two cell lines derived from spontaneous lung metastases showed approximately 1.5-fold greater HER2 protein expression than HuHER2-6. HuHER2-6 cell surface HER2 receptor staining by IF was similar to that of BT-474, a high HuHER2 expressing cell line. The 2 lung cell lines also showed comparable cell-surface staining. ER mRNA levels assessed by qRT-PCR were 20% and 0.05% of the level measured in normal mammary gland for the HuHER2-6 and HuHer2-L2 lines, respectively. The PR levels were also substantially lower (<0.01%) relative to normal mammary gland (MG) tissue. The HuHER2-6 line expressed 1.4 times more HuHER2/neu mRNA than BT-474. The mRNA for E-Cadherin in HuHER2-L1 and -L2 was 20% of MG tissue. The mRNA for TWIST1 and Vimentin were similarly elevated 3-fold and 1.5-fold, respectively, relative to MG. At trastuzumab concentrations ranging from 10 to 500 ug/ml, cell metabolism was decreased to 50%.

These lines show all the hallmarks of highly aggressive, metastatic breast cancer and are being used to establish a left cardiac ventricle injection model of widespread HER2/neu positive metastatic breast cancer to evaluate combination therapy with alpha-particle emitter labeled HER2/neu reactive antibodies.

#4280

**Paralleled workflow for expansion of limited patient material using CRC for** in vitro **/PDX/biological/genetic studies of a low-grade mucoepidermoid carcinoma.**

Ahmad M. Alamri,1 Xuefeng Liu,2 Weisheng Wang,2 Xiaogang Zhong,2 Bhaskar Kallakury,2 Bruce Davidson,3 Priscilla A. Furth2. 1 _Georgetown University, King Khalid University, Washington, DC;_ 2 _Georgetown University, Washington, DC;_ 3 _MedStare Georgetown University, Washington, DC_.

Acquiring live cancer cells preserving original characteristics is a critical step for precision medicine. Here we illustrate a paralleled workflow using a low-grade sublingual salivary mucoepidermoid carcinoma (MEC) as an example. Methods: Working under IRB approval, tissue from two distinct regions of a MEC were obtained and primary epithelial cell cultures established using CRC (F medium with Rho Kinase (ROCK) inhibitor Y-27632 with irradiated Swiss 3T3-J2 mouse fibroblast feeder cells). Epithelial cells were separated from feeders to test under non-CRC conditions including 2D (MammoCult™), colony formation in Matrigel (F medium, Y-27632), patient derived xenograft (PDX) formation (10*6 cells/50:50 matrigel/PBS/mammary fatpad), and RNA and DNA extracted for RNAseq, exome sequencing and PCR/RT-PCR for a CRTC1-MAML2 fusion gene (reported in MEC). Sequences were analyzed for relative transcript abundance, differentially expressed genes (DEGs), and single nucleotide polymorphisms (SNPs) (TopHat/CuffLinks, SAMTOOLS, variant database: dbSNP & 1000G, UCSC hg19) between the two MEC regions and fusion genes (FusionCatcher). Potentially pathophysiological SNPs were identified (OMIM and SNPedia). Gene ontology was performed on transcriptome data (PARTEK Genomic Suite, Pathway Studio). Immunohistochemistry (IHC) was used to compare protein expression of selected DEG and downstream effectors in original cancer and surrounding normal tissue, cell pellets, Matrigel colonies and PDX (confirmed as human using MAB1273). Results: CRC cultures established from both sites grew similarly under all in vitro and in vivo conditions. PDX showed well-differentiated histology comparable to original cancer. One of the 11 DEG was amphiregulin but protein expression was equivalent in the two cell cultures. Amphiregulin, EGFR, p-EGFR, p-AKT, and mTOR were expressed in vitro (CRC and non-CRC), in vivo (PDX) and in original cancer, where they were up-regulated as compared to surrounding normal tissue. Five potentially pathophysiologically significant SNPs were detected (BRCA2 (rs144848), TP53 (rs1042522), AURKA (rs2273535), DBYD (rs1801265), and (SOD2 rs4880)) but no CRCTC1-MAML2 fusion gene. FusionCatcher detected a possible novel fusion product currently under investigation. Conclusion: The paralleled approach provided sufficient material to identify amphiregulin as an upregulated growth factor pathway in a CRTC1-MAML2 fusion gene negative MEC. Previously, amphiregulin upregulation was pathophysiologically linked to the CRTC1-MAML2 fusion gene. Results from the two MEC sites were biologically concordant and genetically similar. In summary, CRC can be used to expand limited patient derived material for more extensive studies under non-CRC conditions. Support: R56DE023259 (PAF) 

## EPIDEMIOLOGY:

### Biomarkers of Endogenous and Exogenous Exposures

#4281

Mitochondrial DNA copy number, urinary isoprostanes and colorectal cancer risk.

Bharat Thyagarajan,1 Renwei Wang,2 Woon-Puay Koh,3 Helene Barcelo,1 Jennifer Adams-Haduch,2 Weihua Guan,1 Roberd M. Bostick,4 Jian-Min Yuan,2 Myron D. Gross1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _University of Pittsburgh Cancer Institute, Pittsburgh, PA;_ 3 _Duke-NUS Medical School, Singapore, Singapore;_ 4 _Emory University, Atlanta, GA_.

Previous epidemiological studies on mitochondrial DNA (mtDNA) copy number and risk of colorectal cancer have yielded mixed results. One study reported a positive association, another reported an inverse association, and a third, a U-shaped association of mtDNA copy number with colorectal cancer risk. Since oxidative stress is one of the proposed underlying mechanisms that may link mtDNA copy number variation to colorectal cancer risk, we conducted a matched case-control study of incident colorectal cancer nested within the Singapore Chinese Health Study. Pre-diagnostic blood and urine samples were obtained from 448 colorectal cancer cases and 869 age- and sex-matched controls. The mtDNA copy number in peripheral white cells was measured using real time PCR and urinary F2-isoprostanes were quantified using a liquid chromatography mass spectrometry based method. Conditional logistic regression was used to estimate associations of mtDNA copy number and urinary F2-isoprostanes with incident colorectal cancer. The odds ratios (OR) and 95% confidence intervals (95% CI) for those in the highest relative to those in the lowest quartiles of relative mtDNA copy numbers and urinary F2-isoprostanes were 1.02 (95% CI 0.72 - 1.43; p for trend = 0.56) and 1.11 (95% CI 0.78-1.59; p for trend = 0.35) respectively. Urinary F2-isoprostanes levels were not associated with the mtDNA copy number among the controls (r=0.03; p=0.33). The results of this prospective study suggest that white blood cell mtDNA copy number or urinary levels of F2-isoprostanes in biospecimens collected several years prior to the diagnosis of colorectal cancer may not be associated with risk of developing colorectal cancer.

#4282

Branched-chain amino acids in the urinary metabolic profile of colorectal cancer patients and associations with muscle mass, BMI, and physical activity in the ColoCare Study.

Jennifer Ose,1 David B. Liesenfeld,2 Juergen Boehm,1 Nina Habermann,3 Robert W. Owen,2 Biljana Gigic,2 Stefanie Skender,2 Johanna Nattenmueller,4 Hans-Ulrich Kauczor,4 Cornelia Ulrich1. 1 _Huntsman Cancer Institute, Salt Lake City, UT;_ 2 _National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany;_ 3 _European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany;_ 4 _Diagnostic and Interventional Radiology, University Hospital, Heidelberg, Germany_.

Background

Branched-chain amino acids (BCAA; e.g., valine, leucine and isoleucine) have been previously linked with survival in colorectal cancer patients. It is unclear whether BCAAs are prognostic biomarkers or a surrogate endpoint for factors related to muscle mass. Thus, we investigated the correlation between BCAAs and muscle mass, physical activity and body mass index (BMI) at multiple time points during the course of disease in a prospective cohort of patients.

Methods

Patients with newly diagnosed colorectal cancer [n=197; (stage I - IV)] from the ColoCare study in Heidelberg, Germany with baseline and follow-up data, and measurements of urinary BCAAs were eligible. Skeletal muscle mass of the dorsal muscle was quantified based on abdominal computed tomography (CT)-scans (vertebral body L3/4 and L4/5). BMI (kg/m2) and physical activity [metabolic equivalent (MET) hours/week] were reported by questionnaire. In addition, minutes of moderate and vigorous physical activity/week were measured by accelerometry. We prospectively monitored the urinary metabolome of these patients at multiple time points after surgery [baseline (n=197), 6-month follow-up (n=107) and 12 month follow-up (n=75)]. BCAAs (valine, leucine and isoleucine) were identified using gas chromatography mass spectrometry (GC/MS). Metabolites were normalized based on the sum intensity of all annotated metabolites for statistical analyses. Pearson and partial correlation coefficients with parameters of energy balance/muscle mass were computed for each time point, adjusted for gender and age at diagnosis.

Results

As expected, urinary valine, leucine and isoleucine were highly correlated, independent of time point (r>0.95, P<0.01). There were no statistically significant correlations between BCAA and parameters of energy balance or muscle mass at baseline, 6 months, or 12 months (ranges of correlations for muscle mass: r=-0.17 to -0.04; for self-reported physical activity, MET hours/week r=-0.006 to -0.04, accelerometry-based moderate physical activity: r=-0.21 to-0.10; BMI: r=-0.05 to 0.09).

Conclusion

The present data suggests that urinary levels of BCAA in colorectal cancer patients do not reflect parameters of energy balance and muscle mass. Thus, they warrant further investigation as potential prognostic biomarkers.

#4283

Relationship between mammographic breast density and measures of terminal duct lobular unit involution among women diagnosed with estrogen receptor positive breast cancer.

Maeve Mullooly,1 Sarah J. Nyante,2 Ruth M. Pfeiffer,1 Renata Cora,3 Jonine D. Figueroa,4 Robert N. Hoover,1 Andrew G. Glass,5 Erin J. Aiello Bowles,6 Louise A. Brinton,1 Amy Berrington de Gonzalez,1 Sherman E. Mark,1 Gretchen L. Gierach1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Department of Radiology, University of North Carolina School of Medicine, Chapel Hill, NC;_ 3 _Independent contractor, CT(ASCP), MB(ASCP), Stamford, CT;_ 4 _Usher Institute of Population Health Sciences and Informatics, University of Edinburgh, Edinburgh, United Kingdom;_ 5 _Kaiser Permanente Northwest Center for Health Research, Portland, OR;_ 6 _Group Health Research Institute, Seattle, WA_.

Introduction: High mammographic breast density (MD) and reduced levels of terminal duct lobular unit (TDLU) involution (the histologic source of most breast cancers) have been associated with increased risk of developing breast cancer. Data relating MD and TDLU involution to breast cancer characteristics and outcomes are sparse. Therefore, we assessed these relationships among women with invasive ER-positive breast cancer.

Methods: The analysis focused on women with ER-positive breast cancers who were diagnosed at Kaiser Permanente Northwest (1990-2008) and followed through the end of 2010. Cases comprised of those who died of breast cancer (n=54) and controls those that did not die of breast cancer (n=180) over similar follow-up. Three reproducible measures that are inversely related to TDLU involution were evaluated in digitized hematoxilin and eosin stained sections in benign breast tissues surrounding the tumors: TDLU counts per unit area, TDLU span and median number of acini per TDLU. Percentage MD was estimated from digitized mammograms using computer-assisted thresholding software (Cumulus). Univariate associations between TDLU measurements and patient characteristics, tumor size, and disease stage at diagnosis, were calculated using Mann Whitney Wilcoxon rank test. TDLU measurements were related to baseline MD using analysis of covariance models and adjusted for age, body mass index (BMI), tumor size, stage, year of diagnosis and smoking.

Results: TDLUs were observed in 95% of cases and 89% of controls. All TDLU measurements declined with age (p<0.001 for each TDLU measurement). Among cases, TDLU measures were not significantly associated with tumor characteristics. Controls with regional spread had greater TDLU span (p=0.05) and median acini counts per TDLU (p=0.03) than those with localized disease; these TDLU metrics also showed a borderline significant association with larger tumor size (>2cm) (p=0.07 and p=0.06, respectfully). All TDLU measures were associated with MD among controls (TDLU count: p=0.04; TDLU span: p=0.06; median acini count per TDLU: p=0.01), whereas among cases only, TDLU span showed a significant association MD (p=0.003).

Conclusion: Preliminarily, our data suggest that among women with non-fatal ER-positive breast cancers, TDLU involution was associated with localized tumor stage, size and MD, whereas these relationships were less evident among women who died of their disease. Ongoing analyses will determine whether measures of MD and TDLU involution are independent predictors of breast cancer outcomes in this patient population.

#4284

Exploratory plasma proteomic analysis in a randomized cross-over trial of aspirin among healthy individuals.

Xiaoliang Wang,1 Yuzheng Zhang,2 Ali Shojaie,1 Paul D. Lampe,2 Lisa Levy,2 Ulrike Peters,2 John D. Potter,2 Emily White,1 Johanna W. Lampe2. 1 _University of Washington, Seattle, WA;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Background: Long-term use of aspirin is associated with lower colorectal cancer (CRC) incidence; however, the mechanism of the chemopreventive effect of aspirin is not fully understood. Animal studies suggest that COX-2, NFκB signaling and Wnt/β-catenin pathways may play a role, but no clinical trials have systematically evaluated the biological response to aspirin in healthy humans. Methods: We assessed the difference in plasma protein levels after 60 days of regular dose aspirin (325 mg/day) compared to placebo in a randomized, double-blinded, placebo-controlled, cross-over trial of 44 healthy non-smoking men and women, aged 21-45 years. Plasma proteomics was analyzed on an antibody microarray with ~3,000 full-length antibodies, printed in triplicate. Moderated paired t-test was performed on individual antibodies, and gene set analyses were performed for KEGG and GO pathways. Results: Among the 3,387 antibodies, significant differences in plasma protein levels were observed for 267 antibodies (p<0.05), the most significant protein being a transcription-factor regulator belonging to a steroid receptor family and found to be differentially expressed in colon cancer cells. Other significant proteins are involved in multiple oncogenic pathways related to colon tumorigenesis. In the pathway analysis, 4 KEGG (among 138) and 69 GO (among 1,089) pathways were found to be significant (p<0.05), including natural killer (NK) cell-mediated cytotoxicity, butanoate metabolism and Wnt signaling pathways. Pathways that modulate cellular protein binding to steroid receptors were also significantly different between aspirin treatment and placebo (p<0.05). None of the results remained statistically significant after correction for multiple testing. Conclusion: Several proteins and pathways, which have previously been reported as playing a role in colorectal carcinogenesis in vitro were found to be differentially expressed after aspirin treatment vs. placebo in healthy human subjects. This study suggests several chemopreventive mechanisms of aspirin; however, larger, confirmatory studies are needed.

#4285

Intra-individual variation in circulating miRNA expression levels in human plasma samples.

Jie Wu,1 Hui Cai,1 Yong-Bing Xiang,2 Charles Matthews,3 Fei Ye,1 Wei Zheng,1 Qiuyin Cai,1 Xiao-Ou Shu1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Shanghai Cancer Institute, Shanghai, China;_ 3 _National Cancer Institute, Bethesda, MD_.

Circulating miRNAs as possible non-invasive biomarkers for disease risk assessment and cancer early diagnosis has attracted increasing interest. Little information, however, is available regarding the intra-individual variation of circulating miRNA levels. We measured miRNA expression in repeated plasma samples that were collected 6 to 12 months apart from 53 healthy individuals participating in a sub-study nested in the Shanghai Women's and Shanghai Men's Health Studies. Total RNA, including miRNA, was isolated from plasma using Qiagen's miRNeasy Serum/Plasma Kit. Three synthetic spike-in RNA oligos (osa-miR414, cel-miR248, and ath-miR159a) were added to control for variances in the starting material and the efficiency of RNA extraction. NanoString's Human v2 miRNA Expression Assay, which includes CodeSets of probes specific for 800 common human miRNAs, was used for the miRNA assays. The derived miRNA counts were first corrected by subtracting the background counts and then were normalized by using the average count from three spike-in oligos and

the top fifty miRNAs that gave most significant hybridization signals. The intra-individual variation was evaluated by an intra-class correlation coefficient (ICC). A total of 185 miRNAs were detected in at least 10% of samples. The detectable rate varied for each miRNA, with 69 and 44 miRNAs being detected in 50% and 75% of samples, respectively. The median ICC was 0.46 for all of the 185 detectable miRNAs. Among them, 41% (75 miRNAs) had an ICC ≥0.5, and 42 miRNAs had an ICC ≥0.6. The ICC is higher for miRNAs with higher expression levels and higher detection rates, when compared to their counterparts with lower expression or detection rates. In summary, we found that 185 miRNAs were expressed in at least 10% of plasma samples, and 69 miRNAs in 50% of samples from healthy Chinese men and women, and that 75 of these miRNAs had an ICC ≥0.5. These results suggest that common circulating miRNAs can serve as reliable biomarkers for epidemiological and clinical research.

#4286

Gene expression profiles in adipose tissue of cancer-bearing breasts differ from that of tumor-free breasts.

Dominique Scherer,1 Isabelle Miran,2 Chafika Mazouni,2 Benjamin Sarfati,2 Marine Bernard,2 Julien Adam,2 Emilie Louvet,2 Francoise Drusch,2 Philippe Vielh,2 Khalid Alhazmi,2 Cornelia M. Ulrich,3 Suzette Delaloge,2 Jean Feunteun2. 1 _National Center for Tumor Diseases, Heidelberg, Germany;_ 2 _Institut Gustave Roussy, Paris, France;_ 3 _Huntsman Cancer Institute, Salt Lake City, UT_.

Introduction

Adipose tissue - long believed to be no more than an energy storage organ - is metabolically active and consists of a variety of cell types. Adipocytes, fibroblasts and macrophages reside in adipose tissue and may have tumor-promoting properties through the release of cytokines and other growth stimulating molecules. Further, adipose tissue has the capacity of storing pollutants and carcinogens. Breast tumors are embedded in adipose tissue and the direct interface between tumor and adipose tissue in the breast defines a micro-environment potentially fostering tumor initiation and/or progression. The aim of this study was to investigate the transcriptome of adipose tissue from cancer-bearing breasts, localized either close to or distant from the tumor, in addition to comparing it to adipose tissue from tumor-free breasts.

Methods

We collected adipose tissue from n= 33 tumor-bearing breasts (a) close to (< 2cm) and (b) distant from (>5cm) the tumor and from n=5 tumor-free breasts to investigate gene expression profiles in these three series of tissues. Gene expression was measured from RNA isolated from fresh frozen adipose tissue using Illumina HT12 bead arrays. Quantile normalized expression values were analyzed between (a) and (b) as well as between (a)+(b) and (c) using t-test. Data was filtered by standard variation of 0.95. P-values ≤0.001 were considered significant.

Results

We did not observe any significant differences in gene expression when comparing adipose tissue close to or distant from the tumor. By contrast, expression profiles in adipose tissue of tumor-free breasts clearly differed from that of cancer-bearing breasts. We observed 81 genes significantly differentially expressed between the two groups. Among the overexpressed genes were the previously identified genes MARCO and VSIG4, which were 1.5-fold upregulated in adipose tissue from diseased patients (both p-value=0.001). Both genes are involved in processes of immunity and inflammation, promoting immune-tolerance in macrophages and T-cells. Other differentially expressed genes also relate to these pathways including CD163, CCL13 and C3. This striking role of inflammatory and immune-modulatory processes was further supported by pathway analyses.

Conclusions

Breast adipose tissues of cancer-bearing breasts show distinct gene expression profiles from that of tumor-free breasts, whereas tumor-distant and tumor-close adipose tissues are similar. However the selected distance of ≤2cm from the tumor may by insufficient to capture the tumor micro-environment. Nevertheless, this rather provocative result raises issues related to the status of breast adipose tissue that may define individually determined cancer fields. Differentially expressed genes are, to a large proportion, involved in immunity-related and inflammatory processes, emphasizing that adipose tissue is an important contributor to one of the hallmarks of cancer.

#4287

Serum metabolomic profiling of prostate cancer risk in the PLCO Cancer Screening Trial.

Alison M. Mondul,1 Stella Koutros,2 Stephanie J. Weinstein,2 Sonja I. Berndt,2 Demetrius Albanes2. 1 _University of Michigan School of Public Health, Ann Arbor, MI;_ 2 _U.S. National Cancer Institute, Bethesda, MD_.

Background: Two recent studies conducted in one cohort (the ATBC Study) identified serum metabolites related to aggressive prostate cancer up to 20 years prior to diagnosis. Lipid and energy metabolites were significantly lower in cases than in controls, including glycerophospholipids, fatty acids, inositol-1-phosphate, alpha-ketoglutarate, and citrate. The present metabolomics investigation aimed to re-examine those associations in another cohort.

Methods: A nested case-control study of prostate cancer in the Prostate, Lung, Colorectal and Ovarian Cancer Screening (PLCO) Trial cohort was conducted based on 380 cases (298 stage 3-4 or Gleason sum 8+; "aggressive disease"), and 380 controls matched on age, race, study center, and date of baseline blood collection (samples were collected between 7am and 4pm). Median time from serum collection to prostate cancer diagnosis was 10.0 years (range, 4.4-17.0), and the majority of cases included in this study were diagnosed after the end of the PLCO screenings. Sera were analyzed on a high resolution accurate mass (HRAM) platform of ultrahigh performance liquid chromatography/mass spectroscopy (LC-MS) and gas chromatography/mass spectroscopy (GC-MS) (Metabolon, Inc.) that identified 722 metabolites. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) of risk associated with a one standard deviation (1-SD) increment in metabolite concentration.

Results: Of the 27 metabolites associated with prostate cancer at p<0.05, 12 were amino acids or dipeptides, and amino acids as a metabolite class were somewhat overrepresented among the top signals for stage 3-4 but not overall prostate cancer (p=0.003 and p=0.08, respectively). Pyroglutamine, gamma-glutamylphenylalanine, phenylpyruvate, N-acetylcitrulline, and stearoylcarnitine yielded the strongest metabolite prostate cancer risk signals (1-SD increment ORs 0.78, 0.76, 0.73, 0.80, and 1.25, respectively; 0.001 < p < 0.006). Findings were similar for aggressive disease. We did not replicate our earlier findings of inverse associations with aggressive prostate cancer for the energy metabolites alpha-ketoglutarate or citrate in the present study (ORs 1.17 and 1.16 (p=0.10 and 0.03), respectively), or the positive associations for trimethylamine-N-oxide (TMAO) or thyroxin (ORs 0.88 and 0.84 (p=0.13 and 0.04), respectively).

Conclusions: In this prospective case-control analysis of serum collected throughout the day without regard to fasting status, we did not find strong prostate cancer associations for lipid or energy metabolites. Rather, amino acids appeared overrepresented in relation to prostate cancer risk. Whether the baseline and subsequent prostate cancer screening with PSA and digital rectal exams or the non-fasting status of the parent cohort contributed to the findings should be examined in other similar studies.

#4288

Association between tumor necrosis factor alpha -308 G/A polymorphism and risk of bladder cancer in Asian population: a meta-analysis.

Wei-Tang Kao,1 Min-Che Tung,1 Kuan-Chun Hsueh,2 Yuan-Hung Wang,3 Chia-Chang Wu4. 1 _Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei City, Taiwan;_ 2 _Department of Surgery, Tungs' Taichung MetroHarbor Hospital, Taichung, Taiwan;_ 3 _Graduate Institute of Clinical Medicine, Taipei Medical University, New Taipei City, Taiwan;_ 4 _Department of Urology, School of Medicine, College of Medicine, Taipei Medical University, Taipei City, Taiwan_.

Previous studies have investigated the association between TNF-α-308 G/A polymorphism and bladder cancer risk in various populations. However, these findings remain inconclusive. Therefore, we performed a meta-analysis to explore the relationship between TNF-α-308 G/A polymorphism and bladder cancer risk. A literature search in PubMed was performed to select eligible studies regarding the association between TNF-α-308 G/A polymorphism and bladder cancer risk. The strength of risk under fixed- and random- effects models were estimated using the odds ratios (ORs) with 95% confidence intervals (CIs). We identified 7 case-control studies including 1153 cases and 1587 controls were included in the present meta-analysis. Compared with subjects carrying the G/G genotype of TNF-α-308 G/A polymorphism, those with the A/A and G/A genotypes had non-significant bladder cancer risks under the fixed effects model (OR=1.037) and the random effects model (OR=1.012). In the recessive model, subjects with the A/A genotype had an increased bladder cancer risk (OR=1.875) compared with those carrying the G/A and G/G genotypes of TNF-α-308 G/A polymorphism. The major finding of this meta-analysis suggest that TNF-α-308 G/A polymorphism is correlated with the risk of bladder cancer in Asian population under the recessive model. We should further investigate the joint effects of environmental risk factors and TNF-α-308 G/A polymorphism on bladder cancer risk.

#4289

C-reactive protein and risk of lung cancer: A pooled analysis of 20 prospective cohorts.

David C. Muller,1 Allison M. Hodge,2 Gianluca Severi,3 Qiuyin Cai,4 Klaus Meyer,5 Kjell Grankvist,6 Arnulf Langhammer,7 Paul Brennan,8 Mattias Johansson,8 Lung Cancer Cohort Consortium (LC3). 1 _Imperial College London, London, United Kingdom;_ 2 _Cancer Council Victoria, Melbourne, Australia;_ 3 _Human Genetics Foundation, Turin, Italy;_ 4 _Vanderbilt University Medical Center, Nashville, TN;_ 5 _Bevital, Bergen, Norway;_ 6 _Umea University, Umea, Sweden;_ 7 _Norwegian University of Science and Technology, Trondheim, Norway;_ 8 _International Agency for Research on Cancer, Lyon, France_.

Background: Inflammation may have an important role in the etiology of lung cancer, and several studies have reported that C-reactive protein (CRP) is associated with risk of lung cancer. To clarify this association, we conducted a pooled analysis of CRP and lung cancer risk using data from 20 prospective cohort studies.

Methods: Within the NCI Cohort Consortium, we designed a prospective nested case-control study. Controls were selected from appropriate risk sets and were matched to cases on smoking status, sex, and age at blood draw. This analysis included 5,299 case-control pairs nested within 20 cohorts. Rate ratios (RR) and their 95% confidence intervals associated with a doubling in concentration of CRP were estimated using conditional logistic regression models.

Results: Overall, higher circulating CRP was associated with an increased risk of lung cancer (RR 1.05, 95% CI [1.03, 1.08]). This association varied strongly by smoking status (p-heterogeneity < 0.01), being similar for current (1.09 [1.05, 1.13]) and former smokers (1.09 [1.04, 1.14]), but not for never smokers (0.95 [0.91, 1.00]). The association was strongest for cancers diagnosed less than 2 years after blood draw (1.21 [1.13, 1.29], p-heterogeneity < 0.01), and weakened as time between blood draw and diagnosis increased. The association was similar across all histological subtypes, with the exception of adenocarcinoma for which we observed no association.

Conclusions: CRP is associated with risk of lung cancer. The fact that the association is restricted to ever-smokers, and is most prominent for cancers diagnosed in the first 2 years of follow-up, strongly suggests that CRP is not a causal risk factor, but rather a distal marker of disease.

#4290

Serum biomarkers of polyomavirus infection and risk of lung cancer in never smokers.

Jyoti Malhotra,1 Tim Waterboer,2 Michael Pawlita,2 Angelika Michel,2 Qiuyin Cai,3 Wei Zheng,3 Qing Lan,4 Nathaniel Rothman,4 Hilde Langseth,5 Tom K. Grimsrud,5 Jian-Min Yuan,6 Woon-Puay Koh,7 Alan A. Arslan,8 Anne Zeleniuch-Jacquotte,8 Paolo Boffetta9. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _German Cancer Research Center (DKFZ), Germany;_ 3 _Vanderbilt University School of Medicine, TN;_ 4 _National Cancer Institute, MD;_ 5 _Cancer Registry of Norway, Institute of Population-based Cancer Research, Norway;_ 6 _University of Pittsburgh Graduate School of Public Health, PA;_ 7 _Duke-NUS Graduate Medical School Singapore, Singapore;_ 8 _New York University School of Medicine, NY;_ 9 _Icahn School of Medicine at Mount Sinai, NY_.

Background: Lung cancer in never smokers is a significant contributor of cancer mortality. As the incidence of lung cancer in never smokers is increasing, there is a need to investigate risk factors for lung cancer in this population. There is data to support that polyomaviruses are potentially carcinogenic in the human lung. We explored the role of polyomaviruses in lung cancer development in never smokers using a multiplex assay to detect serum antibodies to viral capsid proteins.

Methods: We conducted a nested case-control study of never-smoking cases of lung cancer identified from four established prospective cohorts- NYU Women's Health Study (NYU-WHS), Janus Serum Bank, Shanghai Women's Health Study (SWHS) and Singapore Chinese Health Study (SCHS). Controls were matched to cases on gender, never-smoking status, age and calendar period of entry. Serological analysis was performed using a 100 µL pre-diagnostic serum sample at the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) in Heidelberg, Germany using fluorescent bead-based multiplex serology and included antibodies to viral capsid protein-1 and T-antigens of 9 human polyomaviruses: JC virus, BK virus, KI virus, WU virus, Trichodysplasia Spinulosa-associated Polyoma Virus, Merkel Cell Polyoma Virus, Human Polyoma Virus 6, Human Polyoma Virus 7 and Human Polyoma Virus 10.

Results: The final analysis included 511 cases and 508 controls. Mean age of participants was 56.1±11.0 years. SWHS and SCHS included only Asian participants. NYU-WHS and SWHS had only female participants. Nearly 85% of the participants in our pooled analysis were females and 74% were Asians. Seropositivity for each polyomavirus showed significant heterogeneity by study but overall there were no statistical significant differences between cases and controls for any of the polyomaviruses. 72.0% of the cases and 71.5% of the controls were seropositive for JC virus antibody. Seropositivity for BK virus was higher at 89.0% in cases and 89.8% in controls. We did not find any difference in seropositivity between cases and controls in our stratified analysis based on gender, histology or time interval from sample collection to cancer diagnosis. We also performed sensitivity analyses and found the seroprevalence data to be very robust to alterations in the MFI cutpoints.

Conclusions: Our study is the largest epidemiological study in never smokers to investigate the role of polyomaviruses in lung cancer development. We did not find a significant difference in serological measurements of antibodies against each of the polyomaviruses between the cases and controls. Future research to evaluate the relationship between polyomaviruses and lung carcinogenesis should focus on evaluating viral replication in tumor in combination with serological markers of infection especially as antibody reactivities can vary considerably across different populations and geographical areas as demonstrated by our study.

#4291

Vitamin B6 biomarkers and colorectal cancer: Modifications by time.

Björn Gylling,1 Robin Myte,1 Jörn Schneede,1 Göran Hallmans,1 Jenny Häggström,1 Ingegerd Johansson,1 Arve Ulvik,2 Øivind Midttun,2 Per Magne Ueland,2 Bethany van Guelpen,1 Richard Palmqvist1. 1 _Umeå University, UMEÅ, Sweden;_ 2 _University of Bergen, Bergen, Norway_.

Background: Pyridoxal 5'-phospate (PLP) is the most commonly used marker of circulating vitamin B6 status. In prospective case-control studies, individuals with higher plasma concentrations of PLP had a 30-50% decreased risk of colorectal cancer (CRC). This may partly be explained by decreased PLP due to inflammatory activity in persons at an increased risk of CRC. We aimed to investigate different markers of vitamin B6 in relation to CRC risk in a population with a very long follow-up time between screening and diagnosis.

Methods: This was a prospective case-control study of 613 incident CRC cases and 1190 matched controls nested within the population-based Northern Sweden Health and Disease Study. PLP, pyridoxal (PL), pyridoxic acid (PA), 3-hydoxykynurenine (HK) and xanthurenic acid (XA) were measured in plasma by LC-MS/MS. Besides PLP, we investigated the recently introduced metabolite ratios PAr (PA/(PLP+PL)) and HK:XA as markers of vitamin B6 status in relation to CRC risk.

Results: PAr, a marker of vitamin B6 catabolism during inflammation, was linearly associated with CRC risk, with a 50% higher risk in the highest vs. lowest quartile. In the group with a follow up time between screening and diagnosis over 10.5 years no association was observed, while in the groups with a follow up time between 5.8-10.5 years and below 5.8 years risk was approximately twofold in most quartiles vs the lowest quartile. A 50% risk decrease was observed in the in the third vs the lowest quartile of plasma PLP and a similar risk increase was observed in the highest vs the lowest quartile of the functional vitamin B6 marker HK:XA.

Conclusions: PAr may influence later stages of tumor development, but possibly not early tumorigenesis. Poor functional vitamin B6 status and inflammatory activity might influence both the risk of CRC and plasma concentrations of PLP. Our results underscore the need to use several biomarkers for vitamin B6 status together with the already well established plasma PLP. This may be generalizable not only to CRC but also other diseases with an inflammatory aspect.

#4292

Mitochodrial D-loop polymorphisms are associated with colorectal adenoma risk.

Bharat Thyagarajan,1 Weihua Guan,1 Veronika Fedirko,2 Helene Barcelo,1 Myron D. Gross,1 Michael Goodman,2 Roberd M. Bostick2. 1 _University of Minnesota, Minneapolis, MN;_ 2 _Emory University, Atlanta, GA_.

Somatic mutational events in the mitochondria have been detected in adenomatous polyps. However, the contribution of germline variation in the mitochondrial DNA towards risk of colorectal adenomas, well recognized precursor lesions to colorectal cancer, has not been evaluated previously. Hence, we evaluated associations of mitochondrial polymorphisms in the D-loop and non-D loop regions with incident colorectal adenoma in three pooled colonoscopy-based case-control studies (n = 327 colorectal adenoma cases and 420 controls) that used identical methods for case ascertainment and risk factor determination. We sequenced a 1,124 bp fragment to comprehensively identify all genetic variation in the mitochondrial D-loop region, and used the Sequenom platform to genotype 64 previously described tagSNPs in the non-D loop region. We used multivariate unconditional logistic regression to analyze the association between the mitochondrial polymorphisms and colorectal adenoma risk after adjustment for potential confounders. We identified 320 germline mutations in the D-loop region; 30 (9%) had a minor allele frequency (MAF) ≥ 5%. Most of the mutations clustered in the hypervariable regions HV1 (n = 124; 39%) and HV2 (n = 111; 35%) of the D-loop region. Among the nine common polymorphisms (MAF ≥ 5%) in the HV1 region, four polymorphisms (mt16069, mt16278, mt16294, and mt16296) were statistically significantly directly associated with colorectal adenoma risk (odds ratios [OR]: 1.76 - 2.66; p-values: 0.001 - 0.04). In addition, the polyC tract in the HV1 region was inversely associated with colorectal adenoma risk (OR: 0.90; p=0.03). None of the other polymorphisms in the mitochondrial D-loop region tract was associated with colorectal adenoma risk. After correction for multiple comparisons, none of the mitochondrial tagSNPs in the non-D loop region was associated with colorectal adenoma risk. These findings suggest that polymorphisms in the HV1 region of the mitochondrial D-loop may be associated with colorectal adenoma risk and support further investigation in future studies.

#4293

Combination of hepatocyte growth factor, genistein and daidzein concentrations as a biomarker for gastric cancer risk: A nested case-control study.

Jieun Jang,1 Yunji Hwang,1 Choonghyun Ahn,1 Kwang-Pil Ko,2 Keun-Young Yoo,1 Sue K. Park1. 1 _Seoul National University Graduate School, Seoul, Republic of Korea;_ 2 _Gachon University College of Medicine, Incheon, Republic of Korea_.

Backgrounds: Hepatocyte growth factor (HGF) induces ERK pathway which is associated with carcinogenesis by promoting cell proliferation. Inhibition of ERK signal transduction pathways by genistein, one of the isoflavones, has been reported by several in vitro studies. Therefore, gastric cancer risk by HGF and isoflavone concentrations was evaluated in this study.

Methods: Nested case-control study with 93 gastric cancer cases and 1:1 matched controls was conducted within the Korean Multi-center Cancer Cohort (KMCC). HGF and ISF (genistein, daidzein, equol) concentrations in plasma were measured with enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, respectively. Odds ratios (ORs) and 95% confidence intervals (95% CIs) on gastric cancer risk according to HGF and ISF level was calculated using conditional logistic regression models.

Results: Compared to the group with low level of HGF (<315pg/mL) and high level of genistein (≥212.5nmol/L), the group having high level of HGF and lower level of genistein was associated with increased gastric cancer risk (OR=5.08, 95% CI=1.39-18.57). Elevated gastric cancer risk in the subjects having high HGF level and low daidzein level than subjects with low HGF and high daidzein levels was also shown (OR=3.67, 95% CI=1.18-11.45). There was significant interaction between HGF and genistein levels on gastric cancer was found (p for interaction<0.01). Continual increase in gastric cancer risk according to the counts of risky status by HGF, genistein, and daidzein levels was found (p for trend=0.03).

Conclusion: Our study suggests that combination of HGF, genistein and daidzein concentrations has potential as a biomarker for gastric cancer risk.

#4294

One-carbon metabolism and colorectal cancer risk according to molecular subtypes: a Bayesian network learning approach.

Robin Myte,1 Björn Gylling,2 Jenny Häggström,3 Jörn Schneede,4 Per Magne Ueland,5 Göran Hallmans,6 Ingegerd Johansson,7 Richard Palmqvist,2 Bethany Van Guelpen1. 1 _Department of Radiation Sciences, Oncology, Umeå University, Umeå, Sweden;_ 2 _Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden;_ 3 _Department of Statistics, Umeå School of Business and Economics, Umeå University, Umeå, Sweden;_ 4 _Department of Clinical Pharmacology, Pharmacology and Clinical Neurosciences, Umeå University, Umeå, Sweden;_ 5 _Department of Clinical Science, University of Bergen and Laboratory of Clinical Biochemistry, Haukeland University Hospital, Bergen, Norway;_ 6 _Department of Biobank Research, Public Health and Clinical Medicine, Umeå University, Umeå, Sweden;_ 7 _Department of Odontology, Cariology, Umeå University, Umeå, Sweden_.

Background: Colorectal cancer (CRC) is a heterogeneous disease caused by a number of distinct pathways defined by genetic and epigenetic events. These molecular subtypes differ in prognosis and treatment options, and recent studies also imply differences in etiology. One-carbon metabolism has been extensively studied in relation to incident CRC risk because of its involvement in nucleotide synthesis and methylation reactions, yet only a few studies have evaluated if associations differ over CRC subtypes. In this study, we investigated a targeted panel of circulating metabolites and a set of single nucleotide polymorphisms involved in one-carbon metabolism in relation to the risk of overall CRC and CRC subtypes approximated by mutations in the BRAF and KRAS oncogenes, microsatellite instability (MSI), or CpG island methylator phenotype (CIMP).

Methods: This was a prospective population-based nested case control study of 613 CRC cases and 1190 matched controls. The data included plasma concentrations of 14 one-carbon metabolites, 17 related single nucleotide polymorphisms, and other lifestyle-related information for all subjects, and clinical and molecular information for the tumors. In order to account for interactions and dependencies between variables, we applied multivariate Bayesian network learning in combination with more traditional univariate multinomial logistic regression to investigate independent relations to subtype specific CRC risk.

Results: Preliminary findings show that vitamin B6 (Pyridoxal 5-phosphate, PLP) and vitamin B2 (riboflavin) had the strongest independent relations to overall CRC risk in the multivariate analyses. Folate, glycine, vitamin B6, and the RFC1 80G>A polymorphism displayed signs of independent relations to CRC subtypes. In univariate multinomial logistic regression models, plasma folate expressed the strongest etiologic heterogeneity, and was more clearly related to the risk of CIMP low/high tumors, tumors with MSI, or tumors without KRAS mutation, respectively.

Conclusions: This study investigated a large set of prediagnostic circulating metabolites and single nucleotide polymorphisms related to one-carbon metabolism using multivariate statistical learning. We found that some components showed signs of subtype specific relations, of which the association for folate was clearest. Our results suggest that the role of one-carbon metabolism in colorectal carcinogenesis may differ between specific molecular pathways.

#4295

Androgen receptor expression in normal breast TDLUs and subsequent breast cancer risk.

Kevin Kensler,1 Andrew Beck,2 Francisco Beca,2 Laura Collins,2 Stuart Schnitt,2 Aditi Hazra,3 Susan Hankinson,4 Myles Brown,5 Rulla Tamimi3. 1 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _Brigham and Women's Hospital, Boston, MA;_ 4 _University of Massachusetts School of Public Health and Health Sciences, Amherst, MA;_ 5 _Dana-Farber Cancer Institute, Boston, MA_.

Background

Sex steroid hormone signaling is critical in the development and progression of breast cancers, though the role of androgens remains unspecified. Large epidemiologic studies have found a consistent association between circulating androgens and increased breast cancer risk, though it is unknown whether circulating androgens reflect the androgenic milieu in the breast. An interaction between androgen receptor (AR) and estrogen receptor (ER) signaling in the breast has been postulated, wherein AR signaling antagonizes ER signaling in estrogen-rich environments, and AR signaling induces proliferative effects in estrogen-deplete environments.

Methods

We evaluated the association between AR expression and subsequent breast cancer risk in a nested case-control study of women with benign breast disease (BBD) within the Nurses' Health Studies. Cases were women with BBD that subsequently developed breast cancer (median 9 years later) while controls had BBD but did not develop breast cancer. Tissue microarrays were constructed containing normal terminal ductal lobular unit (TDLU) tissue from the BBD biopsy. AR expression was assessed by immunohistochemistry and the percent of positive-staining nuclei was digitally quantified for 61 breast cancer cases and 184 controls. Logistic regression models adjusting for the year of BBD biopsy, age at cancer diagnosis, years since BBD biopsy, and BBD lesion type were used to calculate odds ratios and 95% confidence intervals for the association between the tertile of AR expression and breast cancer risk. We further evaluated the impact of AR and ER co-expression, each dichotomized at the median, in a sub-analysis of 31 cases and 82 controls. Finally, we assessed AR expression as a predictor of subsequent ER tumor status using polytomous logistic regression.

Results

Overall, women in the highest tertile of AR expression experienced non-significant 1.32-fold increased odds of breast cancer (95% CI: 0.64-1.73, p-trend=0.559) compared to the lowest tertile. A significant interaction was detected between AR and ER co-expression in normal breast TDLUs and subsequent breast cancer risk (p-interaction=0.003). Among women with low ER expression, increased AR expression was associated with 2.52-fold increased odds (95% CI: 0.68-9.34) of developing breast cancer. In contrast, among women with high ER expression, high AR expression was associated with a 91% decrease in the odds (OR=0.09, 95% CI: 0.01-0.62) of breast cancer. AR expression was not predictive of subsequent ER tumor status.

Conclusions

There was little evidence for an overall association between AR expression in normal breast tissue and breast cancer risk, though we observed a significant interaction between AR and ER expression. Our findings support the hypothesis that AR interacts with ER to promote cell proliferation in estrogen-deprived environments and inhibit growth in estrogen-rich environments.

#4296

Serum estrogen and estrogen metabolites and smoking among postmenopausal women in the Women's Health Initiative Observational Study.

Hannah P. Yang,1 Garnet Anderson,2 Sally Behan,1 Louise A. Brinton,1 Chu Chen,2 Roni Falk,1 Hannah Oh,1 Ruth Pfeiffer,1 Hilary Tindle,3 Nicolas Wentzensen,1 Britton Trabert1. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Vanderbilt University School of Medicine, Nashville, TN_.

BACKGROUND: A consistent inverse relation between smoking and endometrial cancer risk has been observed, with a meta-analysis demonstrating that smokers are at a 29% (95% confidence interval: 0.65-0.78) lower risk than non-smokers among postmenopausal women. Although smoking may have different effects on other hormone-related cancers, it has been proposed that smoking shifts estrogen metabolism to favor hydroxylation of estrone and estradiol along the 2 as compared with 4 or 16 pathways, thereby presumably reducing estrogenic activity. However, comprehensive studies examining the interrelationships between smoking and estrogen production and metabolism are limited, especially among postmenopausal women.

METHODS: Within the Women's Health Initiative Observational Study, a cohort of 93,676 postmenopausal women recruited between 1993 and 1998, 15 estrogens/estrogen metabolites (jointly referred to as EM) were measured by liquid chromatography/tandem mass spectrometry in pre-diagnostic serum among 974 women (all never/former menopausal hormone therapy users) for a nested case-control study of ovarian and endometrial cancers. Using inverse probability weighted linear regression, we calculated geometric means (GM; pmol/L) of individual EM, adjusted for age at and year of blood draw, time since menopause, race, and body mass index (BMI), by smoking status (never, former, current), intensity (1 pack/day; <5, 5-<20, 20+ pack/years), and duration (<10, 10-19, 20-29, 30+ years). Percent difference was calculated by the difference over the mean EM values across categories of smoking factors. Statistical heterogeneity across categories of smoking factors was assessed using the Wald chi-square test.

RESULTS: Overall, we observed slightly lower, although non-significant EM levels among current compared to never smokers (GM for current vs. never smokers were 331 vs. 352 for estrone, 56.3 vs. 58.8 for estradiol; p-value>0.83), with an indication that this association was limited to women with BMI 25+ kg/m2. We observed similar associations after excluding women defined as cancer cases at time of blood draw. Furthermore, current compared to never smokers had non-significant lower levels in the 2- (GM were 160 vs. 163; percent diff=-2%) as compared with 4- (16.0 vs. 15.7; 2%) or 16- (259 vs. 256; 1%) hydroxylation pathways (p-value>0.82). Among smokers, EM levels tended to increase across categories of packs/day, but we observed no consistent patterns for years of smoking and pack/years.

CONCLUSIONS: Our finding that circulating EM are highest in non-smokers is at odds with the increases in EM seen with smoking intensity among smokers. This suggests that smoking may not be altering estrogen production and metabolism as hypothesized and smoking may be inversely associated with endometrial cancer via other non-hormonal pathways.

#4297

Serum B6 vitamers (pyridoxal-5'-phosphate, pyridoxal, and 4'-pyridoxic acid) and pancreatic cancer risk: Two nested case-control studies in Asian populations.

Joyce Y. Huang,1 Lesley M. Butler,1 Øivind Midttun,2 Per M. Ueland,3 Woon-Puay Koh,4 Yu-Tang Gao,5 Jian-Min Yuan1. 1 _University of Pittsburgh, Pittsburgh, PA;_ 2 _Bevital A/S, Bergen, Norway;_ 3 _University of Bergen, Bergen, Norway;_ 4 _Duke-NUS Graduate Medical School Singapore, Singapore, Singapore;_ 5 _Shanghai Jiaotong University, Shanghai, China_.

Background: Vitamin B6 is an important enzymatic cofactor in the synthesis of one-carbon units, amino acids, and carbohydrates. Epidemiological studies provided inconsistent results on the associations between circulating pyridoxal 5'-phosphate (PLP) and pancreatic cancer risk. Method: Two nested case-control studies were conducted within the Shanghai Cohort Study (SCS) (129 cases and 258 matched controls) and the Singapore Chinese Health Study (SCHS) (58 cases and 104 matched controls). Concentrations of B6 vitamers [PLP, pyridoxal (PL), and pyridoxic acid (PA)] were measured in serum samples that were collected an average of 12.5 years in SCS and 6.8 years in SCHS before pancreatic cancer diagnosis. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using conditional logistic regression with the adjustment for potential confounders. Restricted cubic splines were used to explore potential non-linear relationships between serum B6 vitamer levels and pancreatic cancer risk. Results: The median (5th-95th percentiles) levels of serum PLP were 25.7 (10.0-91.7) nmol/L among SCS controls and 58.1 (20.8-563.0) nmol/L among SCHS controls. There was a statistically significant inverse association between serum PLP levels and pancreatic cancer risk in SCS [comparing highest (&gt35.6 nmol/L) with lowest quartile (&lt18.1 nmol/L): OR=0.44; 95%CI: 0.21, 0.91; P for trend=0.01]. A weaker, nonsignificant association was observed in SCHS [comparing highest (&gt88.2 nmol/L) with lowest quartile (&lt39.2 nmol/L): OR=0.78; 95%CI: 0.27, 2.25; P for trend=0.64]. We combined the data from the two cohorts for the purpose of estimating the serum PLP-pancreatic cancer association using the widest PLP range available. Compared with PLP < 20 nmol/L (defined as vitamin B6 deficiency), PLP ≥ 45 nmol/L was associated with 50% reduced risk of pancreatic cancer (OR=0.50; 95%CI: 0.26, 0.97). Although the linear trend test was statistically significant (P = 0.04), the spline curve modeling revealed a non-linear relationship between serum PLP and pancreatic cancer, where the strongest inverse relationship was seen among individuals with serum PLP of below approximately 30 nmol/L, while a weak association was seen for those with higher PLP levels. The inverse association between PLP and pancreatic cancer risk remained statistically significant after excluding cases whose blood samples were collected within 2 years prior to cancer diagnosis. No statistically significant association was found between serum level of PL or PA and pancreatic cancer risk. Conclusion: This study is consistent with the notion that higher circulating levels of PLP may protect against the development of pancreatic cancer. The inverse association between circulating PLP and pancreatic cancer risk may be masked in studies without a wide range of PLP levels, or enough subjects in deficient range.

#4298

Cytokines and adipokines in breastmilk of black and white women.

Jeanne Murphy,1 Mark E. Sherman,1 Ruth M. Pfeiffer,1 Hannah P. Yang,1 Ana I. Caballero,2 Eva P. Browne,2 Gretchen L. Gierach,1 Kathleen F. Arcaro2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _University of Massachusetts, Amherst, MA_.

Introduction: Giving birth may be associated with a transient increase in breast cancer risk post-delivery and with elevated risk of basal breast cancers, especially in the absence of breastfeeding, whereas parity is related to lower risk of postmenopausal ER-positive tumors. Black women develop more early-onset and basal breast cancers than White women, but factors contributing to this disparity are poorly understood. Accordingly, we compared levels of 15 proteins with hypothesized roles in breast cancer risk in breastmilk from healthy Black and White women.

Methods: We tested breastmilk donated by 130 Black and 162 White women, annotated with breast cancer risk factor questionnaire data. Following pilot testing using the MesoScale Discovery system (Rockville, MD), the following analytes were measured in breastmilk, adjusted for total protein concentration: interferon-γ, IL1-β, IL-6, IL-8, TNF-α, FLT-1, TIE-2, PlGF, VEGFC, VEGFD, adiponectin, leptin, and FAS-L. M30-apoptosis and Bradford protein were measured with Molecular Devices VersaMax reader (Sunnyvale, CA). Univariate associations of race with women's characteristics were computed using chi square tests and t-tests. Multivariable logistic regression models with a random effect to account for plate effects were used to assess the associations (odds ratios (ORs) and 95% confidence intervals (CIs)) of analyte levels with: race, family history of breast cancer, smoking, baby's age in days, body mass index at time of sample donation, age, menarche, age at first birth, return of menses, parity, and over-the-counter pain medication.

Results: Of the 15 analytes measured, all but VEGFC and TIE2 were detectable and reliably measured. Black women compared to White women had higher BMI (p = 0.05) and higher parity (p = 0.02), and were younger age at menarche (p = 0.005) and younger age at first birth (p = 0.01). White women had higher levels of smoking (p = 0.01) and more frequently had a first degree relative with breast cancer (p = 0.03). Compared with White women, Black women had significantly higher levels of IL1-β (OR 1.77, 95% CI 1.13 - 2.77, p = 0.01) adjusting for the baby's age in days. Black women had significantly higher levels of leptin (OR 2.01, 95% CI 1.28 - 3.15, p = 0.002) and leptin/adiponectin ratio (OR 2.36, 95% CI 1.49 - 3.72, p = 0.0004), even after adjusting for body mass index.

Discussion: Preliminary data demonstrate differences in levels of putative markers of breast cancer risk between White and Black women, including IL1-β, leptin and the leptin/adiponectin ratio. These data suggest that further analyses of biomarkers in breastmilk may be useful for understanding breast cancer risk and to identify possible factors that may be associated with racial disparities in early onset and basal breast cancers.

#4299

Short telomeres are associated with increased risk for arsenic-related skin lesions in Bangladesh.

Chenan Zhang, Muhammad Kibriya, Farzana Jasmine, Shantanu Roy, Habibul Ahsan, Brandon L. Pierce. _University of Chicago, Chicago, IL_.

Chronic exposure to arsenic, a human carcinogen, has been shown in epidemiologic studies to increase the risk of arsenical skin lesions (keratosis and melanosis) as well as cancer (including non-melanoma skin cancer). Recent studies suggest that the mechanism of arsenic's toxicity may be related to telomere dysfunction, including altered telomere length (TL), and differential expression of genes involved in telomere maintenance. However, prior studies of arsenic exposure, TL, and skin lesion tend to be small and cross-sectional in nature. In this study, we investigated the association between arsenic exposure and TL (measured by qPCR as T/S ratio), as well as TL and subsequent risk of skin lesion in individuals who are exposed to naturally contaminated drinking water with a wide range of arsenic levels in the Health Effects of Arsenic Longitudinal Study (HEALS) in Araihazar, Bangladesh (2000-2009). In all laboratory analyses, we attempted to randomize samples across plate positions, and in statistical analyses, we controlled for any residual confounding by plate or plate position. All TL data generated showed a strong inverse association with age. In a random sample of the baseline cohort (n=649), we observed a positive association between water arsenic levels and TL (Ptrend=0.035), with beta estimates of 0.001, 0.023, and 0.025 for water arsenic levels of 10.1-50ug/L, 50.1-150ug/L and 150+ug/L, respectively, compared to <10ug/L. However the association was not replicated in an independent random sample (n=1181, Ptrend=0.176) with beta estimates of 0.007, -0.004, and -0.017. In a nested case-control study comparing skin lesion incident cases diagnosed across a nine year period with sex- and age-matched controls (448 cases and 462 controls), we observed higher incident skin lesion risk with shorter TL (Ptrend=4.6E-5), with odds ratios of 3.05, 1.30, and 1.21 for the first (shortest), second, and third TL quartiles compared to the longest. Our findings show that arsenic exposure is not strongly associated with TL, indicating that TL is likely not the main mode of action for arsenic's carcinogenic toxicity. However, TL is inversely associated with skin lesion risk, suggesting that short TL may be a biomarker of susceptibility for arsenical skin lesions independent of arsenic exposure.

#4300

Novel metabolism pathway in pancreatic cancer: findings from targeted metabolomics study.

Li Jiao,1 Cristian Coarfa,1 Kimal Rajapakshe,1 Suman Maity,1 Liang Chen,1 Feng Jin,1 Vasanta Putluri,1 Lesley Tinker,2 Haleh Sangi-Haghpeykar,1 Hashem B. El-Serag,1 Nagireddy Putluri1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Fed hutchinson Cancer Research Institute, WA_.

Background: Early diagnosis of pancreatic cancer holds the promise for improving the prognosis of this disease. We aimed to identify individual or combination of metabolites in pre-diagnostic blood that can be used as early diagnostic markers to differentiate pancreatic cancer from normal controls.

Methods: Using targeted approach, we conducted a two-phase case-control metabolomics study in the Women's Health Initiative Study using pre-diagnostic fasting blood. Each cancer-free control was individually matched to each pancreatic cancer case by age (± 1 year), ethnicity, history of treated type 2 diabetes, body mass index (± 5 kg/m2), study arm, current use of hormone treatment, blood draw time in year (± 1), blood draw time in hour (± 3). All samples from cases were collected less than one year before the diagnosis of pancreatic cancer. We used triple quadrupole mass spectrometer LS/GC-MS to quantitate 480 metabolites in sera of 30 cases and 30 controls. The selected candidate metabolites were validated in sera of additional 18 cases and 18 controls. Each metabolite was normalized against the standards. We used the paired t-test to compare the concentrations of each metabolite among all cases and controls. Unadjusted P value less than 0.25 was used for selecting candidate metabolites for validation and false discovery rate (FDR) adjusted Q-value < 0.25 was considered statistical significant in the validation phase. Log fold change (FC) was used to indicate the magnitude of difference between cases and controls.

Results: Blood samples were collected 241 days (SD: 94 days) before pancreatic cancer diagnosis on average. A total of 181 metabolites were detected in the study samples with 28 metabolites found to be significantly differentially expressed in cases versus controls with unadjusted P value < 0.25. Further validation in the pooled 48 cases and 48 controls found the metabolites 1-methyltryptophan was significantly higher in controls compared to cases with pancreatic cancer (FC: -0.23; FDR Q-value = 0.01). The levels of acadesine (FC: 0.29), aspartic acid (FC: 0.44), and nicotinamide (0.34) were higher among cases than controls and the level of citrulline (FC: -0.22), sn-glycerol 3-phosphate (FC: -0.25), and ketoglutarate (FC: -0.20) were significantly higher among controls than cases (FDR Q-value < 0.25).

Conclusion: Metabolites associated with perturbed amino acid metabolism were detected by metabolomics approach in pre-diagnostic blood of postmenopausal women who developed pancreatic cancer. In particular, depletion of tryptophan along kynurenine pathway has been associated with immunosuppression in tumor microenvironment in pancreatic cancer. Further study should determine the use of the metabolites of amino acid metabolism in early diagnosis of pancreatic cancer, in addition to lipid and choline metabolites that were identified by previous studies.

#4301

Serum vitamin B12 and development of non-cardia gastric cancer: a prospective study.

Eugenia Miranti,1 Rachael Stolzenberg-Solomon,1 Stephanie Weinstein,1 Jacob Selhub,2 Satu Männistö,3 Philip R. Taylor,1 Neal D. Freedman,1 Demetrius Albanes,1 Christian Abnet,1 Gwen Murphy1. 1 _National Cancer Institute, Rockville, MD;_ 2 _Tufts University, Boston, MA;_ 3 _National Institute for Health and Welfare , Helsinki, Finland_.

Background: The pathogenesis of non-cardia gastric adenocarcinoma (NCGA) begins with Helicobacter pylori (Hp) infection, which induces atrophic gastritis, a pre-neoplastic state characterized by gland loss and achlorhydria. In parallel, vitamin B12 absorption requires intact gastric mucosa which can produce acid and intrinsic factor. Several previous studies have suggested that Hp infection, the primary risk factor for NCGA, impairs vitamin B12 absorption. Other studies have shown that previous diagnosis of pernicious anemia, the most common form of vitamin B12 deficiency and autoimmune atrophic gastritis, predicts later NCGA. We hypothesized that decreased serum vitamin B12 would predict subsequent incidence of NCGA in a dose-dependent fashion.

Methods: To examine the relationship between serum vitamin B12 and risk of NCGA, we conducted a nested case-control study within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of male Finnish smokers aged 50-69 at baseline (1985-1988). Vitamin B12 concentrations were measured in serum samples that were collected at study enrollment. Subjects were followed for 17 years for cancer development (mean time to NCGA diagnosis = 7.5 years). 177 NCGA cases were matched 1:1 on age and date of serum collection with cancer-free controls. We calculated odds ratios (OR) and 95% confidence intervals (CI) by conditional logistic regression, controlling for Hp infection, smoking, fruit and vegetable intake, and other potential confounders.

Results: Baseline mean pre-diagnostic serum vitamin B12 concentrations were 16% lower in subjects who subsequently developed NCGA (Cases: 435 pg/mL, SE=129; Controls: 516 pg/mL, SE=207; p = 0.0001; serum vitamin B12 levels >200 pg/mL are clinically normal.) Lower serum vitamin B12 at baseline was associated with subsequent incidence of NCGA during follow-up (OR = 7.24; 95% CI=2.12 to 24.73 for lowest quartile compared to highest, p-trend=0.002). This association remained after restricting the analysis to subjects who developed cancer more than 10 years after baseline serum vitamin B12 measurements.

Conclusion: Lower serum vitamin B12 concentrations were associated with an increased risk of NCGA even among subjects who were diagnosed more than 10 years after their enrollment in the study. Our findings suggest a possible role for serum vitamin B12 as a biomarker for the atrophic gastritis that precedes NCGA.

#4302

Associations of leg length with increased colorectal cancer incidence in the atherosclerosis risk in communities (ARIC) study.

Guillaume C. Onyeaghala,1 Pamela L. Lutsey,1 Ellen W. Demerath,1 Kristin E. Anderson,1 Aaron R. Folsom,1 Corinne E. Joshu,2 Elizabeth A. Platz,2 Anna E. Prizment1. 1 _University of Minnesota, Minneapolis, MN;_ 2 _John Hopkins Bloomberg School of Public Health, Baltimore, MD_.

Purpose

There is growing evidence that taller people have an increased risk of colorectal cancer, which is thought to be explained by an increased production of growth hormones during puberty and/or a larger pool of at-risk colonic cells. Long bone growth during puberty is, in part, promoted by insulin-like growth factor-1, a higher circulating level of which is a risk factor for colorectal cancer. Thus, we hypothesized that long-bone growth indicated by leg length would be the component of height that is more strongly associated with colorectal cancer. To address this hypothesis, we evaluated the association of leg length, sitting height and total height with colorectal cancer risk in the Atherosclerosis Risk in Communities (ARIC) prospective cohort.

Methods

The ARIC participants included 14,605 men and women who were cancer-free at baseline (1987-1989), and followed until 2006. Information about demographic, lifestyle, and anthropometric characteristics was obtained at baseline. Leg length was estimated as standing height minus sitting height. Incident colorectal cancers (n=344) were ascertained by linkage to cancer registries and supplemented by hospital records.

Statistical analysis

Cox proportional hazards regression was used to estimate hazard ratios (HR) of colorectal cancer and 95% confidence intervals (CI) across quartiles of height, leg length, and sitting height. The final models were adjusted for age, sex, race, study center, education level, waist-to-hip ratio, female hormone replacement therapy use, and smoking status.

Results

Participants with longer legs tended to be male, African-American, and had higher education. Leg length was correlated with total (Pearson r=0.83), and sitting height (Pearson r=0.52). Participants in the highest quartile of leg length were at a 42% greater risk of colorectal cancer, relative to the lowest quartile (95% CI, 1.01-2.00, p-trend=0.04). After stratification by gender, leg length remained associated with colorectal cancer risk in men (HR=1.83, 95% CI, 1.11-3.05) but not in women (HR=1.14, 95% CI, 0.70-1.83) for the highest versus the lowest quartile of leg length (p-interaction=0.16). The HRs for the highest quartiles of total height and sitting height compared to the lowest quartiles were also increased, but the associations were weaker (HR=1.25, 95% CI, 0.90-1.74, p-trend=0.10 and HR=1.30, 95% CI, 0.95-1.79, p-trend=0.18 respectively).

Conclusion

In this prospective study, longer leg length was more strongly associated with an increased risk of colorectal cancer than overall height or sitting height. While we cannot rule out that the association between taller height and colorectal cancer risk may be explained by a larger pool of colonic cells in taller people, these findings support the prior observation that this association is driven by biological mechanisms correlated to long bone growth during puberty, such as insulin-like growth factor-1.

#4303

Arsenic exposure and mosaic loss of the Y chromosome among Bangladeshi men.

Brandon L. Pierce,1 Muhammad Kibriya,1 Farzana Jasmine,1 Joseph Graziano,2 Habibul Ahsan1. 1 _University of Chicago, Chicago, IL;_ 2 _Columbia University, NY_.

Background: Exposure to drinking water contaminated with arsenic, a known carcinogen, is a serious global health issue, increasing risk for several types of cancer, as well as overall mortality. Somatic mosaic loss of the Y chromosome (LoY) is the most frequent known somatic chromosomal aberration in men, and LoY in peripheral blood cells has been associated with exposure to cigarette smoke, as well as cancer risk and overall mortality. In this work, we examine the correlates of LoY among 2,588 male members of an arsenic-exposed Bangladeshi cohort (mean age: 42 years).

Methods: DNA was extracted from peripheral blood at baseline interview, and genome-wide SNP data was generated using Illumina arrays. We assessed LoY using the median Log R Ratio (mLRR) for 2,882 SNPs located in the male-specific region of chromosome Y. Within each of five different genotyping batches, we classified individuals have having LoY if their mLRR-Y value fell below the 2nd percentile of an mLRR distribution simulated assuming no LoY. Arsenic exposure was measured as the arsenic concentration in participants' primary drinking wells (reported at baseline). All association estimates were adjusted for age, BMI, and smoking status.

Results: We detected LoY for a total of 305 men across 5 different genotyping batches, with rates of LoY ranging from 7% to 18% across batches. Age showed a consistent positive association with LoY across all batches, consistent with prior studies. Among the two genotyping batches representing random samples of the cohort (n=391 and n=344), quartiles of increasing arsenic concentration in drinking water were associated with increased LoY (P-trend=0.05 in both batches). In a comparison of 297 individuals with incident arsenical skin lesions (the most common sign of arsenic toxicity) and 673 control individuals (randomly selected from the cohort), LoY was significantly more common in cases than controls (P=0.001); however, this association was attenuated after adjusting for arsenic exposure (P=0.22). In an analysis of all 2,588 individuals (including non-randomly-sampled case groups), current smoking showed a suggestive association with increased LoY (P=0.06), consistent with a prior studies of older populations.

Conclusion: In this population-based study of arsenic-exposed Bangladeshi men of a wide range of ages, increasing exposure to arsenic, a known carcinogen, was associated with increased risk of LoY, and LoY was more common among men with subsequent arsenic toxicity (i.e., arsenical skin lesions). This study provides evidence that a carcinogenic environmental exposure causes the most common form of genetic mutation in men. Additional research is needed to determine if LoY is causally involved in arsenic-induced carcinogenesis or if LoY is simply a marker of exposure.

#4304

Prevalence and triggers of drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) in a cancer patient cohort.

Nancy K. Gillis,1 Gillian C. Bell,2 Howard L. McLeod,3 Amy J. Brandt3. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Mission Health, Asheville, NC;_ 3 _Moffitt Cancer Center and Research Institute, Tampa, FL_.

SJS and TEN are extremely rare (approximately 2-7 cases per million per year) hypersensitivity reactions most commonly attributed to medications. The life-threatening nature of SJS/TEN necessitates early diagnosis and immediate identification and withdrawal of causative agents. Some of the most commonly associated culprits include sulfonamide antibiotics, aromatic anticonvulsants (phenytoin, carbamazepine, lamotrigine), -oxicam NSAIDs (e.g., meloxicam), allopurinol, and nevirapine. Individual case reports of SJS/TEN have been associated with anti-cancer agents, but a comprehensive assessment has not been performed. A large-scale retrospective analysis of the prevalence of SJS/TEN in cancer patients treated at Moffitt Cancer Center (MCC) between Jan 1, 2002 - Dec 31, 2014 was conducted. A total of 104,917 cancer patients were screened for SJS/TEN-related ICD-9 codes in the MCC health research informatics database. SJS/TEN diagnoses were identified in 121 patients. Manual chart review of physician, dermatology, and pathology notes confirmed SJS/TEN in 47 patients (in-patient + historical) and possible SJS/TEN in an additional 17 patients, corresponding to an overall prevalence of 0.06%. Confirmed in-patient cases of SJS/TEN were more common in hematologic malignancies compared to solid tumors (n = 12 vs. n=7, respectively). Notably, 5 of the 19 (26.3%) confirmed cases were observed in patients with acute myeloid leukemia. Physician-reported culprits for SJS/TEN diagnoses included antibiotics, immunomodulators, anticonvulsants, and anti-cancer agents. The observed prevalence of SJS/TEN was higher in cancer patients than previous reports from the general USA population. There were 19 confirmed in-patient diagnoses of SJS/TEN and an additional 45 historical and possible cases of SJS/TEN in 104,917 cancer patients, corresponding to an overall prevalence of 0.018% to 0.06%, an approximately 90-fold increase when compared to the general population. Possible explanations for increased risk in cancer patients include increased exposure to culprit medications, cancer disease process, immunocompromised state, or synergy between risk factors. A thorough understanding of the factors that increase risk to SJS/TEN in cancer patients is critical to facilitate culprit withdrawal and maximize patient outcomes and survival.

#4305

Short-term outcomes of abiraterone in community settings.

Grace L. Lu-Yao,1 Dirk F. Moore,1 Yong Lin,1 Timothy R. Rebbeck,2 Kitaw Demissie,1 David Rotter,1 Kimberly A. McGuigan,1 Anthony V. D'Amico2. 1 _Rutgers-The Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Dana Farber Cancer Institute, Boston, MA_.

Introduction: About 32% of men with high-risk prostate cancer have comorbidities, conditions that may be under-represented in randomized clinical trials (RCTs). Because men enrolled on RCTs are often excluded if they have multiple comorbidities, these patients may be at higher risk for serious toxicities from cancer therapies that the results of RCTs may not reflect. This study was undertaken to test the hypothesis that 90-day mortality rates in men undergoing treatment with abiraterone (a novel agent for treating castration-resistant prostate cancer) in the community settings is higher than that observed in the pivotal trial.

Methods: SEER-Medicare linked data with Medicare Drug Event File during the period 2006-2012 were used to identify patients who filled prescriptions for abiraterone during the study period and these patients were followed through 12/31/2013 for overall mortality. Ninety-day mortality after abiraterone initiation among patients with Charlson score 0 was compared to that in the pivotal abiraterone trial published in the New England Journal of Medicine 2011.

Results: We identified 1,106 patients who took abiraterone during the study period. Among them, 484 (43.8%) were age 75 or older at the time of abiraterone initiation and 61 (5.5%) had comorbidity score 2 or higher. Hospitalization rates among those who had abiraterone were 0.09 per person month during the 6-month window before the initiation of abiraterone and 0.12 per person month within 3 months of drug initiation. The top five diagnoses for hospital admission after drug initiation were general symptoms (ICD-9 780), malignant neoplasm of prostate (ICD-9 185), respiratory abnormality (ICD-9 786), care involving rehabilitation (ICD-9 V57), and urinary tract infection (ICD-9 599). The 90-day all-cause mortality after drug initiation was 14.5% (95% 12.4% - 16.5%) in the entire study cohort, 13.0% (95% CI 10.4% - 15.7%) among those under age 75 and 16.3% (13.0% - 19.6%) among those 75 or older. The 90-day mortality was 15.5% (95% CI 12.2% - 18.8%), 12.8% (95% CI 6.2% - 19.3%), and 18.0% (95% CI 8.3% - 27.8%) among patients with comorbidity score 0, 1, and 2+ respectively. The 90-day mortality among patients with comorbidity 0 in the community settings is substantially higher than those found in the pivotal trial abiraterone arm (risk ratio 2.03; 95% CI 1.48 - 2.78) and control arm (risk ratio 1.43; 1.01 - 2.03).

Conclusion: This first US national study shows that 90-day mortality after abiraterone among relatively healthy patients in the community settings is twice that observed in the pivotal trial abiraterone arm. Further studies are needed to improve selection of eligible patients and post-treatment monitoring.

#4306

Free IGFBP3 and the risk of liver cancer in a nested case-control study.

Yasushi Adachi,1 Masanori Nojima,2 Mitsuru Mori,1 Yasutaka Matsunaga,1 Noriyuki Akutsu,1 Shigeru Sasaki,1 Takao Endo,3 Youichi Kurozawa,4 Kenji Wakai,5 Akiko Tamakoshi6. 1 _Sapporo Medical Univ., Sapporo, Japan;_ 2 _The University of Tokyo, Tokyo, Japan;_ 3 _Sapporo Shirakaba-dai Hosp., Sapporo, Japan;_ 4 _Tottori Univ., Yonago, Japan;_ 5 _Nagoya Univ., Nagoya, Japan;_ 6 _Hokkaido Univ., Sapporo, Japan_.

Background: Insulin-like growth factor-1 (IGF1) is a potent mitogen, whereas IGF-binding protein-3 (IGFBP3) binds and inhibits IGF1. High circulating IGF1 and low IGFBP3 are associated with increased risk of several cancers. Here we examined relations of those serum levels with the risk of hepatoma in a prospective case-control study nested in the JACC Study.

Methods: A baseline survey was conducted from 1988 to 1990 and 39,242 subjects donated blood samples. Those who had been diagnosed as hepatoma were regarded as cases for nested case-control studies. Ninety-one cases and 263 controls are eligible for the present analysis. A conditional logistic model was used to estimate odds ratios (OR) for incidence of hepatoma associated with serum IGF1 and IGFBP3 levels.

Results: Both IGF1 and molar ratio of IGF1/IGFBP3 were not correlated with the risk of hepatoma. After adjustment for hepatitis-viral infection, body mass index, smoking, and alcohol intake, higher molar difference of (IGFBP3 - IGF1) was associated with decreased risk of hepatoma (p for trend < 0.001), and peoples in the highest quintile had a lower risk (OR = 0.098; 95%CI = 0.026-0.368). In subgroup analyses, both in male and female, high molar difference of (IGFBP3 - IGF1) was associated with decreased risk of hepatoma (p for trend < 0.001). Both in the elderly and non-elderly, it was correlated inversely with risk of hepatoma (p for trend = 0.001, < 0.001, respectively).

Conclusions: Molar difference of (IGFBP3 - IGF1) might associate inversely with the incidence of hepatoma.

#4307

Influence of age on androgen deprivation therapy-associated Alzheimer's disease.

Kevin Thomas Nead,1 Greg Gaskin,2 Cariad Chester,2 Samuel Swisher-McClure,1 Joel T. Dudley,3 Nicholas J. Leeper,2 Nigam H. Shah2. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Stanford University School of Medicine, Stanford, CA;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY_.

Androgen deprivation therapy (ADT) is a mainstay of treatment for prostate cancer with approximately 500,000 males currently receiving ADT in the United States. ADT decreases androgen levels with 20-30% of patients experiencing prolonged suppression. Concerningly, low testosterone has been shown to precede Alzheimer's disease (AD) and ADT increases β-amyloid protein levels. Here, extending prior work, we examine the association of ADT with the development of AD in an age-stratified analysis among men with prostate cancer.

We analyzed electronic medical record data at Stanford University and Mt. Sinai. Individuals with prostate cancer and follow-up ≥ 180 days after diagnosis without a history of dementia, stroke or chemotherapy were eligible. Covariates, extracted from coded and textual data, were age at prostate cancer diagnosis, race, smoking status, use of anti-platelet, anti-coagulant, anti-hypertensive and statin medications, and a history of cardiovascular disease, diabetes or malignancy. We examined the association of ADT with AD stratified by age at diagnosis. Age 70 years was selected as the cut-off given existing management guidelines for cancer patients older than 70. Hazard ratios (HRs) were calculated using 1:5 propensity score matched and traditional multivariable Cox proportional hazards models to test the association of use versus non-use of ADT, and ADT duration (no-ADT, <12 months, ≥ 12 months), on risk of AD. Kaplan–Meier survival functions were compared among individuals < 70, non-ADT users ≥ 70, and ADT users ≥ 70 years in the propensity score matched cohort using Cox tests for equality. A 2-sided p-value < 0.05 was considered significant.

Baseline patient characteristics were well balanced after propensity score matching in the full and age ≥ 70 years subgroup (p>0.07). The median follow-up period was 2.7 years (interquartile range, 1.0-5.4 years). Kaplan-Meier survival functions demonstrated a lower probability of remaining AD-free for age ≥ 70 versus < 70 years (p<0.001) and between ADT and non-ADT users ≥ 70 years (p=0.034). There was a statistically significant association between ADT use and AD among those ≥ 70 years using propensity score matched (HR=1.84; 95% confidence interval [CI], 1.07-3.17; p=0.027) and traditional multivariable Cox regression analysis (HR=2.04; 95% CI, 1.23-3.40; p = 0.006). Among those ≥ 70 years a longer duration of ADT was associated with a greater risk of AD (HR=1.41; 95% CI 1.01-1.96, p=0.043).

Among men aged 70 years or older the utilization and duration of ADT is associated with an increased rate of AD. This further supports the association between ADT and cognitive dysfunction and suggests that older men may be most susceptible. Limitations of this study include inadequate power to conduct subgroup analysis in the < 70 years cohort. Prospective studies are required to determine the mechanism of this association in order to develop preventative strategies.

#4308

Multiplicative interaction between HIV infection status and FIB-4 in prediction of hepatocellular carcinoma risk.

Lesley S. Park,1 Janet P. Tate,2 Vincent Lo Re,3 Adeel A. Butt,4 Cynthia Gibert,5 Matthew Bidwell Goetz,6 Sheldon T. Brown,7 Joseph Lim,8 David Rimland,9 Jennifer S. Lee,10 Amy C. Justice,2 Robert Dubrow11. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _Veterans Affairs Connecticut Healthcare System, West Haven, CT; Yale School of Medicine , New Haven, CT;_ 3 _Perelman School of Medicine, University of Pennsylvania; Philadelphia Veterans Affairs Medical Center, Philadelphia, PA;_ 4 _Hamad Healthcare Quality Institute, Hamad Medical Corporation, Doha, Qatar; VA Pittsburgh Healthcare System, Pittsburgh, PA;_ 5 _Washington DC Veterans Affairs Medical Center; George Washington University School of Medicine and Health Sciences, Washington, DC;_ 6 _Veterans Affairs Greater Los Angeles Healthcare System; David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA;_ 7 _James J. Peters Veterans Affairs Medical Center, Icahn School of Medicine at Mt. Sinai, New York, NY;_ 8 _Yale School of Medicine, New Haven, CT;_ 9 _Atlanta Veterans Affairs Medical Center; Emory University School of Medicine, Atlanta, GA;_ 10 _Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA; Stanford University School of Medicine, Stanford, CA;_ 11 _Yale School of Public Health; Yale School of Medicine, New Haven, CT_.

Purpose: FIB-4 is an established marker of liver fibrosis and cirrhosis, calculated from platelet count, alanine transaminase, aspartate transaminase, and age. We previously found baseline FIB-4 to be strongly associated with hepatocellular carcinoma (HCC) risk among HIV-infected (HIV+) patients in the US; a similar finding was reported among persons who consume alcohol or are chronic hepatitis B virus (HBV) carriers in South Korea. Longitudinal associations between FIB-4 and HCC risk have not yet been explored. We aimed to expand our prior investigation by including uninfected patients, using time-updated FIB-4, and testing for multiplicative interaction between HIV status and FIB-4 in the prediction of HCC risk. We hypothesized that the relationship between FIB-4 and HCC risk would differ by HIV status.

Methods: We tested this hypothesis in the Veterans Aging Cohort Study, an open cohort that enrolls HIV+ veterans when they begin HIV care in the Veterans Health Administration and matches two uninfected patients by age, sex, race/ethnicity, and clinical site. We used proportional hazards regression models with time-varying covariates to calculate hazard ratios (HR) and 95% confidence intervals (CI) for FIB-4, adjusted for HCC risk factors (age, sex, race, hepatitis C virus (HCV) infection, HBV infection, smoking, alcohol, BMI, and diabetes). We used the counting process to create time-updated FIB-4 intervals and examined one-, three-, and five-year lagged FIB-4. We identified incident HCC cases from the VA Central Cancer Registry and determined hepatitis C virus and hepatitis B virus status from laboratory results. We defined low (3.25) as previously established.

Results: Between 2000 and 2012, among 37,158 HIV+ subjects, 202 developed HCC. Among 78,339 uninfected subjects, 207 developed HCC. There was a significant multiplicative interaction between HIV status and one-year lagged FIB-4 (interaction p=0.0015). High FIB-4 was a stronger predictor of HCC in the uninfected than in HIV+. Among uninfected, the adjusted HR was 6.9 (95% CI: 3.4, 12.5) for intermediate FIB-4 and 40.0 (95% CI: 22.3, 71.8) for high FIB-4 compared to uninfected with low FIB-4. Among HIV+, with the same reference group (uninfected with low FIB-4), the adjusted HR was 2.1 (95% CI: 1.0, 4.4) for low FIB-4, 6.4 (95% CI: 3.5, 11.7) for intermediate FIB-4, and 23.7 (95% CI: 13.1, 42.9) for high FIB-4. There was no interaction between FIB-4 and HCV status (p=0.92). Results were qualitatively similar using a three- or five-year lag.

Conclusions: Calculated from routine, non-invasive laboratory tests, FIB-4 is a strong, independent HCC risk factor in both HIV+ and uninfected subjects after adjustment for other HCC risk factors. FiB-4 appears to be a stronger risk factor in uninfected than in HIV+.

#4309

Rare protein truncating and missense variants in ATM, CHEK2, PALB2, but not XRCC2, confer increased breast cancer risks.

Brennan Decker, Jamie Allen, Craig Luccarini, Karen A. Pooley, Paul PD Pharoah, Alison M. Dunning, Douglas F. Easton. _University of Cambridge, Cambridge, United Kingdom_.

Breast cancer (BC) is the most common malignancy in women and has a major heritable component that is driven by both common SNPs and rarer variants in susceptibility genes. However, the risks associated with most rare susceptibility alleles are not well estimated. We sequenced the coding regions of ATM, CHEK2, PALB2, and XRCC2 in 13,087 BC cases and 5,488 controls from East Anglia to characterize their role in BC risk in the largest sequencing study of these genes to date.

We performed multiplexed microfluidic PCR target enrichment of 221 amplicons, covering 99.3% of the coding bases. Amplified DNA was barcoded, pooled, and sequenced on the Illumina HiSeq2000. Reads were demultiplexed, trimmed, and aligned to the hg19 reference sequence. Variants were called with GATK UnifiedGenotyper and filtered for missing data. Retained variants were annotated with CADD and VEP. Truncating variants, missense alleles, and other functional variant groups were assessed for association with BC risk using logistic regression.

Truncating variants in ATM, CHEK2, and PALB2 increased overall BC risk, whereas XRCC2 variants did not (Table 1). The estimated relative risk was higher for PALB2 than ATM or CHEK2. While 88% of CHEK2 variant alleles were attributable to CHEK2*1100delC, other truncating alleles appeared to confer similar risks. CHEK2 truncating alleles were more strongly associated with risk of estrogen receptor (ER) positive disease than ER negative or triple negative disease. In contrast, PALB2 protein-truncating variants conferred similar relative risks for all subtypes. Analysis of rare missense variants suggested that specific subset of alleles in ATM (P=0.012), CHEK2 (P=0.009), and PALB2 (P=0.017) may contribute to risk. Localization within known functional domains were a predictor of association among these variants.

These results confirm that truncating variants in ATM, CHEK2, and PALB2 are associated with moderate BC risks, and exclude a large effect on risk from XRCC2 variants.

Truncating Variant Risk

---

|

ATM | ATM | CHEK2 | CHEK2 | PALB2 | PALB2 | XRCC2 | XRCC2

|

OR | P-Value | OR | P-Value | OR | P-Value | OR | P-Value

SEARCH Cases | 3.26 | 2.11E-05 | 3.34 | 5.18E-11 | 5.42 | 4.45E-08 | 0.94 | 0.9232

ER Positive | 3.19 | 1.04E-04 | 3.71 | 9.11E-12 | 4.32 | 2.73E-05 | 0.87 | 0.8419

ER Negative | 1.28 | 0.6743 | 1.56 | 0.2365 | 5.58 | 3.62E-04 | 0.88 | 0.9102

Triple Negative | 2.85 | 0.1488 | 0.42 | 0.3221 | 7.50 | 0.0022 | NA | NA

## PREVENTION RESEARCH:

### Diet and Cancer

#4310

Regular intake of high-fat diet exacerbates oxidative stress-mediated DNA hypermethylation and activation of Nrf2-Keap1 signaling in UVB-induced skin tumors in SKH-1 hairless mice.

Tripti Singh, Ram Prasad, Santosh K. Katiyar. _University of Alabama at Birmingham, Birmingham, AL_.

Generation of reactive oxygen species (ROS) is an essential metabolic process for maintaining normal cellular homeostasis; however elevated and sustained levels of ROS contribute in many human diseases, including cancer. Sustained and excessive cellular ROS result in genetic as well as epigenetic alterations, leading to deregulation of oncogenes and tumor suppressor genes. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a ubiquitous transcription factor, capable of eliciting the coordinated induction of cytoprotective genes in response to oxidative stress. Upon oxidative insult, Nrf2 rapidly translocate to nucleus, activates genes encoding anti-oxidative enzymes such as heme oxygenase 1 (HO-1) and superoxide dismutase (SOD). Earlier, we have shown that regular intake of high-fat diet (HF-diet) enhances tumor development in UV-exposed SKH-1 hairless mouse skin compared to control-diet (C-diet) fed mice. Administration of HF-diet along with exposure of UVB radiation (180 mJ/cm2, 3x/week for 24 weeks) resulted in sustained oxidative stress as evident by depletion in antioxidant defense capabilities. The levels of reduced glutathione (40%, p<0.001), glutathione peroxidase (49%, p<0.001) and SOD (47%, p<0.001) were noted, while the level of lipid peroxidation was significantly increased (160%, p<0.001) in skin tumors of HF-diet fed mice compared with C-diet fed mice. Epigenetic alterations in association with oxidative stress were analyzed by estimation of global DNA methylation, DNA methyltransferase (Dnmt) activity and histone modifications. We observed significant increase of Dnmt activity (3 fold, p<0.001) and global DNA methylation level (2 folds, p<0.001) along with Dnmt proteins and methylated histone in tumors of HF-diet fed mice compared with tumors of C-diet fed mice. Oxidative stress disrupts Nrf2-Keap-1 binding by DNA hypermethylation and epigenetic silencing of Keap1 gene leading to its inactivation, release and translocation of Nrf2 to nucleus. Our RT-PCR data showed elevated levels of methylated Keap-1 and lower levels of un-methylated Keap-1 gene in skin tumors from HF-diet fed mice. Disruption of Keap-Nrf2 binding resulted in higher nuclear translocation of Nrf2 and activation of its target genes NQO-1 and HO-1 as confirmed by western blot analyses. Taken together, this study shows that chronic exposure to UVB along with administration of HF-diet induces oxidative stress, causes epigenetic modifications and loss of Keap1 function leading to nuclear accumulation of Nrf2 which creates a susceptible environment for skin tumor growth.

#4311

Oligomeric proanthocyanidins inhibit Hippo-YAP pathway and prevent colorectal cancer stem cell formation.

Shusuke Toden, Ajay Goel. _Baylor University Medical Center, Dallas, TX_.

Background and Aims: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality rate in the United States, but can be prevented through lifestyle and dietary modifications. Over the last two decades, anti-tumorigenic properties of various botanicals have been studied extensively, and currently selected compounds are being exploited as potential chemopreventive agents. Proanthocyanidins are the most abundant dietary polyphenols present in many fruits and vegetables. Although anti-tumorigenic properties of proanthocyanidins have been investigated substantially, emerging evidence indicates that oligomeric form of proanthocyanidins (OPCs) has more potent anti-tumorigenic properties, but the underlying mechanisms remain unclear. A small subset of cancer cells, termed "cancer stem cells" (CSCs), are believed to be the major contributors of tumor initiation and metastasis. Despite complex molecular network associated with CSCs, several botanicals have shown promising efficacy against CSCs. We aimed to investigate the protective role of OPCs, focusing on CRC CSC formation, using cell lines and animal models. Methods: The effects of OPCs on cellular proliferation, apoptosis and cell-cycle dynamics were examined in a series of in-vitro experiments. Spheroid derived CSCs (SDCSCs) were generated from CRC cell lines to determine whether OPCs can suppress sphere formation capacity. The role of OPCs on major signaling pathways driving self-renewal capability (Hippo-YAP, Polycomb repressive complex (PRC) and intestinal stem cell markers) was systematically assessed. Finally, chemopreventive efficacy of OPCs was tested in a mouse xenograft model. Results: OPCs inhibited cellular proliferation and induced apoptosis in a dose-dependent manner. Cell cycle analysis revealed that OPC-treated CRC cells underwent G2/M arrest and subsequently showed reduced cellular proliferation. Intriguingly, OPCs suppressed SDCSC formation at a significantly low dose, suggesting that these could effectively inhibit CSC formation. Mechanistically, OPCs suppressed Hippo-YAP pathway, a major pathway driving cellular proliferation and stem cell reprogramming, through simultaneous inhibition of multiple Hippo-YAP associated proteins. In CRC cell lines with high YAP expression, OPCs preferentially inhibited YAP, while a downstream regulator TAZ was targeted in cells with low YAP expression. Furthermore, we showed that OPCs suppressed CRC CSC markers (CD44, CD133, ALDH1 and Notch1) and BMI1, EZH2 and SUZ12, key oncogenic PRC subunits. In addition, using SDCSC derived mouse xenograft model, we demonstrated that OPCs significantly inhibited tumor growth. Conclusion: Collectively, our data unravels previously unrecognized mechanisms for OPCs to target CSCs and Hippo-YAP pathway in CRC. These data highlight the therapeutic potential of OPCs as chemopreventive agents through inhibition of CSC formation.

#4312

Preneoplastic activity of dietary poly unsaturated fatty acid (PUFA) regulation of organ and tissue microenvironments in an iso-caloric pair-fed mouse model.

Saraswoti Khadge,1 Paul Black,2 Concetta DiRusso,2 Geoff Thiele,1 Lynell W. Klassen,1 J Graham Sharp,1 Timothy R. McGuire,1 Michael J. Duryee,1 Holly C. Britton,1 Alicia Dafferner,1 Jordan Beck,2 James E. Talmadge1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _University of Nebraska Lincoln, Lincoln, NE_.

Diets containing omega-3 (ω3) PUFAs have health benefits due to their anti-inflammatory activity and lower risk of chronic conditions including cardiac, autoimmune and neoplastic diseases. This contrasts with ω6 PUFA containing Western diets, which are pro-inflammatory. Balancing the dietary ω6:ω3 ratio has been suggested to have cancer preventive and potentially therapeutic activity; however, the majority of these studies have not differentiated dietary PUFA content from obesity. Herein we examined the effects of the ω6:ω3 ratio in iso-caloric diets that were pair fed. These diets had 35.5% of calories from fat with an ω6:ω3 ratio in the ω6 and ω3 based diets of 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet and fish oil substituting for 70% of olive oil. The ω3 diet contained eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid at a 3:1 ratio, while the ω6 diet contained linoleic acid as the predominant ω6 PUFA and a low level of the ω3 PUFA, linolenic acid. After establishing female BALB/c mice on these diets, weight gain and diet consumption were monitored for 12 weeks, the mice sacrificed and the dietary effects on mammary gland and hepatic histopathology, leukocyte phenotypes and organ and tissue lipidomics determined. In association with pair feeding there were no differences in diet consumed (ω-3 diet used as baseline) and weight gain between cohorts. The cohort on the ω6 diet had depressed numbers of marrow progenitor cells and increased splenic subcapsular extramedullary hematopoiesis consistent with an inflammatory response, increased hepatocyte lipidosis, hypertrophy and multinucleation and increased hepatic vascularity with thicker intima. The ω-6 dietary cohort also had mammary fat pads (MFPs) with increased adipocytes in the tubular epithelium, stromal cellularity and epithelial tissue density. These mammary gland and hepatic histopathologic changes and subclinical inflammation are consistent with pre-neoplastic lesions and were accompanied by organ specific lipodemic changes. This included significant increases in arachidonic acid (AA) in the plasma, spleen and liver, but not MFPs, of the ω-6 cohort, and a significant increase in EPA and DHA levels in the plasma, spleen, MFPs and liver of the ω-3 cohort. In addition, the liver and MFPs had a significant increase in the ω3 PUFA, docosapentaenoic acid. In summary, our studies demonstrate that the dietary ratio of ω6:ω3, independent of obesity can regulate mammary gland and hepatocyte proliferation and subclinical inflammation in association with significant, organ specific increases in AA levels contributing to microenvironmental preneoplastic hyperplasia.

#4313

Periodic fasting mimicking diet delays cancer development and progression.

Sebastian Brandhorst, Gerardo Navarrete, Min Wei, Todd E. Morgan, Valter D. Longo. _University of Southern California, Los Angeles, CA_.

Dietary intake and composition can have profound effects on aging and age-related pathologies including cancer. As such, studies of fasting (the complete withdrawal of food) and dietary restriction (DR, generally described as a 20- 40% reduction in calories) have contributed to the understanding of the relationship between nutrients, cellular aging, cancer development and cancer cell survival. DR may only be feasible in delaying tumor development but might not present an effective treatment modality due to the time required to achieve beneficial effects. Further, chronic DR may also have negative impacts on wound healing, which could be detrimental in patients undergoing surgery. Our findings in yeast indicated that starvation decreases age-dependent mutations whereas in mouse xenograft tumor models fasting alone can be as effective as chemotherapy drugs in delaying the progression of certain tumor types, in part through the reduction of extracellular glucose and IGF-1 concentration/signaling. Fasting, although results indicate efficacy as a treatment option, on the other hand may not be feasible for patients at risk for undernutrition or cachexia.

To address the need for a dietary based intervention that both delays cancer development and progression, we developed a fasting-mimicking diet (FMD) to ensure micronutrient supply and tested its effectiveness in rodent models. Periodic bi-weekly FMD cycles had a significant impact on the development and progression of multiple cancer models while allowing the preservation of lean body mass even at old age and extending longevity in healthspan study. As a treatment modality, FMD cycles alone significantly reduced tumor progression and sensitized various cancer models to chemotherapeutic drugs.

#4314

Using antioxidant to enhance liver cancer preventive effect of n-3 PUFAs.

Ying Fu,1 Angela Y. Bai,1 Marcin Dyba,1 Jing X. Kang,2 Fung-Lung Chung1. 1 _Georgetown University Medical Center, Washington, DC;_ 2 _Laboratory for Lipid Medicine and Technology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

In various animal models, n-3 polyunsaturated fatty acids (PUFAs) have demonstrated cancer preventive effects. This protective effect is associated with reduced levels of inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2). On the other hand, an endogenous DNA adduct formed from the peroxidation of ω-3 PUFAs, γ-hydroxy-1, N2-propanodeoxyguanosine (γ-OHPdG), is known to be mutagenic. In light of these observations, we hypothesized that the combination of n-3 PUFAs with an antioxidant to inhibit lipid peroxidation (e.g. α-lipoic acid) will result in an enhanced cancer preventive effect than n-3 PUFAs alone. The chemical carcinogen diethylnitrosamine (DEN) induced HCC model is employed in this study. The DEN-induced HCC model has a histology and genetic signature similar to that of human HCCs, such that it has poor prognosis and recapitulates a dependence on inflammatory signaling. It also reflects the same gender disparity seen in human HCCs. Fat-1 transgenic mice were used because they express a Caenorhabditis elegans desaturase converting n-6 to n-3 PUFAs endogenously without using dietary supplementation. This model underlies the importance of dietary control of PUFAs intake and ratios due to the inability of mammalian cells to generate anti-inflammatory n-3 PUFAs from pro-inflammatory n-6 PUFAs, which are a major component of the standard Western diets. The results showed that antioxidant can efficiently suppress the elevated level of γ-OHPdG in fat-1 mice. The tumor incidence, multiplicity, and size are monitored continuously. This work was supported by the NCI grant: RO1-CA-134892. Y.F. thanks the Prevent Cancer Foundation for his fellowship (Marcia and Frank Carlucci Charitable Foundation Award in Cancer Prevention and Early Detection)

#4315

Effects of increased dietary fat consumption on prostate cancer progression in genetically engineered mice.

Marco A. De Velasco,1 Yurie Kura,1 Yuji Hatanaka,1 Takashi Oki,1 Yutaka Yamamoto,1 Koichi Sugimoto,1 Yasunori Mori,1 Kazuhiro Yoshimura,1 Masahiro Nozawa,1 Kazuhiro Yoshikawa,2 Kazuto Nishio,1 Hirotsugu Uemura1. 1 _Kinki University Faculty of Medicine, Osaka-Sayama, Japan;_ 2 _Aichi Medical University, Nagakute, Japan_.

Prostate cancer is a highly heterogeneous disease and evidence indicates that lifestyle and environmental factors can contribute to cancer-related deaths. Accumulating data also suggests that increased dietary fat consumption may promote and increase the risk of prostate cancer-related morbidity and mortality. However, data between studies is inconsistent and a direct link between increased consumption of dietary fat and the promotion of prostate cancer remains unclear. Here, we present our findings from a preclinical study of increased dietary fat consumption and its effects on prostate tumor growth and progression using genetically engineered mouse models of prostate cancer. We determined the effects of a high fat diet on tumor cell proliferation and apoptosis in vivo using a well-established mouse model of prostate cancer. Twenty-week-old mice, with conditional biallelic deletion of PTEN, harboring castration-naïve or castration-resistant prostate tumors were randomized and assigned to a standard laboratory diet (4% fat) or a high-fat diet (HFD, 13.6% fat) for 8 weeks. In both tumor models, mice fed a HFD had increased expression levels of proteins associated with proliferation and decreased levels of apoptosis markers. To determine the long term effects of a HFD, we used a mouse model of advanced prostate cancer. Mice with concomitant inactivation of PTEN and p53 develop tumors that progress quickly and are characterized with increased metastatic development and decreased survival compared to PTEN knockout mice. PTEN/p53 double knockout mice were randomized and assigned to control or HFD experimental groups after weaning. Our findings revealed that mice fed a HFD resulted in obesity that led to decreased progression-free survival and overall survival rates compared to mice fed a standard diet. Our results provide evidence to support the effects of high dietary fat consumption and increased prostate cancer risk, and provide a platform to investigate chemoprevention strategies.

#4316

Pharmacological targeting of the TGF-beta-induced epithelial-mesenchymal transition by anthocyanidins in glioblastoma.

Amira Ouanouki, Evelyne Muhire, Sylvie Lamy, Borhane Annabi. _Université du Québec à Montréal, Montreal, Quebec, Canada_.

Introduction: Glioblastoma multiforme (GBM) is the most common primary brain tumor, known for its invasiveness and high resistance to standard treatments. Therefore, better understanding of the mechanisms that promote mesenchymal changes in GBM are of great clinical importance. Epithelial to mesenchymal transition (EMT) is a reversible biological process in which epithelial cells adopt mesenchymal properties. During EMT, migration, adhesion and cellular morphology are altered allowing benign tumor cells to infiltrate surrounding tissues and to metastasize to distant sites. Several growth factors have been shown to trigger EMT in tumor development; the Transforming Growth Factor-beta (TGF-b) is the most prominent inducer of EMT. Epidemiological studies have shown that diets rich in fruits and vegetables play an important role in preventing cancer due to their polyphenol content. Among polyphenols, the anthocyanidins, found in berries, red grapes, and other pigmented foods, plants and vegetables, have anti-inflammatory, cardioprotective, anti-angiogenic and anti-carcinogenic properties. Despite the well-known role of TGB-b in tumor progression, the impact of anthocyanidins on TGF-b-induced EMT remains misunderstood. Objectives: 1) Characterize the pharmacological effects of anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin and petunidin) on EMT, 2) identify the molecular targets leading to the inhibition of EMT modulated by anthocyanidins, and 3) characterize their effect on cellular activities involved in this tumor process. Methodology: The human U-87 glioblastoma (U-87 MG) cells were treated with anthocyanidins in pre-, co- and post-treatment with TGF-b. Results: Depending on the treatment, anthocyanidins affect differently the EMT induction by inhibiting U-87 MG cell migration. This inhibition resulted in a down-regulation of mesenchymal markers (fibronectin, snail), phosphorylation of Smad2 proteins and the mitogen-activated protein kinases (ERK, JNK) in a dose-dependent manner. Overall, delphinidin was the most potent inhibitor for all treatments. Conclusion: This study highlights a new antimetastatic action of anthocyanidins that supports the beneficial health and chemopreventive effects of dietary-based strategies against cancer.

#4317

Black raspberries show potent activity in prevention of experimental squamous cell esophageal cancer compared to a combination of selective COX-2 and iNOS inhibitors.

Ni Shi,1 Kenneth M. Riedl,1 Steven J. Schwartz,1 Xiaoli Zhang,2 Steven K. Steven,1 Tong Chen1. 1 _The Ohio State University Comprehensive Cancer Center, Columbus, OH;_ 2 _The Ohio State University Center for Biostatistics, Columbus, OH_.

Esophageal squamous cell carcinoma (SCC) remains a major health threat worldwide and preventive strategies are needed. We previously reported that overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are correlated with nitrosamine-induced squamous carcinogenesis of the esophagus and the selective iNOS and COX-2 inhibitors significantly inhibit incidence and progression. We also demonstrated anti-cancer activity of black raspberries (BRB) in a rodent model of esophageal SCC. The objective of the current study was to compare the impact of BRB versus the combination of celecoxib, a selective COX-2 inhibitor, and S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, in inhibition of SCC of the esophagus, and to elucidate molecular mechanism of an effective cancer prevention. We found that BRB is superior to the combination of two drugs in suppression of premalignant tissue growth in the rat esophagi. Moreover, rats fed BRB have lower tumor multiplicity than those fed celcecoxib + PBIT. Our data indicated that BRB also shows a potent inhibitory effect on esophageal iNOS and COX-2, in addition to modulating several associated oncogenic cell signaling pathways. We further conducted parallel mechanistic studies in vitro using BRB anthocyanins and the above two drugs. Our findings demonstrated that dietary BRB is superior to the combination of two chemopreventive pharmaceutical agents in prevention of experimental squamous cell esophageal cancer, suggesting the potential value of additional translational studies in developing food-based products using BRB for the prevention of esophageal SCC carcinogenesis in humans (Supported by NIH/NCI R01 CA131073).

#4318

Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR.

Ornella I. Selmin, Andreas J. Papoutsis, Donato F. Romagnolo. _The University of Arizona Cancer Center, Tucson, AZ_.

Sporadic breast cancers, which represent the vast majority (~90%) of breast tumor cases, do not have mutations in the tumor suppressor gene, BRCA-1 but have absent or markedly reduced levels of BRCA-1 similar to those observed in hereditary BRCA-1 tumors. Therefore, understanding the non-mutational mechanisms that contribute to repression of BRCA-1 has important implications for the prevention of both hereditary and sporadic breast tumors. Agonists of the aromatic hydrocarbon receptor (AhR) are ubiquitous in the environment and include dietary compounds, metabolites of fatty acids, industrial pollution, and photoproducts generated in the skin from ultraviolet radiation. In cultured estrogen-receptor (ER)α-positive MCF-7 breast cancer cells, we found that activation of the AhR with the prototype ligand 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) repressed by 50% estradiol-induced BRCA-1 expression. Conversely, the post-treatment with the soy isoflavone genistein and tea (-)-epigallocatechin-3-gallate (EGCG) at physiological doses (1 μM) rescued within 24 to 48 h BRCA-1 expression to levels ~2.0 to 3.0-fold higher than those measured in cells treated with estradiol alone. Genistein and EGCG reversed BRCA-1 promoter hypermethylation in AhR-activated MCF-7 cells. In ERα-negative and AhR-overexpressing UACC-3199 breast cancer cells, genistein (1 μM) reactivated BRCA-1 expression. The post-treatment of AhR-activated MCF-7 cells with genistein and EGCG antagonized the recruitment of DNA-methyl transferase-1 (DNMT-1) enzyme to the BRCA-1 promoter. These results suggest the involvement of epigenetic mechanisms in the repression of BRCA-1 and reversal effects of genistein and EGCG against breast tumorigenesis associated with activation of the AhR.

#4319

Associations of dietary inflammation scores with incident, sporadic adenoma.

Ashley C. Holmes, Roberd M. Bostick. _Emory University, Atlanta, GA_.

It is now well-accepted that inflammation is causal in colorectal carcinogenesis and that reducing inflammation reduces risk for colorectal neoplasms. The ability to characterize specific dietary components as having pro- or anti-inflammatory effects is a promising area of cancer prevention. To assess associations of diet with inflammation and diet-associated inflammation with cancer risk, we created a new food-based inflammation (FBI) score based on associations of whole food dietary components (processed meats, red meat, white meat, fish, nuts, coffee, tea, high- and low-fat dairy products, refined and whole grains, and 10 botanical categories) with a panel of biomarkers of inflammation (plasma C-reactive protein [CRP] and interleukins-6, -8, and -10) among 590 chronic disease diagnosis-free participants (47% male, 53% female, 57% white, 43% black) in the REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort study. A previously reported Dietary Inflammation Index (DII) is a primarily nutrient-based score developed for the same purposes. We investigated associations of our new FBI score, a DII that includes contributions from foods and nutritional supplements (Food + Supplements DII), and a DII that includes contributions only from foods (Food-only DII) with incident, sporadic adenoma in a case-control study (CPRU; n=2,195) conducted in the Twin Cities area of Minnesota from 1990 - 1994. Diet was assessed using a Willett food frequency questionnaire. Associations of the scores with adenoma were estimated using unconditional logistic regression models adjusted for age, sex, hormone replacement therapy use, family history of colorectal cancer, regular non-steroidal anti-inflammatory drug (NSAID) use, total energy intake, smoking, physical activity level, alcohol consumption, and body mass index (BMI). The multivariable-adjusted odds ratios (OR) and 95% confidence intervals (CI) for those in the highest relative to those in the lowest score quartiles were 1.28 (95% CI 0.95, 1.72) for the FBI, 1.23 (95% CI 0.82, 1.85) for the Food + Supplements DII, and 1.14 (95% CI 0.85, 1.53) for the Foods-only DII. These results suggest that 1) a more pro-inflammatory diet may be associated with higher risk of colorectal adenoma, and 2) support further development and investigation of a food-based inflammation score for investigating associations of diet-related contributions to systemic inflammation with colorectal and other cancers and chronic diseases.

#4320

Circulating vitamin D2 and D3 levels and single nucleotide polymorphism associations with lung cancer status: A case-control study.

Majda Haznadar, Kristopher W. Krausz, Ezra Margono, Elise D. Bowman, Brid M. Ryan, Frank J. Gonzalez, Curtis C. Harris. _National Cancer Institute, Bethesda, MD_.

Vitamin D is an essential micronutrient, required for normal physiological function and classically recognized for its role regulating calcium metabolism. Recent work is beginning to demonstrate a role of vitamin D in chronic illnesses, such as cancer, but questions addressing how depletion of this nutrient affects cancer risk remain largely unanswered. In 2011, the Institute of Medicine report highlighted the need for more research to explore the role of vitamin D in non-skeletal health outcomes. Previously, vitamin D has largely been measured using radio- and chemiluminescence immunoassays, unable to distinguish between D2 (from plant dietary sources and supplements) and D3 forms. We measured circulating serum levels of both, inactive (25(OH)D), and active (1,25(OH)2D) forms of D2 and D3 using sensitive liquid chromatography coupled to mass spectrometry in 406 lung cancer cases and 437 controls. We observed that levels of inactive D3 are associated with decreased lung cancer risk across quartiles, after adjustment for age, gender, race, smoking status, pack years, interview year and blood collection month (ORadjusted = 0.5 (95% CI = 0.2, 0.9), P < 0.02; Ptrend <0.0001) , compared with the lowest quartile. The observed associations remained significant upon stratifications on race, with ORadjusted ranging from 0.4 to 0.3 (95% CI = 0.1, 0.9), P < 0.04 in 2nd and 3rd quartiles, respectively, in African Americans, and ORadjusted = 0.5, P = 0.04 in 4th quartile in European Americans. Increasing levels of active D3 were associated with decreased lung cancer risk (ORadjusted ranging from 0.2 to 0.04 (95% CI = 0.01, 0.7), P < 0.01; Ptrend < 0.0001). High levels of inactive D2 were associated with decreased lung cancer risk only in the highest when compared to the lowest quartile (ORadjusted = 0.5 (95% CI = 0.2, 0.9), P = 0.05); no significant observations were seen for the active D2. Additionally, we conducted comprehensive genotyping of single nucleotide polymorphisms (SNPs) in the genes from the vitamin D metabolism pathway (CYP2R1, CYP27A1, CYP27B1, CYP24A1, and VDR), with a minor allele frequency of ≥5%, using a Fluidigm platform. Of 261 SNPs, 21 were associated with lung cancer risk. Four SNPs in VDR were found to modulate vitamin D3 levels in the controls: alleles associated with increased risk were associated with decreased vitamin D levels and vice versa, suggesting their functional significance in the context of lung cancer. We are the first group to investigate physiological levels of both, inactive and active forms of D2 and D3 in cancer patients. Our findings suggest a protective role of vitamin D in lung cancer, with stronger associations observed for D3, and reinforce the importance of maintaining optimal levels of this crucial vitamin for human health. Additional research to illuminate the mechanism through which vitamin D contributes to lung cancer carcinogenesis is warranted.

#4321

The pro-tumorigenic effects of obesity are reversed by severe weight loss via chronic or intermittent calorie restriction but not weight normalization via a low-fat diet.

Laura W. Bowers, Emily L. Rossi, Meghana G. Shamsunder, Stephen D. Hursting. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Background: Breast cancer outcomes in both pre- and postmenopausal women are negatively impacted by obesity, but the reversibility of these pro-cancer effects via weight loss has not been well established. We examined how weight loss via four different dietary interventions affects mammary tumor progression in a mouse model of chronic obesity and pre-menopausal basal-like breast cancer.

Methods: Six week old C57BL/6 mice were randomized to a control (Con, 10% kcal from fat) or diet-induced obesity (DIO, 60% kcal from fat) regimen and maintained on these diets for 15 weeks. DIO mice were then randomized to remain on DIO diet (Obese) or begin one of four weight loss regimens: low-fat control (formerly obese, FOb), high-carb 30% calorie-restricted (HCCR), low-carb 30% CR (LCCR), or intermittent energy restriction (IER). IER mice received a 14% CR diet 5 days/week and a 70% CR diet on 2 non-consecutive days/week, such that their average daily kcal intake and macronutrient distribution was equivalent to the LCCR group. The Con mice received the same control diet through the end of the study to maintain a normal weight. Mice were orthotopically injected with E0771 mouse mammary tumor cells at week 25. All mice were euthanized when one tumor reached 1.5 cm in diameter.

Results: At study endpoint, Obese mice had a higher average body weight and body fat percentage in comparison to all other diet groups. These phenotypic measures were statistically equivalent in Con and FOb mice, indicating that the FOb mice had returned to a normal weight and body fat percentage, but they were significantly lower in the 3 CR groups. The CR mice all lost and then maintained an equivalent amount of weight following the diet switch at 15 weeks, regardless of the diet timing or macronutrient distribution. Tumor volume and weight at sacrifice were significantly greater in the Obese mice relative to Con mice and all CR mice, but not FOb mice, despite their weight loss to Con levels. In contrast, tumors in the HCCR and IER, but not LCCR, mice were significantly smaller than Con. Serum insulin and leptin levels were statistically equivalent in Con, FOb, HCCR, LCCR, and IER, but serum insulin-like growth factor 1 remained significantly elevated in FOb mice, with levels statistically equivalent to Obese mice.

Conclusions: These results suggest that weight normalization via a low-fat control diet does not reverse the mammary tumor-promoting effects of chronic obesity. However, severe weight loss through various forms of calorie restriction successfully reverses these effects. An improved understanding of the energy responsive mechanisms mediating the anticancer effects of calorie restriction in obese mice may lead to the identification of new intervention targets and strategies to reduce the impact of obesity on breast cancer outcomes.

#4322

Maternal obesity increases tamoxifen resistance in female rat offspring.

Xiyuan Zhang,1 Idalia Cruz,1 Hansheng Zhang,2 Leena Hilakivi-Clarke1. 1 _Georgetown University Medical Center, Washington, DC;_ 2 _Poolsville High School, Poolsville, MD_.

More than 50% of pregnant women in the USA are overweight or obese, and over 40% gain more weight than recommended by the Institute of Medicine. We investigated if maternal obesity before and during pregnancy affects mammary cancer risk in the offspring, or alters response of the mammary tumors to antiestrogen therapy in a preclinical rat model of estrogen receptor positive (ER+) breast cancer.

Female Sprague-Dawley rats were fed an obesity-inducing high fat (OIHF) or control diet before and during pregnancy. Their offspring were all switched to a control AIN93G diet upon birth. Female offspring of control (n=35) or OIHF (n=40) diet fed dams were treated with a carcinogen DMBA on postnatal day 50 to induce ER+ mammary tumors. When mammary tumors reached a size of 13 mm in diameter, 337 ppm TAM citrate was added to the offspring's diet, resulting a daily intake of 15 mg/kg TAM. Responses of the tumors were categorized as de novo resistant (tumor kept growing), partial (size decreased but did not disappear), and complete (tumor disappeared). The animals with complete response were taken off from TAM and monitored for an additional 20 weeks to determine the risk of local recurrence.

The risk of developing mammary tumors was non-significantly increased in the OIHF group. Further, the OIHF exposed offspring had significantly more de novo resistant tumors than the control offspring. Although the percentage of completely responding tumors was similar in the two groups, local recurrence in the OIHF offspring was significantly higher than in the control offspring (90% vs 29%, respectively). To investigate the possible mechanisms of increased recurrence, we measured the protein levels of the members of unfolded protein response (UPR), inflammation and tumor immune pathways. We found that de novo TAM resistant and recurring tumors in the OIHF offspring exhibited significantly increased levels of Perk and Beclin-1, indicating activation of UPR. However, Nbr1 and p62 were also significantly increased in the OIHF offspring, suggestive of inhibition of autophagy in their mammary tumors. The levels of CD8a, a marker of cytotoxic T cells, were significantly reduced, whilst ERα, erBb2/Her2 and Vegfr2 were increased. Changes in the expression of these receptors could indicate increased inflammation.

We conclude that maternal obesity increased TAM resistance and the risk of mammary cancer recurrence in the female offspring. In addition, it induced changes in the UPR, autophagy and tumor immune responses in the offspring's mammary tumors.

#4323

Dietary omega-3 suppress mammary tumor growth, metastasis and enhances survival in an iso-caloric pair-fed mice model.

Saraswoti Khadge,1 Geoffrey M. Thiele,1 Lynell W. Klassen,1 Graham J. Sharp,1 Timothy R. McGuire,1 Michael J. Duryee,1 Holly C. Britton,1 Alicia J. Dafferner,1 Jordan Beck,2 Paul Black,2 Concetta DiRusso,2 James E. Talmadge1. 1 _University of Nebraska MEdical Center, Omaha, NE;_ 2 _University of Nebraska-Lincoln, Lincoln, NE_.

Omega-6 (ω6) and omega-3 (ω3) poly unsaturated fatty acids (PUFA) are pro- and anti-inflammatory respectively. Regulating their dietary ratio provides an approach for cancer prevention and potentially therapy; however, most studies have not separated the inflammatory effects of dietary fatty acids (FA) from that of obesity. Herein we studied the effects of the ω6:ω3 ratio in iso-caloric diets containing 35.5% of calories from fat on mammary tumor growth and metastasis. The ω6:ω3 ratio in ω6 and ω3 based diets were 42:1 and 1:1 respectively (confirmed by gas chromatography-mass spectrometry) using the liquid, Lieber DeCarli diet with fish oil substituting for 70% of olive oil. The ω3 diet contained 3:1 ratio of eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) as major ω3 FA with small amount of linolenic acids. The ω6 diet contained linoleic acid as predominant ω6 FA and small level of linolenic acid as ω3 FA. The diets were pair-fed to avoid the tumor promoting effects of obesity. Two weeks after establishing female BALB/c mice on the diets, they were orthotopically injected with 4T1 mammary tumors. Outcomes included body weight, diet consumption, tumor growth, metastasis and survival. In association with pair feeding there were no significant differences in the diet consumed (ω3 diet used as baseline) and weight gain between animals; however, the time to tumor growth was significantly more rapid in mice fed the ω6 diet versus the ω3 diet cohorts. The ω6 diet fed cohort had significantly higher numbers of pulmonary and hepatic metastases and significantly shortened survival. Further, ω6 diet fed mice had an unusual metastasis profile including an increase in ovarian and renal metastases and an increase in posterior paralysis that in prior studies was associated with demineralization, osteolysis, marrow metastases and spontaneous long bone fractures in mice fed ω6-enriched diet. Histological analysis of tissues supported the observation of more frequent unusual metastasis sites (ovaries, kidneys, heart) along with systemic changes to myeloid cells infiltration in the tissues of mice fed ω6 diet. Further, ω6 and ω3 diets had identical total and fat calories and were pair fed; however, the tumors and livers of ω6 diet fed cohorts had more lipid content in adipocytes. Our results were replicative and also found to be consistent when the tumor growth and survival experiments were compared between young (22 weeks) versus old (58 weeks) mice in independent studies. In summary, our studies demonstrate that the dietary ratio of ω6:ω3 regulates tumor growth and metastasis.

#4324

Undenatured whey protein isolate exhibit chemopreventive activity against HPV-16 induced carcinogenesis of CD271+ oral mucosa stem cells.

Sora Sandhya,1 Rika Tsuchida,2 Sukanya Gayan,2 Rashmi Bhuyan,2 Joyeeta Talukdar,1 Bidisha Pal,2 Seema Bhuyan,1 Jugal Ch Das,1 Amal Ch Kataki,3 Debabrata Baishya,4 Bikul Das2. 1 _KaviKrishna Laboratory, Guwahati Biotech Park, Guwahati, India;_ 2 _Forsyth Institute, Cambridge, MA;_ 3 _B Borooah Cancer Institute, Guwahati, India;_ 4 _Gauhati University, Guwahati, India_.

Oral cancer, and associated HPV-16 infection, and tobacco smoking is a major public health concern in the Kamrup district of India. To facilitate research on oral cancer chemoprevention, we have set up a collaborative research team between Forsyth Institute, an oral medicine specialized center affiliated to Harvard School of Dental Medicine, and KaviKrishna Laboratory, a non-for profit private laboratory set up located in Guwahati, the major city of Kamrup district, India. To study the cellular and molecular mechanism of HPV-16 mediated carcinogenesis, we have developed an in vitro model of HPV-16 mediated carcinogenesis of oral squamous stem cells. In this model (Bhuyan et al. The potential role of oral mucosa stem cell altruistic behavior as the initiating event of malignant transformation. AACR abstract Control # 16-A-6618-AACR), the treatment of oral mucosa cells derived from healthy volunteer with HPV-16 derived E6 protein led to expansion of a p53 deficient CD271+ expressing oral mucosa stem cells (OMSC). Noted that CD271 cell surface marker was recently identified as a putative marker for OMSCs. Using this in vitro carcinogenesis model, we evaluated the potential chemopreventive role of dietary whey protein. The E6 treated p53 deficient CD271+ cells were treated with DMEM/F12 media containing 0.2% of Immunocal, an undenatured whey protein (Tsai WY et al. Nutr Cancer 2000. PMID: 11525598). The p53 status as well as in vitro self-renewal activity of the CD271+ cells were examined by ELISA, transcriptional activity assay, and methylcellulose based clonogenic assay. We also fed Immunocal 20 gm/daily for 6 months to 5 individual with oral leukoplakia lesion with HPV-16 positivity. The CD271+ cells obtained from the leukoplakia lesion of clinical subjects were subjected to p53 status, and clonogenic assay. The data was compared with CD271+ cells obtained from leukoplakia lesion of subjects taking regular diet without whey protein supplements. We found that addition of Immunocal whey protein led to 8-fold decrease in the expansion of CD271+ cells following E6 treatment in the in vitro model of OMSC culture. Importantly, Immunocal treatment prevented the suppression of p53 in OMSCs. In the preliminary clinical study, the dietary intake of Immunocal led to complete loss of leukoplakia lesion in the 4/5 individual. In contrast, the control subjects exhibited the presence of CD271+ cells in the leukoplakia lesion having low p53 status, and high clonogenic activity. We are incorporating a larger number of subjects to study the potential chemopreventive activity of whey protein, using the locally available affordable whey protein extracts. Conclusion: This study indicate that whey protein extract, which is available in the local villages of Kamrup district could serve as a chemopreventive agent against oral cancer.

#4325

High-fat diet enhances and monocyte chemoattractant protein-1 deficiency reduces bone loss in mice with pulmonary metastases of Lewis lung carcinoma.

Sneha Sundaram, Lin Yan. _ARS USDA, Grand Forks, ND_.

Bone is adversely affected by metastasis and metastasis-associated complications. Obesity is a risk factor for both bone and cancer. Adipose tissue is an endocrine organ that produces pro-inflammatory adipokines, such as monocyte chemotactic protein-1 (MCP-1), that contribute to obesity and obesity-related diseases. This study (2x2 factorial design) investigated the effects of a high-fat diet (45% of energy from corn oil) and MCP-1 deficiency on bone micro-structural changes, analyzed by micro-computed tomography, in male C57BL/6 mice bearing lung metastases of Lewis lung carcinoma (LLC). Subcutaneous injection of LLC cells resulted in lung metastasis which was significantly enhanced by the high-fat diet and attenuated by MCP-1 deficiency. Micro-computed tomographic measurement showed significant reductions in bone volume fraction (BV/TV), trabecular number (Tb.N) and bone mineral density (BMD) in right femurs and vertebrae of LLC-bearing mice compared to non-tumor-bearing controls. In LLC-bearing mice, compared to the low-fat diet (16% of energy from corn oil), the high-fat diet significantly reduced BV/TV, Tb.N and BMD in femurs but not in vertebrae. Furthermore, compared to wild-type mice, MCP-1 deficiency significantly increased BV/TV and Tb.N in both femurs and vertebrae and BMD in vertebrae in LLC-bearing mice. These results demonstrate that metastasis causes bone loss which is enhanced by the high-fat diet and attenuated by MCP-1 deficiency. Overall, it indicates that the high-fat diet and MCP-1 contribute positively to metastasis-associated bone deterioration.

#4327

Dietary tomato reduces castration-resistant prostate cancer progression in TRAMP mice.

Joshua W. Smith,1 Joe L. Rowles,1 Rita J. Miller,1 Steven K. Clinton,2 William D. O'Brien,1 John W. Erdman1. 1 _University of Illinois at Urbana-Champaign, Urbana, IL;_ 2 _The Ohio State University, Columbus, OH_.

Epidemiological data support the hypothesis that consumption of diets rich in tomato products are associated with a reduced risk of prostate cancer. Castration-resistant prostate cancer (CRPC) represents the late and lethal phase of human prostate carcinogenesis, and is defined as cancer progression after surgical castration or pharmacologic reduction of serum testosterone through androgen deprivation therapy. Mechanisms involved in the transition to CRPC include cancer cells' acquired capacity for androgen synthesis or activation of ligand-independent signaling. We have previously shown that dietary tomato can inhibit prostatic androgen signaling and expression of androgen biosynthetic genes, as well as reduce primary cancer incidence in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Therefore, we examined whether dietary tomato might be an effective inhibitor of CRPC progression in mice. We hypothesized that lifelong dietary intake of tomato, as well as dietary tomato intervention following castration, would reduce tumor burden and growth rate in a mouse model of CRPC. TRAMP mice were fed an AIN-93G diet (CON) for one week and then randomized to the CON diet (n=28) or a similar diet with 10% w/w lyophilized tomato paste (TP; n=27) from 4 wks of age until euthanization. A third group, modeling dietary intervention, consumed CON from 4 to 12 wks of age, and the 10% w/w lyophilized tomato paste diet from wk 12 until euthanization (TP-I; n=25). All animals were castrated at 12 wks of age. Beginning at 10 wks of age, mice were monitored longitudinally with ultrasound for tumor detection, tumor volume estimation, and calculation of tumor growth rate. Serial 2D image slices were used to generate a 3D volume estimate. Mice were euthanized after 5 consecutive tumor scans, or if no tumor had been detected by 30 weeks of age. Tumor growth area under the curve (AUC) was reduced approximately 46% by tomato intervention (TP-I) and 27% by lifelong tomato consumption (TP). At euthanization, TP-I reduced tumor weight by approximately 26%. Additionally, incidence of gross metastases to distant organs (lung, liver, kidney) was strongly inhibited by TP-I, compared to CON (0% and 26%, respectively). TP diet reduced incidence of distant organ gross metastasis to <10%. The consumption of tomato products following castration inhibits CRPC progression in the TRAMP model. Our findings support the development of human studies of dietary tomato interventions in combination with anti-androgen therapy for men with advanced prostate cancer.

#4328

Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells.

Sanjay Kumar,1 James Stokes,1 Karyn Scissum Gunn,1 Selvarangan Ponnazhagan,2 Upender Manne,2 Manoj K. Mishra1. 1 _Alabama State University, Montgomery, AL;_ 2 _University of alabama, Birmingham, AL_.

Background: Nitric oxide (NO) is an important signaling molecule in the immune, nervous and cardiovascular systems. NO involves in the regulation of cellular behavior as well as cytotoxic events. Studies demonstrated that resveratrol (RES) (a polyphenolic compound in the skin of red fruits) at high concentration (≥50µM) induces NO production in endothelial F-2 cells and in human umbilical vein endothelial cells. However, the impact of RES on the biological functions of NO are still poorly understood. Therefore, the goal of this study was to investigate the impact of RES on NO-mediated oxidative stress at mitochondria level in mouse prostate cancer (PCa) cells.

Experimental Procedures: The transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (mirrors the pathogenesis of human prostate cancer) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 IU/ml, streptomycin 100 μg/ml, 0.005mg/ml bovine insulin, and 10nM Dehydroisoandrosterone in 5% CO2 at 370C. Cells were harvested by trypsinization after 80% confluency (at 4-5 pass), counted (105/ml), and incubated with RES (50uM), RES (50uM) + Deta-NONOate (750uM), Deta-NONOate (750uM), RES (50uM) +L-NMMA (750uM), and L-NMMA (750uM). Deta-NONOate was used as nitric oxide donor while L-NMMA was used as nitric oxide inhibitor. Thereafter, cell viability, NO-estimation in culture supernatant, mitochondrial membrane potential (MMP), total ROS-production in mitochondria and cytoplasm, and apoptotic assay (Annexin-V and caspase-3/7) were carried out.

Results and Conclusion: PCa cells incubated with RES showed decreased cell viability, decreased NO production in culture supernatant, decreased MMP, and decreased level of ROS in mitochondria. However, PS-externalization, and total ROS in cytoplasm were found high after normalization with control cells. Further, treatment with Deta-NONOate results in decreased cell viability, decreased total ROS in the cytoplasm, and decreased MMP, while mitochondrial ROS and NO production in culture supernatant were observed highly significant as compared control cells. Furthermore, cells were also exposed to the combination of RES and NO which suggest that RES reduced NO-mediated cell killing and reactive oxygen species generation in TRAMP cells. However, no significant changes were observed after L-NMMA exposure to TRAMP cells. The result demonstrates that RES protect cells from NO-mediated cell killing and induced NO-independent apoptosis in PCa cells. This suggests that role of RES as a potential therapeutic agent to control cancer. However, further investigation is needed to explore potential mechanism of action of RES in controlling cancer proliferation.

#4329

The consequence of obesity and obstructive sleep apnea in liver cancer.

Nathan W. Sweeney, Cecil J. Gomes, Sairam Pathasarthasy, Jesse D. Martinez. _University of Arizona, Tucson, AZ_.

Primary liver cancer is the 3rd leading cause of cancer mortality worldwide. Eighty-five percent of liver cancers is comprised of hepatocellular carcinoma (HCC). Risk of developing HCC increases when patients have obesity related comorbidities, involving non-alcoholic steatohepatitis (NASH), hepatic inflammation, or lipid accumulation. Additionally, obesity is generally regarded as a prime risk factor for the development of obstructive sleep apnea (OSA), a repeated obstruction of the upper airway during sleep leading to oxygen deficiency (OD), which also increases the incidence of NASH. The connection of these morbidities presume that patients suffering from obesity induced OSA have an increased risk of developing HCC. To examine obesity and OSA's role in HCC we combined three generally accepted methods: A one-time injection of tumor initiator diethylnitrosamine followed by continuous administration of the promoter phenobarbital, a high fat diet, and a hypoxia chamber to house the mice for regulation of their oxygen intake, allowing us to mimic the OD seen in OSA. Utilizing these treatment methods and applying microCT imaging to non-invasively and repeatedly monitor the liver, we have been able to examine the effects of obesity and/or OD. Preliminary result indicate that a high fat diet results in obesity, lipidosis and liver damage. Interestingly, OD seems to inhibit obesity while still generating hepatic inflammation. Furthermore, we have observed that obesity and OD together cause severe damage to the liver, enhancing the risk of developing HCC. This study will be a significant preclinical step in distinguishing mechanisms that may explain obesities and OSA's combined consequence in HCC.

#4330

A high-fat diet promotes tumorigenesis in two mouse models of K-ras-driven lung cancer.

Jeffrey Norris,1 Krista Pearman,1 Regan Memmott,2 Kristin Lastwika,3 Joell Gills,4 Phillip Dennis5. 1 _Midwestern University, Glendale, AZ;_ 2 _Banner University Medical Center, Phoenix, AZ;_ 3 _Fred Hutchison Cancer Research Center, Seattle, WA;_ 4 _Johns Hopkins Medical Institute, Baltimore, MD;_ 5 _Astrazeneca, Gaithersburg, MD_.

Lung cancer is the leading cause of cancer-related mortality worldwide, and 85% of lung cancer cases are associated with tobacco use. Within this group, activating mutations in K-ras have been identified in ~25% of lung adenocarcinomas. Using a mouse model of k-ras-driven lung tumorigenesis, we previously demonstrated that deletion of the IGF-1 gene or reduction of systemic IGF-1 levels using the antidiabetic drug metformin markedly reduced tumor burden. Since preclinical and clinical studies suggest that diet composition is the best predictor of IGF-1 levels, we hypothesized that diets high in fat or carbohydrate would promote lung tumorigenesis by increasing systemic IGF-1 levels.

To assess the effect of diet on systemic IGF-1 levels, 9 week old C57Bl/6J and A/J mice were fed standard cereal, high-carbohydrate, or high-fat (HFD) diets for 12 weeks. At the conclusion of the study plasma, liver, and lung samples were collected. Compared to the cereal-fed control mice, IGF-1 and insulin levels were increased in both strains of mice only with HFD. Average body weight only increased for the C57Bl/6J group that was fed HFD.

We investigated the effect of HFD on lung tumorigenesis using two mouse models of lung cancer. In the first, C57Bl/6LA2 mice, which are genetically modified with a K-ras mutation present in human smokers, were fed either cereal diet or HFD for 10 weeks following weaning. Lung tumor burden in the mice fed HFD was increased 2.7-fold compared to littermates fed cereal diet. In the second model, the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone (NNK) was given by IP injection to A/J mice beginning at 6 weeks of age. This carcinogen causes lung tumor formation by inducing K-ras mutations. Following once weekly injections of NNK for 3 weeks, the mice were randomized to cereal diet or HFD. 10 weeks later, mice fed HFD were found to have a 60% increase in lung tumor burden. In both studies, there was no relationship between the final body weight of the mice and tumor burden.

These studies show that HFD promotes lung tumor growth resulting from a mutation commonly observed in smokers. Therefore, dietary modification may slow the progression of tumorigenesis resulting from smoking-related genetic changes through IGF-1. Chemopreventative drugs, like metformin, may also have greater efficacy in a HFD model due to the increased insulin and IGF-1 associated with this model. Finally, understanding the molecular mechanisms by which HFD promotes tumor growth may help to identify new targets for cancer prevention.

## CANCER CHEMISTRY:

### Drug Design and Delivery

#4331

Discovery of potent 2-Indole-acylsulfonamide Mcl-1 inhibitors using structure guided fragment-based methods.

Subrata Shaw, James Chris Tarr, Nicholas Pelz, Zhiguo Bian, Johannes Belmar, Bin Zhao, Craig Goodwin, Allison Arnold, John Sensintaffar, Olivia Rossanese, Taekyu Lee, Edward Olejniczak, Stephen Fesik. _Vanderbilt University, Nashville, TN_.

Mcl-1 (Myeloid cell leukemia 1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and is one of the major acquired resistance mechanisms for many anticancer therapies. Targeting Mcl-1 with small molecules is very challenging because Mcl-1 exerts its effects through protein-protein interactions. Here we describe our continuing efforts in the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. We previously described the discovery of small-molecule Mcl-1 inhibitors containing a 2-indole-carboxylic core that primarily bind to the P2 pocket. In order to enhance potency, we extend our molecules to occupy the entire BH3 binding interface. A number of P4 site hits were identified using a fragment-based screening while the P2 pocket of Mcl-1 was saturated with our previously reported lead. P4 site ligands were then linked to our initial lead guided by the ternary Mcl-1 structure containing both P2 and P4 units using an acylsulfonamide as a synthetic handle that retained the critical charged-charged interaction with R263. X-ray co-crystal structures of new inhibitors occupying both P2 and P4 sites bound Mcl-1 provided detailed information on how these small-molecules bind to the target and guided subsequent potency optimization. From the efforts, we discovered a novel series of 2-indole-acylsulfonamide containing Mcl-1 inhibitors that exhibit low nanomolar binding affinities to the target and greater than 500-fold selectivity over Bcl-xL. These compounds represent useful starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

#4332

Discovery of BAY 1163877 - A pan-FGFR inhibitor: De novo structure-based design and lead optimization of benzothiophenyl-pyrrolotriazines.

Marie-Pierre L. Collin,1 Mario Lobell,1 Walter Huebsch,1 Dirk Brohm,1 Mélanie Héroult,2 Klemens Lustig,1 Sylvia Gruenewald,2 Ulf Boemer,2 Rolf Jautelat,1 Holger Hess-Stump,2 Stefan Jaroch,2 Michael Brands,2 Karl Ziegelbauer1. 1 _Bayer HealthCare, Wuppertal, Germany;_ 2 _Bayer HealthCare, Berlin, Germany_.

Fibroblast growth factors (FGFs) orchestrate a variety of cellular functions by binding to their transmembrane tyrosine-kinase receptors (FGFR1-4) and activating downstream signaling pathways. Alterations in FGFR encoding genes are frequently observed in a variety of solid tumors including lung, gastric, breast and urothelial cancer. Therefore, targeting FGFRs using selective FGFR inhibitors is an attractive therapeutic approach to treat cancer patients.

BAY 1163877 is an orally active, highly potent and selective small molecule FGFR-1, -2 and -3 kinase inhibitor. We disclose for the very first time its discovery and chemical structure. BAY 1163877 was derived from a de novo structure-based design approach and medicinal chemistry optimization. Data on the structure activity relationship and the pharmacokinetic profile of the benzothiophenyl-pyrrolotriazine structure class will be presented. Based on its favorable preclinical profile, BAY 1163877 is currently being investigated in a Phase 1 clinical trial (NCT01976741).

#4333

Identification and optimization of the first highly selective GLUT-1 inhibitors.

Marcus Bauser, Bernd Buchmann, Holger Siebeneicher, Arwed Cleve, Hartmut Rehwinkel, Roland Neuhaus, Iring Heisler, Thomas Mueller, Charlotte Christine Kopitz. _Bayer Healthcare, Berlin, Germany_.

Increased glycolysis rates combined with an insufficient cellular respiration in tumor cells have been known for decades as the Warburg effect (1930). One of the key players in this metabolic alteration of cancer cells is glucose transporter 1 (or GLUT-1), which facilitates the transport of glucose across the plasma membranes of mammalian cells. Many tumor biopsies underline the crucial importance of GLUT-1 overexpression for the vitality of many tumor entities. Moreover the glucose uptake dependency of cancer cells can be visualized by the widespread clinical application of 18fluorodeoxyglucose (FDG) uptake in PET tumor imaging. Therefore, inhibition of glucose uptake for the treatment of cancer seems to be an interesting approach.

Based on our first identified GLUT-1 inhibitors (1H-pyrazolo[3,4-d]pyrimidines) with modest selectivity profile, a high throughput screen (HTS) was performed to identify new potent lead structures with unique selectivity against the other members of the GLUT-1 family (GLUT-2,-3 and -4). Among several lead structures we identified from the HTS the quinoline carboxamides with the most promising selectivity profile. Extensive SAR elaboration by synthesizing more than 1600 new derivatives of the lead structure clearly revealed the essential moieties for high activity, potency, and selectivity. Additionally, we were able to improve the in vitro and in vivo PK properties permitting in vivo animal studies. We are able to report for the first time the preclinical profile and structure of the very potent and highly selective GLUT-1 inhibitor BAY-876 representing an ideal tool to further evaluate the scope and limitations of GLUT-1 inhibition as new therapeutic option.

#4334

A novel linker to enable alcohol-containing payloads for the preparation of antibody-drug conjugates.

Robert V. Kolakowski, Karl Haelsig, Scott Jeffrey, Peter Senter. _Seattle Genetics, Bothell, WA_.

Antibody-drug conjugates (ADCs) have emerged as important therapeutics for treating cancer as evidenced by the recent FDA approval of brentuximab vedotin Adcetris™ and trastuzumab emtansineKadcyla™, and the large number of ADCs (>35) currently in clinical trials. ADCs are composed of two core units: the targeting antibody and the drug-linker. The drug-linker typically consists of a cytotoxic payload, a moiety for covalent attachment to the antibody, and a conditionally cleavable linker that is stable in circulation but releases the cytotoxic payload upon internalization into a target cell. The released payload will possess a residual chemical functional group that is used for attachment of the drug to the cleavable linker. Functional groups that have been exploited within the ADC field for this purpose include amines (primary and secondary), thiols, and carboxylic acids. Unfortunately, many potential ADC payloads do not possess any of these functional group 'handles' that allow for stable conjugation and release of the drug in an unmodified form. Use of these payloads often requires modification of the drug structure to introduce a handle for conjugation. To avoid this, we have developed new linker chemistry, the methylene-alkoxy-carbamate (MAC), which enables direct conjugation of drugs through alcohol functional groups. Such groups are present on a diverse array of synthetic drugs and cytotoxic natural products, thus, expanding the potential chemical space available for payload selection. As proof-of-concept, we have linked Auristatin E through the secondary alcohol of the norephedrine residue and characterized the stability and in vivo activity of the resulting ADCs. To further define the potential applicability of the MAC unit, we have examined its stability and release properties with a series of model systems including primary, secondary, and tertiary alcohols as well as a phenol. We have also explored the effect of substitution of the MAC nitrogen on these properties. This novel linker chemistry is currently being applied to new payloads that were inaccessible with previous technology.

#4335

Nanoformulations of PARP inhibitors Olaparib and Talazoparib for targeted cancer therapy.

Paige Baldwin,1 Anders Ohman,2 Jeremy Thong,2 Shifalika Tangutoori,1 Anne van de Ven,1 Rajiv Kumar,1 Daniela Dinulescu,2 Srinivas Sridhar1. 1 _Northeastern University, Boston, MA;_ 2 _Brigham and Women's Hospital, Boston, MA_.

Introduction: Poly(ADP-ribose) Polymerase (PARP) plays an important role in a number of DNA repair pathways. PARP inhibitors (PARPi) such as Olaparib and Talazoparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. These drugs are currently only available in oral form which results in limited bioavailability, poor tumor accumulation, and systemic toxicity. Here we report the development of novel nanoformulations of Olaparib and Talazoparib to allow intravenous or intraperitoneal delivery, providing greater bioavailability and tumor accumulation, while limiting systemic toxicities.

Methods: Nanoparticle formulations of Olaparib and Talazoparib were synthesized and tested in vitro and in vivo. Short-and long-term dose response with a panel of ovarian cancer cell lines were conducted. These cell lines include KURAMOCHI, SKOV3, OVSAHO, JHOS2, PA1, COV318, 403 and 404, derived from BRCA2-/-, PTEN-/-, TP53mut mice, and 4306 and 4412, developed from conditional LSL-K-rasG12D/+, PTENloxP/loxP mice. Radiosensitization with NanoOlaparib was tested in the radiation resistant prostate cancer cell line FK01, derived from Ptenpc-/-;Trp53pc-/- mice. In vivo, NanoOlaparib was tested in an IP spread model using 404 cells. Animals were treated IP with NanoOlaparib alone, and in combination with cisplatin. Radiosensitization with NanoOlaparib in vivo was tested in a xenograft model using FK01 cells to mimic castration resistant prostate cancer. Animals were treated biweekly with NanoOlaparib before and after radiation treatment.

Results: The murine cell lines 403 and 404 were highly sensitive to this treatment due to the mutations in BRCA2, PTEN, and TP53. 4412 and 4306 showed comparable sensitivity, suggesting that a PTEN deletion confers similar sensitivity to PARP inhibitors as a BRCA2 deletion. PA1 demonstrated high sensitivity to NanoOlaparib which may be attributed to genetic instability. NanoTalazoparib is more potent than NanoOlaparib, resulting in a similar relationship in cell line sensitivity with overall lower IC50's. Strong synergistic radiosensitization was observed in FK01 cells with NanoOlaparib.

Bioluminescence imaging illustrated that NanoOlaparib administered IP daily resulted in a greater inhibition of tumor growth than those treated with oral Olaparib daily. The FK01 xenografts are highly radioresistant with little difference between untreated and radiation only animals. NanoOlaparib delays tumor growth, while the combination of radiation and NanoOlaparib clearly shrinks tumors.

Conclusions: Robust nanoparticle formulations of NanoTalazoparib and NanoOlaparib have been successfully developed for in vitro and in vivo studies. These results show that NanoOlaparib and NanoTalazoparib amplify the therapeutic efficacy of PARP inhibition and imply a very promising role for the nanoformulation in ovarian and prostate cancers.

#4336

Spliceosome inhibition as a novel therapeutic option in acute leukemia.

Anna Wojtuszkiewicz,1 Rocco Sciarrillo,1 Gerrit Jansen,1 Yehuda G. Assaraf,2 Kazunori Koide,3 Robert K. Bressin,3 Upamanyu Basu,3 Edwin Sonneveld,4 Godefridus J. Peters,1 Gertjan J L Kaspers,1 Jacqueline Cloos1. 1 _VU Medical Center, Amsterdam, Netherlands;_ 2 _Technion - Israel Institute of Technology, Amsterdam, Israel;_ 3 _University of Pittsburgh, Pittsburgh, PA;_ 4 _Dutch Childhood Oncology Group, The Hague, Netherlands_.

Spliceosome targeting is a novel therapeutic strategy, showing promising results in solid tumors and hematological malignancies. Aberrant splicing of genes involved in apoptosis regulation and drug metabolism was shown to confer chemoresistance in various tumor cells, including acute leukemia. Moreover, increased expression of abnormal splice variants cause reduced sensitivity of leukemic cells to crucial components of current treatment protocols, such as glucocorticoids or methotrexate. Therefore, targeting the spliceosome holds potential to modulate drug resistance-related splicing and to eradicate cells, which do not respond to conventional therapy. In this study, we assessed the in vitro sensitivity of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells to spliceosome inhibitors, including meayamycin B (MAMB), pladienolide B (PB) and spliceostatin A (SSA).

First, the growth inhibitory activity of MAMB was determined using a 72 h MTT assay in a panel of ALL and AML cell lines, including sublines with acquired resistance to conventional chemotherapeutics. Mechanistically, the effect of MAMB, PB and SSA on splicing profiles, cell cycle distribution and apoptosis induction was assessed in time course experiments. Finally, we compared MAMB sensitivity between 10 primary ALL, 10 AML specimens, and 6 healthy bone marrow (BM) specimens.

Remarkably, both ALL and the notoriously apoptosis-resistant AML cell lines responded to subnanomolar concentrations of MAMB, with IC50 values (50% growth inhibition in the MTT assay) ranging between 0.07 and 0.16 nM. Moreover, MAMB retained full sensitivity towards leukemic sublines resistant to conventional chemotherapeutics with various modes of action, including methotrexate, dexamethasone, bortezomib and imatinib. MAMB, PB and SSA-induced growth inhibition was associated with time and dose-dependent alterations in splicing profiles of selected apoptosis-related genes (including Mcl-1, Bcl-X, FAS and Casp2), concomitant cell cycle arrest (in G1 and G2/M phases) and apoptosis induction (up to 40% after 24 h exposure to 1nM MAMB). Consistent with cell line observations, both primary ALL and AML specimens showed remarkable response to MAMB (mean LC50=0.42 nM, range: 0.26-0.69 nM and 0.43 nM, range: 0.33-0.44 nM, respectively), with a significantly lower MAMB sensitivity of healthy BM samples (mean lethal concentration causing 50% cell kill [LC50] value 0.57, range: 0.39-1.13 nM, p=0.02).

Collectively, this is the first study to demonstrate that spliceosome inhibition constitutes a promising therapeutic option for both ALL and AML patients, including those with acquired resistance to other anti-leukemic drugs.

Financial support by KiKa (Children Cancer Free)

#4337

Novel potent and selective orally available CDK8/19 kinase inhibitors.

Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Maria Isabel Albarran, Antonio Cebria, Borja Barrera, Enara Aguirre, Maria del Carmen Rodriguez de Miguel, Manuel Urbano, Ana Isabel Hernandez, Cristina Gomez de la Oliva, Virginia Rivero, Rosario Concepcion Riesco, Sonia Martinez Gonzalez, Joaquin Pastor. _CNIO, Madrid, Spain_.

CDK8 and its paralog CDK19 are cyclin-dependent kinases and together with CDK7 and CDK9 belongs to the group of C-terminal domain (CTD) kinases that phosphorylate the CTD of RNA polymerase II, thus regulating transcription. CDK8 together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. CDK8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes. As Mediator independent roles, CDK8 has been shown to act as part of a separate complex behaving as a histone kinase. Moreover, CDK8 has been identified as a major kinase in the response to IFN signaling mediated STAT1-S727 phosphorylation.

Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer being a marker of poor prognosis. Moreover, in gastric cancer CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Recently, gene amplification of CDK8, CDK19, CCNC and MED13 in breast cancers has been related with poor response to adjuvant therapy. These results suggest that CDK8 inhibitors may become a unique class of anticancer drugs that could increase the efficacy of cancer therapy by blocking chemotherapy-induced production of tumor-promoting secreted factors.

We have carried out a medium throughput screening campaign which led to the discovery of several low nanomolar hits belonging to 3 different chemical series. Later on, we have performed a Hit Generation phase, designing novel inhibitors within a patentable chemical space. Here, we have taken into account information from the chemical structures of the screening hits, X-ray structure of CDK8 protein co-crystalized with sorafenib, docking studies, chemical feasibility and intellectual property.

We have identified a novel chemical series of CDK8/19 inhibitors. After a HtL exploration we have reached lead compounds with potency in the picomolar range and high selectivity versus a panel of 468 protein kinases. The leads display cellular activity by blocking βcatenin reporter activity and STAT1 phoshorylation in the low nanomolar range. Lead compounds have been screened in a panel of tumoral cell lines, showing a defined profile in terms of sensitivity with GI50s which range from nanomolar to low micromolar values. These inhibitors induce cell death in a dose response manner. The novel chemical series show a drug-like profile in terms of solubility, permeability, CYP450 inhibition and hERG. The identified lead compounds are orally bioavailable, with good clearance, volume of distribution and exposure levels. A selected lead compound has been used in in vivo PK-PD studies showing positive results.

## CLINICAL RESEARCH:

### Clinical Qualification of Impactful NGS-based Biomarkers

#4338

Advanced neoplasia detection in colorectal cancer screening using multiple stool DNA markers and haemoglobin.

Linda J.W. Bosch,1 Veerle Melotte,2 Sandra Mongera,3 Kathleen L.J. Daenen,2 Veerle H.M. Coupe,3 Sietze T. van Turenhout,3 Esther M. Stoop,4 Thomas R. de Wijkerslooth,5 Chris J.J. Mulder,3 Ernst J. Kuipers,4 Evelien Dekker,5 Michael Domanico,6 Graham P. Lidgard,6 Barry M. Berger,6 Beatriz Carvalho,1 Manon van Engeland,2 Gerrit A. Meijer1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Maastricht University Medical Center, Maastricht, Netherlands;_ 3 _VU University Medical Center, Amsterdam, Netherlands;_ 4 _Erasmus Medical Center, Rotterdam, Netherlands;_ 5 _Academic Medical Center, Amsterdam, Netherlands;_ 6 _Exact Sciences Corporation, Madison, WI_.

Purpose of the study: Molecular tests have the potential to improve current non-invasive faecal immunochemical test (FIT) screening for colorectal cancer (CRC) and advanced precancerous lesions. We examined the performance of a panel of faecal DNA (sDNA) markers and FIT in archival samples from an invitational CRC screening population.

Methods: Whole stool samples were prospectively collected from individuals participating an invitational primary colonoscopy-screening program (COCOS trial). Only participants that provided stool, performed FIT (OC-Sensor) and underwent colonoscopy were selected. The sDNA panel included quantitative molecular assays for KRAS mutations and for aberrant NDRG4 and BMP3 methylation. The performance of the sDNA plus FIT panel was compared to the FIT results alone, by Receiver Operator Characteristic (ROC) analyses.

Results: A total of 1047 individuals (51% male) with a median age of 60 years (range 50-75) were included, of which 7 (0.7%) had colorectal cancer and 104 (9.9%) had advanced precancerous lesions (advanced adenomas or sessile serrated polyps ≥ 1 cm).

The combination of sDNA and FIT was more sensitive than FIT alone for detecting advanced precancerous lesions (49% (50/102) and 25% (26/102), respectively). Specificities among individuals with non-advanced or negative findings (controls) were 89% and 96% for sDNA and FIT testing, respectively.

ROC analysis of CRC and advanced precancerous lesions compared to controls revealed an Area Under the Curve (AUC) of 0.75 for the sDNA plus FIT test, compared to 0.68 for FIT alone. At an equal specificity of 95%, advanced precancerous lesions were detected with a higher sensitivity by the sDNA plus FIT test compared to FIT alone (36% vs 28%, p=0.08).

Conclusions: In an invitational colorectal cancer screening cohort, combining stool DNA markers with FIT detected more advanced neoplasia than FIT alone, primarily due to detecting more advanced adenomas.

#4339

Prognostic significance of copy number alteration burden in unfavorable intermediate-risk prostate cancers harboring intraductal carcinoma and cribriform architecture.

Melvin Lee Kiang Chua,1 Jure Murgic,1 Melania Pintilie,1 Emilie Lalonde,2 Charlotte Kweldam,3 Winnie Lo,1 Alejandro Berlin,1 Alan Dal Pra,1 Michele Orain,4 Valerie Picard,4 Helene Hovington,4 Alain Begeron,4 Yves Fradet,4 Bernard Tetu,4 Julie Livingstone,2 Alice Meng,1 Jun Yan Zhang,1 Gaetano Zafarana,1 Neil Fleshner,5 Mike Fraser,1 Paul Boutros,2 Robert Bristow,1 Theodorus van der Kwast5. 1 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Erasmus MC, Rotterdam, Netherlands;_ 4 _Universite Laval, Quebec city, Quebec, Canada;_ 5 _University Health Network, University of Toronto, Toronto, Ontario, Canada_.

Aim: Men with intermediate-risk prostate cancer represent a heterogeneous group of patients with varying prognoses. Intraductal carcinoma (IDC), cribriform architecture (CA), and high copy number alteration burden are novel pathological and genomic indices that predict aggressive disease and inferior clinical outcomes in patients with localised prostate cancer. We aimed to test the independent prognostic effect of these indices in a cohort of intermediate-risk prostate cancers.

Experimental methods: We defined a set of clinical and genomic indices to test for their prognostic significance in a cohort of individuals with NCCN-defined intermediate-risk prostate cancer, who were treated with radical prostatectomy (RadP) or radiotherapy (RT) (N = 173, RadP; N = 358, RT). Clinical indices include primary T-category, pre-treatment PSA level, Gleason's score and pattern, and presence of IDC and/or CA. Pathological features were reviewed centrally by two expert pathologists. Copy number alteration burden was assessed in 215 tumours of the cohort using SNP array profiling (Affymetrix Oncoscan), and reported as percent genome aberration (PGA). Our primary endpoint was to test if a model incorporating IDC/CA and PGA stratifies patients with intermediate-risk disease for risk of biochemical relapse after primary treatment.

Results: Biochemical relapse was associated with PGA on univariable and multivariable analysis for the sub-cohort of 215 patients (HR of High vs Low PGA defined by median = 1.61, 95% CI = 1.04-2.49, Wald's p = 0.033). Based on modelling of the clinical indices, presence of IDC/CA was associated with biochemical relapse-free rate (bRFR) on univariable and multivariable analyses (HR = 1.90, 95% CI = 1.34-2.69, p = 0.00034), along with PSA level. Risk stratification considering both IDC/CA and PGA indicated an additive prognostic effect of IDC/CA to PGA for early biochemical relapse, with 18-month bRFR of 83% in High PGA, present IDC/CA vs 94% in Low PGA, absent IDC/CA subgroups (HR = 2.55, 95% CI = 1.49-4.39, p = 0.00069). A comparison of risk stratification models revealed that inclusion of PGA to IDC/CA yielded the strongest model for predicting 18-month bRFR in patients with intermediate-risk prostate cancer following treatment (area under the curve, AUC = 0.580, 95% CI = 0.456-0.675 [T-category, PSA, and IDC/CA] vs 0.649, 95% CI = 0.476-0.776 [T-category, PSA, IDC/CA, and PGA]).

Conclusions: We herein demonstrate for the first time a novel risk stratification model integrating pathological (IDC/CA) and genomic (PGA) indices to identify patients with unfavourable intermediate-risk prostate cancer, who may benefit from intensification to conventional definitive treatment.

#4340

DNA repair genes aberrations in germline DNA in metastatic castration-resistant prostate cancer patients.

JOAQUIN MATEO,1 Suzanne Carreira,2 Helen Mossop,2 Pasquale Rescigno,1 Michael Kolinsky,1 Elena Castro,3 Ada Balasopoulou,1 Jo Hunt,1 Desamparados Roda,1 Claudia Bertan,2 Jane Goodall,2 Susana Miranda,2 Penny Flohr,2 Nuria Porta,2 Zsofia Kote-Jarai,2 David Olmos,3 Christopher J. Lord,2 Emma Hall,2 Ros Eeles,1 Johann S. de Bono1. 1 _The Institute of Cancer Research & The Royal Marsden NHS Foundation Trust, London, United Kingdom; _2 _The Institute of Cancer Research, London, United Kingdom;_ 3 _CNIO - Spanish National Cancer Research Center, Madrid, Spain_.

INTRODUCTION: DNA repair defects are found in mCRPC and are therapeutically actionable; germline BRCA mutation-associated (gBRCA) prostate cancer has a poor prognosis. We hypothesized that metastatic castration resistant prostate cancer (mCRPC) is enriched for germline DNA repair mutations and that these may be relevant to patient outcome.

METHODS: Targeted-sequencing for DNA repair genes was conducted in germline DNA from patients consenting to 3 clinical trials between 2013-2015. Germline DNA was extracted from saliva or buccal swabs using the Oragene kit; libraries were constructed using a customized Qiagen panel and sequenced using the Illumina MiSeq. Family history and clinical data were prospectively collected. For time to event analyses unadjusted Cox regression models were used and comparisons were made using log-rank tests.

RESULTS: Germline samples from 154 mCRPC patients were available. Median age at diagnosis was 61years (y), median time to castration-resistance was 14.5 months (m) and median overall survival (OS) from initial diagnosis of prostate cancer was 106.8m; 69% (91/131; 24 N/A) of patients were initially diagnosed with Gleason≥8 tumors. 130/154 (84.4%) and 131/154 (85.0%) received Docetaxel and Abiraterone respectively. Of 154 patients, 4 were previously known to be gBRCA2 mutation carriers and were removed from the prevalence analysis but included in the clinical analyses; 22/150 (14.7%, 95%CI 9.4-21.4%) harboured a truncating or frameshift mutation in a DNA repair gene (9 BRCA2, 6%; 4 ATM, 2.7%; 2 PALB2, 1.3%, 1 each for CHEK2, FANCI, MRE11A, NBN, RAD51C, RAD51D and MSH6). Overall, patients with any germline DNA repair aberrations had a worse median OS (75.8 vs 106.8 m; log-rank p=0.04). Time to resistance to primary hormonal ablation was shorter specifically for gBRCA2 mutations carriers (11.0 vs 14.8 m; log-rank p=0.01) but not for non-BRCA2 repair aberrations. Age at diagnosis was similar in patients with or without DNA repair germline mutations (median 61.3 vs 61.7y, Mann-Whitney p=0.41) as well as frequency of Gleason≥8 tumors (16/21 [76%] vs 75/109 [68%]; Mann-Whitney p=0.23) Response rates to Docetaxel (14/18 [77.8%] vs 64/94 [68.1%]; Fisher exact p=0.67) and Abiraterone (10/21 [47.6%] vs 44/94 [46.8%]; Fisher exact p=0.73) were similar among individuals with and without mutations. Family cancer history was collected in 125/154 cases (81%). While having cases of ovarian/prostate/breast/pancreas cancers in these patients' families associated with a higher likelihood of finding a germline mutation (Odds Ratio 3.36, p=0.03), 5 of 68 (7.4%) men with no cases of ovarian/prostate/breast/pancreas cancers registered in their families carried a germline mutation in a DNA repair gene.

CONCLUSIONS: mCRPC is enriched for patients with germline mutations in DNA repair genes (15%), with 6% having gBRCA2 mutation. Germline DNA repair aberrations are associated with a worse prognosis from mCRPC.

#4341

Employing the epigenetic field effect to detect prostate cancer in biopsy-negative patients.

Bing Yang,1 Johnathon McCormick,1 Nathan Damaschke,1 Anastasia Montgomery,1 Glen Leverson,1 Wei Huang,1 Daniel W. Lin,2 David F. Jarrard1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _University of Washington, Seattle, WA_.

Purpose: Prostate cancer (PCa) is typically multifocal supporting the principle that molecular alterations can occur in histologically normal tissues in patients that have cancer. We have recently demonstrated using DNA methylation microarrays that epigenetic differences define a widespread field defect in the normal prostate tissue from men with cancer. The purpose of this study is to determine whether a novel panel of DNA methylation markers can predict the presence of PCa using fresh and paraffin-embedded histologically normal biopsy cores from multiple medical sites.

Materials and Methods: Encode HG18 DNA methylation arrays, containing 385,000 probes tiling the biologically significant pilot regions of the human genome, were used to identify methylation alterations between NTA and TA tissues. Bisulfite Pyrosequencing confirmed alterations at numerous loci. Methylation was assessed using quantitative pyrosequencing in a training set consisting of 65 non-tumor associated (NTA) and tumor associated (TA) prostate tissues. A multiplex model was generated using multivariate logistic regression and externally validated in a blinded fashion using a set of 47 NTA and TA biopsy specimens from an external institution.

Results: The methylation field defect was not significantly different between adjacent and distant tissues from tumor foci indicating this was not a peritumor effect. DNA methylation was analyzed in a subset of significant loci in NTA and TA tissues. Robust methylation differences were observed for all genes at all CpGs assayed (p<0.0001) in the 47 fresh-frozen specimens. Regression models incorporating individual genes (EVX1, CAV1, and FGF1) and a gene panel (EVX1 and FGF1) discriminated between NTA and TA tissues in the original training set (AUC 0.796-0.898, p<0.001). Upon external validation, uniplex models incorporating EVX1, CAV1, or FGF1 discriminated between TA and NTA biopsy-negative specimens with an AUC of 0.702, 0.696, and 0.658, respectively (p<0.05). Furthermore, a multiplex panel (EVX1 and FGF1) identified PCa patients with an AUC of 0.774 (p=0.001) and had a negative predictive value of 0.909. Further results will be reported from an ongoing multicenter trial involving 160 FFPE specimens.

Conclusions: A widespread epigenetic field defect can be utilized to detect the existence of PCa in patients with histologically negative biopsies. This assay is unique in that it detects alterations in non-tumor cells at distance from tumor foci. With further validation, this marker panel (EVX1 and FGF1) has the potential to decrease the need for repeated prostate biopsies, a costly procedure associated with pain and complications.

#4342

Ultra-deep next generation sequencing (NGS) of plasma cell-free DNA (cfDNA) from patients with advanced lung cancers: results from the Actionable Genome Consortium.

Bob T. Li,1 Filip Janku,2 Pasi A. Janne,3 Gordon B. Mills,2 Kiran Madwani,2 Ryan S. Alden,3 Cloud P. Paweletz,3 Marc Ladanyi,1 Alex Aravanis,4 Byoungsok Jung,4 Sante Gnerre,4 Amy J. Sehnert,4 David B. Solit,1 Gregory J. Riely,1 Geoffrey R. Oxnard3. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _MD Anderson Cancer Center, Houston, TX;_ 3 _Dana-Farber Cancer Institute, Boston, MA;_ 4 _Illumina, Inc., San Francisco, CA_.

Introduction: Noninvasive genotyping using plasma cfDNA from cancer patients has the potential to obviate the need for some biopsies while also characterizing disease heterogeneity. This study was undertaken to develop an ultra-deep plasma NGS panel for patients with non-small cell lung cancers (NSCLC).

Methods / Results: Plasma was prospectively collected from 51 patients with advanced, progressive NSCLC and a known oncogenic driver from prior tumor genotyping. We performed ultra-deep NGS on extracted cfDNA using a customized Illumina library preparation, hybrid capture panel covering 37 lung cancer related genes (complete exons and partial introns), and ultra-deep sequencing (HiSeq4000). Mean sequencing depth was ~50,000X (150 million, 150bp reads per sample). After specialized consensus-based error correction for low allele frequency (AF) genomic alterations, the median unique DNA molecules per position were ~3,500. The mean sequence error rate was reduced by 20-fold to 0.002%, enabling the confident call of a driver mutation as low as 0.03%. In a subset of cases, paired plasma droplet digital PCR (ddPCR) was performed for common EGFR and KRAS mutations using a validated assay.

Blinded to tumor genotype, plasma NGS detected SNVs (EGFR, KRAS, BRAF), indels (EGFR, ERBB2), and fusions (ALK, ROS1) as well as significant copy number gains (CNG) (ERBB2, MET). Sensitivity of cfDNA for the detection of known oncogenic drivers was 88% (45/51). A single false positive driver mutation was identified in a case with a known EGFR mutation in tumor; plasma NGS found both EGFR exon 19 del (0.88% AF) and KRAS G12D (2.65% AF), and plasma ddPCR confirmed the presence of both mutations (2.2% and 2.0% AF). Evaluation for an occult second primary is ongoing. In 22 EGFR, ALK, or ROS1 cases with acquired resistance to targeted therapy, plasma genotyping detected a range of potential resistance mechanisms: EGFR T790M and C797S, ALK F1174C, ERBB2 CNG, MET CNG. In 16 cases with paired resistance biopsies, concordance for EGFR T790M status was 94% (15/16).

18 cases with known EGFR or KRAS mutations underwent paired ddPCR. In 14 cases the driver mutation was detected using both assays with high concordance of the %AF (r=0.91). The remaining 4 cases were negative with ddPCR but 3 were positive with NGS at low AF (0.04%, 0.08%, and 0.29%), and the specificity for each driver was 100%.

Conclusions: Ultra-deep plasma NGS can detect a wide range of oncogenic drivers in NSCLC and may be more sensitive than established ddPCR assays. In the setting of acquired resistance to targeted therapy, plasma NGS reliably captured EGFR T790M and additional somatic alterations as potential resistance mechanisms.

#4343

Comparison of over 10,000 clinical NGS circulating tumor DNA profiles to tissue-derived genomic compendia.

Oliver A. Zill, Kimberly C. Banks, Coyt Jackson, Stefanie Mortimer, Arthur Baca, Becky Nagy, Richard B. Lanman, Helmy Eltoukhy, AmirAli Talasaz. _Guardant Health, Inc., Redwood City, CA_.

Analysis of cell-free circulating tumor DNA (ctDNA) enables non-invasive and serially repeatable genomic profiling of advanced cancer patients, providing options when tissue biopsy is contra-indicated or of insufficient quantity (20-25% of solid tumor patients). Liquid biopsy studies to-date have been limited to modest-sized cohorts and case studies. Here we present genomic profiling results from liquid biopsies of >10,000 advanced cancer patients in whom Guardant360TM (G360) was ordered for clinical care.

G360 provides deep-coverage (15,000x average) and highly accurate sequencing of ctDNA across a 70-gene target-capture panel. It detects single nucleotide variants, indels, gene amplifications, and fusions across all NCCN-recommended solid tumor genomic alterations with analytic specificity >99.9999% and analytic sensitivity <0.1% mutant allele frequency. Consecutive commercial test results (6/2014-11/2015) were analyzed for yield and patterns of genomic alterations by tumor type. ctDNA mutation patterns and frequencies were compared to published tumor tissue sequencing results (TCGA, ICGC).

Of the >10,000 patients (>50 solid cancer types), 80% of patients had at least one somatic alteration detected in ctDNA (median = 3; mean = 4.3 alterations). The most common cancers were lung (32%), gastrointestinal (23%), and breast (14%). Mutational spectra across the 70 analyzed genes were similar to TCGA results with the notable exception of increased prevalence of resistance mutations in the liquid biopsy cohort, likely owing to ongoing/prior therapy. Canonical drivers in NSCLC patients were generally mutually exclusive, although activating EGFR or RAS mutations were observed in 12.5% of patients with fusions (5/40). Colorectal cancers showed both mutual exclusivity among KRAS, NRAS, and BRAF drivers, and convergent evolution toward downstream pathway activation under anti-EGFR therapy, with some samples showing multiple resistance mechanisms. Mutation patterns for the most frequently mutated genes were generally well correlated between ctDNA and published tissue data, including for commonly altered tumor suppressor genes (for TP53: Pearson r=0.94, Spearman ρ=0.80).

G360 is ordered prior to initial treatment when biopsy tissue is exhausted, unobtainable or under-genotyped, but more often is ordered upon progression to identify evolving resistance mechanisms that may be targetable. Thus, relative frequencies of genomic alterations were expected to differ from published frequencies established in unselected, early-stage, treatment-naïve cohorts. Nonetheless, the specific patterns of alterations in ctDNA from this unprecedented liquid biopsy cohort largely recapitulated patterns observed in published tissue sequencing studies. Moreover, ctDNA sequencing clearly and robustly identified convergent evolution of drug resistance occurring under therapy.

#4344

Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC).

Cristina Teixidó,1 Noemí Reguart,2 Ana Giménez-Capitán,1 Miguel Ángel Molina-Vila,1 Patricia Galván,2 Sonia Rodriguez,1 Laia Paré,2 Santiago Viteri,3 Vicente Peg,4 Zaira Yeste,1 Núria Viñolas,2 Rafael Rosell,5 Aleix Prat2. 1 _Pangaea Biotech, Barcelona, Spain;_ 2 _Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain;_ 3 _Dr Rosell Oncology Institute, Barcelona, Spain;_ 4 _Vall Hebron Hospital, Barcelona, Spain;_ 5 _Catalan Institute of Oncology, Barcelona, Spain_.

Background: Targetable rearrangements in anaplastic lymphoma kinase (ALK), ROS1, and RET genes are present in approximately 7% of patients (p) with advanced NSCLC. Current methods for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHC) or RT-PCR. However, these tests have disadvantages in terms of sensitivity, cost and throughput, and often show discrepancies. The nCounter platform allows simultaneous detection with no enzymatic reaction, within 72 hours, of several fusion genes in formalin-fixed paraffin-embedded (FFPE) samples using a transcript-based method.

Methods: A custom set of 5´and 3´ probes and fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was designed and evaluated. A panel of ALK-ROS1-RET positive cell lines (H2228, H3122 [EML4-ALK], SUDHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS1], BaF3 pBABE [CD74-ROS1], LC2/ad [RET]) and negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) was used to validate the technique. Then, a total of 70 FFPE samples was analyzed, 49 of them positive by FISH, IHC and/or qRT-PCR for ALK (n=30), ROS1 (n=17) and RET (n=2). Total RNA was isolated from cell lines and FFPE and < 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and four house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative translocations were defined by two criteria: (1) a 3'/5' ratio score of > 2.0 and ≤ 2.0 respectively or/and (2) a signal for a fusion-specific probe above background. Response to crizotinib by RECIST criteria was retrospectively collected in p with ALK-positive NSCLC by any technique.

Results: nCounter sensitivity to detect fusion transcripts ALK, ROS1 and RET in cell lines was 100% using the two criteria (3'/5' and direct probes) and specificity was also 100%. Among 20 ALK-FISH-positive p, ALK 3'-5' scoring was positive in 18 (95%). One p was non-evaluable (NE) by ALK 3'-5' scoring. Among 48 ALK-FISH-negative p, nCounter score was positive in 13 (27%). All p positive for ALK by nCounter were either positive or NE for ALK by IHC. A total of 17 p were treated with crizotinib, 16 of whom responded to treatment and were positive by nCounter. Regarding FISH, five p responding to crizotinib were negative and one was NE. Finally, one p not responding to crizotinib was positive by RT-PCR but negative by nCounter.

Conclusion: The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening assay that identifies ALK-FISH-negative/NE NSCLC patients who could benefit from treatment with ALK inhibitors.

## EPIDEMIOLOGY:

### Endogenous and Exogenous Factors in Cancer Epidemiology throughout the Life Course

#4345

Association of smoking with breast cancer risk by estrogen and progesterone receptor status: the multiethnic cohort.

Inger T. Gram,1 Song-Yi Park,2 Laurence N. Kolonel,2 Gertraud Maskarinec,2 Lynne R. Wilkens,2 Christopher Haiman,3 Loic Le Marchand2. 1 _Univ. of Tromsø, Tromsø, Norway;_ 2 _Univ. of Hawaii, Honolulu, HI;_ 3 _Univ. of Southern California, Los Angeles, CA_.

Background: The 2014 report of the Surgeon General put forward seven major summary points for the smoking and breast cancer relationship that needed to be addressed. One of these was that there was insufficient evidence to conclude that the risk of breast cancer from smoking differs between women diagnosed with ER+ tumors and those diagnosed with ER-tumors. The purpose of the present study was to examine whether the previously-reported association between risk of breast cancer and smoking in the Multiethnic Cohort (MEC) differed by ER+, ER-, PR+ or PR-tumor status.

Methods: From 1993 to 2010, we followed 83,300 women who were enrolled in the MEC at 45-75 years of age. We identified cancer cases via linkages to the statewide Hawaii and California cancer registries through December 2010. We used Cox proportional hazards regression with age as the underlying time scale to estimate multivariate-adjusted hazard ratios (HR) and 95% confidence intervals (CI) for the associations between different measures of smoking exposure and the four types of tumors according to hormone receptor status. The included covariates were selected a priori. For parous women, we also estimated breast cancer risk by category

of age at smoking initiation in relation to first childbirth (after or within 1 year before first childbirth, 1-5 years before, or >5 years before) compared with parous never smokers for each tumor type.

Results: At cohort entry, the mean age of the participants was 62 years. Of these women, 45% reported at baseline that they had ever smoked. During a mean follow-up of 15 years, 4,484 women developed invasive breast cancer of which 2,417 (53.9 %) had known status for at least one hormone tumor receptor. Altogether, 1,939 women were diagnosed with ER+, 469 with ER-, 1,548 with PR + and 688 with PR- hormone receptor status. The HRs associated with ever versus never smoking were similar for the tumor subtypes: ER+(1.05, 95% CI: 0.95, 1.15), ER-(0.99, 95% CI: 0.82, 1.20), PR+ (1.06, 95% CI: 0.96, 1.18), PR- (1.00, 95% CI: 0.85, 1.17). Compared with never smokers, ever smokers who had smoked more than 20 pack-years had a 17% a higher risk of ER+ (1.17, 95% CI 1.01-1.35) and an 11% higher risk of ER- (1.11, 95% CI 0.83-1.49) breast cancer.

Among parous women, those who had smoked more than 5 years before their first live childbirth had an increased risk of ER+ breast cancer of 45% (95% CI 1.18-1.77) and a non-statistically significant increase for ER- breast cancer of 13% (95% CI 0.72-1.76) than never smokers. The respective values for women with PR+ and PR- tumors were 37% (95% CI 1.09-1.73) and 43% (95% CI 1.03-2.01).

Conclusions: Parous women who had smoked more than five years before their first childbirth seem to have the most noticeable risk regardless of tumor type. The current data do not support any difference for the smoking and breast cancer association by hormone receptor status although this may be due to lack of power.

#4346

Adolescent fiber intake and mammographic density in premenopausal women.

Lusine Yaghjyan,1 Gabriella Ghita,1 Maryam Farvid,2 Bernard Rosner,2 Kimberly A. Bertrand,3 Rulla Tamimi2. 1 _University of Florida, Gainesville, FL;_ 2 _Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA;_ 3 _Boston University, Boston, MA_.

Background: Some, but not all, previous studies have suggested that higher adolescent fiber intake may be associated with a reduced risk of breast cancer. Consistently, adolescent fiber intake has been suggested to reduce breast cancer risk in adulthood in some of the previous studies. To date, there is limited epidemiologic evidence that diet has a strong influence on mammographic density and inconsistency in results for specific dietary factors. While some earlier studies demonstrated an inverse association between adolescent fiber intake and breast density (defined as parenchymal patterns), a more recent study did not find significant associations. We investigated the associations of adolescent fiber intake with mammographic density, a strong and consistent predictor of breast cancer, in premenopausal women. This is by far the largest study that investigated these associations.

Methods: This study included 743 premenopausal women within the Nurses' Health II cohort with no history of breast or other cancer. Percent breast density, absolute dense and non-dense areas were measured from digitized film mammographic images using a computer-assisted thresholding technique. Adolescent and adult diet was assessed with a food frequency questionnaire and energy-adjusted nutrient intakes were estimated for each of the questionnaires. Information regarding breast cancer risk factors was obtained from baseline or biennial questionnaires, closest to the mammogram date. We used generalized linear regression to quantify associations between quartiles of adolescent fiber intake and each of the breast density measures, adjusting for age, body mass index, and other known and suspected predictors of mammographic density. Absolute measures were square root-transformed in analyses to improve normality. Associations were examined separately for total fiber intake; fiber from fruits, vegetables, legumes, and cereal; and food sources of fiber (fruits, vegetables, and nuts).

Results: In multivariable analyses, total fiber intake during adolescence was not associated with percent breast density (p for trend=0.64), absolute dense area (p for trend=0.80), or non-dense area (p for trend=0.75). Similarly, neither consumption of nuts, fruits, or vegetables nor specific sources of fiber intake (fruits, vegetables, legumes, or cereal) during adolescence were associated with any of the mammographic density phenotypes.

Conclusions: Our findings do not support the hypothesis that adolescent fiber intake is associated with premenopausal mammographic density. If observed associations with breast cancer risk are causal, then the effect may not be mediated through mammographic density.

#4347

A randomized phase 2 clinical trial of PEITC on detoxification of tobacco-specific and non-specific carcinogens and toxicants.

Jian-Min Yuan,1 Jian-Min Yuan,2 Irina Stepanov,3 Sharon E. Murphy,3 Sharon E. Murphy,4 Renwei Wang,1 Steven G. Carmella,3 Heather H. Nelson,3 Heather H. Nelson,5 Dorothy Hatsukami,3 Stephen S. Hecht3. 1 _University of Pittsburgh - Division of Cancer Control and Population Sciences, Pittsburgh, PA;_ 2 _University of Pittsburgh - Department of Epidemiology, Graduate School of Public Health, Pittsburgh, PA;_ 3 _University of Minnesota - Masonic Cancer Center, Minneapolis, MN;_ 4 _University of Minnesota - Department of Biochemistry, Molecular Biology and BioPhysics, Minneapolis, MN;_ 5 _University of Minnesota, Minneapolis, MN_.

Abstract

Background: Cigarette smoke contains the tobacco-specific lung carcinogen NNK and other carcinogens and toxicants. 2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in cruciferous vegetables, can inhibit the metabolic activation and lung carcinogenicity of NNK in rodents. Intake of a broccoli sprout beverage containing high levels of sulforaphane can increase urinary excretion of benzene and acrolein metaboites. Glutathione S-transferases (GST) catalyze the conjugation of carcinogens and toxicants with glutathione enhancing their urinary excretion, thus functional GST genotypes may modify the effect of PEITC.

Methods. The goal of this randomized phase 2 clinical trial with a crossover was to determine if PEITC inhibits the metabolic activation of NNK and enhances the detoxification of benzene, acrolein and crotonaldehyde. Eighty-two smokers were completed the trial. The smokers smoked cigarettes containing deuterium labeled [pyridine-D4]NNK to allow measurement of metabolic activation. The subjects were randomly assigned to one of two arms: PEITC-placebo, or placebo-PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 ml of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. Three 24h urine samples were collected during each of the 2 treatment periods. The NNK metabolic activation ratio: [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL, and the mercapturic acids of benzene, acrolein and crotonaldehye in urine were quantified using validated LC-ESI-MS/MS methods. The linear mixed model with random effect was used to examine the PEITC effect.

Results. Overall, PEITC treatment significantly reduced the NNK metabolic activation ratio by 7.7% (P = 0.023), and increased the detoxification metabolites of benzene by 24.6% (P = 0.002) and acrolein by 15.1% (P = 0.005), but had no effect on crotonaldehyde (5.5%, P = 0.148). PEITC had stronger effects in subjects with GST null genotype. PEITC reduced the NNK metabolic activation ratio by 15.6% (P = 0.039),

and increased detoxification metabolites of benzene by 95.4% (P <0.001), acrolein by 32.7% (P = 0.034), and crotonaldehyde by 29.8% (P = 0.006) among the subjects with null genotype of both GSTM1 and GSTT1, but had no effect in those possessing both genes (Pinteraction < 0.05).

Conclusion. The results of this trial demonstrate that PEITC inhibits the metabolic activation of NNK in smokers, and has a stronger effect on the detoxification of environmental carcinogens and toxicants such as benzene, acrolein, and crotonaldehyde. A more pronounced effect of PEITC in subjects lacking both GSTM1 and GSTT1 genes supports the epidemiological findings of stronger protection of dietary isothiocyanates against the development of lung cancer in such individuals. [Grant support: NCI R01CA122244].

#4348

A prospective study of smoking habit and risk of synchronous colorectal cancers.

David A. Drew,1 Reiko Nishihara,2 Paul Lochhead,1 Aya Kuchiba,3 Zhi Rong Qian,2 Kosuke Mima,2 Katsuhiko Nosho,4 Kana Wu,5 Molin Wang,5 Donna Spiegelman,5 Edward L. Giovannucci,5 Charles S. Fuchs,2 Shuji Ogino,2 Andrew T. Chan1. 1 _Massachusetts General Hospital, Boston, MA;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _National Cancer Center, Tokyo, Japan;_ 4 _Sapporo Medical University School of Medicine, Sapporo, Japan;_ 5 _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: Synchronous colorectal cancers (CRC) (2 or more distinct primary carcinomas simultaneously identified in one patient) may arise as a result of a "field effect" or shared etiologic factors. Synchronous CRC has been associated with somatic epigenetic changes; cigarette smoking has been associated with DNA methylation in CRC. Thus, we examined the association between cigarette smoking and risk of synchronous CRC.

Methods: Among men and women enrolled in the Health Professionals Follow-up Study and Nurses' Health Study over 24 and 32 years, respectively, we examined the association of smoking with CRC. We examined the differential risk of developing synchronous CRC compared with solitary CRC, classified based on review of pathology reports, through duplication-method Cox proportional hazards regression.

Results: During 3,477,211 person-years of follow-up of 45,691 men and 88,614 women, we documented 1,960 individuals with solitary CRC and 45 individuals with synchronous CRC. The association of smoking with CRC differed significantly according to the presence of synchronous compared with solitary tumors (Pheterogeneity=0.0008). Compared with never smokers, current smokers experienced a higher risk of synchronous CRC (multivariable HR=5.26; 95% CI, 2.08-13.32). In contrast, current smokers did not experience an elevated risk of solitary cancer (multivariable HR=0.97; 95% CI, 0.83-1.13). Similar differences in the association of cumulative pack-years smoked with CRC according to tumor synchronicity status were also observed (Pheterogeneity=0.006). Smoking cessation for ≥10 years was significantly associated with reduced risk of synchronous tumors (multivariable HR=0.44; 95% CI, 0.20-1.00), but not solitary tumors (multivariable HR=1.07; 95% CI, 0.91-1.25) (Pheterogeneity=0.003).

Conclusion: Current cigarette smoking is associated with an increased risk of synchronous but not solitary CRC. These data support a model in which smoking contributes to an etiologic field effect predisposing individuals to the development of synchronous CRC.

#4349

Identifying causal risk factors of metabolic syndrome for renal cell carcinoma. A Mendelian randomization approach.

Robert Carreras-Torres,1 Mattias Johansson,1 Ghislaine Scelo,1 Philip Haycock,2 Mark Purdue,3 Xifeng Wu,4 Richard Houlston,5 Stephen Chanock,3 Paul Brennan1. 1 _IARC, Lyon, France;_ 2 _MRC Integrative Epidemiology Unit, University of Bristol., Bristol, United Kingdom;_ 3 _National Cancer Institute., Rockville, MD;_ 4 _MD Anderson Cancer Center, Houston, TX;_ 5 _Institute of Cancer Research, London, United Kingdom_.

Epidemiological studies have convincingly demonstrated that several factors of the metabolic syndrome (MetS) are associated with the risk of Renal Cell Carcinoma (RCC)These factors often occur together and it is therefore challenging to disentangle their individual causal relevance in the etiology of RCC. In order to circumvent this limitation, we have applied a Mendelian randomization (MR) approach whereby genetic markers are evaluated in relation to RCC risk as unconfounded markers of the individual MetS factors.

We focused on MetS parameters from which genetic instruments could be identified from large-scale genome-wide association studies (GWAS). The following MetS factors were instrumented using multiple gene-variants: general and central obesity (body mass index (BMI) and waist-to-hip ratio), elevated blood pressure (systolic and diastolic blood pressure), dyslipidemia (high and low density cholesterol, total cholesterol, and triglycerides), hyperglycemia (fasting glucose and glucose levels at 2 hours after glucose intolerance tests), and hyperinsulinemia (fasting insulin). Genetic proxies for these parameters were identified from GIANT, ICBP, GLGC and MAGIC Consortia.

Summary statistics on RCC risk for instrumental SNPs of each MetS factor, including OR estimates and standard errors, were available from GWAS coordinated by the International Agency for Research on Cancer, the US National Cancer Institute, the Institute for Cancer Research UK, and the MD Anderson Cancer Center US. Together these studies comprised a total of 12,104 RCC cases and 24,999 controls from European origin that were genotyped using different genotyping platforms. Imputation was conducted on each dataset and only SNPs with an imputation quality higher than 0.6 were considered for the analyses. The causal effect of each MetS parameter on RCC risk was subsequently estimated using the MR likelihood-based approach, assuming a linear relationship between the risk factor and the outcome and a bivariate normal distribution for the genetic association estimates.

The MR risk analysis using genetic instruments of the individual MetS factors indicated that elevated BMI (P: 1x10-08) and fasting insulin (P: 7x10-04)increased the risk of RCC, whereas elevated post-load glucose levels were associated with a lower risk (P: 2x10-3). The odds ratio per standard deviation increase were estimated at 1.58 (95% CI: 1.35-1.86) for BMI, 1.77 (95%CI: 1.27-2.46) for fasting insulin, and 0.62 (95%CI: 0.46-0.83) for post-load glucose. No associations were seen for genetic instruments of blood pressure or lipids.

These results provide a clear support for a causal role of obesity in RCC etiology, and suggest that factors related to hyperglycemia and/or hyperinsulinemia may be involved in the causal pathway. This study may guide future efforts aiming to clarify the biological mechanisms whereby the metabolic syndrome influences RCC pathogenesis.

#4350

Human oral microbiome and prospective risk for pancreatic cancer: a population based, nested case control study.

Xiaozhou Fan,1 Alexander V. Alekseyenko,2 Jing Wu,1 Eric J. Jacobs,3 Susan M. Gapstur,3 Mark P. Purdue,4 Christian C. Abnet,4 Rachael Stolzenberg-Solomon,4 George Miller,1 Jacque Ravel,5 Richard B. Hayes,1 Jiyoung Ahn1. 1 _New York University School of Medicine, New York, NY;_ 2 _Medical University of South Carolina, Charleston, SC;_ 3 _Epidemiology Research Program, American Cancer Society, Atlanta, GA;_ 4 _Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 5 _Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#4351

A neighborhood-wide association study (NWAS) in prostate cancer: A new methodologic approach.

Shannon Lynch,1 Nandita Mitra,2 Michelle Ross,2 Craig Newcomb,2 Karl Daily,2 Tara Jackson,2 Charnita Zeigler-Johnson,3 Harold Riethman,4 Charles Branas,2 Timothy Rebbeck5. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _University of Pennsylvania, Philadelphia, PA;_ 3 _Jefferson, Philadelphia, PA;_ 4 _Old Dominion University, Norfolk, VA;_ 5 _Harvard University, Boston, MA_.

Background: Cancer results from complex interactions of multiple variables at biological, individual and social levels. However, comprehensive, empirical methods to assess social and neighborhood-level effects are limited. We propose a Neighborhood-Wide Association Study(NWAS), analogous to a genome-wide association study(GWAS), to comprehensively identify neighborhood signatures associated with disease. Methods: Pennsylvania State Cancer Registry data from 1995-2005 were linked to the 2000 U.S. Census data. In a successively more stringent multiphase approach, we evaluated the association between neighborhood exposures(n=14,663, standard deviation adjusted census variables) and prostate cancer aggressiveness (n=6,416 aggressive Stage>3/Gleason grade >7 cases vs. n=70,670 non-aggressive Stage<3/Gleason grade<7 controls) in White men. Each phase accounted for age, year of diagnosis, spatial correlation, and multiple testing. We used generalized estimating equations in Phase 1 and Bayesian mixed effects models in Phase 2 to calculate odds ratios(OR) and credible intervals(CI). Variables meeting statistically significant thresholds after Bonferroni adjustment were identified and used in subsequent phases. In Phase 3, principal components analysis grouped correlated variables.

Results: From 14,663 census variables, 17 variables that were most significantly associated with prostate cancer aggressiveness were identified. The top two hits related to transportation (OR=1.05;CI=1.001-1.09) and poverty (OR=1.07;CI=1.01-1.12). Significant associations between prostate cancer and income, housing, employment, and immigration were also found.

Conclusions: This NWAS methodology addresses gaps in neighborhood and cancer research by proposing a standardized, agnostic evaluation of neighborhood using readily accessible data from the U.S. Census. This approach has implications for health disparities research and can be applied broadly to large-scale, public health data.

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Identifying Targets and Combinations through Novel Approaches

#4352

Discovery of the first cell-active inhibitors of poly(ADP Ribose) glycohydrolase through high-throughput screening and computational approaches.

Bohdan Waszkowycz,1 Dominic James,1 Steven Durant,2 Nicola Hamilton,1 Cliff Jones,2 Stuart Jones,1 Allan Jordan,1 Alan Lau,2 Alison MGonagle,1 Mark O'Connor,2 Kate Smith,1 Alex Stowell,1 Julie Tucker,3 Ian Waddell,1 Donald Ogilvie1. 1 _CRUK Manchester Institute, Manchester, United Kingdom;_ 2 _Oncology iMed, AstraZeneca, Manchester, United Kingdom;_ 3 _Newcastle University, Newcastle, United Kingdom_.

DNA repair is a critical process for the survival and normal proliferation of healthy cells. However, given the enhanced levels of cellular stress and genomic instability, these repair processes are even more critical to the survival of malignant cells, where rates of DNA damage are considerably increased. Given this, inhibitors of DNA damage repair have seen a resurgence of interest in recent years in an effort to exploit tumour cell vulnerabilities. One such example of this approach has resulted in the recent approval of the PARP inhibitor olaparib (Lynparza™) for women with advanced ovarian cancer associated with defective BRCA genes.

Olaparib acts against the poly(ADP-ribose)polymerase (PARP) enzymes, more recently re-defined as the ARTD (Diphtheria toxin-like human ADP-ribosyltransferase) enzyme class. Whilst PARP is widely known to play critical and well-understood roles in DNA repair, poly(ADP ribose) glycohydrolase (PARG) is less well known but equally essential for effective DNA repair, degrading PAR chains and facilitating effective DNA repair. However, its inhibition may offer several key advantages over PARP inhibition. Most critically, whilst there are 18 isoforms , there exists only a single PARG protein, offering a specific point of therapeutic intervention. However, due to the open nature of the PARG binding cleft and the nature of the binding site, this protein has been considered to be difficult to inhibit with small, drug-like small molecules, particularly in the cellular context.

This poster will describe our efforts to overcome these challenges against this challenging target and report our early successes achieved through innovative computational chemistry strategies. These efforts have delivered several credible, drug-like startpoints for further medicinal chemistry optimisation.

#4353

Preclinical evaluation of the PARP inhibitor niraparib and cytotoxic chemotherapy alone or in combination in a panel of 25 triple-negative breast cancer PDX models: Relevance of BRCA mutations, HRD status and other biomarkers.

Olivier Deas,1 Stefano Cairo,1 Keith Wilcoxen,2 Keith Mikule,2 Truong-An Tran,1 Kirsten Timms,3 Jean-Gabriel Judde1. 1 _Xentech, Evry, France;_ 2 _Tesaro, Waltham, MA;_ 3 _Myriad, Salt Lake City, UT_.

Poly-ADP-ribose polymerase 1 (PARP) inhibitors are therapeutically effective in a subset of breast and high-grade ovarian cancers harboring deleterious BRCA1/2 mutations. Recently, several studies have tried to identify other molecular markers of HRD in BRCA1/2 wild-type (wt) breast and ovarian cancers to identify patients that could benefit from PARP inhibitor treatment. Most clinical studies of single-agent PARP inhibitors are therefore focused on breast or ovarian cancer with BRCA1/2 mutations and/or identified as HRD. Preclinical evidence also indicates that PARP inhibitors have therapeutic potential in combination with genotoxic chemotherapy. However, the toxicity associated with such combinations has generally imposed dose reduction of the chemotherapy, possibly explaining why the clinical activity observed with the combinations has in general not been superior to that of the corresponding single agents given at full dose.

In a panel of 25 triple negative breast cancer (TNBC) PDX models, we investigated the antitumor efficacy of the PARP inhibitor niraparib used alone or in combination with cyclophosphamide, a backbone genotoxic drug of TNBC standard chemotherapy. Responses were correlated with both BRCA1/2 mutations and HRD status as defined by the myChoice HRD™ test from Myriad Genetics and with other HRD markers.

Responses to cyclophosphamide ranged from progression to partial or complete tumor regression. Responses to niraparib were also variable and occurred only in tumors with BRCA mutations or a high HRD score. High sensitivities to niraparib and cyclophosphamide were tightly associated, suggesting that they may both be a result of a defect in a BRCA1/2-related pathway. We tested if niraparib could potentiate the efficacy of cyclophosphamide in TNBC models that initially regressed on cyclophosphamide but eventually relapsed. We chose a gapped sequential design in which treatment with niraparib was initiated 14 days after a full dose of cyclophosphamide, to avoid toxicity associated with concomitant administration. Potentiation with inhibition of tumor relapse was observed in tumors sensitive to single-agent niraparib, but not in tumors refractory to single-agent niraparib. In responder tumors, the cyclophosphamide-niraparib sequential combination was superior to sequential cycles of cyclophosphamide in preventing tumor relapse.

These results show that a gapped sequential combination of full dose cyclophosphamide and niraparib is well tolerated and demonstrates remarkable efficacy in a subset of TNBCs. The data will be discussed with regard to potential adjuvant treatment strategies with PARP inhibitors in TNBC and the value of myChoice HRD™ as a biomarker in predicting response to PARP inhibition alone and in combination with chemotherapy.

#4354

CRISPR/Cas9 genome-wide gRNA library for target identification.

Donato Tedesco, Paul Diehl, Mikhail Makhanov, Sylvain Baron, Dmitry Suchkov, Costa Frangou, Alex Chenchik. _Cellecta, Inc., Mountain View, CA_.

Genome-wide loss-of-function screening is a fundamental method to identify genes responsible for driving biological responses, and complex pooled lentiviral-based libraries expressing large numbers of genetic disruptors, such as shRNAs, make large-scale cell screening practical. While RNAi-based approaches have proven to be an effective strategy for these screens, recent work suggests CRISPR technology offers an effective alternative. Although shRNA and sgRNA pooled library screens are similar in concept, the gene interruption with the two techniques occurs by a very different mechanism so some divergence may be expected when comparing results obtained using one method versus the other.

To investigate the potential difference in the two methodologies, we performed parallel dropout viability screens to identify essential genes in a pair of primary isogenic CML cell lines using a CRISPR/Cas9 knockout library and an RNA interference (RNAi) library targeting the same set of 6,300 genes with the same number of targeted effectors (sgRNA or shRNA) for each gene. The results showed significant, but not complete, overlap in the essential genes identified by each assay in each cell line indicating that both approaches are effective to identify the majority of essential genes in a cell system. However, analysis did indicate that a small number of essential targets were only identified with CRISPR and certain unique targets seemed to show up only in the RNAi screen results. By combining data from the two screening methodologies, a consistent number of viability genes and pathways could be identified and subsequently validated by independent cell based assays at a very high confirmation rate.

#4355

Elucidation of the different roles of CDK8 and CDK19 in colorectal cancer (CRC) using CRISPR gene editing technology.

Maria J. Ortiz Ruiz,1 Olajumoke Popoola,1 Aurelie Mallinger,1 Sharon Gowan,1 Will Court,1 Gary Box,1 Melanie Valenti,1 Alexis De Haven Brandon,1 Robert Te-Poele,1 Paul Workman,1 Kai Schiemann,2 Cristina Esdar,2 Dirk Wienke,2 Julian Blagg,1 Paul Clarke,1 Suzanne A. Eccles1. 1 _Institute of Cancer Research, Sutton, United Kingdom;_ 2 _Merck, Darmstadt, Germany_.

Background

CDK8 and CDK19 are two highly homologous cyclin-dependent kinases involved in gene transcription via the Mediator complex. Individual kinase module paralogs (CDK8 or CDK19) in complex with cyclin C, Med13 and Med12 regulate transcription through phosphorylation of the C-terminal domain of RNAPol II. Despite their structural similarities, emerging data suggest possible distinct transcriptional regulatory functions. There are few studies of CDK19 function, but CDK8 has been described as an oncogene in CRC, required for β-catenin mediated transcription and tumor cell proliferation. In addition, CDK8 knock down reportedly inhibited the growth of HT29 and Colo205 CRC xenografts.

Results

Recently, we identified a series of 3,4,5-trisubstituted pyridines (exemplified by CCT251545) as small molecule chemical probes of CDK8 and CDK19.1 Although our compounds bind selectively to these closely related biochemical targets, it is not known whether both are equally inhibited, whether inactivation of one (or both) is required for tumor growth control or if there is compensation in terms of kinase function and/or other roles in the Mediator complex. Using single and double CRISPR/Cas9 knockout clones, we aim to elucidate the roles of CDK8 and CDK19 in human CRC cells. We used the SW620 CRC cell line due to its sensitivity in vivo to our CDK8/19 inhibitor CCT251921. Single and double homozygous knockout (KO) clones were generated after CRISPR/Cas9 transfection of plasmids targeting CDK8 and CDK19. KO clones were identified by western blotting and validated by qPCR and exome sequencing analyses. Functional assays were performed in vitro and PD/PK and efficacy studies with CCT251921 in vivo using selected single and double KO clones. Substrates including pRNAIISer5, E2F1Ser375 and pSTAT1Ser727were also quantified by several independent assay platforms in in vitro and ex vivo tumor samples. mRNA profiling of ex vivo samples from PD/PK and efficacy studies was also undertaken.

CRISPR deletion of neither CDK8 (in contrast to literature reports using RNAi in different CRC xenografts) nor CDK19 inhibited the growth of SW620 CRC cells in vitro or in vivo. Interestingly, the CDK19 knockout cells remained responsive to two chemically distinct probes in vitro and/or in vivo, with clear inhibition of the pSTAT1Ser727 engagement biomarker, whereas the CDK8 and CDK8/19 knockouts were far less responsive and showed significantly lower levels of pSTAT1Ser727. Our data suggest that there are certain independent functions of CDK8 and CDK19 that might not be simply interchangeable and that the presence or absence of one may also impact on the behaviour of the other.

References

1.

Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980.

#4356

Identification of the anthrax toxin receptor (ANTXR1) as the high affinity cellular receptor for Seneca Valley Virus (SVV).

Linde A. Miles, J.T. Poirier, Charles M. Rudin. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Small cell lung cancer (SCLC) is an extremely aggressive and almost universally lethal form of lung cancer, which accounts for approximately 15% of all lung cancer diagnoses annually. Novel and more effective therapies are desperately needed for SCLC, as the standard treatment has not improved in almost three decades. Recently, oncolytic viruses have shown strong anti-cancer efficacies in tumors with high levels of selectivity for cancer cells.

The oncolytic picornavirus, Seneca Valley Virus (SVV), has previously been shown to infect and lyse cancer cells with neuroendocrine features, notably SCLC and pediatric brain tumors. Pre-clinical mouse models and early phase clinical trials have confirmed the ability of SVV to home directly to the tumor through the vasculature, specifically infect tumor cells without affecting normal cells, and replicate to high viral titers intratumorally even after the production of neutralizing antibodies. However, the clinical development of SVV has been hindered by the inability to identify patients who would benefit from SVV virotherapy because essential host proteins for viral entry, including the cellular receptor, have not been characterized.

Using genome wide loss-of-function CRISPR screens in the SCLC cell line H446 and haploid cell line HAP1, we identified the anthrax toxin receptor 1 (ANTXR1) as an essential host protein for SVV. Interestingly, ANTXR1 is also one of the receptors for the toxin of Bacillus anthracis and has been previously described as being upregulated in tumor endothelial cells. Secondary screens performed in both cell lines confirmed gene knockout of ANTXR1 conferred resistance to SVV. We established that in multiple permissive SCLC and pediatric cancer cell lines that loss of ANTXR1 protein expression using CRISPR gene knockout leads to a loss of SVV permissivity. Re-expression of ANTXR1 in ANTXR1 knockout cell lines was sufficient to rescue SVV permissivity. Furthermore, the expression of the ANTXR1 protein in two non-permissive SCLC cell lines led to a conversion in SVV permissivity. A direct and high affinity interaction was confirmed between SVV and ANTXR1 using co-immunoprecipitation assays. Additionally, we discovered that ANTXR1 is the major binding determinant for SVV on cells using a binding assay with fluorescently labeled SVV. Lastly we determined using previously published gene expression data from the Cancer Cell Line Encyclopedia (CCLE) that non-permissive lines which do express ANTXR1 can be explained by the presence of an intact and active innate immune response pathway. These findings identify ANTXR1 as the high-affinity cellular receptor for SVV. Furthermore, these data identify a predictive biomarker for SVV permissivity that could lead to defined selection criteria for subsequent clinical trials testing SVV in both SCLC and pediatric brain cancer patients.

#4357

Harnessing system xCT- to target mutant p53 cancer cells.

David SH Liu,1 Matthew Read,1 Klas G. Wiman,2 Sue Haupt,1 Cuong P. Duong,1 Lars Abrahmsen,3 Ygal Haupt,1 Nicholas J. Clemons,1 Wayne A. Phillips1. 1 _Peter MacCallum Cancer Ctr., Melbourne, Australia;_ 2 _Karolinska Institutet, Stockholm, Sweden;_ 3 _Aprea AB, Sweden_.

p53, a critical tumour suppressor is mutated in over half of all human cancers. The loss of wild-type p53 activity together with oncogenic gain-of-function, secondary to aberrant accumulation of mutant p53 protein frequently results in aggressive tumour phenotype, resistance to conventional therapies and poor survival. Therefore, effective therapeutic strategies to target mutant p53 cancer cells remain an urgent and unmet medical need. Here we show that mutant p53 accumulation across multiple tumour types represses the transcription of SLC7A11, a key component of system xCT-, resulting in reduced cystine uptake, lowering endogenous glutathione stores and predisposing cells to oxidative damage. Notably, genetic knockdown or pharmacological inhibition (erastin and sulfasalazine) of system xCT- preferentially induces apoptosis in cancer cells with mutant p53 accumulation. Moreover, we found that APR-246 (PRIMA-1met), a first-in-class reactivator of mutant p53 currently in early clinical trials, depletes cellular glutathione and induces significantly higher amounts of reactive oxygen species in mutant p53 cancer cells compared with normal cells. This leads to lipid peroxidation of mitochondrial membranes and the release of matrix contents, culminating in apoptotic cell death. Conversely, APR-246-induced cytotoxicity could be rescued by cysteine or glutathione replacement, or with lipophilic antioxidants. In extension, we identified and functionally validated SLC7A11 expression as a specific predictive biomarker for APR-246. Importantly, we demonstrate that antagonising system xCT- activity in combination with APR-246 selectively and synergistically inhibit mutant p53 cancer cells. Together, our findings propose that accumulation of mutant p53 protein in cancer cells, through its repressive effects on SLC7A11 expression, creates an 'Achilles heel' that can be targeted by further perturbations of the glutathione pathway.

#4358

Neutralization of BCL2/XL enhances the cytotoxicity of T-DM1 in vivo.

Jason J. Zoeller,1 Roderick T. Bronson,1 Deepak Sampath,2 Joel Leverson,3 Joan S. Brugge1. 1 _Harvard Medical School, Boston, MA;_ 2 _Genentech, San Francisco, CA;_ 3 _AbbVie, Chicago, IL_.

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody-drug conjugate, T-DM1, composed of trastuzumab (T) linked to the cytotoxic maytansinoid (DM1). T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. We previously identified BCL2 upregulation as a critical component and biomarker of the adaptive response to inhibition of PI3K/mTOR or HER2, and thus examined whether BCL2/XL combinatorial strategies could improve the initial efficacy of T-DM1. Here, we demonstrate that combined inhibition of BCL2/XL plus T-DM1 significantly enhances the cytotoxicity of T-DM1 in vivo. The effectiveness of T-DM1 plus BCL2/XL inhibition was evaluated in two patient-derived xenograft (PDX) models of advanced HER2+ER- resistant disease (PDX8;PDX12). Animals were randomized into one of four groups: T-DM1, ABT-737, T-DM1 + ABT-737 or vehicles. Our initial results after a 14d treatment period indicate that combined treatment with T-DM1 and ABT-737, the dual BCL2/XL inhibitor, confers an exceptional tumor cell cytotoxic advantage characterized by widespread elimination of the tumor cells. To evaluate whether ABT-263, the clinically relevant BCL2/XL inhibitor, mimics ABT-737, we randomized animals into one of four groups: T-DM1, ABT-263, T-DM1 + ABT-263 or vehicles. To minimize thrombocytopenia that is induced by ABT-263, we included a fifth group that received pulsed treatment of ABT-263 + T-DM1. Notably, unlike continuous treatment, pulsed administration of ABT-263 reduced weight loss to vehicle levels and allowed recovery of platelets. Evaluation of pathological responses by H&E staining indicated that T-DM1 + ABT-263 mimics T-DM1 + ABT-737. To better distinguish tumor cells from stromal elements, we used epithelial membrane antigen IHC to specifically visualize tumor cells and Trichrome stain to visualize stromal content and scored the tissue sections blindly. ABT-263 had no observable effect. T-DM1 induced a 38.75% and 20% average reduction in tumor cell content in the two PDX models, whereas the combined treatment caused a 74% and 54% average reduction after the 14d treatment period. The loss of tumor cell content was associated with an increased stromal reaction at the tumor bed. T-DM1 treated tumors contained 27.5% and 47.5% average stromal content, whereas combination treated tumors contained 86% and 85.6% average stromal content. Importantly, T-DM1 + pulsed ABT-263 elicited a similar response as continuous treatment in the PDX8 model, but was not as effective in PDX12. The dramatic improvement in tumor regression observed in these preclinical studies, together with the safety benefits of modified dosing of ABT-263, provides substantial rationale for the clinical investigation of this drug combination.

## IMMUNOLOGY:

### Potentiating Immunotherapy Responses with Next Generation Agents and Combinatorial Partners

#4359

Rescue of exhausted CD8 T cells by PD-1 targeted therapies is CD28-dependent.

Alice O. Kamphorst,1 Andreas Wieland,1 Shu Yang,1 Tahseen Nasti,1 Ruan Zhang,2 Daniel L. Barber,1 Bogumila T. Konieczny,1 Lydia Koenig,1 Ke Yu,1 Gabriel Sica,1 Taofeek K. Owonikoko,1 Rathi Pillai,1 Suresh S. Ramalingam,1 Arlene H. Sharpe,3 Gordon J. Freeman,3 Laurence A. Turka,2 Koichi Araki,1 Rafi Ahmed1. 1 _Emory University, Atlanta, GA;_ 2 _Massachusetts General Hospital and Harvard Medical School, Boston, MA;_ 3 _Harvard Medical School, Boston, MA_.

T cell exhaustion was first described in mice during chronic lymphocytic choriomeningitis virus (LCMV) infection and later shown to occur in humans during persistent infections and cancer. Sustained expression of the inhibitory receptor programmed cell death-1 (PD-1) is a hallmark of T cell exhaustion. Therapeutic blockade of PD-1 rescues proliferation, cytokine production and cytotoxicity of exhausted T cells. PD-1 targeted therapies have shown efficacy for multiple cancer types and changed the landscape of cancer treatment, but the requirements for T cell rescue by blockade of the PD-1 pathway remain elusive. T cell activation ensues when positive signals overcome negative signals. CD28 is the most prominent positive co-receptor for T cell activation, and human CD8 T cells become CD28neg upon continuous stimulation. We analyzed tumor specimens from non-small cell lung cancer (NSCLC) patients by flow cytometry and found heterogeneous CD28 expression on tumor-infiltrating CD8 T cells. In most NSCLC patients, CD28neg cells were prevalent among CD8 T cells co-expressing inhibitory receptors (such as PD-1 and Tim-3). To address the role of the CD28/B7 pathway on CD8 T cell rescue by PD-1 targeted therapies, we used the LCMV chronic infection model. When B7 signals were impeded by CTLA-4-Ig or B7 blocking antibodies, blockade of the PD-1 pathway failed to rescue virus-specific CD8 T cells. To investigate a cell intrinsic requirement of CD28 on exhausted CD8 T cells, we used an adoptive transfer strategy of cells genetically deficient in CD28. We found that CD28-deficient LCMV-specific CD8 T cells do not expand after blockade of the PD-1 pathway. Furthermore, to ensure appropriate development and differentiation of CD8 T cells, we used mice in which the CD28 gene can be conditionally deleted. We confirmed that CD28neg exhausted CD8 T cells failed to proliferate after blockade of the PD-1 pathway even when CD28 gene deletion was induced on already exhausted T cells. These data demonstrate that CD28 engagement is required for CD8 T cell rescue, and CD28 status may constitute a predictive biomarker for PD-1 targeted therapies.

#4360

Inhibition of HSP90 enhances T cell-mediated antitumor immune responses through expression of interferon-alpha response Genes.

Rina M. Mbofung,1 Jodi A. McKenzie,1 Shruti Malu,2 Chengwen Liu,1 Leila Williams,1 Weiyi Peng,1 Zhe Wang,1 Satyendra Tripathi,1 Trang Tieu,1 Shuping Zhao,1 Seram Devi,1 Isere Kuiatse,1 Emily Ashkin,1 Leah Bailey,1 Jason Roszik,1 Samir Hanash,1 Timothy Heffernan,1 Richard E. Davis,1 Rodabe N. Amaria,1 Patrick Hwu1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Dana Farber Cancer Instituite, Boston, MA_.

Recently, T cell based immunotherapies have moved to the forefront of cancer immunotherapy with the success of Adoptive T cell therapy (ACT) and Immune checkpoint blockade. ACT, where patients are treated with tumor infiltrating T cells (TILs), conferred a clinical response rate of ~50%. Treatment with anti-CTLA4 therapy, Ipilimumab, conferred response rates of 10-20%, greatly improving the overall survival of patients with advanced melanoma. Despite the encouraging outcomes, there are relatively low response rates coupled with the delay of weeks to months before tumor shrinkage can be appreciated. Thus, understanding mechanisms of resistance to immune therapies, to improve response rates, shorten time to treatment effect and developing predictive biomarkers of response are vital to the care of melanoma patients. In order to identify possible resistance mechanisms to immunotherapy, a high-throughput in vitro screen with 850 different bio-active compounds (Selleckchem), was designed to search for agents that could either increase or decrease the resistance of melanoma tumor cells to T cell mediated killing. Paired tumor samples and TILs from melanoma patients were used to assess which compounds when used to treat the melanoma cell lines can enhance the cytotoxic activity of the TILs against the paired melanoma sample, using a flow cytometry based assay in which active caspase 3 was used as a read out of apoptosis. We identified heat shock protein 90 (HSP90) inhibitors amongst compounds that improved T cell mediated cytotoxicity. We show that treatment with the HSP90 inhibitor ganetespib (Synta) greatly improves T cell mediated cytotoxicity of both human and murine cancer cells lines in vitro. Furthermore, in vivo murine studies using the MC38/gp100 tumor model show that ganestespib in combination with anti-CTLA4, resulted in superior antitumor effect and survival compared to either treatment alone ( Average tumor volume at day 21 of treatment: Vehicle 294.3mm3, α-CTLA4 193 mm3, Ganetespib 237.5 mm3 and Ganetespib + α-CTLA4 105.8 mm3, P < 0.0001) . Microarray analysis of human cell lines treated with ganetespib in vitro revealed an increase in interferon alpha (IFN-α) response genes including IFIT1, IFIT2, IFIT3 and IFIH1. Silencing IFIT2 abrogated the synergy observed with ganetespib treatment and T cell mediated killing, suggesting that the IFN-α response pathway plays an important role in this combination therapy. We are further elucidating the role of these genes in the synergy observed. This will enable the emergence of a new combination therapy of HSP90 inhibitors and anti-CTLA4 for the treatment of melanoma patients that will increase the percentage of patients responding to immunotherapy and achieving long term responses.

#4361

Timing of PD-1 blockade is critical to successful synergy with OX40 costimulation in preclinical mammary tumor models.

David J. Messenheimer, Zipei Feng, Keith W. Wegmann, Shawn M. Jensen, Carlo B. Bifulco, Bernard A. Fox. _Earle A. Chiles Research Institute, Portland, OR_.

With the recent success of cancer immunotherapies targeting specific inhibitory receptors like PD-1 and CTLA-4, there is great interest in how to combine these drugs with other novel therapies targeting costimulatory receptors that could further augment an anti-tumor response. Recently, we observed that orthotopically-transplanted MMTV-PyMT tumor-bearing mice that received anti-OX40 treatment saw a significant delay in tumor growth (p < 0.001) compared to untreated mice. However when combined concurrently with anti-PD-1 blockade, instead of a synergistic effect, we noted no additive benefit, and in fact saw a significant attenuation (p < 0.05) in survival. These results fit a similar pattern to what we have seen in the 4T1 tumor model, where the anti-tumor effect provided by vaccine plus anti-OX40, was significantly attenuated (p < 0.001) when anti-PD-1 was added concurrently.

We hypothesized that treating with anti-PD-1 antibody would be more effective during the contraction phase of the T cell boost generated by anti-OX40. Thus we delayed anti-PD-1 treatment until after anti-OX40 dosing was complete and saw a significant delay (p < 0.01) in tumor growth and subsequent increase (p < 0.01) in survival in the PyMT transplant model (compared to anti-OX40 alone), with some of the tumors reaching full regression. We had also previously seen a similar significant antitumor effect (p < 0.001) in the 4T1 tumor model. These results were reproduced using delayed treatment with an anti-PD-L1 antibody combined with anti-OX40, demonstrating that blocking either side of the PD-1-PD-L1 interaction is sufficient. Also supporting this hypothesis, we noted a significant increase (p < 0.05 compared to untreated) in PD-1 expression on CD4+ T cells during and after anti-OX40 treatment.

Investigating the effects of the concurrent combination, we noticed a striking increase in IFN-γ in the serum compared to treatment with single agent (p < 0.001 after 3 doses). Serum levels of other cytokines TNF, IL-4, IL-6, and IL-10 were also elevated in the combination treated group compared to anti-OX40 alone. Interestingly, we observed a large increase in PD-L1 expression on both CD4+ and CD8+ T cells and we also noted significant increases (p < 0.05) in inhibitory receptors LAG3, TIM3 and CTLA4 on CD4+ and CD8+ splenic T cells. These may provide additional escape mechanisms for the tumor to evade immune destruction and potentially offer other targets to enhance combination therapy.

Our results demonstrate that the sequence of antibody treatment targeting both costimulatory and inhibitory receptors is critical to success of the combined therapy. These data offer a strong rationale for delaying PD-1 blockade until after costimulation has provided an initial immune boost.

#4362

T cell repertoire diversification is associated with immune related toxicities following immune checkpoint inhibition in metastatic cancer patients.

David Y. Oh,1 Jason Cham,1 Li Zhang,1 Grant Fong,1 Mark Klinger,2 Malek Faham,2 Lawrence Fong1. 1 _University of California, San Francisco, San Francisco, CA;_ 2 _Adaptive Biotechnologies, South San Francisco, CA_.

Immune checkpoint inhibitors can induce clinical responses to a broad range of tumor types by presumably enhancing T cell responses against the tumor. These treatments are also associated with certain side effects that are also thought to be immune mediated (termed immune-related adverse events, or IRAEs). The mechanism by which IRAEs occur, and biomarkers that may predict IRAE development, are unknown. In prior work (Cha et al. Sci Transl Med 2014), we demonstrated that treating metastatic cancer patients with checkpoint inhibitors blocking CTLA-4 can lead to T cell repertoire diversification. However, we found that prolonged survival was not associated with the induction of high frequency clonotypes, but rather the maintenance of a pre-existing T cell response. Here, we examined whether treatment-induced TCR diversification is related to another clinical outcome: the development of IRAEs. We find that IRAEs are specifically associated with a more diverse T cell repertoire and with an increase in the number of T cell clonotypes, including the generation of de novo clones. This broadening of the circulating T cell repertoire occurred early with treatment, preceding the development of IRAEs. While IRAE patients demonstrate markedly greater diversity in their CD4+ T cells, they demonstrate a greater degree of change in clonal frequencies in their CD8+ T cells after ipilimumab, which may have key implications in the pathogenic mechanism of IRAE development. Finally, clinical response to checkpoint blockade is also associated with increased diversity. These results indicate that immune repertoire diversity following immune checkpoint inhibition can be both detrimental and beneficial to patients with metastatic cancer.

#4363

Loss of PTEN promotes resistance to T cell-mediated immunotherapy.

Weiyi Peng,1 Jie Qing Chen,1 Chengwen Liu,1 Shruti Malu,1 Caitlin Creasy,1 Michael Tetzlaff,1 Chunyu Xu,1 Jodi McKenzie,1 Chunlei Zhang,1 Xiaoxuan Liang,1 Leila Williams,1 Wanleng Deng,1 Guo Chen,1 Rina Mbofung,1 Alexander Lazar,1 Carlos Torres-Cabala,1 Zachary Cooper,1 Pei-Ling Chen,1 Trang Tieu,1 Stefani Spranger,2 Xiaoxing Yu,1 Chantale Bernatchez,1 Marie-Andree Forget,1 Cara Haymaker,1 Rodabe Amaria,1 Jennifer McQuade,1 Isabella Glitza,1 Tina Cascone,1 Haiyan Li,1 Lawrence Kwong,1 Timothy Heffernan,1 Jianhua Hu,1 Roland Bassett,1 Marcus Bosenberg,3 Scott Woodman,1 Willem Overwijk,1 Gregory Lizée,1 Jason Roszik,1 Thomas Gajewski,2 Jennifer Wargo,1 Jeffrey Gershenwald,1 Laszlo Radvanyi,1 Michael Davies,1 Patrick Hwu1. 1 _M.D. Anderson Cancer Center, Houston, TX;_ 2 _University of Chicago, Chicago, IL;_ 3 _Yale University School of Medicine, New Haven, CT_.

T cell-mediated immunotherapies are promising cancer treatments. However, most patients still fail to respond to these therapies. The molecular determinants of immune resistance are poorly understood. Here, we interrogated the role of loss of expression of the tumor suppressor, PTEN, in immune resistance.

In preclinical studies, we found that silencing PTEN in tumor cells inhibited T cell-mediated tumor killing and decreased T cell trafficking into tumors. In clinical studies, we observed that tumors with loss of PTEN had significantly less CD8+ T cell infiltration than PTEN-present tumors. In addition, 26% of melanomas that did not yield successful TIL growth demonstrated PTEN loss, which was more frequent than was observed in tumors that yielded successful TIL growth (11%). We further validated the association between reduced number and impaired function of TIL with PTEN loss using another independent cohort, TCGA dataset for SKCM. More importantly, we analyzed clinical outcomes of metastatic melanoma patients treated with the FDA-approved anti-PD-1 antibodies. Our analysis demonstrates that a greater reduction in tumor burden was achieved by PD-1 blockade in PTEN present patients, when compared with PTEN absent patients.

To decipher the factors mediating the immunosuppressive effects of PTEN loss, we determined the expression profiles of tumor cells with or without PTEN expression. Our results indicated that PTEN loss increased the production of immunosuppressive factors, including CCL2 and VEGF. Anti-VEGF blocking antibody improved anti-tumor activity of transferred tumor-reactive T cells and enhanced tumor infiltration of transferred T cells in PTEN-silenced tumors. These results suggest that loss of PTEN can facilitate the resistance of T cell-mediated immune responses by increasing the expression of immunosuppressive factors.

Given that PTEN loss results in activation of the PI3K pathway, we evaluated the efficacy of immunotherapy in combination with a selective PI3Kβinhibitor to treat spontaneously developed BRAF mutant, PTEN null melanomas in genetically engineered mouse models. Our result showed that the combination of PI3Kβ inhibitor and anti-PD-1 significantly delayed tumor growth in tumor-bearing mice. Mice treated with this combination had a median survival time of 28 days, which is longer than the survival time of mice treated with either therapy. Increased numbers of T cells at tumor sites were found in mice receiving the combination therapy compared with mice receiving either agent alone.

Taken together, our results demonstrate that PTEN loss contributes to the generation of immunosuppressive tumor microenvironment. Notably, this study provides the first direct clinical evidence to support the association between PTEN loss and poor clinical outcome in immunotherapy treated patients. In addition, our study indicates that inhibition of the PI3K-AKT pathway can improve the efficacy of immunotherapy in cancer.

#4364

Adenosine A2a receptor blockade as a means of enhancing immune checkpoint inhibition and adoptive T-cell therapy.

Robert D. Leone, Judson M. Englert, Chih-Hsien Cheng, Jiayu Wen, Min-Hee Oh, Im-Hong Sun, Chirag Patel, Ian A. Bettencourt, Jonathan D. Powell. _Sidney Kimmel Comprehensive Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, MD_.

Adenosine signaling through the A2a receptor (A2aR) has pleiotropic immunosuppressive effects on a broad range of immune cells. Extracellular adenosine levels, which are typically very low in normal tissues, are elevated in the tumor microenvironment, allowing increased signaling through the A2aR. Previously, our lab and others have shown increased rejection of tumors in A2aR-null mice, demonstrating that adenosine signaling through A2aR limits immunologic response to tumors.

Present work in our lab demonstrates the ability of a potent and specific, orally administered A2aR antagonist, CPI-444, to enhance immunologic response in mice. First, A2aR blockade with CPI-444 (1 mg/kg) in the peri-vaccination period led to significant expansion of antigen-specific T cells over untreated controls. Interestingly, antigen-specific CTLs from CPI-444 treated mice showed significantly lower expression of inhibitory checkpoint receptors, including PD-1 and TIM-3. Second, in an MC38 tumor model, daily treatment with CPI-444 (100 mg/kg) led to tumor growth suppression and survival benefit compared with vehicle-treated controls. Notably, tumor infiltrating regulatory T cells had significantly lower CTLA-4 and FoxP3 expression in CPI-444 treated mice.

These initial immunologic findings of enhanced early CTL expansion and suppression of inhibitory co-signaling have important implications for applying A2aR blockade to immunotherapy in cancer. First, given the ability of CPI-444 to augment CTL expansion, we investigated its application to a therapeutic adoptive T cell transfer system using OVA-expressing B16 melanoma cell line. In mice with established tumors, daily CPI-444 (100 mg/kg) treatment initiated on the day of adoptive transfer of activated OVA-specific OT-I T cells led to suppressed tumor growth and increased survival versus vehicle treated controls. Second, in light of our data demonstrating its ability to suppress PD-1 in CTLs, we theorized that CPI-444 may lower the threshold for effective anti-PD-1 therapy and achieve synergistic effects as combination therapy. Indeed, daily CPI-444 (100 mg/kg) treatment combined with anti-PD-1 treatment showed robust antitumor effect in the CT26 tumor model, with complete tumor rejection in 7/9 mice (vs. 2/10 mice in the anti-PD-1 alone group). Survival was markedly improved in mice receiving combination treatment versus either CPI-444 or anti-PD-1 alone. In as much as increased adenosine in the tumor microenvironment is partly a consequence of dysregulated cancer cell metabolism, we hypothesized that targeting tumor metabolism would enhance the efficacy of A2aR antagonism. To this end, inhibition of tumor glutamine metabolism potently augmented the efficacy of CPI-444 in a CT26 model. Overall, our data suggest that A2aR antagonism can enhance the efficacy of immunotherapy in the form of checkpoint blockade and adoptive cellular therapy.

#4365

A phase II trial of high-dose Interleukin-2 (HDIL-2) with recombinant MAGE-A3 protein combined with adjuvant system AS15 in patients with unresectable or metastatic melanoma.

Jennifer Leigh McQuade,1 Carlos Antonio Torres-Cabala,1 Rashmi Murthy,1 Marihella L. James,1 Jade Homsi,2 Luis M. Vence,1 Wen-Jen Hwu1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Arizona Cancer Center, Tucson, AZ_.

Background: As shown in a phase III study, active immunization against gp100 tumor antigen can significantly potentiate the antitumor activity of HDIL-2 as well as improve the disease-specific survival of patients (pts) with metastatic melanoma (MM) (Schwartzentruber NEJM 2011). In addition, a phase II study of MAGE-A3 immunotherapy demonstrated modest clinical benefit in pts with MM, and identified a gene signature (GS) associated with response (Kruit JCO 2013). Hypothesis: We hypothesized that the combination of HDIL-2 and MAGE-A3 immunotherapy would improve the clinical efficacy with reduced toxicity in pts with MM. We also wanted to evaluate whether pretreatment GS is predictive of response to the combination. Methods: We conducted a phase II trial of MAGE-A3 immunotherapy and HDIL-2 in pts with MAGE-A3 positive MM (NCT01266603). Pts' archived or fresh tumor tissue was screened for MAGE-A3 expression using RT-PCR. Mandatory baseline biopsy was performed for gene expression profiling for the previously described GS by RT-PCR. Optional tumor biopsies were performed at wk 8 and at end of treatment (EOT). Serum and PBMC were collected on d 1 of each cycle, following cycle 1 of HDIL-2, and at EOT for circulating T-cell subsets, interferon and interferon-induced factors. In the induction phase, 300 μg of recMAGE-A3 and AS-15 immunostimulant were given every 2 wk x 6, and then every 3 wk x 6. HDIL-2 was initiated on the day following MAGE-A3 immunotherapy on wk 1,3,9,11,18,21,27 and 30 at 720,000 IU/kg every (q) 8 hr for up to 14 doses/cycle. Following completion of HDIL-2, pts who remained on study were continued on maintenance phase with MAGE-A3 immunotherapy alone, q 6 wk x 4, then q 12 wk x 4, and then q 24 wk x4. Response was assessed by RECIST v1.1 every 8 wk. Results: The combination of MAGE-A3 immunotherapy and HDIL-2 was well tolerated, with expected toxicities from HDIL-2. Sixteen pts were evaluable for response. Four pts had partial response (PR) as the best response during induction phase: 1 pt was rendered free of disease after resection, and remaining 3 pts achieved complete response (CR) during maintenance phase. The overall response rate was 25%, and an additional 6 patients had durable stable disease for a disease control rate of 63%. GS was not predictive of response. Conclusions: The combination of HDIL-2 and MAGE-A3 immunotherapy shows robust antitumor activity compared to historical HDIL-2 monotherapy and a comparable safety profile. MAGE-A3 immunotherapy maintenance was able to convert some PRs to durable CRs and thus may be a strategy to limit exposure to the toxicities of HDIL-2. GS is not predictive of response to the combination therapy. Ongoing exploration of the tumor microenvironment and circulating immune biomarkers as predictors of response will be presented.

Funding source: GlaxoSmithKline Biologicals SA

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Genomic Alterations and Their Functional Consequences

#4366

Identifying novel recurrent mutations reveals candidate actionable mutations.

Matthew T. Chang,1 Sizhi Paul Gao,1 Tripti Shrestha Bhattarai,1 Tambudzai Shamu,1 Cyriac Kandoth,1 Saurabh Asthana,2 Jocelyn S. Chapman,2 JianJiong Gao,1 Nicholas D. Socci,1 Adam B. Olshen,2 David M. Hyman,1 Michael F. Berger,1 David B. Solit,1 Nikolaus D. Schultz,1 Barry S. Taylor1. 1 _Memorial Sloan Kettering, New York, NY;_ 2 _UCSF, San Francisco, CA_.

Mutational hotspots indicate selective pressure across a population of tumor samples and drive the dysregulation of proliferation, invasion, and apoptosis in many human tumors. We recently performed a comprehensive analysis of hotspot mutations in 11,000 retrospectively characterized primary untreated cancers, revealing widespread lineage diversity and mutational specificity. Yet, as broad-based clinical sequencing has begun to guide the routine care of patients, most clinically sequenced cancer patients present with recurrent or metastatic solid tumors. Furthermore, such tumors likely possess distinct mutational patterns driven by prior treatment exposure or treatment-associated evolution after diagnosis that are undetectable in primary untreated samples. We therefore sought to expand our analysis to incorporate such prospectively characterized active cancer patients as part of an ongoing initiative at Memorial Sloan Kettering Cancer Center. With nearly 10,000 patients profiled thus far, this growing prospective dataset with extensive clinical and treatment history is a transformative resource and the largest of its kind. Here, we combined these prospective data with retrospective studies to analyze the somatic mutational landscape of 21,000 cancer patients across 74 cancer types representing the broadest range of human malignancies characterized to date. We developed and applied a biologically and statistically principled computational model to identify hotspots mutated more frequently than would be expected in the absence of selection. This analysis uncovered hundreds of new biologically and therapeutically relevant candidate driver mutations for which targeted agents are currently being investigated in early phase trials. Indeed, we identify several hotspots in key effectors of PI3K and MAPK signaling with differential response in vitro to current investigational therapies suggesting mutant allele-specific therapeutic decisions may be warranted. In total, over half of all hotspot mutations identified were novel, including several novel hotspots in small GTPase RAC1 that are lineage specific and functionally distinct. Understanding the distinct molecular function of these mutations, and the specific mutant alleles at individual codons, is a necessary translational prerequisite for enabling precision oncology through clinical decision support for patients sequenced at the point of care.

#4367

Accelerating prediction of tumor vulnerabilities using next-generation cancer models.

Yuen-Yi Tseng,1 Andrew Hong,2 Paula Keskula,1 Shubhroz Gill,1 Jaime Cheah,3 Grigoriy Kryukov,1 Aviad Tsherniak,1 Francisca Vazquez,1 Glenn Cowley,1 Coyin Oh,1 Anson Peng,1 Abeer Sayeed,1 Rebecca Deasy,1 Peter Ronning,1 Philip Kantoff,2 Levi Garraway,2 Mark Rubin,4 Calvin Kuo,5 Sidharth Puram,6 Adi Gazdar,7 Filemon Dela Cruz,8 Adam Bass,2 Nikhil Wagle,2 Keith Ligon,2 Katherine Janeway,9 David Root,1 Stuart Schreiber,1 Paul Clemons,1 Aly Shamji,1 William Hahn,1 Todd Golub,1 Jesse S. Boehm1. 1 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _Massachusetts Institute of Technology, Cambridge, MA;_ 4 _Weill Cornell Medical College, New York, NY;_ 5 _Stanford University, Palo Alto, CA;_ 6 _Massachusetts General Hospital, Boston, MA;_ 7 _University of Texas Southwestern Medical Center, Dallas, TX;_ 8 _Columbia University, New York, NY;_ 9 _Children's Hospital, Boston, MA_.

The mapping of cancer genomes is rapidly approaching completion. The genomic information encoded by individual patients' tumors should, in principle, provide a guide for predicting dependencies, but our ability to do so is suboptimal. The challenge stems from the absence of clinical data relating genotypes with dependencies since most cancer mutations are rare and our arsenal of cancer drugs is incomplete. If it was possible to build a preclinical 'cancer dependency map' at a scale that captured the genomic diversity of cancer (for instance, models of all genotypes tested for genetic and small-molecule dependencies), it should be feasible to improve dependency predictions. New technologies (e.g. CRISPR/Cas9 libraries) make such an effort now feasible. However, we lack a sufficient diversity of cancer models derived directly from patient samples to reflect the genetic diversity of cancer and the ability to systematically create functional data for each cancer patient to expand the map.

In an attempt to overcome these obstacles, we have established an industry-scale pipeline to generate new cancer models directly from patient samples, a "Cancer Cell Line Factory". We have processed over 620 samples from 400 patients across 16 cancer types through this pipeline with a 25% success rate overall. To optimize conditions for each tumor type, we have systematically compared published cell line generation methods with standard approaches and captured all information with a data management system that will enhance the ability to predict optimal ex vivo propagation conditions for future samples. In all, we report the successful derivation of over 100 new genomically confirmed cancer and normal cell lines, including a series of unique pediatric cancer models derived from rare tumors.

We hypothesized that novel patient-derived cultures could be used to enhance dependency predictions. To test this hypothesis, we tested dependencies of 65 of these novel cultures against an identical set of 440 small molecules that were previously tested against 860 existing cancer cell lines. Our results suggest that dependency data generated with novel cell cultures is potentially backwards-compatible with existing small molecule dependency datasets. Finally, we demonstrate proof-of-concept that such new models can successfully used in CRISPR-Cas9 screens and integrate results with small molecule sensitivities to uncover CDK4 and XPO1 dependencies in a rare pediatric undifferentiated sarcoma. In aggregate, these proof-of-concept studies demarcate a path by which pre-clinical dependency maps may enhance clinical dependency predictions from genomic data alone.

#4368

High-throughput phenotyping of lung cancer somatic mutations.

Alice H. Berger,1 Angela N. Brooks,2 Xiaoyun Wu,1 Yashaswi Shrestha,1 Candace Chouinard,1 Federica Piccioni,1 Mukta Bagul,1 Atanas Kamburov,1 Marcin Imielinski,3 Larson Hogstrom,4 Cong Zhu,1 Xiaoping Yang,1 Sasha Pantel,1 Ryo Sakai,5 Nathan Kaplan,6 David Root,1 Rajiv Narayan,1 Ted Natoli,1 David Lahr,1 Itay Tirosh,1 Pablo Tamayo,7 Gad Getz,1 Bang Wong,1 John Doench,1 Aravind Subramanian,1 Todd R. Golub,1 Matthew Meyerson,1 Jesse S. Boehm1. 1 _Broad Institute, Cambridge, MA;_ 2 _University of California, Santa Cruz, CA;_ 3 _Weill Cornell Medical College, New York, NY;_ 4 _MIT, Cambridge, MA;_ 5 _KU Leuven, Belgium;_ 6 _Dana-Farber Cancer Institute, Boston, MA;_ 7 _University of California, San Diego, CA_.

Recent cancer genome sequencing and analysis has identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood, limiting the use of this genetic knowledge for clinical decision-making. Here we describe a new high-throughput approach, expression-based variant impact phenotyping (eVIP), which uses gene expression changes to infer somatic mutation impact. We generated a lentiviral expression library representing 53 genes and 194 somatic mutations identified in primary lung adenocarcinomas. Next, we introduced this library into A549 lung adenocarcinoma cells and 96 hours later performed gene expression profiling using Luminex-based L1000 profiling. We built a computational pipeline, eVIP, to compare mutant and wild-type expression signatures to infer whether variants were gain-of-function, change-of-function, loss-of-function, or neutral. Overall, eVIP identified 69% of mutations as impactful whereas 31% appeared functionally neutral. A very high rate, 92%, of missense mutations in the KEAP1 and STK11 tumor suppressor genes were found to inactivate or diminish protein function. As a complementary approach, we assessed which mutations are epistatic to EGFR or capable of initiating xenograft tumor formation in vivo. A subset of the impactful mutations identified by eVIP could induce xenograft tumor formation in mice and/or confer resistance to cellular EGFR inhibition. Among these mutations were 20 rare or non-canonical somatic variants in clinically-actionable or -relevant oncogenes including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and PIK3CA E600K. eVIP can, in principle, characterize any genetic variant, independent of prior knowledge of gene function. Further application of eVIP should significantly advance the pace of functional characterization of mutations identified from genome sequencing.

#4369

Genome-wide copy number dependency analysis identifies partial copy loss of SF3B1 as a novel cancer vulnerability.

Brenton R. Paolella,1 William J. Gibson,1 Laura M. Urbanski,1 John A. Alberta,1 Travis I. Zack,1 Pratiti Bandopadhayay,1 Caitlin A. Nichols,1 Pankaj K. Agarwalla,2 Meredith S. Brown,1 Rebecca Lamothe,1 Yong Yu,3 Peter S. Choi,1 Esther A. Obeng,1 Dirk Heckl,1 Benjamin L. Ebert,1 Guo Wei,4 Belinda Wang,1 William C. Hahn,1 Francisca Vazquez,4 Barbara A. Weir,4 Charles D. Stiles,1 Robin Reed,3 Rameen Beroukhim1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Harvard Medical School, Boston, MA;_ 4 _Broad Institute of MIT and Harvard, Cambridge, MA_.

Genomic instability is a hallmark of cancer resulting in widespread somatic copy number alterations. We integrated a genome-scale shRNA viability screen and copy number profiles from 179 cancer cell lines to perform an unbiased analysis of copy-number associated gene-dependency interactions. We found most copy-number associated gene dependencies result from losses of genetic material rather than gains. Strikingly, the most enriched class of these dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes. Hemizygous loss of CYCLOPS genes sensitizes cancer cells to their further suppression. One of the "top hits" from the analysis was the pre-mRNA splicing factor SF3B1, which is also frequently mutated in cancer. We then sought to evaluate SF3B1 as a CYCLOPS gene. Cancer cells with hemizygous SF3B1 copy-loss were uniquely sensitive to partial SF3B1 suppression by RNAi compared to cells with normal SF3B1 gene dosage. Mechanistically, cancer cells harboring partial SF3B1 copy-loss lack a reservoir of excess SF3b complex, a precursor complex of the U2 snRNP, which protects cells with normal SF3B1 copy number from cell death upon SF3B1 suppression. Our data highlight the prevalence of CYCLOPS dependencies in cancer and establish the spliceosome as a frequent CYCLOPS target. Further, these data indicate targeting wild-type SF3B1 as a novel cancer dependency in cells with hemizygous SF3B1 copy-loss.

#4370

Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between PKMYT1 and WEE1 in patient-derived glioblastoma stem-like cells.

Chad Toledo,1 Ding Yu,1 Pia Hoellerbauer,1 Ryan Davis,1 Ryan Basom,1 Emily Girard,1 Philip Corrin,1 Hamid Bolouri,1 Jerry Davison,1 Qing Zhang,1 Do-Hyun Nam,2 Jeongwu Lee,3 Steven Pollard,4 Jeffery Delrow,1 Bruce Clurman,1 James Olson,1 Patrick Paddison1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _Institute for Refractory Cancer Research, Samsung Medical Center, Seoul, Republic of Korea;_ 3 _Lerner Research Institute, Cleveland Clinic, Cleveland, OH;_ 4 _Edinburgh CRUK Cancer Research Centre and MRC Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, United Kingdom_.

Glioblastoma multiforme (GBM) is the most aggressive and common form of brain cancer in adults. There are currently no effective therapies for GBM. Even with standard of care treatments, such as surgery, radiation, and chemotherapy, ~90% of adult patients die within 2 years of diagnosis. To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 "knockout" (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality. From a biological standpoint, our results help re-discover PKMYT1 function in human cells. From a cancer standpoint, these results suggest that targeting PKMYT1 is a glioma-lethal gene and that tandem or sequential use of PKMYT1 and WEE1 inhibitors may illicit therapeutic responses in GBM. In addition to these results we will also present retest data for other candidate glioma-lethal genes and networks in multiple patient samples.

#4371

Integrated molecular characterization of pheochromocytoma and paraganglioma including a novel, recurrent and prognostic fusion gene.

Lauren Fishbein,1 Ignaty Leshchiner,2 Vonn Walter,3 Ludmila Danilova,4 A Gordon Robertson,5 Amy Johnson,6 Tara Lichtenberg,7 Bradley A. Murray,2 Hanse K. Ghayee,8 Tobias Else,9 Shiyun Ling,10 Stuart R. Jefferys,3 Aguirre A. de Cubas,11 Brandon Wenz,12 Esther Korpershoek,13 Antonio L. Amelio,3 Liza Makowski,14 W Kimryn Rathmell,11 Anne-Paule Gimenez-Roqueplo,15 Thomas J. Giordano,16 Sylvia L. Asa,17 Arthur S. Tischler,18 The Cancer Genome Atlas Pheochromocytoma and Paraganglioma Analysis Working Group, Karel Pacak,19 Katherine L. Nathanson,12 Matthew D. Wilkerson20. 1 _Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 2 _The Eli and Edythe L. Broad Institute of MIT and Harvard University, Cambridge, MA;_ 3 _Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC;_ 4 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University, Baltimore, MD;_ 5 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 6 _Department of Nutrition, University of North Carolina, Chapel Hill, NC;_ 7 _The Research Institute at Nationwide Children's Hospital, Columbus, OH;_ 8 _Department of Medicine, University of Florida, Gainesville, FL;_ 9 _Department of Internal Medicine, University of Michigan, Ann Arbor, MI;_ 10 _University of Texas MD Anderson Cancer Center, Houston, TX;_ 11 _Lineberger Comprehensive Cancer Center, Department of Medicine, University of North Carolina, Chapel Hill, NC;_ 12 _Department of Medicine, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA;_ 13 _Department of Pathology, Erasmus MC University Medical Center; ENSAT, Rotterdam, Netherlands;_ 14 _Department of Nutrition, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC;_ 15 _INSERM; Hôpital Européen Georges Pompidou; Paris-Descartes University; European Network for the Study of Adrenal Tumors (ENSAT), Paris, France;_ 16 _Department of Pathology, University of Michigan Medical School, Ann Arbor, MI;_ 17 _Department of Pathology, University Health Network, University of Toronto, Toronto, Ontario, Canada;_ 18 _Department of Pathology and Laboratory Medicine, Tufts Medical Center, Boston, MA;_ 19 _Endocrinology and Tumor Genetics Affinity Group, Eunice Kennedy Shriver NICHD, NIH, Bethesda, MD;_ 20 _Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina; Department of Anatomy, Physiology, & Genetics, Uniformed Services University, Bethesda, MD_.

Pheochromocytomas (PCC) and paragangliomas (PGL) are tumors of the autonomic nervous system; 25% are metastatic or locally aggressive. Characterization of the inherited basis of disease has identified a variety of underlying germline mutations; however, understanding of somatic alterations remains limited. As part of The Cancer Genome Atlas, we performed the most comprehensive genomic characterization of PCC/PGL to date, by applying eight genomic profiling assays to 173 patients. Despite having a low overall mutation rate per tumor, we observed remarkable diversity in genomic alterations. 27% of patients had a pathogenic germline mutation among eight known familial PCC/PGL susceptibility genes, thus making PCC/PGL the tumor type with the greatest rate of germline mutations in The Cancer Genome Atlas. 38% of patients possessed a somatic driver mutation across 12 genes. RET, NF1 and VHL were affected by both germline and somatic mutation, albeit with different mutation site tendencies. We identified a new somatic driver gene, CSDE1, which had coordinated intron splicing defects, DNA copy number loss, and RNA under-expression, suggesting a loss of function consequence. Most notably, we discovered the first fusion genes in PCC/PGL from RNA and DNA sequencing (7% of patients), demonstrating for the first time that inter-chromosomal translocation and gene fusion is a method of molecular pathogenesis in this disease. Recurrent, novel MAML3 fusion genes spanned three isoforms and were activating based on over-expression of MAML3 and on fusion transcript exonic expression. MAML3 fusion positive tumors had concomitant dual focal DNA amplification of the fusion gene partners and a significantly divergent methylation profile. Another novel driver gene in PCC/PGL, BRAF, was affected by a hotspot somatic mutation and by an activating fusion gene. Through integrated platform analysis, four statistically significant molecular subtypes of PCC/PGL were detected and found to represent divergent molecular etiology – the kinase signaling subtype, the pseudohypoxia subtype, the Wnt-altered subtype, and the cortical admixture subtype. In particular, MAML3 fusions and CSDE1 mutations defined the new Wnt-altered expression subtype of PCC. Adding to the limited set of prognostic markers in PCC/PGL, three molecular markers were positively associated with clinically aggressive disease: germline mutations in SDHB, somatic mutations in ATRX and fusions involving MAML3. Nearly all somatic driver mutations, germline driver mutations and fusion genes were mutually exclusive across the cohort and covered a large portion of the cohort (69%). Our study provides important novel insights into PCC/PGL biology and identifies potential markers for aggressive disease and therapeutic intervention.

#4372

MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism.

Lori Ramkissoon,1 Pratiti Bandopadhayay,1 Payal Jain,2 Guillaume Bergthold,1 Adam Resnick,2 Rameen Beroukhim,1 Keith Ligon1. 1 _Dana-Farber Cancer Institute, Boston, MA;_ 2 _Children's Hospital of Philadelphia, Philadelphia, PA_.

Diffuse pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children. While BRAF mutations and MYBL1 rearrangements have recently been identified as diagnostic and treatment related oncogenic drivers in pediatric gangliogliomas and diffuse astrocytomas, respectively, for the majority of diffuse PLGGs the oncogenic driver(s) remain unknown. Recent efforts to identify new oncogenic drivers lack sufficient power to determine the true frequency of alterations in genes or to associate specific alterations with rare histological subtypes, such as Angiocentric gliomas (AGs). To address this issue, we performed genomic analysis of new and published data from 249 PLGGs including 19 AGs. We identified MYB-QKI fusions as a specific, recurrent, single candidate driver event in AGs. Although MYB is expressed during early brain development in a subset of progenitor cells, it is not known to play a role in normal cortical brain where AGs frequently occur. In vitro functional studies show MYB-QKI rearrangements promote tumorigenesis in mouse neural stem cells (mNSCs) through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression, and hemizygous loss of the tumor suppressor QKI. Expression of the MYB-QKI fusion increases mNSC proliferation and activates known MYB target genes including KIT and CDK6. H3K27ac enhancer profiling of AG tumors demonstrate active enhancer elements are translocated proximally to the MYB promoter while in vitro promoter assays confirmed that QKI enhancer sequences activate the MYB promoter. Together with functional studies demonstrating that MYB-QKI can also bind and activate the MYB promoter, these data support a positive auto-regulatory feed-back loop model in which active MYB-QKI is able to drive its own expression through enhanced MYB-promoter-activation. Furthermore, when injected orthotopically into immunodeficient mice, expression of truncated MYB or MYB-QKI is sufficient to induce tumor formation that recapitulate histologic and immunophenotypic characteristics of AGs. Analysis of AG specific MYB-QKI fusion represents the first example of how a single driver rearrangement simultaneously transforms cells via three genetic and epigenetic mechanisms in a cancer.

### Oncogenic Cell Signaling: Mechanisms and Translational Insight

#4373

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ.

Sathiya Dharmaiah,1 Lakshman Bindu,1 Peter Frank,1 William Gillette,1 Dominic Esposito,1 Dwight Nissley,1 Frank McCormick,2 Andrew Stephen,1 Dhirendra Simanshu1. 1 _Frederick National Laboratory for Cancer Research, Frederick, MD;_ 2 _Frederick National Laboratory for Cancer Research and University of California San Francisco, San Francisco, CA_.

KRAS is the most frequently mutated member of RAS superfamily in all human cancers. Post-translational farnesylation and methylation of KRAS at its C-terminal end plays an important role in its trafficking to proper subcellular compartments for cell signaling events. PDEδ acts as a chaperone for farnesylated members of the RAS superfamily and plays a key role in their trafficking from the place of biosynthesis or post-translational modification to a transport vesicle or plasma membrane. We solved the structures of wild-type full-length KRAS4b (farnesylated and methylated) in complex with PDEδ in two different crystal forms at 2 Ang resolution. Our structure showed that only the last six amino acids of hypervariable region (and not the G-domain) of KRAS4b interact with PDEδ. Farnesyl and methyl groups present on Cys185 bind tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we could see all amino acids present in the hypervariable region of KRAS4b, thus providing atomic details of hypervariable region of KRAS4b for the first time. Comparison of the two crystal forms suggests how PDEδ could bind to both farnesylated as well as geranylgeranylated KRAS4b. The structure provides the rationale for nucleotide-independent binding of KRAS4b to PDEδ and its interaction with other farnesylated but not palmitoylated members of RAS superfamily.

#4374

Activation mechanism of oncogenic C-helix shifting mutations in BRAF, EGFR, and HER2.

Scott Foster,1 Dan Whalen,1 Aysegul Ozen,1 Matt Wongchenko,1 JianPing Yin,1 Ivana Yen,1 Gabriele Schaefer,1 John Mayfield,2 Juliann Chmielecki,2 Phil Stephens,2 Lee Albacker,2 Yibing Yan,1 Kyung Song,1 Georgia Hatzivassiliou,1 Charles Eigenbrot,1 Christine Yu,1 Andrey Shaw,1 Gerard Manning,1 Nicholas Skelton,1 Sarah Hymowitz,1 Shiva Malek1. 1 _Genentech, Inc., South San Francisco, CA;_ 2 _Foundation Medicine, Cambridge, MA_.

Kinase domain mutations are frequent drivers of many different types of cancer. While the effect of hotspot point mutations (such as BRAF V600E or EGFR L858R) is well described, the mechanism of deletion mutations, such as the recurrent EGFR exon 19 deletions is not fully understood. In this work, we have discovered and characterized analogous deletions in BRAF mutant patient samples of varying tumor types, with the highest frequency (15 patients) in pancreatic cancer representing ~5% of KRAS wildtype patients in this data set. In addition, we have identified additional patients (3) with similar deletions in HER2 that have been previously reported at low frequency in breast cancer. The crystal structure of the most frequent BRAF deletion, molecular modeling of lower frequency BRAF deletions, and extensive molecular modeling in conjunction with molecular dynamic simulations of the most frequent EGFR deletion highlight the commonality of the mechanism of activation of this class of oncogenic alterations. These deletions truncate the loop between the β3 strand and αC-helix (β3-αC loop) of the kinase domain, forcing αC into the active ("in") conformation, and constitutively activating these kinases. Similar to BRAF V600E mutations, constitutive activity of these BRAF β3-αC deletions is CRAF-independent and dimer-independent. Because these deletions genetically restrict the flexibility of this region of the kinase domain, they render these kinases resistant to inhibitors such as vemurafenib (for BRAF) or lapatinib (for EGFR and HER2) that bind in the αC "out" conformation. Functional characterization of the full spectrum of deletion lengths explains the high prevalence of 5 amino acid deletions in BRAF, EGFR, and HER2 in various cancers, with deletions of this length resulting in the strongest activation of kinase activity likely due to the optimal positioning of αC. This work exploits the selective power of oncogenic mutations to highlight a conserved mechanism of kinase activation and underscores the importance of conformation-specific kinase inhibitors to target mutationally activated kinases in cancer.

#4375

Pediatric low-grade gliomas with CRAF fusions respond differentially to targeted therapeutics based on their dimerization profiles.

Payal Jain,1 Tamara Fierst,2 Amanda Silva,3 Jake Budlow,1 Harry Han,1 Phillip B. Storm,3 Angela Waanders,1 Adam Resnick1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Temple University, Philadelphia, PA;_ 3 _The Children's Hospital of Philadelphia, Philadelphia, PA_.

INTRODUCTION: Recent studies have identified QKI-RAF1 and SRGAP3-RAF1 as CRAF (or RAF1) fusions in pediatric low-grade gliomas (PLGGs). CRAF fusions, like BRAF fusions are activating mutations driving the mitogen activated protein kinase (MAPK) pathway. Our previous findings suggest effective inhibition of BRAF fusion driven tumors using second-generation RAF inhibitor, PLX8394 and downstream MEK inhibitors (MEKi). We sought to investigate the mechanistic basis of response of CRAF fusions to clinically relevant RAF inhibitors and downstream pathway inhibitors, studying effect on CRAF dimerization profiles.

METHODS: Heterologous cell model systems with stable expression of CRAF fusions were generated and used for testing downstream signaling pathways via immunoblotting. Soft agar assays and flank xenografts in immuno-compromised mice were used to characterize the oncogenic properties of CRAF fusions. Pathway activation and oncogenicity were further assessed in the presence of first - and second-generation RAF inhibitors, PLX4720 and PLX8394 respectively, MEKi, and mTOR inhibitors as single agents or in combination. Myc- and Flag-tagged constructs of CRAF fusions were used in co-immunoprecipitation assays to assess dimerization profiles of CRAF fusions with or without inhibitors.

RESULTS: CRAF fusions activated the MAPK and PI3K pathways. CRAF fusions can homo-dimerize as well as hetero-dimerize with full-length BRAF, CRAF and the N-terminal fusion partner protein. Compared to BRAF fusions, CRAF fusions were not found to be responsive to any RAF inhibitors tested, including the paradox breaker PLX8394. We found that while PLX8394 decreased BRAF fusion-mediated dimerization, dimerization of CRAF fusion is unaffected. Clinically relevant MEKi AZD6244 and GSK1120212 both suppressed CRAF fusion driven pathways and growth in vitro but in mice flank xenografts, GSK1120212 partially inhibited CRAF fusion driven tumors. Since the CRAF fusions also activate the PI3K pathway, combinatorial targeting using MEKi and mTOR inhibitor, GSK1120212 and RAD001 respectively, was found to show robust tumor inhibition in vivo.

CONCLUSIONS: This study demonstrates that CRAF fusions do not respond to RAF inhibitors, show partial response to single-agent MEKi, and respond robustly to combinatorial targeting of both MAPK and PI3K pathways via GSK1120212 and RAD001. Since PLX8394 does not affect CRAF fusion mediated dimerization, this provides a mechanistic basis for unresponsiveness to RAF inhibitors. Additionally, the N-terminal fusion partner also contributes to dimerization of CRAF fusions. Therefore, CRAF fusions are distinct from BRAF fusions in terms of responsiveness to targeted therapies due to dimerization profiles. These findings suggest molecular classification of PLGGs prior to treatment and provide preclinical rationale for combination therapy of CRAF fusion expressing PLGGs.

#4376

Beyond AKT: Critical pathways for PI3K-dependent transformation.

Arturo Orlacchio, Antonio Di Cristofano. _Albert Einstein College of Medicine, Bronx, NY_.

Genetic alterations activating the PI3K/AKT pathway have been identified in most human cancers, including those originating in the thyroid gland, particularly follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC).

However, although PI3K and AKT represent relevant targets for therapeutic purposes, the number of essential roles played by this pathway in normal cells poses the problem of reaching therapeutic efficacy without harming healthy tissues. Ideally, the toxicity associated with wide-range inhibitors could be reduced by targeting only molecules directly involved in the transformation process.

The PDK1 kinase is a master regulator of PI3K downstream effectors belonging to the AGC kinases family, such as AKT and S6K1. Additional AGC kinases are controlled by PDK1 independently of PI3K activity. If PDK1-controlled AGC kinases contribute to PI3K-dependent transformation, they would clearly represent novel important therapeutic targets.

PDK1 possesses a substrate-docking site, the "PIF pocket", required for the phosphorylation of most AGC kinases (including S6K, PKC, SGK, and RSK) but not of AKT. The L155E mutation in PDK1disrupts the PIF pocket, without affecting AKT activation.

This differential mechanism of substrate selection allows us to clearly define the relative contribution of AKT and other PDK1 targets to the neoplastic transformation process.

Using in vivo as well as ex vivo and in vitro genetic and pharmacological approaches, we have shown that Akt activation is not sufficient to transform thyroid epithelial cells.

Mice in which the PI3K pathway is constitutively activated in the thyroid epithelium through loss of Pten show thyroid hyperplasia at birth that progresses to invasive and metastatic follicular carcinoma. While Pdk1 deletion, as expected, completely rescue the phenotype observed in Pten-/- thyroids, we found that the simple impairment of the PIF-pocket is able to completely abrogate neoplastic transformation, despite AKT and mTOR being constitutively activated.

We show that members of two families of AGC kinases, SGK (1and 3) and RSK (1-3) are essential components of this PDK1-dependent pathway required for PI3K-dependent transformation.

Genetic and pharmacological SGK and RSK inhibition strongly reduces cell proliferation in Pten-/- as well as Pik3ca mutant cell lines, both in 2d and in 3D systems, with SGK1 and RSK3 being the most critical isoforms. Furthermore, genetic inhibition of SGK and RSK drastically impairs tumor growth in syngeneic allograft and metastasis models.

Taken together, our data identify two new signaling cascades that are essential for neoplastic transformation of thyroid epithelial cells, and thus novel strong candidates for drug development.

#4377

AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications.

Brajendra K. Tripathi,1 Tiera Grant,1 Philipp Mertins,2 Xiaolan Qian,1 Dunrui Wang,1 Alex G. Papageorge,1 Steven A. Carr,2 Douglas R. Lowy1. 1 _National Institutes of Health, Bethesda, MD;_ 2 _The Broad Institute of MIT and Harvard, Cambridge, MA_.

Many human cancers have high AKT activity and constitutive up-regulation of Rho-GTP, which is a major factor in the neoplastic process, but AKT is not known to regulate Rho-GTP. In this study, we show that AKT positively regulates Rho-GTP by directly phosphorylating tumor suppressor DLC1 and attenuating the Rho-GAP activity of DLC1, which catalyzes the conversion of active Rho-GTP to inactive Rho-GDP. Interestingly, AKT was found to increase Rho-GTP and its downstream activities in DLC1-positive cancer lines whether the DLC1 was endogenous or transfected, but not in DLC1-negative lines. Similarly, when epithelial cell lines were stimulated with EGF, they activated AKT, which increased Rho-GTP in a DLC1-dependent manner. Three Serines (S298, S329, S567) in DLC1 have AKT consensus motifs and were phosphorylated by AKT in vitro and in vivo. Their phosphorylation attenuated the Rho-GAP and tumor suppressor activities of DLC1 - including decreased cell migration, focal adhesion turnover, anchorage-independent growth, and tumor growth in mice - as did the combined phosphomimetic mutant DLC1-3D. By contrast, the combined Serine to Alanine mutant DLC1-3A was even more active than wild type DLC1 (DLC1-WT). Remarkably, an AKT inhibitor stimulated the tumor suppressor and Rho-GAP activities of DLC1-WT, but did not influence DLC1-3A or DLC1-3D. The N-terminal half of DLC1 (amino acids 1-600) bound the DLC1 Rho-GAP domain (amino acids 609-878), as determined by in vivo complex formation and by microscale thermophoresis, which measures protein interactions in close-to-native conditions. There was increased binding and decreased Rho-GAP activity when the three Serines in the N-terminus were phosphorylated or carried the 3D mutations. Conversely, there was decreased binding and increased Rho-GAP activity when the Serines were not phosphorylated or carried the 3A mutations. In a xenograft mouse model, AKT inhibitor treatment of DLC1-positive palpable tumors reduced their size, their Rho-GTP level, and their Rho-dependent signaling, but did not affect these three parameters in isogenic DLC1-negative tumors. Similarly, in the MMTV-PyMT breast cancer model, which had high AKT activity, high Rho-GTP, and expressed DLC1, an AKT inhibitor reduced the size of palpable tumors, and reduced Rho-GTP and its downstream signaling. AKT inhibition reduced DLC1 phosphorylation in both tumor models. We conclude: AKT can increase Rho-GTP by phosphorylating three N-terminal Serines in DLC1, which attenuates its Rho-GAP and tumor suppressor functions; the N-terminus of DLC1 is an auto-inhibitory domain that reversibly binds the Rho-GAP domain; AKT attenuates DLC1 functions by phosphorylating the Serines in the N-terminus, which increases its binding the Rho-GAP domain; and AKT inhibition has greater anti-tumor activity in DLC1-positive tumors than in DLC1-negative tumors.

#4378

Discovery and characterization of two novel drivers of hepatocellular carcinoma.

Charlotte R. Feddersen, Adam J. Dupuy. _University of Iowa, Iowa City, IA_.

Liver cancer is globally the sixth most commonly diagnosed and the third most deadly cancer. Hepatocellular carcinoma (HCC) is the most common form of the disease and a difficult cancer to treat due to inadequately understood pathways involved in tumorigenesis and progression. To obtain a better understanding of key HCC driver mutations, we performed a forward genetic screen using Sleeping Beauty transposon mutagenesis in a damaged liver microenvironment. Briefly, T2Onc3; Rosa26-SB11lsl/lsl;Alb-Cre mice were treated with carbon tetrachloride (CCl4) during ongoing transposition. We then sequenced the resulting liver tumors using linker mediated PCR (LM-PCR) and Illumina sequencing. We analyzed the sequenced data using gene-centric common insertion site (gCIS) analysis that allowed us to determine common insertion sites across tumors. These common insertion sites are referred to as candidate driver mutations.

This screen demonstrated the role of Glioma associated protein-2 (Gli2) and Fidgetin (Fign) as candidate driver mutations in a significant subset of HCC tumors (20% and 10% respectively). Gli2 is a key member of the Hedgehog signaling pathway, acting as a transcription factor for genes promoting cell proliferation. Fign is a relatively uncharacterized protein in the AAA-ATPase family with the ability to sever microtubules in vitro. We validated Gli2 as a driver of HCC by generating a hepatocyte specific Gli2-overexpression mouse model using hydrodynamic tail vein injection of Gli2 cDNA into FAH-/- mice. Mice underwent rapid tumorigenesis, regardless of CCl4 treatment, demonstrating that Gli2 is a strong oncogene. Additionally we have demonstrated that overexpression of Fign drives cell proliferation and promotes stabilization of the primary cilium in hepatocytes, a novel finding that provides insight into the mechanism of Fign-driven HCC.

The discovery of two novel HCC candidate driver mutations, validation of Gli2 as a bona fide HCC oncogene, and mechanistic insight into these commonly mutated genes in HCC will allow for future research into understanding HCC tumorigenesis and potential therapeutic options.

#4379

Exosomes isolated from ovarian cancer cells transfer oncogenic features to the target cells promoting epithelial to mesenchymal transition.

Miharu Kobayashi,1 Laura Cohelo,1 Dominic Guanzon,2 Katherin Scholz-Romero,1 Melissa Brown,3 Richard Kline,4 Li Li,5 Gregory Rice,1 Carlos Salomon1. 1 _Exosome Biology Laboratory, UQ Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, The University of Queensland, Brisbane, Australia;_ 2 _School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia;_ 3 _Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Australia;_ 4 _Maternal-Fetal Medicine, Department of Obstetrics and Gynecology, Ochsner Clinic Foundation, New Orleans, LA;_ 5 _Translational Research, Colon and Rectal Surgery, the Ochsner Clinic Foundation, New Orleans, LA_.

Introduction: Recent studies establish the role of exosomes in cell-cell communication in cancer. As adaptation to hypoxia is a critical step in tumor progression, the aim of this study was to test the hypotheses that hypoxia promotes epithelial to mesenchymal transition (EMT) and that exosomes isolated from hypoxic cancer cells transfer oncogenic properties to target cells.

Methods: CaOV-3 cell line was used as model of ovarian cancer. Cells were cultured under 8% O2 (normoxia) and 1% O2 (hypoxia) for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. Exosomes were characterised by the presence of TSG101 using Western Blot, size distribution (Nanosight™) and morphology by electron microscopy. Exosomal protein and miRNA content were determined using liquid chromatography mass spectrometry (5600 Triple TOF, AB Sciex,) and an Illumina NextSeq 500 Platform, respectively. The effects of exosomes released from CaOV-3 incubated under 1% O2 on EMT induction in CaOV-3 cells cultured under 8% O2 were assessed by the measuring the ratio of E-cadherin (epithelial marker) to N-cadherin (mesenchymal marker) by Wester Blot and the expression of 84 key genes involve in the EMT (RT² Profiler™ PCR Array, QIAGEN).

Results: Exosomes were identified as spherical vesicles with a typical cup-shape, diameters ranging from 50 to 100 nm with the expression of TSG101. Exosome release from ovarian cancer cells was ~4-fold higher under hypoxic than normoxic conditions (p <0.001). Hypoxia-specific exosomal proteins and miRNAs were identified. Hypoxia induced EMT (increased N-cadherin/E-cadherin ratio) on CaOV-3 cells compared to cells cultured under 8% O2. Interestingly, exosomes isolated from hypoxic conditions mimic the effect of hypoxia on cells cultured under 8% O2, involving the unregulated of a set of transcription factors associated with EMT such as SNAIL1/SNAIL2, bHLH (E47, E2-2, and TWIST1/TWIST2), and ZEB1/ZEB2.

Conclusion: The data obtained is consistent with the hypothesis that exosomes released from cancer cells modify the phenotype of target cells by transferring pro-oncogenic molecules inducing cancerous phenotype of receipt cells, contributing to tumour growth and metastasis.

## MULTIDISCIPLINARY:

### New "Cool Tools" for Cancer Discovery

#4380

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-sequencing.

Benjamin Izar,1 Itay Tirsh,2 Sanjay Prakadan,3 Marc Wadsworth,3 Daniel Treacy,4 John Trombetta,2 Asaf Rotem,1 Christine Lian,5 George Murphy,5 Mohammad Fallahi-Sichani,6 Ken Dutton-Regester,2 Jia-Ren Lin,6 Judit Jane-Valbuena,2 Orit Rozenblatt-Rosen,2 Charles Yoon,5 Alex Shalek,7 Aviv Regev,7 Levi Garraway1. 1 _Dana-Farber Cancer Institute, Broad Institute of Harvard and MIT, Boston, MA;_ 2 _Broad Institute of Harvard and MIT, Cambridge, MA;_ 3 _MIT, Cambridge, MA;_ 4 _Dana-Farber Cancer Institute, Boston, MA;_ 5 _Brigham and Women's Hospital, Boston, MA;_ 6 _Harvard Medical School, Boston, MA;_ 7 _MIT, Broad Institute of Harvard and MIT, Cambridge, MA_.

Tumors are heterogeneous ecosystems composed of genetically and epigenetically distinct cancer cell populations embedded in an intricate tumor microenvironment. The complexity and cell-to-cell interactions within this system pose a tremendous therapeutic challenge and opportunity. Due to technical constraints, current profiling technologies only provide average signals that do not reflect this intrinsic genetic and phenotypic variability.

Here, we applied single-cell RNA-sequencing to examine 4,645 single cells isolated from 19 freshly procured melanomas, profiling malignant, immune and stromal cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, stem-like cells, spatial context, and a drug treatment resistance program. All tumors harbored malignant cells from two distinct transcriptional cell states, such that treatment-sensitive "MITF-high" tumors also contained drug-resistant "AXL-high" tumor cells; similar heterogeneity was present in 18 established melanoma cell lines. The frequency of AXL-high cells increased in post-relapse resistant tumors following treatment with BRAF/MEK inhibitors. Using multiplexed, quantitative single-cell immunofluorescence analysis and FACS, we validated these observations in melanoma cell lines treated with BRAF±MEK inhibitors. Signatures of cell types identified from single-cell analysis revealed distinct patterns of the tumor microenvironment. We inferred cell-to-cell interactions between stromal, immune and malignant cells, and identified factors, including known secreted gene products (e.g. CXCL12) and several complement factors. We validated the association between cancer-associated fibroblast (CAF)-expressed complement factor 3 (C3) and TIL infiltration in an independent set of 308 melanomas. Finally, analysis of TILs revealed T-cell activation dependent and independent exhaustion programs that varied among patients dependent on their exposure to treatment with immune checkpoint-inhibitors. In addition to co-expression of several known co-inhibitory receptors, including PD1, CTLA-4, and TIM-3, we identified common markers associated with cytotoxicity-independent T-cell exhaustion across patients. To identify potential T-cell clones, we classified single T-cells by their isoforms of the V and J segments of the alpha and beta TCR chains, allowing us to identify expanded T-cell clones. We found that clonally expanded T-cells expressed a strong exhaustion program, while non-expanded T-cells lacked this phenotype.

This study represents the most comprehensive single-cell genomics analysis in humans to date and begins to unravel the cellular ecosystem of tumors. Single-cell genomics offer new insights with implications for both targeted and immune therapies by simultaneously profiling numerous aspects of a tumor with a single assay.

#4381

**High-throughput** in vivo **systems biology using implantable microdevices.**

Oliver Jonas. _Massachusetts Institute of Technology, Cambridge, MA_.

This abstract has been withheld from publication due to its inclusion in the AACR Annual Meeting 2016 Official Press Program. It will be posted online following its presentation.

#4382

Intravital imaging of endogenous BRAFV600E melanoma reveals plasticity of tumor response and resistance to trametinib over time.

Hailey E. Brighton, Steven Angus, Tao Bo, David Darr, Norman E. Sharpless, Gary L. Johnson, James E. Bear. _UNC Chapel Hill, Chapel Hill, NC_.

Although the number of therapies entering clinical trials continues to increase, the success rate for targeted therapy studies is quite low due, in part, to a lack of relevant preclinical models. The use of targeted therapy has become a popular method for cancer treatment against mutant BRAF melanoma, as studies show that clinical use of selective inhibitors against MEK and BRAF extend life expectancy. However, melanoma becomes refractory to treatment. Since the tumor and stromal factors that contribute to drug resistance remain poorly understood, our ability to treat patients with advanced disease remains limited. Current genetically engineered murine (GEM) models in melanoma research have enhanced our understanding of tumor biology and therapeutic response, but fail to mimic tumor progression in clinical settings as most lack spatial and temporal control of endogenous tumor initiation. To better mimic human disease, we have developed a novel tamoxifen application method that reproducibly induces local tumor formation on the mouse ear, and we have incorporated a tdTomato fluorescent reporter allele (tdTomatoLSL) into an existing tamoxifen-inducible GEM model of BRAFV600E/PTEN-null melanoma. The tdTomato allele serves as a visual marker of Cre recombination in endogenous melanocytes and allows disease progression to be followed through macroscopic and multiphoton intravital imaging.

To investigate therapeutic effects at the cell level, we have performed imaging studies of primary tumors before and after treatment with a selective MEK1/2 inhibitor (MEKi), Trametinib, which is a FDA approved therapy for malignant melanoma. Our melanoma induction method enables longitudinal studies of drug response that ultimately give rise to fully resistant tumors (up to 90 days on MEKi). Our studies show that this treatment causes a striking morphological change of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining tumor cells are highly spatially correlated with bundled collagen structures detected by second harmonic signal, suggesting that cellular milieu strongly influences drug response. Interestingly, our current studies with MEKi suggest that association of tumor cells with collagen fibrils initially provides a protective effect against this drug, however once true resistance emerges, this dependency on extracellular matrix in the tumor stroma diminishes. Furthermore, in parallel with our imaging studies, we have performed molecular analysis of tumor response using Mib/MS and mRNA-seq. By coupling our imaging studies with transcriptome and kinome reprogramming analysis, we hope to identify properties that promote resistance. Using these novel approaches, we have developed a model that enables direct observation of endogenous tumor development, plasticity, and resistance to targeted therapy at the cell level in situ for the first time.

#4383

SOCRATES: integrating ex vivo and in silico analysis to identify optimal drug combinations for patients.

Elizabeth A. Coker,1 Adam Stewart,1 Anna Minchom,2 Mary O'Brien,3 Timothy Yap,2 Paul Workman,1 Udai Banerji,2 Bissan Al-Lazikani1. 1 _The Institute of Cancer Research, London, United Kingdom;_ 2 _The Institute of Cancer Research and The Royal Marsden, London, United Kingdom;_ 3 _The Royal Marsden, London, United Kingdom_.

Recent work on cancer heterogeneity and evolution has greatly enhanced our understanding of drivers of drug resistance, but this has also highlighted the need to move away from generic, "one-size-fits-all" treatment regimes. It is clear that we should tailor therapy to individual patients at different stages of disease based on the current behaviour of their tumour cells, yet detailed experimental characterisation and analysis of patient tissue is impractical and costly. Predictive computational models that can produce patient-specific recommendations of drug combinations would therefore be of great value to the field of personalised medicine. In silico modelling has already been used to successfully predict synergistic drug combinations in single cell lines in the lab, but such approaches have not yet been translated to dynamic, real patient data in the clinic.

The SOCRATES project (Signalling output to rationalise combinations of targeted anticancer therapies) integrates experimental data and computational analysis to predict context-specific synergistic drug combinations. Here we present ambitious computational modelling of dynamic signalling network changes in response to ex vivo exposure of seven targeted drugs used singly at clinically appropriate concentrations. We also apply our adaptive modelling to genomics and proteomics data from 54 proteins in over 30 non small-cell lung cancer cell lines and ten patient samples to predict and experimentally validate personalised, context specific drug combinations. We will describe our adaptive, in silico modelling for predicting response to drug combinations, including novel methods utilizing network topology. We have predicted over 50 synergistic drug combinations and will present our predictions alongside initial results of experimental validation in the lab. We will also illustrate the differences observed in various cell lines and patient derived cells depending on genetic context.

SOCRATES is, to our knowledge, the first project of its kind and is a prototype for the future of adaptive, individualised cancer treatment.

#4384

Selective cross-cohort discovery of transcriptional mechanisms presiding over high-risk neuroblastoma subtype state maintenance.

Presha Rajbhandari,1 Gonzalo Lopez,1 Jiyang Yu,2 Ruth Rodriguez-Barrueco,3 Mariano Alvarez,1 Daniel Martinez,4 Mark Yarmarkovich,4 Jo Vandesompele,5 Pieter Mestdagh,5 Jose M. Silva,3 Anna Lasorella,1 Antonio Iavarone,1 John M. Maris,4 Andrea Califano1. 1 _Columbia University, New York, NY;_ 2 _Pfizer Inc., Pearl River, NY;_ 3 _Icahn School of Medicine at Mount Sinai, New York, NY;_ 4 _University of Pennsylvania, Philadelphia, PA;_ 5 _Ghent University, Ghent, Belgium_.

BACKGROUND: Neuroblastoma (NBL) is the most common extracranial solid tumor in children. High-risk NBLs progress to metastatic disease and have 5-year survival of only ~40%, despite intensive multimodal therapy. This malignancy is characterized by significant heterogeneity, both clinical and molecular, which is still poorly understood.

Rather than focusing on its initiating genetic events, which are highly idiosyncratic, we focused on the core regulatory machinery responsible for implementation and maintenance of tumor state. This approach led to elucidating three molecularly distinct subtypes of high-risk NBLs, as well as the core regulatory machinery responsible for their implementation and stability, including canalization and integration of mutational events and regulation of the genetic programs that represent the hallmarks of this disease.

METHODS: We dissected large-scale gene expression profiling data available from TARGET and NRC Consortium by clustering algorithm and established three subtypes of high-risk NBL, followed by identification of master regulators (MR)s of each subtype by Master Regulator Inference algorithm (Lefebvre, C. et al, 2010). We performed extensive experimental validation of MRs by both in-vitro and in-vivo RNAi mediated screening, using cell viability as readout. We then used a variety of experimental assays to elucidate the modular logic controlling disease state and to identify novel NBL subtype specific dependencies.

RESULTS: We identified unique MR protein modules for three distinct molecular subtypes of high-risk NBL, which were conserved across independent cohorts. Experimental MR validation identified a TEAD4-MYCN positive feedback loop as the key NBL state maintenance mechanisms in the MYCN amplification associated subtype. Jointly, MYCN and TEAD4 regulate 90% of inferred MR proteins and causally implement 70% of the subtype gene expression signature. Biologically, MYCN repressed differentiation and TEAD4 activated proliferation, two hallmarks of MYCN-amplified NBL. Specifically, TEAD4 was shown to induce MYCN-independent proliferation by transactivating key genes implicated in high-risk NBL pathogenesis, including cyclin-dependent kinases, cyclins, E2Fs, DNA replication factors, checkpoint kinases and ubiquitin ligases. Consistently, TEAD4 inhibition induced loss of NBL cell viability, thus providing novel therapeutic targets. TEAD4 activity was an outstanding predictor of survival, independent of outcome-related variables.

CONCLUSION: Our results show that the inference of transcriptional regulators driving distinct molecular subgroups when combined with functional analyses is valuable to uncover the regulatory modules required for sustaining the tumor subtypes. This approach can be used to successfully identify the functional bottlenecks of other cancer subtypes.

#4385

Live-imaging the interface between homeostasis and cancer initiation.

Cristiana Pineda,1 Catherine Martone,1 Katie Suozzi,1 Samara Brown,1 Slobodan Beronja,2 Valentina Greco1. 1 _Yale University, New Haven, CT;_ 2 _Fred Hutchinson Cancer Center, Seattle, WA_.

While cancer-associated mutations are most frequently found within tumors, it has been established that these same mutations can be present in even phenotypically normal organs, such as the skin. This raises the question as to how the switch from normal to malignant is initiated within the interface between homeostasis and cancer. One challenge to understanding the initiating events towards malignancy is the inability to follow the same cells over time in an intact mammal. Specifically, this roadblock hinders the ability to understand both the role of specific cells and how their location contributes to their growth, whether that growth be normal or cancerous. To overcome this, we have established a novel live imaging approach to track cellular behaviors in normal, healthy skin. This approach has previously enabled our lab to demonstrate that location dictates the fate and behaviors of hair follicle stem cells during skin regeneration (Rompolas et al. Nature 2013). We are now utilizing this approach to capture the emergence of the cellular behaviors that lead to Hras-driven skin tumors in live mice.

To begin to characterize the initiating events of skin cancer, we utilized a model of oncogenic Hras (HrasG12V), which has been established to give rise to skin papillomas. Using targeted, inducible genetic drivers, we activated HrasG12V at low levels and restricted the activation to the cycling, stem-cell rich portion of the hair follicle. Strikingly, we have observed that the presence of HrasG12V cells within the cycling portion of the hair follicle are not only tolerated, but can also be eliminated, supporting the idea that the surrounding wild-type tissue has the capability to both integrate and overcome the presence of a single oncogenic hit. Furthermore, within this targeted genetic system, we found that the initial position of the mutant cell impacted the resultant phenotype of the tissue. To next understand these behaviors at the cellular level, we utilized a viral approach to directly activate and fluorescently tag HrasG12V at clonal levels throughout the skin epithelium allowing us to observe interactions between wild-type and mutant cells. Furthermore, within the upper portion of the hair follicle and epidermis, which (unlike the cycling portion) is immune permissive, we observed the release of exosomes by mutant cells and their subsequent clearing by both epithelial and immune cells. Currently, we are introducing a second mutation, loss of TGFβ signaling, to the oncogenic Hras system which is a mutational combination well established to produce cutaneous Squamous Cell Carcinoma (cSCC). This system will provide us with a spectrum of phenotypes from wild-type, to benign yet aberrant (papillomas), to malignant (cSCC) and allow us to interrogate how interactions among different cell types (wild-type, single mutants, and double mutants) within particular niches influence the overall fate of the tissue to initiate disease.

#4386

CETSA as a new strategy to understand efficacy, adverse effects and resistance development of anticancer drugs.

Pär Nordlund,1 Sara Lööf,1 Henritte Laursen,1 Anette Öberg,1 Johan Lengqvist,1 Rozbeh Jafari,1 Lingyun Dai,2 Ka DIam Go,2 Nayana Prabhu,2 Radoslaw Sobota,3 Andreas Larsson,2 Anna Jansson,2 Chris Heng Tan Soon,3 Lekshmy Sreekumar,2 Yan Ting Lim,2 Daniel Martines Molina1. 1 _Karolinska Institutet, Stockholm, Sweden;_ 2 _Nanyang Technological University, Singapore, Singapore;_ 3 _IMCB, ASTAR, Singapore, Singapore_.

A key step of the action of most drugs is their binding (engagement) of the target protein(s). However, limitations in the available methods for directly accessing this critical step have added uncertainties in many stages of drug development.

We have developed a generic method for evaluating drug binding to target proteins in cells and tissues (Martinez Molina et al. Science, 341:84). The technique is based on the physical phenomenon of ligand-induced thermal stabilization of target proteins; the method is therefore called the cellular thermal shift assay (CETSA). The technique allows for the first time to directly measure the biophysical interactions between a drug and protein target in non- engineered cells and tissues. We show that using CETSA a range of critical factors for drug development can be addressed at the target engagement level, including drug transport and activation, off-target effects, drug resistance as well as drug distribution in cells, patient and animal tissues. Using quantitative mass-spectrometry, proteome-wide CETSA has been established which allows for off-target effects as well as downstream biochemistry to be discovered (Savitsk et al. Science, 346, 6205:1255784). Together the data supports that CETSA is likely to become a valuable tool for developing and understanding the action of cancer drugs in the future.

## TUMOR BIOLOGY:

### Therapeutic Studies in Cell Culture and Mouse Models

#4387

Alterations of TP53 mediate resistance to abiraterone in castration-resistant prostate cancer.

Min Zou, Roxanne Toivanen, Antonina Mitrofanova, Michael M. Shen, Cory Abate-Shen. _Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY_.

Prostate cancer remains the second-leading cause of cancer death in men. The first line treatment for advanced prostate cancer is hormone deprivation therapy (ADT), which although it is initially effective, usually leads to progression to more aggressive and often lethal castration resistant prostate cancer (CRPC). Nonetheless, CRPC remains dependent on androgen receptor (AR) signaling through various mechanisms. Therefore, drugs that target androgen synthesis or AR signaling, such as abiraterone acetate and enzalutamide, have been employed as second line therapy. Despite the fact that both drugs improve the overall survival for CRPC patients, neither of them is curative, suggesting intrinsic or acquired resistance. Here we have focused on elucidating resistance mechanisms for abiraterone acetate, an inhibitor of androgen biosynthesis, using preclinical analyses of relevant genetically engineered mouse models (GEMMs).

Given the high frequency of co-alteration in PTEN and p53 in mCRPC, we generated conditional GEMMs based on deletion of either PTEN alone or PTEN plus p53 to obtain the NP and NPp53 GEMMs, respectively. Both NP and NPp53 mice develop CRPC, and cross-species gene set enrichment analysis (GSEA) suggests that they have molecular programs that are well-conserved with human CRPC. In preclinical studies, we find that the NP mice are highly responsive to tumor inhibition by abiraterone, whereas the NPp53 mice do not respond. In fact, the NPp53 mice display a marked acceleration of tumor progression following abiraterone treatment. The disparate phenotypic responses of the NP and NPp53 mice to abiraterone are evident by analyses of their tumor histology, immunohistochemical staining, as well as the change in tumor volume by MRI imaging. We further validated the results in a genetically modified human prostate cancer cell line. In particular, a xenograft model based on 22RV1 cells that are depleted for both PTEN and p53 also displayed an acceleration of the tumor phenotype following abiraterone treatment. Notably, expression profiling of the abiraterone- or vehicle-treated "responder" NP tumors versus "non-responder" NPp53 tumors identified a unique set of cancer driver genes that are enriched for genes found in human neuroendocrine prostate cancer (NEPC). These findings were confirmed by immunohistochemistry, which demonstrated a significant increase in synaptophysin-expressing cells in CRPC in the NPp53 mice but not the NP mice.

Our findings suggest that loss of p53 in the context of PTEN deletion promotes resistance to abiraterone while CRPC in the NPp53 mice partially resemble human NEPC. Given the high prevalence of co-alteration of PTEN and p53 in advanced human mCRPC, and observed occurrence of treatment induced NEPC-like phenotypes observed following treatment with AR targeting agents in patients, our finding suggest that abiraterone may not be suitable for patients with alterations in both PTEN and p53.

#4388

The role of neoantigens in immunotherapy of cutaneous melanoma.

Renee M. Thomas,1 Chi-Ping Day,2 Zoe Weaver Ohler,3 Rajaa El Meskini,3 Carri Graff-Cherry,4 Sung Chin,4 Aleksandra Michalowski,2 Ji Luo,2 Terry Van Dyke,5 Glenn Merlino2. 1 _National Institute of Health, Bethesda, MD;_ 2 _National Cancer Institute, NIH, Bethesda, MD;_ 3 _Leidos Biomedical Research Inc., National Frederick Laboratory for Cancer Research, Frederick, MD;_ 4 _National Frederick Laboratory for Cancer Research, Frederick, MD;_ 5 _Center for Cancer Research, National Cancer Institute, Frederick, MD_.

Melanoma is increasing in incidence by up to 3% annually, and metastatic melanoma, which is resistant to most conventional therapies, has a poor prognosis with a 5-year survival rate of less than 20%. Immunotherapy is able to yield durable response in melanoma; however, the response rate is limited (20-30%). It has been shown that tumor immunogenicity is highly correlated to the response to immunotherapy. The mutational load and the characteristics of the neoantigens displayed by the tumor cells are theorized to play an important role in the immunogenicity of the tumor. We used genetically engineered preclinical mouse models to study the role of these neoantigens in melanoma response to immune therapy. Three syngeneic C57BL/6 mouse melanoma models were developed: UV induced melanoma in albino BRAF(V600E);PTEN-/- mice (1), DMBA induced melanoma in pigmented HGF-tg;CDK4(R24C) mice (2), and UV induced melanoma in pigmented HGF-tg mice. These three models were found to have varying degrees of tumor immunogenicity based on vaccination studies. The UV induced BRAF(V600E);PTEN-/- model displayed poor immunogenicity, while the DMBA induced HGF-tg;CDK4(R24C) model exhibited moderate immunogenicity and the UV induced HGF-tg model showed the highest immunogenicity. Exome sequencing results showed that high immunogenicity is associated with mutations of genes in DNA damage responses and frameshift mutations. To test the role of neoantigens, RNA from each of the melanoma models was sequenced and analyzed for mutational and expression profiles. We will be comparing the response of these three mouse models to correlate immunogenicity with response to anti-CTLA-4 therapy. We plan to analyze the pathways in which mutated genes were enriched, allowing the identification of "druggable" targets to enhance therapeutic efficacy of immune checkpoint inhibitors. CRISPR/Cas9-based screening approaches will be used to identify specific neoantigens able to influence response to immune therapy. In conclusion, our preclinical mouse models show that tumor immunogenicity may be correlated to the carcinogenic mechanisms, and RNA sequence has provided further insight into the role of mutational load and neo-epitopes on immune-based therapeutic response.

(1) Provided by Marcus Bosenberg (Yale School of Medicine, New Heaven, CT), Martin McMahon (Huntsman Cancer Institute, Salt Lake City, UT).

(2) Provided by Thomas Tueting (University Hospital Bonn, Bonn, Germany).

#4389

Therapeutic opportunities of RANK pathway in breast cancer.

Hector Perez Montoyo,1 Jorge Gomez Miragaya,1 Eva M. Trinidad,1 Antonio Martinez-Aranda,2 Maria Teresa Soler Monso,3 Anna Petit,3 Angels Sierra,2 Eva Gonzalez Suarez1. 1 _IDIBELL, L´Hospitalet de Llobregat, Spain;_ 2 _Institut d'Investigacions Biomèdiques August Pi i Sunyer-IDIBAPS, Barcelona, Spain;_ 3 _University Hospital of Bellvitge IDIBELL, L´Hospitalet de Llobregat, Spain_.

RANK pathway, key in bone remodeling and metastasis, is essential for mammary gland development. Using genetic mouse models we have previously shown that RANKL is the main mediator of the protumorigenic role of progesterone in the mouse mammary gland. RANK overexpression in non-transformed human mammary cell lines induces epithelial to mesenchymal transition and stemness, and promotes tumorigenesis and metastasis in breast cancer lines. RANK is expressed in a subset of basal and luminal human adenocarcinomas.

Our goal is to determine a putative association of RANK and RANKL expression in breast adenocarcinomas with clinical parameters and to evaluate the functional role of RANK signaling using patient derived orthoxenografts of breast cancer. We have analyzed RANK and RANKL expression levels in an extensive collection of human breast adenocarcinomas collected from multiple centers, enriched in metastatic tumor samples. From the 318 patients with breast adenocarcinomas 138 developed metastasis. RANK expression is found on tumor cells but also on stromal cells, including tumor associated macrophages. RANKL is strongly expressed in some tumors, although the frequency is low. Analyses of RANK and RANKL mRNA and protein expression has been performed in multiple models of patient derived breast orthoxenografts. Several models with variable levels of RANK as well as RANKL expression have been identified and established in the laboratory. Functionality of the pathway was corroborated analyzing NF-kB activation upon in vitro and in vivo treatments with RANKL, in order to activate RANK signaling, or with RANK-Fc to inhibit it. We found that RANKL treatment induced NF-kB activation even in tumor cells with low levels of RANK expression.

#4390

SVV-001 prolongs animal survival in PDOX adult GBM models.

Huiyuan Zhang,1 Lin Qi,1 Yuchen Du,1 Mari Kogiso,1 Frank K. Braun,1 Sibo Zhao,1 Holly Lindsay,1 Nabil Ahmed,1 Akash J. Patel,2 Patricia A. Baxter,3 Jack M. Su,3 Xiao-nan Li1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Jan and Duncan Neurological Research Institute, Houston, TX;_ 3 _Texas Children's Cancer Center, Houston, TX_.

Background: Glioblastoma (GBM) is the most malignant and aggressive brain tumor, and survival of patients affected by GBM has remained virtually unchanged over numerous years. GBM is only minimally responsive to aggressive standard therapies including radical surgery and concurrent chemo-radiation treatment with temozolomide (TMZ). Seneca Valley virus (SVV-001) is a non-pathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. In this study, we developed a new panel of patient derived orthotopic xenograft (PDOX) models to examine the therapeutic efficacy of SVV-001 combined with irradiation. In addition, we explored the mechanism of tumor cell infection with SVV-001 in malignant gliomas.

Material and Methods: Surgical GBM tumor samples were obtained from 17 patients and directly implanted into the right cerebrum of NOD/SCID mice (1x105 cells suspended in 2 uL growth medium). Once tumor formation was confirmed, we performed H&E staining and IHC to determine the pathologic characteristics of the PDOX models over serial in vivo passages and compared with the matched patient tumors. In vitro antitumor activities of SVV-001 were examined in primary cultures, pre-formed neurospheres, and monolayer cells derived from PDOX models. In vivo therapeutic efficacy was examined by single I.V. injection of SVV-001 alone or administered in combination with radiation treatment of pre-formed xenograft tumors in permissive models.

Results: 8 out from 17 samples had confirmed tumor formation in mouse brains; 4 of these samples have since been serially sub-transplanted for a total of 5 generations, while 4 intracerebral xenografts are in generation 2. The tumorigenicity of 8 additional samples is pending. These xenograft models precisely replicated histopathologic characteristics of their parental human tumors. SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, pre-formed neurospheres, and monolayer glioma cells derived from adult glioma xenograft models in vitro. A single I.V. injection of SVV-001 (1 ×1011 viral particles/kg) administered immediately after radiation or 1 week after radiation led to the infection of orthotopic xenografts without harming normal mouse brain cells and resulted in significantly prolonged survival in permissive mouse models. Additionally, SVV-001 injected immediately after radiation significantly improved the overall survival of animals compared to administration of the virus 1 week after radiation, indicating that dose timing was crucial for full efficacy of the combinatory therapy.

Conclusion: Our results demonstrated that SVV-001 possesses potent anti-tumor activity against adult malignant high-grade gliomas. Because this study was performed in a panel of patient tumor-derived orthotopic xenograft models, it provides a pre-clinical rationale that supports the consideration of SVV-001 for clinical trials against adult gliomas.

#4391

Overcoming primary ibrutinib resistance in mantle cell lymphoma.

Leo Zhang, Lan Pham, Hui Zhang, Jingmeng Xie, Wenjing Tao, Taylor Bell, Zhihong Chen, Krystle Nomie, Bingliang Fang, Michael Wang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Mantle cell lymphoma (MCL) is a rare and incurable subtype of B-cell lymphoma. In a phase II study of ibrutinib in MCL patients, most of the patients responded and had long durable remissions; however, 22.7% of patients were considered to be non-responsive to ibrutinib, and an additional 22/110 patients displayed initial positive responses to ibrutinib but also experienced disease progression within 12 months of treatment, with both responses classified as primary ibrutinib resistant. Therefore, understanding the mechanisms mediating primary ibrutinib resistance may identify new prognostic and predictive markers and potential therapeutic targets.

Bruton's tyrosine kinase (BTK) plays an important role in B-cell development, activation, and differentiation. Upon B-cell receptor (BCR) activation, BTK is phosphorylated and activated by SYK. Phosphoinositide 3-kinase (PI3K) is recruited to the BCR, and the induction of these signaling activities leads to the downstream activation of multiple effector proteins, including nuclear factor-kB (NF-kB), AKT, RAS, mTOR and mitogen-activated protein kinase (MAPK). BTK and PI3K have been shown to function independently to mediate BCR signaling, suggesting that PI3K signaling activities may underlie ibrutinib resistance independently of BTK. PI3K has also been associated with primary ibrutinib resistance. To further elucidate the mechanisms underlying ibrutinib resistance, we conferred primary ibrutinib resistance using both MCL cell lines and MCL-bearing patient-derived xenograft (PDX) mouse models and used whole exome sequencing (WES) and reverse phase protein analysis (RPPA) to identify any genetic and expression changes associated with primary ibrutinib resistance. WES did not reveal any mutations in BTK or within the proximal BCR pathway, consistent with WES data on primary ibrutinib resistant MCL cases. RPPA analysis showed a significant increase in the PI3K/AKT/mTOR/MCL-1 compensatory pathway component levels in ibrutinib-resistant cell lines when compared with their parental cells as well as ibrutinib-resistant PDXs. To determine whether inhibiting these pathways would overcome primary ibrutinib resistance, we tested various therapeutic combinations targeting these pathways. Ibrutinib plus the PI3K inhibitor idelalisib, the AKT inhibitor ACP-319, the mTOR inhibitors AZD8055 or BEZ235 as well as the proteasome inhibitor carfilzomib inhibited tumor growth in vitro and in vivo in the PDX mouse models, demonstrating that targeting these alternative pathways may overcome ibrutinib resistance. This work strongly suggests that targeting the PI3K/AKT/mTOR/MCL-1 compensatory pathway successfully inhibits the viability of ibrutinib-resistant MCL tumor cells both in vitro and in vivo and identified potential therapies that can be used to treat ibrutinib-resistant patients in clinical practice.

#4392

Yes1 is the key molecule for the resistance to trastuzumab in breast cancer, and dasatinib overcomes the resistance.

Hiromasa Yamamoto, Tatsuaki Takeda, Hirotaka Kanzaki, Ken Suzawa, Kei Namba, Hiroki Sato, Hidejiro Torigoe, Mototsugu Watanabe, Yuho Maki, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi, Yoshihisa Kitamura, Toshiaki Sendo, Shinichi Toyooka. _Okayama University Hospital, Okayama, Japan_.

Background: Overexpression of human epidermal growth factor receptor 2 (HER2) was observed in approximately 15-23% of breast cancers and they are classified as HER2-positive breast cancer. Trastuzumab is a therapeutic drug for the first choice for HER2-positive breast cancer, showing good response. However, acquired resistance to trastuzumab is one of the critical clinical issues in breast cancer treatment, especially in the patients with recurrent breast cancer. Therefore, it is necessary to develop the effective therapy to overcome the resistance. In this study, we established a trastuzumab-resistant breast cancer cell line from a trastuzumab-sensitive cell line with HER2 amplification (BT-474). We analyzed the mechanisms of resistance to trastuzumab and demonstrated the anti-tumor effect of dasatinib.

Methods: Trastuzumab-resistant breast cancer cell line (BT-474-R) was established by treating BT-474 cells for long-term exposure with increasing doses of trastuzumab (from 0.1 µg/mL up to 40 µg/mL). Expression and activation of HER2 and its related molecules were investigated using western blotting and real-time PCR. Cell viability was evaluated using MTS assay. Cell cycle was analyzed using flow cytometry.

Results: Proto-oncogene tyrosine-protein kinase Yes1, which is one of the Src family members, was amplified, overexpressed and activated in BT-474-R. HER2 and Akt were also activated. Silencing of Yes1 by siRNA induced BT-474-R to recover sensitivity to trastuzumab. Pharmaceutical inhibition of Yes1 by Src inhibitor dasatinib was also effective to recovery sensitivity to trastuzumab in BT-474-R. Combination treatment of dasatinib and trastuzumab induced down-regulation of signaling molecules such as HER2 and Akt. Moreover, these combination treatments induced G1-phase cell-cycle arrest and apoptosis.

Conclusion: Yes1 plays an important role in acquired resistance to trastuzumab in HER2-positive breast cancer. Our data also suggest that pharmacological inhibition of Yes1 may become the new strategy to overcome resistance to trastuzumab.

#4393

A pooled shRNA screen in 3D cultures of primary tumor propagating cells identifies regulators of innate chemoresistance in KRAS-driven NSCLC.

David Richard Simpson,1 Dedeepya Vaka,2 Yanyan Zheng,1 John Tamaresis,1 Alejandro Sweet-Cordero1. 1 _Stanford University School of Medicine, Stanford, CA;_ 2 _UCSF, San Francisco, CA_.

Despite advances developing targeted therapies for non-small cell lung cancer (NSCLC), patients carrying activating KRAS mutations still have no effective targeted therapeutics and respond poorly to chemotherapy. Discovering drug targets that work in combination with existing chemotherapeutics to inhibit innate chemoresistance and prevent re-initiation of tumor growth could reduce the likelihood of relapse and extend patient survival. Heterogeneous tumor propagating cell (TPC) populations in NSCLC have been shown to have increased chemoresistance, an enhanced ability to re-seed tumors in immunocompromised mice, and express genes associated with poor prognosis. To target chemoresistance in TPCs, we have developed a 3D culture model for functional genomic screens using primary tumor cells from a mouse model of NSCLC driven by activation of KRAS and p53 loss. Using pooled shRNA libraries, we have performed a focused screen in 3D TPC cultures of over 3,000 shRNAs targeting 600 genes associated with chemotherapeutic response in TPCs, TPC gene expression, DNA damage response, or bulk tumor resistance. We selected targets for validation, characterization, and in vivo studies if knockdown of a gene selectively sensitized TPCs to platinum-based chemotherapy or inhibited growth of TPCs. Multiple DNA damage response pathways were implicated in chemoresistance including known regulators, such as ATR and the translesion synthesis pathway. Many genes expressed selectively by TPCs alone or in response to chemotherapy were required for chemoresistance, including regulators of mitochondrial transport, RNA export, and DNA replication. A parallel screen using the KRAS-mutant LKR10 cell line was used as a control for shRNA representation and to determine which targets are required for TPCs to survive in 3D but not in 2D cultures. 3D-specific vulnerabilities include genes that are expressed highly in TPCs or in chemoresistant tumors and represent potential KRAS vulnerabilities that exist in the TPC population. Most chemoresistance targets and KRAS vulnerabilities are not shared by LKR10 cells grown in 2D suggesting growth in a multidimensional matrix exposes therapeutic opportunities that are distinct from cells grown as monolayers. Although functional genomic screens using primary tumor cells in 3D have limited throughput, focused screens utilizing primary tumor cells have the potential to discover therapeutic opportunities that exist in vivo that have not been uncovered in screens utilizing cell lines in 2D.

### Tumor-supporting Microenvironment

#4394

Phenotypic heterogeneity of disseminated tumor cells is predetermined by primary tumor hypoxic microenvironments.

Georg Fluegen,1 Alvaro Avivar-Valderas,1 Yarong Wang,2 Michael R. Padgen,3 James K. Williams,3 Vladislav V. Verkhusha,2 David Entenberg,2 Kevin W. Eliceiri,4 James Castracane,3 Patricia J. Keely,4 John S. Condeelis,2 Julio A. Aguirre-Ghiso1. 1 _Department of Medicine and Department of Otolaryngology, Tisch Cancer Institute, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY;_ 2 _Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Integrated Imaging Program, Albert Einstein College of Medicine, Bronx, NY;_ 3 _Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY;_ 4 _Laboratory for Optical and Computational Instrumentation, Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI_.

Heterogeneity of primary tumors (PTs) is reflected by genetic and epigenetic diversity, as well as diverse PT microenvironments. Whether PT microenvironments might influence the fate of disseminating tumor cells (DTC) has never been explored in situ. We previously defined, in breast cancer, a dormancy signature (DS) associated with longer metastasis-free periods. Key genes in the DS induce quiescence and are also regulated by hypoxia. Interestingly, a main response of tumor cells to hypoxia is growth arrest, while clinical evidence links hypoxic tumors to increased therapy resistance and a worse outcome. We hypothesized that hypoxic PT microenvironments may spawn a subpopulation of DTCs that, by virtue of becoming dormant, might escape therapies and eventually fuel incurable metastasis. We used H2B-GFP inducible HEp3 HNSCC and photo-switchable H2B-Dendra2 (green-to-red fluorescence) expressing MDA-MB-231 and ZR-75-1 human breast cancer cell lines to identify cells from hypoxic microenvironments. To initiate spatially defined hypoxic microenvironments in vivo in primary tumors we implanted induction NANo IntraVItal Devices (iNANIVIDs) carrying a hypoxia-mimetic agent (desferrioxamine - DFOM) in T-HEp3 tumors or exposed cultured MDA-MB-231 or ZR-75-1 cells in vitro to 21% or 1% O2. The regions influenced by the DFOM-iNANIVID displayed significant upregulation of p27, NR2F1 and DEC2 (dormancy genes), as well as induction of hypoxia markers (GLUT1, HIF1α). Human HNSCC PT samples showed the same link between spontaneous hypoxic regions and upregulation of dormancy markers. We found a significant increase in quiescent lung DTCs of hypoxia induced T-HEp3 or MDA-MB-231 cells, traceable >2 weeks after extravasation by fluorescent label retention. Significantly more single, non-proliferating HEp3 DTCs originating from the iNANIVID induced hypoxic regions showed a dormant profile compared to DTCs originating from a normoxic milieu. Only the hypoxic pre-treated group was able to form micrometastases at 10 days after injection, suggesting the presence of a more aggressive sub clone in this group. Analysis in 3D culture models revealed that ZR-75-1 cells (ER+) were more prone to enter a prolonged quiescent state after exposure to hypoxia (1% O2) while this response was not observed in MDA-MB-231 (TN). The induction of quiescence in ZR-75-1 was NR2F1 dependent. Lastly, using a spontaneously metastatic PyMT driven Dendra2-tagged breast cancer model in immunocompetent mice, we found that ~75% of dormant DTCs upregulate the dormancy marker NR2F1 at or soon after reaching the lung, suggesting a rapid induction of dormancy upon reaching target organs. We propose that hypoxic PT microenvironments increase phenotypic heterogeneity of DTCs and lead to the expression of the DS. These DTCs may be more prone to enter dormancy, evade anti-proliferative therapies and eventually fuel metastasis.

#4395

Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK.

Mei-Fong Pang,1 Melody Stallings-Mann,2 Michael J. Siedlik,1 Victor D. Varner,1 Siyang Han,1 Derek C. Radisky,2 Celeste M. Nelson1. 1 _Princeton University, Princeton, NJ;_ 2 _Mayo Clinic, Jacksonville, FL_.

The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche.

Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples.

Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs.

#4396

Circulating CAFs and CAF-secreted factors may be indicative of breast cancer metastasis.

Sanket H. Shah, Phil Miller, Leah Machlin, Kelsie Medina-Saenz, Ritesh Parajuli, Marc E. Lippman, Dorraya El-Ashry. _University of Miami, Miami, FL_.

Tumor metastasis is the main cause of breast cancer mortality. Increasing evidence demonstrates stromal cells play pivotal roles in promoting breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). CAFs secrete various growth factors and cytokines that promote breast cancer progression and metastasis; one of these factors is SDF-1 (CXCL12), a soluble chemokine that is promotes chemotaxis and motility, and facilitates cancer cell motility and angiogenesis. CAFs also secrete soluble factors that activate ERK 1/2 MAPK signaling in breast cancer cells, which has been shown to promote loss of estrogen receptor (ER) in luminal breast cancer cells. Hyperactivation of MAPK signaling (hMAPK) also associates with aggressive, basal-like and HER2-positive breast cancer and poor prognosis. Recently, we identified a patient-derived hMAPK-microRNA signature indicative of poor clinical outcome that contains microRNAs known to regulate breast cancer associated genes. The vast majority of ER- breast cancers display this microRNA signature, as do a subset of ER+ breast cancers with poorer clinical outcome. We also discovered that the breast cancers that exhibit this microRNA signature display high stromal and immune infiltrate scores, suggesting that breast cancer stroma provides important contributions to this microRNA signature and the poor clinical outcomes associated with it.

To study the role of CAFs and CAF-secreted factors in breast cancer progression and metastasis, we established primary breast CAF lines from 'indolent' breast cancers (Luminal A), and from 'aggressive' breast cancers (ER-/HER2 amplified; triple negative). We have demonstrated that these CAFs differentially express several members of the hMAPK-microRNA signature compared to cultured primary breast cancer cells, supporting the contribution of stroma to the signature. Importantly hMAPK-microRNAs secreted from "aggressive" CAFs can be taken up by breast cancer cells, whereupon they repress their targets. Normal human mammary fibroblasts (HMFs) and 'indolent' CAFs do not secrete these microRNAs.

We identified a novel class of circulating cells in the blood of breast cancer patients with metastases -CAFs (cCAFs). Patients without metastases did not have these cCAFs - suggesting cCAFs may be "aggressive" CAFs that facilitate breast cancer metastasis. Patients with overt metastasis and elevated counts of cCAFs have significantly higher levels of circulating SDF-1 in their plasma, as well as differential circulating microRNAs, and specifically hMAPK-microRNAs also found secreted by "aggressive" CAFs. Our results suggest there is a hierarchy of CAFs whereby "aggressive" tumors establish "aggressive" CAFs to facilitate metastasis. We also establish a clear link between circulating CAFs and CAF-secreted factors such as SDF-1 and microRNAs with breast cancer metastasis.

#4397

Mesenchymal stem cell-derived collagen I plays a role in organizing breast cancer cell migration and metastasis.

Maria E. Gonzalez, Emily Martin, Caroline Arellano-Gracia, Arjun Lama, Celina G. Kleer. _University of Michigan Comp. Cancer Center, Ann Arbor, MI_.

Background: Accumulating evidence suggests that mesenchymal stem cells (MSCs) are recruited to the tumor microenvironment and play roles in tumor progression; however the underlying mechanisms by which MSCs promote breast cancer migration and invasion need further investigation. Studies have demonstrated that collagen plays an important role in breast tumorigenesis by activating signaling pathways. We hypothesize that MSCs may promote breast cancer progression through regulating collagen-induced signaling in breast cancer cells.

Methods: We isolated carcinoma-associated MSCs (CA-MSCs) from human breast cancer metastasis to lymph node (LNM) and liver (LM). CA-MSCs were subjected to multilineage differentiation assays and labeled with Ds-Red. COAL I expression and its receptor discoidin domain receptor 2 (DDR2) were downregulated in the CA-MSCs-DsRED using specific shRNA. We established single culture and co-cultures of CA-MSCs with GFP labeled breast cancer cells (BCCs) MDA-MB-231 and MCF10ACA1a which were used for Live Imaging Microscopy, IHC, RT-PCR, WB, immunofluorescence, 3D proliferation and invasion assays, and in vivo xenograft experiments.

Results: CA-MSCs had spindle morphology, normal karyotype, were nontumorigenic in vivo, and possessed tri-lineage differentiation ability (osteoblast, adipocyte, and chondrocyte). CA-MSCs exhibited high mRNA and protein levels of collagen I (COAL I) and its receptor DDR2. ShRNA-mediated knockdown of COAL I or DDR2 in CA-MSCs induced a change in morphology towards epithelial, decreased expression of epithelial to mesenchymal transition (EMT) markers, and impaired migration. Co-culture of CA-MSCs with BCCs led to increased BCC proliferation, EMT, invasion, and increased DDR2 expression in BCCs compared to single cultures of BCCs, which was blocked by COAL1 and DDR2 shRNA in CA-MSCs. Live imaging studies revealed that shCOAL1 and shDDR2 was sufficient to completely disrupt the organized migration pattern of BCCs aligned with CA-MSCs. In vivo, xenografts derived from MDA-MB-231 cells co-cultured with shControl CA-MSCs exhibited increased collagen I deposition in the tumor microenvironment, increased tumor growth, and metastasis compared to the single cultures of MDA-MB-231 cells. Remarkably, shDDR2 in CA-MSCs reduced tumorigenesis and metastasis.

Conclusion: We successfully isolated and characterized CA-MSCs, confirming their presence in human breast cancer metastasis. Our findings suggest that collagen I and its receptor DDR2 play a role in directional migration of breast cancer cells in alignment with CA-MSCs, a function that may be implicated in breast cancer invasion and metastasis. Downregulation of collagen I expression and signaling reduces tumor growth and metastasis in vivo. Modifying tumor microenvironment by manipulating collagen I and/or DDR2 levels in MSCs might be therapeutically useful in preventing metastasis.

#4398

Impact of Interleukin-22 on K-ras mutant lung tumor microenvironment and stemness properties.

Nasim Khosravi, Amber M. Cumpian, Soudabeh Daliri, Cynthia De La Garza, Mauricio S. Caetano, Seyed Javad Moghaddam. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Oncogenic K-ras mutations found in ~ 30% of all non-small cell lung cancers are associated with chemoresistance and poor prognosis. Using a K-ras induced lung cancer mouse model, CC-LR, we previously showed that K-ras mutant lung tumors have intrinsic inflammatory characteristics with activation of NF-kB pathway, release of inflammatory cytokines IL-6, and activation of the IL-6 responsive transcription factor STAT3. We have further shown that IL-6/STAT3 pathway, and IL-17 producing CD4 helper T cells (Th17 cells) through their main cytokine, IL-17A, play critical roles in promotion of lung cancer in this model. IL-22 is another effecter molecule secreted by Th17 cells which is highly expressed in our K-ras mutant mouse model. IL-22 is a unique cytokine, which seems to act exclusively on nonhematopoietic cells, with basal IL-22R expression in the epithelial cells and fibroblast, and mostly signals through STAT3 pathway. Here we found that genetic ablation of IL-22 in CC-LR mice (CC-LR/IL22-KO mice), causes significant reduction in lung surface tumor numbers by ~54% (2.1-fold). Histopathological analysis of lung sections confirmed a reduction in number and size of tumors in CC-LR/IL22-KO mice, which was associated with significantly lower tumor cell proliferation, angiogenesis and STAT3 activation. IL-22 ablation also reduced the numbers of inflammatory cells in bronchoalveolar lavage fluid, and decreased the expression of pro-tumor inflammatory cytokines such as IL-6, IL-17 and TNFα. This was associated with increased expression of anti-tumor Th1 cells -specific transcription factor (Tbet) and their activation markers, IFNγ, and GZB, and decreased expression of pro-tumor Th17- (RORƳ) and T regulatory (FOXP3+) specific transcription factors. Recent studies have shown an association between IL-22 and stem-cell like properties in colon cancer. In lung cancer, cell populations expressing NANOG, SOX2, Oct4 and/or aldehyde dehydrogenase activity are enriched with stemness properties. Interestingly, in CC-LR/IL22-KO mice we found significant reduction in expression of these stemness genes. Thus, we conclude that IL-22 promotes K-ras mutant lung tumorigenesis by inducing a pro-tumor inflammatory microenvironment with proliferative and angiogenic properties as well as protecting stemness characteristic in epithelial/tumor cells. Therefore, we propose pharmacological targeting of IL-22 as a potential therapeutic strategy in combination with conventional cytotoxic therapy, immune check point blockade, or other targeted therapies (e.g. MEK inhibition) for lung cancer patients with K-ras mutation.

#4399

Hypoxia as a driver of myeloid-derived suppressor cell recruitment in hepatocellular carcinoma via CCL26/CX3CR1.

David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Larry Lai Wei, Hui-Yu Koh, Regina Cheuk-Lam Lo, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. _The University of Hong Kong, Hong Kong, Hong Kong_.

Background and Objective: Myeloid-derived suppressor cells (MDSCs) accumulate in tumors and highly pro-tumorigenic. These MDSCs exhibit inhibitory functions against effector T cells and natural killer cells in tumor sites, as well as secrete pro-angiogenic factors or differentiate to endothelial cells to promote angiogenesis and metastasis. While it is appreciated that depletion of MDSCs could bring tumoricidal effects, there are significant gaps in knowledge about the underlying mechanisms responsible for MDSC recruitment to tumor sites. Hypoxia, O2 deprivation, is an important factor in the tumor microenvironment of HCC that modifies the stromal components. Using hepatocellular carcinoma (HCC) as a model, we investigated whether hypoxia is a driver of MDSC recruitment in HCC.

Experimental Procedures: Gene profiling of HCC cells exposed to hypoxia and normoxia were analyzed by transcriptome sequencing to identify potential hypoxia-induced chemokines for MDSC recruitment. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. Boyden chambers were used to evaluate the invasive ability of MDSCs. Flow cytometry was used to detect the frequencies of tumor-associated MDSCs in orthotopic HCC mouse models.

Results: We observed that MDSCs preferentially infiltrated into hypoxic regions in human HCC tissues and hypoxia-induced MDSC infiltration was dependent on hypoxia-inducible factors (HIFs). HIFs activated the transcription of chemokine (C-C motif) ligand 26 (CCL26) in HCC cells to recruit chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing MDSCs to the primary tumor. Knockdown of CCL26 in HCC cells profoundly reduces MDSC recruitment, angiogenesis, and tumor growth. Therapeutically, blockade of CCL26 production in HCC cells by HIF inhibitor, digoxin, or blockade of CX3CR1 in MDSCs by CX3CR1 neutralizing antibody could substantially suppress MDSC recruitment and tumor growth.

Conclusion: This study unprecedentedly reveals a novel molecular mechanism by which HCC cells direct MDSC homing to primary tumor and suggests that targeting MDSC recruitment represents an attractive therapeutic approach against HCC.

#4400

Hematopoietic age at onset of breast cancer dictates disease aggressiveness and progression.

Jaclyn Sceneay,1 Timothy Marsh,1 Irene Wong,1 Yuanbo Qin,1 Andreas Sjödin,2 Björn Nilsson,3 Sheila A. Stewart,4 Sandra S. McAllister1. 1 _Brigham and Women's Hospital, Boston, MA;_ 2 _Harvard School of Public Health, Boston, MA;_ 3 _Broad Institute, Boston, MA;_ 4 _Washington University School of Medicine, St Louis, MO_.

Aging is strongly associated with increased risk of developing breast cancer. Young age at diagnosis is typically associated with more aggressive disease, while breast cancer is generally more indolent in older women. Among the different breast cancer subtypes, triple negative breast cancer (TNBC) accounts for ~15% of invasive breast cancer cases and TNBC patients have a poor prognosis due to lack of targeted therapies and limited sensitivity to chemotherapy. Emerging studies indicate that older women with TNBC have a better outcome than younger women for reasons that are unclear. Nevertheless, older patients tend to suffer co-morbidities associated with chemotherapy and often forego treatment, resulting in disease recurrence and a poor outcome for these patients. Treatment of TNBC therefore presents age-specific challenges.

Here, we investigated how age affects the composition and function of pro-tumorigenic bone marrow cells using TNBC models that enable us to interrogate the impact of bone marrow derived cells on both tumor cells and the tumor microenvironment. Consistent with clinical observations, we found that disease progression was significantly more aggressive in young mice relative to aged mice. We identified age- and tumor-dependent molecular and functional changes to bone marrow derived hematopoietic cells that impact TNBC progression. A specific population of Sca1+/cKit- bone marrow cells that expresses CSF1R and secretes the growth factor granulin to support stromal activation and robust tumor growth in young mice, fails to promote tumor outgrowth in aged mice, resulting in maintenance of disease indolence. Importantly, bone marrow cells from young mice are sufficient to activate a tumor-supportive microenvironment and rescue tumor progression in aged mice.

The results presented here indicate that hematopoietic age is an important determinant of TNBC aggressiveness and provide rationale for investigating age-stratified therapies designed to prevent activation of tumor-promoting bone marrow cells.

### Advocates Poster Session 2

#ADV21

Clinical trials - breaking through the barriers.

Dawn K. Aldrich. _SOLUTIONS Cancer Resource Center, Inc., New Rochelle, NY_.

SOLUTIONS Cancer Resource Center, Inc. exists to provide information and assistance to cancer diagnosed clients, their families and friends. Its mission is to provide a bridge that spans the gulf between the physicians' lack of time for individual attention and the patients' need for further information and support to bolster a client's ability to make informed and confident decisions in the care of their condition. We offer support to the minority community through informational sessions on the importance of prevention and early detection as well as where to find the low cost or free screenings, clinical trials, peer connections, conduct research through surveys, informational and nutritional sessions. The challenge of treating minorities with cancer is one that is questioned with skepticism as the current statistics indicates that the mortality rates are higher within the minority population. There is a call to focus on the effective treatment of those with Multiple Myeloma. Although we see the progress in the survival rates of those with Multiple Myeloma, the process of gaining access to clinical trials is discouraging. Additional barriers include - trust, education and financial considerations.

#ADV22

Addressing oral health disparities in Washington, DC.

Everett E. Dodson. _Georgetown Lombardi Comp. Cancer Ctr., Silver Spring, MD_.

Disparities in dental care represent a crisis in Washington- perhaps the worst example of oral health disparity in the nation. Based on the information from the D.C. cancer registry, the District of Columbia has one of the highest incidence and lowest survival rates of oral cancer in the nation. The goal of the current project is to identify factors that impede oral health, cancer prevention, behavior change and identify effective education and communication strategies.Other research studies includes and exercise randomized clinical trial targeting African American women with metabolic syndrome and high risk of breast cancer, environmental studies, human papilloma virus (HPV) educational intervention studies, an exercise clinical trial for Latinas and an exercise study for men.

#ADV23

Bridging research advocacy with patient advocacy to increase awareness in the African American community.

TeMaya Eatmon. _Advocate, Atlanta, GA_.

The mission of Susan G. Komen is to save lives and end breast cancer forever by empowering others, ensuring quality care for all and energizing science to find the cure. Atlanta Affiliate Mission: Enable our community to detect and survive breast cancer by facilitating access to quality care, providing education and supporting research. As an independent advocate, I have the opportunity to work with Susan G. Komen on national and local level, and assist both in having an impact on the African American community through the participation of Advocates in Science and Sisters of Promise. The Advocates in Science program (AIS) is a community of dedicated volunteer advocates who work to reduce the burden of breast cancer in their communities. Research advocates bring the patient voice to research, ensuring that the unique and valuable perspectives of breast cancer patients, survivors, and co-survivors are integrated into the scientific dialogue and decisions, which impact progress toward ending breast cancer. The tools/opportunities for the advocates given by Susan G. Komen are through the participation in the AAADV meeting, the San Francisco Breast Cancer Symposium, and serving on the Komen Advocacy Advisory Taskforce. The Atlanta Affiliate clearly identified the urgency in reaching the African American community and created Sisters of Promise. Sisters of Promise was designed to bring influential African American women in Atlanta that embrace Komen's mission to become ambassadors who have a voice in all communities, with particular focus on the African American community, to spread awareness and to raise funds for Komen Atlanta to help provide the services necessary to change the statistics impacting African American women in our community. Being part of the Komen Speaker's Bureau and facilitating educational sessions in settings designed for African Americans in their communities achieve this. The Atlanta Affiliate has chosen these items to combat the high African American mortality rates from breast cancer.

#ADV24

More than my mutated BRCA genes: the psychosocial impact of hereditary breast and ovarian cancer.

Brandi Forbes. _FORCE: Facing Our Risk of Cancer Empowered, Cincinnati, OH_.

FORCE: Facing Our Risk of Cancer Empowered is the only national nonprofit dedicated to improving the lives of individuals and families affected by hereditary breast and ovarian cancer (HBOC). FORCE was founded on the principle that no one should have to face hereditary breast and ovarian cancer alone. Our mission is to improve the lives of those affected by hereditary breast, ovarian and related cancers. We accomplish this mission by creating awareness, supplying information and support to our community, advocating for and supporting research and working with the research and medical communities to help people dealing with hereditary breast, ovarian and related cancers. Hereditary cancer accounts for about 10% of all cancer diagnoses and, while this may seem small, the affect hereditary cancer has on individuals and families can be devastating. BRCA-mutation carriers have a lifetime risk of developing breast cancer of up to 87%, ovarian cancer up to 60% and increased risks for melanoma and uterine, pancreatic, prostate and colon cancer. The HBOC-community is often overlooked and underrepresented. Genetic counseling and testing can provide individuals and families with knowledge and a sense of empowerment. Once diagnosed, however, the decisions that must follow are often overwhelming, daunting and, even with the best support, sometimes debilitating. A sub-group of the HBOC-community is young women; those under the age of 30. Many have already undergone genetic testing, know their positive mutation status and increased cancer risk, and have begun making decisions about their future that no one their age should have to make. They face ongoing cancer screening surveillance, which may produce anxiety, risk-reducing surgeries, which alter their body image, and are forced to make choices about their future and family planning. The psychosocial impact of a BRCA-mutation diagnosis in young women is immensely overlooked, understudied and largely ignored by medical professionals.

#ADV25

Cancer prevention in Anguilla.

Jennifer Gumbs. _Anguilla Cancer Society / Health Authority of Anguilla, The Valley, Anguilla_.

In March 2011, the Health Authority of Anguilla, a statutory organisation of the Government of Anguilla saw the need to promote cancer awareness in Anguilla. A committee was set up under the name Cancer Awareness Steering Committee (CASC). Later in May 2012, with the guidance of several organisations including the Foxchase Cancer Center, Anguilla Community College and the Anguilla Community Foundation the Anguilla Cancer Society (ACS) was established. The Anguilla Cancer Society service is extended to the entire population. Anguilla is 35 square miles located in the Caribbean. Population approx: 14,000.00 people. Our Theme is: Cancer is Everybody's Business.Purpose: To create cancer awareness among the general public by promoting early detection through educational programmes, encouraging and promoting a healthy lifestyle through diet and exercise, and providing a network of support to persons affected by cancers.Objectives: To promote early detection among 90% of the population, to encourage and promote a healthy lifestyle among 95 % of the population , to provide a network of support to 85 % of persons affected by cancer.

#ADV26

Kim Hall Jackson, from patient to advocate. Recovery was the beginning..

Kim H. Jackson. _KHJ Enterprises, Philadelphia, PA_.

Kim Hall Jackson, proprietor of KHJ Enterprise, LLC is a certified meeting planner and events professional with more than 20 years experience in hospitality, customer service and event production and management. This promising career was almost cut short at the age of 45, when Kim was diagnosed with Stage III Colorectal Cancer. Through immense support from family, friends, and health professional, Kim emerged from this fight victorious and cancer-free. Now Kim is using her experience and strength for events for education, advocacy and patient support.

#ADV27

BCAN: leading the way to awareness and a cure.

Neil J. Kurtz. _Bladder Cancer Advocacy Network, Port Jefferson, NY_.

Founded in 2005, BCAN is the only national advocacy organization devoted to advancing bladder cancer research, providing information and support for those impacted by the disease, and raising awareness. BCAN provides patients, caregivers and the medical community with the educational resources and support services needed to navigate their bladder cancer journey. In addition to working with medical and research professionals dedicated to the prevention, diagnosis and treatment of bladder cancer, BCAN also seeks to empower the patient community to share experiences and participate in building awareness of the need for a cure. Additionally, BCAN raises funds and awards research grants to support the most promising research, providing the greatest opportunity to advance the understanding of bladder cancer and how to improve management and treatment. The grants include the Young Investigators Awards, Bladder Cancer Research Innovation Awards and John Quale Travel Fellowships.

#ADV28

"Backstage" patient advocacy.

Ian Liston. _Patient Advocate, Bolney, United Kingdom_.

As an independent patient advocate Ian Liston has made a speciality in recent years of engaging with those involved "behind the scenes" in the bench to bedside approach of cancer care; meeting especially with people in labs and research centres who would never usually get to meet or talk with a cancer patient survivor who has participated successfully in several clinical trials. Clinical research in the cancer community can be greatly enhanced by those 'on the bed' feeding back directly to those working 'on the bench' who may never come into contact with a patient. - Not forgetting those who work in call centres and fund raising organisations, battling to raise vital funds for cancer research: they can be hugely encouraged by meeting a patient survivor. A primary objective is to enhance this experience with a two-way feedback to give advocates a better understanding of the science and methodology behind cancer research in this fast changing and rapidly evolving area of medicine, especially in the field of genetics. Structured science-based foundation courses to help patient advocates are beginning to appear in several countries and are becoming a necessity in the landscape of cancer care. This 'poster' will review the situation so far and look towards future possibilities.Meeting and sharing experiences with advocates from all over the world is an enormously valuable exercise for all those involved. This value is enhanced by being able to report back and share experiences with fellow patients, patient advocates and colleagues in affiliated organisations in the UK.

There is currently relatively little international exchange between patient advocates. This conference is an opportunity to forge and maintain valuable links.

#ADV29

Advocating for cancer research in Japan.

Yoshiyuki Majima. _NPO Pancreatic Cancer Network Japan, Tokyo, Japan_.

NPO PanCAN Japan is a Japanese affiliate of Pancreatic Cancer Action Network in California. We have 3 missions -- Advance Research, Support Patients, Create Hope. In recent years, thanks largely to US and EU cancer research efforts we have seen new discoveries, and some of them have given us hope that we are within reach of cure for certain types of cancer. At the same time, there are cancer types that are recalcitrant to treatments and we are quite ways to go in order to find diagnostic tools for them. In Japan, we are dependent on US and EU based research and clinical trials for the development of new therapies. However, there are certain cancer types which are more common in East Asia but not so common in US and EU. As a result, the development of new therapies are lagging behind those types of cancer that are prevalent in US and EU. This poster disucusses first, which types of cancer are more prevalent in Japan. Secondly, the reason why cancer research in Japan is important for Japanese patients. Lastly, this poster explores the possibility of collaboration between east and west to find a cure.

#ADV30

Local procedures for treating extracranial, nonsymptomic lesions in oligometastatic breast cancer.

Joan Mancuso. _SHARE: Self-help for Breast and Ovarian Survivors, Doylestown, PA_.

Local procedures for treating extracranial, nonsymtomatic lesions in oligometastatic breast cancer Oligometastatic, which refers to metastatic cancer that is not widely disseminated throughout the body, is a term that two radiologists at The University of Chicago used in a 1995 article to propose that patients with limited metastatic spread could benefit from local procedures such as radiation. That idea continues to resonate today, as was seen at the American Association for Cancer Research's 2015 annual meeting in a session on oligometastasis. Even though metastatic breast cancer is systemic, local treatments for treating extracranial tumors with no symptoms in cases of limited metastatic spread are often bypassed in favor of multiple lines of chemotherapy. An alternative is to combine those therapies with local procedures, which also include surgery and interventional radiology. Such a protocol is particularly conceivable in the age of biologic drugs. As a volunteer and breast cancer advocate at SHARE Cancer Support, my goals are aligned with SHARE's mission to "create and sustain a supportive network and community that empowers breast and ovarian cancer survivors." I believe strongly in educating those living with cancer to become better self-advocates and understand potential treatment options. To that point, oligometastatic is a term that few of those living with metastatic breast cancer have ever heard. The poster covers whether oligometastasis has a biological basis, potential biomarkers for those who might benefit from local procedures, and clinical trials. SHARE supports survivors with a helpline in English and Spanish, support groups, educational programs and webinars, as well as a Latina section that reaches out to Hispanics, "ambassadors" that connect with the Black community, and a breast cancer survivor who facilitates a Japanese-language support group. SHARE supports those living with metastatic breast cancer by also providing nationwide, weekly telephone supports groups.

#ADV31

Life and Breath.

Linnea Olson. _Patient Advocate, Lowell, MA_.

I was diagnosed with lung cancer in 2005 when there were very few treatment options and one targetable mutation (EGFR). I am also fortunate to receive my treatment at a hospital that is a major cancer research center. Found to be positive for an ALK mutation in 2008, timing and location have allowed me access to cutting edge treatments. I am currently enrolled in my third phase one clinical trial and have also participated in trials for circulating tumor cells and circulating DNA. My poster shall be a timeline of developments/discoveries in the treatment of thoracic cancers along with a corresponding timeline of my cancer and the therapies I have received. This poster will reflect the mission of my blog, www.outlivinglungcancer.com, which has been to share my personal experience in such a way as to provide information but also hope to those impacted by lung cancer

#ADV32

FCNA-MH.

Cherry Sloan-Medrano. _Filipino Cancer Network of America-Metro Houston, Missouri City, TX_.

Filipino Cancer Network of America-Metropolitan House: All about us

Founded in 2008 as non-profit organization whose mission is to serve not only Filipino- Americans but the community in general, assisting in cancer care navigation at M D Anderson Cancer Center with appropriate referrals, assisting in appointments and education of families and caregivers on available cancer resources and other issues related to cancer care. This organization has served the Metropolitan Houston area cancer survivors and caregivers/ families by providing cancer education symposia, cancer support group meetings quarterly, and a celebration of the gift of life get together for education, fund-raising and cancer survivors' testimonials in spring. FCNA-MH holds an annual retreat for all cancer support facilitators, survivors and families every fall and a health fair/ picnic for the same population with the celebration of life of all cancer survivors once again and remembering those who succumbed to cancer by releasing red (live) and white (deceased) balloons at the event.

This organization has gained national attention and is now a cancer initiative of the Philippine Nurses Association of America (PNAA), Community Outreach Committee. All 50 chapters are encouraged and assisted in developing their own cancer initiatives by FCNA-MH. The goal of this organization is not limited to cancer service to Houston but throughout Texas, the nation and globally.FCNA-MH has nurse volunteers who are fully dedicated to cancer prevention, cancer education and cancer support to all who want to access its services. It has served various ethnic groups since its existence and has assisted many in the quest for a premier cancer care in our city.

#ADV33

Advocacy can take many forms.

Patrick Sullivan. _Team Finn Foundation, Vancouver, British Columbia, Canada_.

The TeamFinn / Ac2orn poster will foster on the role of advocacy in advancing a specific pan-Canadian pediatric genomics initiative led by the Terry Fox Research Institute. The purpose of the poster is to demonstrate the various important roles that cancer research advocacy did play in this project which focusses on young Canadians with relapsed, refractory and hard to treat cancer.

#ADV34

LGBT Thriving after Cancer.

J. Catherine Sumner. _Patient Advocate, Pleasant Hill, CA_.

The poster will discuss: Outreach to LGBT, including additional outreach regarding screening for female-specific cancers to those who present themselves as more masculine; LGBT Specialized Care and Concern through Treatment, including finding doctors with some knowledge and connection to the LGBT community; LGBT Thriving after Cancer .

#ADV35

Ovarian cancer- Why did I survive.

Kerie Berkowitz. _OCNA: Ovarian Cancer National Alliance, Moorpark, CA_.

In 1990 my life would change forever. I was diagnosed with stage 1B epithelial ovarian cancer at the age of 35. The road from diagnosis and constant pain to diagnosis would take 6 months.. I did not know anyone with ovarian cancer with the exception of one celebrity who was in the fight for her own life, Gilda Radner from Saturday Night Live. Her book, "It's Always Something" saved my life.

It held my hand to become my own advocate through what I now know to be a life saving read.OCNA~ Ovarian Cancer National Alliance 1) Advances the interests of ovarian cancer through advocacy, awareness and fund raising, 2) Programs include research advocacy and the STS Program- Survivors Teaching Students Its constituencies include women with ovarian cancer and their families. Ovarian cancer long term survival comes with its own set of challenges .Early menopause, emotional well being, relationships and our own sexuality are just a sample of surgically induced menopause due to ovarian cancer. Most of us are not given the tools from our doctors to deal with a host of symptoms after we survive 25 plus years. How can we best approach our doctors for a better sense of what we can do to improve our quality of life? What are the symptoms of recurrence after hysterectomy? How much do doctors actually know about long term survival ? What is the key to not going from doctor to doctor when a health related issue from ovarian cancer does arise? The battle never really ends even after 25 years. There is always the fear of recurrence. Do I have the responsibility to test for BRCA ? Osteoporosis , cardiovascular disease, libido and intimacy are sometimes lifelong health factors. There can be anxiety and concerns about speaking to your doctor when it comes to sexuality and intimacy after cancer. The pros and cons of estrogen are still an issue today. Why are women are still being prescribed medications that may be harmful to cancer survivors ? Where are we in 2016 and is anybody listening to long term survivors? Through advocacy programs like OCNA, the Ovarian Cancer National Alliance, The OCRF The Ovarian Cancer Research Fund , STS, Survivors Teaching Students, we can begin again. When feeling like "YOU" after your journey allows you to become your own best advocate, you can find some of the answers. Participation through advocacy is key if we are going to make strides against ovarian cancer. 

# Wednesday, April 20, 2016

## MOLECULAR AND CELLULAR BIOLOGY / GENETICS:

### Cell Signaling

#4401

Disclosing the Hippo signaling networks in castration-resistant prostate cancer.

Damien Ready,1 Yuksel Yildiz,2 Vincent Funari,3 Serdar Bozdag,1 Bekir Cinar4. 1 _Marquette University, Milwaukee, WI;_ 2 _Adnan Menderes University, Aydin, Turkey;_ 3 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 4 _Clark Atlanta University, Atlanta, GA_.

Prostate cancer often evolves as a metastatic castration-resistant disease, which is lethal. Despite recent advances, the molecular mechanisms contributing to this lethal disease are largely unknown. Our initial studies have indicated that the loss-of-function of the STK4-encoded MST1 protein kinase, a key component of the Hippo tumor suppressor pathway, may play a prominent role in the etiology of prostate cancer progression. Here, we establish cytoplasmic-, membrane- or nuclear-localized MST1 cell models to elucidate a possible mode of actions by which MST1 control the disease progression. We show that membrane or nuclear MST1 confers superior growth inhibitory effects on castration-resistant prostate cancer cells ex vivo and in vivo in comparison with cytoplasmic MST1, though they express the similar levels of MST1 protein. Consistently, a number of differentially expressed genes (2-fold with FDR ≤ 0.05) regulated by membrane or nuclear MST1 protein are much greater than cytoplasmic MST1, as assessed by RNA-sequencing and bioinformatics analysis. Functional annotation clustering reveals that genes affected by cytoplasmic MST1 protein are mostly associated with cell-cell interactions and membrane trafficking. However, expression of genes altered by membrane or nuclear MST1 involves much broader cellular biology that includes cell signaling, steroid biogenesis, lipid and cholesterol metabolisms, and developmental processes. In addition, Ingenuity Pathway analysis indicates that several genes effected by ectopic MST1 protein the defined cell location interact with androgen receptor pathway signaling, a critical regulator of prostate cancer pathogenesis. The results of this study suggest that the Hippo pathway signaling restricts prostate cancer progression by modulating the array of signaling networks, indicating important therapeutic implications for human disease.

#4402

Glycosyltransferase ST6Gal-I enables cancer cells to survive upon growth factor deprivation.

Colleen Britain, Susan Bellis. _University of Alabama at Birmingham, Birmingham, AL_.

Tumor growth often outpaces the tumor's ability to form new vascularization leading to a tumor microenvironment containing areas with limited access to serum-derived growth factors (GFs). How tumor cells are able to proliferate and survive in the absence of GFs is not completely understood. Recent work from our group has uncovered a novel mechanism by which ST6Gal-I, a glycosyltransferase upregulated in many cancers, enables cells to evade GF deprivation-mediated apoptosis. ST6Gal-I is a trans-Golgi sialyltransferase that catalyzes the addition of a bulky, negatively charged sialic acid in an α2-6 manner to the termini of N-linked glycans on glycoproteins bound for the plasma membrane. This sialic acid addition can modulate protein conformation and function, similar to phosphorylation. Prior studies from our group show that ST6Gal-I activity confers a cancer stem cell phenotype as well as enhances chemoresistance, leading us to hypothesize that ST6Gal-I promotes tumor cell survival, invasion and metastasis. To better understand the role ST6Gal-I plays in tumor cell survival upon GF deprivation, we monitored the response of ovarian and pancreatic cancer cells with either ST6Gal-I overexpression or knockdown to serum starvation. Upon serum starvation, cells overexpressing ST6Gal-I preferentially survived and continued to proliferate whereas cells with ST6Gal-I knockdown underwent cell death more readily, as evidenced by live-dead cell staining and cell proliferation assays. Furthermore, in the absence of serum, ST6Gal-I activity promotes the activation and/or expression of multiple pro-survival proteins, such as Akt, p70-S6K, NFκB, and cIAP2, as well as cell cycle regulators including CyclinD2. Interestingly, upon long-term GF deprivation, immunoblotting analysis reveals a selective enrichment for ST6Gal-I in the remaining live cells providing evidence that cells with high ST6Gal-I expression selectively survive GF deficiency. Collectively these data suggest that upregulation of ST6Gal-I in tumor cells may promote cell survival in areas of the tumor microenvironment that have decreased access to a blood supply.

#4403

SMYD2-mediated lysine methylation regulates nuclear transport of beta-catenin in human cancer cells.

Xiaolan Deng, Yusuke Nakamura, Ryuji Hamamoto. _University of Chicago, BSD, Chicago, IL_.

The canonical Wnt-signal pathway is a major signaling system in cancer cells as well as stem/progenitor cells. It is well-known that binding of Wnt molecules to cell surface receptors, including a Frizzled (Fz) protein and LDL receptor-related protein 6, initiates a transduction cascade leading to stabilization of the transcription coactivator β-catenin, which then enters the nucleus to form a transcriptional complex with T cell factor (TCF) or lymphoid enhancer factor (LEF) to activate the expression of TCF/LEF downstream genes, such as cyclin D1 (CCND1) and c-Myc. However, the molecular mechanisms that regulate nuclear localization of β-catenin and its transcriptional activation are not fully understood. Here we demonstrate that the protein lysine methyltransferase SMYD2 methylates β-catenin at lysine 133, which is located in an amino-terminal domain (NTD), and regulates nuclear transport of β-catenin. After treatment with SMYD2-specific siRNAs in hepatocellular carcinoma SNU475 and SNU449 cells, we fractionated cell components and conducted western blot analysis and found that nuclear localization of β-catenin was significantly decreased after knockdown of SMYD2; we also confirmed this result by immunocytochemistry. In addition, we prepared a lysine 133-substituted β-catenin mutant expression vector and transfected it into 293T cells with a SMYD2 expression vector. We observed that protein amount of lysine 133-substituted β-catenin in the nucleus is significantly lower than that of wild-type β-catenin by western-blot and immunocytochemical analyses. These results indicate that SMYD2-mediated methylation plays a critical role in the nuclear transport of β-catenin in cancer cells. This is a novel mechanism of the canonical Wnt-signal pathway regulated by lysine methylation.

#4404

NFAT-5/STAT-3 interaction mediates synergism of high salt with IL-17 towards induction of VEGF-A expression in breast cancer cells.

Suneetha Amara,1 Dalal Alotaibi,2 Venkataswarup Tiriveedhi3. 1 _Mercy Hospital, St Louis, MO;_ 2 _Tennessee State University, Brentwood, TN;_ 3 _Tennessee State University, Nashville, TN_.

Chronic inflammation has been considered an important player in cancer proliferation and progression. High salt (sodium chloride) has been considered a potent inducer of chronic inflammation. In this study, we investigated the synergistic role of high salt with IL-17 towards induction of inflammatory and angiogenic stress factor VEGF. Stimulation of MCF-7 breast cancer cells with high salt (0.2 M NaCl) and sub-minimal IL-17 (1 ng/mL) enhanced the expression of VEGF-A (2.9 and 2.6 fold, respectively p<0.05) compared to untreated cells. Further, co-treatment with both high salt and sub-minimal IL-17 demonstrated a 5.9 fold increase in VEGF-A expression (p<0.01), thus suggesting a synergistic role of these both factors. VEGF-A promoter analysis and specific siRNA knock-down of transcription factors revealed that high salt induces VEGF-A through NFAT5, while IL-17 induced VEGF-A via STAT-3 signaling mechanisms. Treatment of the supernatant from activated MCF-7 cells on normalized human aortic endothelial cells induced enhanced cell migration and expression of migration specific factors VCAM, β1-integrin and CD31. Based on these data, we conclude that high salt synergizes with pro-inflammatory IL-17 to potentially induce cancer progression and metastasis through VEGF-A expression. Therefore, low salt diet, anti-NFAT5 and anti-STAT3 therapies might provide novel venues for enhanced efficiency of current cancer therapy.

#4405

Over-expression of PARP is associated with an aggressive phenotype and can be synergistically targeted using combination of PARP and XIAP inhibitors in breast cancer.

Maqbool Ahmed, Azhar R. Hussain, Shaham Beg, Saravanan Thangavel, Rafia Begum, Dahish Ajarim, Fouad Al-Dayel, Abdul Khalid Siraj K. Siraj, Khawla S. Al-kuraya. _King Faisal Specialist Hospital & Res. Ctr., Riyadh, Saudi Arabia_.

Breast cancer is the most common cancer in females and despite improvement in treatment modality with the introduction of hormonal targeted therapy, these cancers still remains a major cause of morbidity and mortality in females. Poly ADP ribose polymerase (PARP) is an enzyme which plays a critical role for repairing single-strand breaks. The main function of PARP1 is dependent upon cellular stress responses and directs cells towards DNA repair based on the strength of the stress stimulus. PARP has been found to be over-expressed in various tumors including ovarian cancer; breast cancer and lung cancer, however, its role in the pathogenesis of breast cancer of Middle Eastern origin have not been completely elucidated. Therefore, we examined the expression of PARP in a large cohort of more than 1000 breast cancer by IHC in a tissue microarray format and found PARP was over expressed in 44.7% (451/1008) and was found to be significantly associated with aggressive markers such as Triple negative phenotype, high grade tumors, large tumor size, stage 4 disease and had a poor 5 year overall survival of 72.6 months as compared to 85.4 months (p=0.0005). Interestingly PARP expression was also found to be significantly associated with aggressive molecular markers such as activated AKT, XIAP and proliferative marker; ki67. Next, in vitro effect of inactivation of PARP was evaluated using a PARP inhibitor; olaparib on a panel of BC cell lines. Olaparib treatment alone did not affect the cell viability and apoptosis at doses up to 10μM concentration. However, when BC cells were treated with sub-optimal dose of Olaparib (1μM) and Embelin (XIAP inhibitor)(5μM) in combination for 48 hours, there was appreciable inhibition of cell viability via induction of apoptosis. Combination of Olaparib and Embelin induced apoptosis via activation and cleavage of caspases-9, -3 and PARP in BC cells. These studies highlight the importance of targeting PARP in combination with either small molecular inhibitors or chemotherapeutic agents in BC can improve the management and survival of this aggressive disease.

#4406

Inducible knockdown of insulin receptor substrate I desensitizes ER alpha response to both agonist (estradiol) and antagonist (fulvestrant) in MCF-7L breast cancer cells.

Xihong Zhang, Sidhant Varma, Douglas Yee. _University of Minnesota, Minneapolis, MN_.

Insulin receptor substrate (IRS) proteins are adaptor proteins phosphorylated by insulin and insulin-like growth factor (IGF) receptors. In addition to their roles in regulating IGF and insulin responses, IRS proteins function in other pathways in normal and malignant physiology. Our data suggested that IRS1 enhanced IGF/insulin stimulated proliferation while IRS-2 was important in regulating motility. Therefore IRS proteins could be potential therapeutic target for cancer therapy. We have previously shown that reduced IRS1 can hinder cancer cell growth and tumorigenicity stimulated by IGF-I, IGF-II and insulin. In this study, we evaluated whether inducible IRS1 suppression affected estrogen receptor-alpha (ER) responsiveness to ER agonist estradiol and antagonist fulvestrant. We evaluated several doxycycline inducible shRNA IRS1 constructs (Thermo Scientific pTRIPZ inducible Lentiviral IRS1 shRNA) expressed in the ER+ MCF-7L cell line. Doxcycline treatment reduced IRS1 mRNA and protein levels confirmed by qRT-PCR and immunoblot in several independently selected clones. We used a representative clone (3G5) to evaluate effects on ER function. After IRS1 inducible knockdown, estradiol (E2) stimulated growth was suppressed. To investigate if reduced IRS1 regulated E2 stimulated binding of ER to the pS2 estrogen response element (ERE), chromatin immunoprecipitation (ChIP) assays were performed. Compared to non-induced MCF-7L cells, inducible IRS1 suppression demonstrated significantly reduced binding of ER to this promoter. The pharmacologic antagonist of IRS proteins, NT157, also resulted in diminished ER binding to pS2 promoter. mRNA of several ER regulated genes, such as PGR, TFF1, etc, showed downregulation in response to E2 when IRS-1 was suppressed. We have shown that the pure steroidal ER antagonist fulvestrant activates EGFR signaling in an ER dependent manner through upregulation of EGFR ligands. However, inducible IRS1 suppression blocked this ER-dependent activation of EGFR phosphorylation by fulvestrant yet EGFR phosphorylation remained intact when stimulated by addition of EGF. These data suggest that regulation of EGFR ligands by fulvestrant also requires IRS-1 expression. We conclude that ER function is affected by IRS-1 expression. Thus, IRS-1 may also be a target in endocrine responsive and resistant breast cancers.

#4407

Inhibition of STAT3 in primary trastuzumab-resistant HER2-positive cells leads to decrease cell viability.

Xousaen M. Helu, Alfida Fernandez, Daisy D. De Leon. _Center for Health Disparities and Molecular Medicine, Loma Linda University, Loma Linda, CA_.

Breast Cancer is the second leading cause of cancer related death in women. Approximately, 20%-30% of breast cancer patients overexpress Human Epidermal Growth Factor Receptor 2 (HER2). HER2 Is a Tyrosine Kinase receptor involved in proliferation and survival and its overexpression makes it a good target for patients with HER2 positive breast cancer. Trastuzumab (TRA) is a recombinant monoclonal antibody that binds HER2 receptor and inhibits its signaling. Unfortunately, clinical studies have demonstrated that 30% of patients that overexpress HER2 do not respond to Trastuzumab treatment. Therefore, understanding the molecular mechanisms underlying Trastuzumab resistance is essential to develop new therapies to treat these breast cancer patients. Previous studies in our lab demonstrated that a decrease in cell growth of HER2 overexpressing cells occurred when IGF2 and HER2 were inhibited. Nevertheless, these cells overcame the growth inhibition and the resulting proliferative growth coincided with an increase in STAT3 activation. STAT3 is a transcription factor that promotes cell proliferation and can regulate apoptosis in cancer cells. These findings lead us to hypothesize that blocking STAT3 together with IGF2 and HER2 will inhibit proliferation and cell viability of Trastuzumab resistant breast cancer cells. We treated Trastuzumab-resistant HER2 positive JIMT1 cells with HER2, IGF2, and/or STAT3 inhibitors. After 24 hours of treatment, there was a marked difference in cell morphology observed between the treatment groups. Cell viability was measured using WST-1, a reagent that measures mitochondrial activity. A significant decrease in cell viability was observed when cells were treated for 24 hours with a commercially available STAT3 inhibitor (Sttatic). There was no difference between the treatment groups when STAT3 was inhibited by Stattic or when STAT3 was inhibited in combination with IGF2 and/or HER2 inhibitors. These findings suggest that in Trastuzumab-resistant HER2 positive JIMT1 cells, STAT3 activation overcomes the inhibition of the HER2 and IGF2 proliferation pathways resulting in cell death prevention. Our study demonstrate that Trastuzumab-resistant HER2 positive JIMT1 cells can activate STAT3 as an alternative pathway to evade apoptosis and promote chemoresistance. Our data also illustrate the challenge of treating cancer based on single therapies since tumors have many alternative pathways to promote growth.

#4408

Mutant ABL1 is a genetic dependency in non-small cell lung cancer amenable to pharmacological intervention.

PEDRO Torres-Ayuso, Ewelina Testoni, Natalie L. Stephenson, Anna A. Marusiak, Eleanor W. Trotter, Andrew Hudson, Cassandra L. Hodgkinson, Christopher J. Morrow, Caroline Dive, John Brognard. _Cancer Research UK Manchester Institute, Manchester, United Kingdom_.

Non-small cell lung cancer (NSCLC) is the most frequent lung cancer subtype and it affects near 1.5 million patients worldwide annually. The lack of actionable mutations in NSCLC patients presents a significant hurdle in the administration of targeted therapies for this disease, and their identification is an urgent unmet need. Here we identify somatically mutated ABL1 as a genetic dependency that is required to maintain NSCLC cell survival. We demonstrate that NSCLC cancer cells with ABL1 mutations are sensitive to ABL inhibitors and we verify that the drug-induced effects on cell viability are specific to pharmacological inhibition of the ABL1 kinase. Consistent with structural modeling, we demonstrate that mutations in ABL1 identified in primary NSCLC tumors and a lung cancer cell line increase downstream pathway activation compared to wild-type ABL1. Finally, we show that imatinib suppresses lung tumor growth in vivo, specifically in lung cancer cells harboring a gain-of-function (GOF) mutation in ABL1. In summary, our results suggest that NSCLC patients with ABL1 mutations could be stratified for treatment with imatinib in combination with other therapies.

#4409

Assessment of sonic hedgehog pathway inhibition in a novel orthotopic xenograft bladder cancer murine model.

Peter A. Raven,1 Sebastian Frees,2 Betty Zhou,1 Claudia Chavez-Munoz,2 Michael Cox,2 Alan So1. 1 _University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Purpose of the study:

To develop a reliable and reproducible bladder cancer murine model for the evaluation of the SHH pathway.

Introduction:

The sonic hedgehog (SHH) signaling pathway has been shown to play an integral role in the maintenance and progression of bladder cancer. Smoothened inhibitors are currently used in the clinic for treatment of some skin cancers, however they have failed to be effective in bladder cancers in vitro. Blocking alternative downstream molecules of the SHH pathway may be an efficacious strategy for bladder cancer treatment. In vitro assays possess a number of limitations such as environmental differences, loss of heterogenicity and vascularization and artificial levels of growth factors and cytokines within the cell culture media. To overcome this animal models are needed to facilitate the study of carcinogenesis mechanisms. A new bladder cancer model is now required to evaluate experimental therapeutics as previous animal models have utilized the mistakenly identified cervical cancer cell line KU7.

Methods:

A novel orthotopic murine bladder cancer model was developed to assess the role of the SHH signalling pathway in tumor growth and disease progression. The model comprises the instillation of luciferase-expressing UM-UC3 human transitional cell carcinoma cells into the bladder of athymic nude mice following a poly-L-lysine irrigation of the bladder. Tumors are measured by bioluminescent imaging after intra-peritoneal injection of luciferin. Antisense oligonucleotides (ASO) targeted to the SHH pathway transcription factors, Gli family zinc fingers 1 and 2 (Gli1, Gli2), were assessed in this model. Immunohistochemistry (IHC) of proteins in the SHH pathway were evaluated and compared to our in-house tissue microarray (TMA) of clinical bladder cancer samples.

Results:

Tumors in this model grew in an average period of time of 40 days with high rates of engraftment and reproducibility. In vitro analysis of SHH pathway inhibition showed a decrease in bladder cancer cell viability and increase in apoptosis following Gli ASO treatment. This effect bypasses smoothened protein regulation which was found to be ineffective in some cell lines. Gli ASO treatment reduced tumor size, and prevented further growth, of UM-UC3 bladder cancers in this mouse model. IHC confirmed Gli2 knock-down and showed increased apoptosis and decreased proliferation in the in vivo tumors. Comparison of modelled tumors to clinical samples showed an increased and diffused Gli2 staining throughout the tissue in both cases.

Conclusions:

A murine intravesical model consisting of human tumors provides a robust and effective in vivo system for the assessment of cancer inhibiting treatments. Gli2 ASO proved to be a promising treatment for bladder cancer by inhibiting tumor growth and disease progression.

#4410

ZEB2 drives cell motility and metastasis in ER+ breast cancer cells through a novel, E-cadherin independent pathway.

Hope E. Burks,1 Lyndsay V. Rhodes,1 Elizabeth C. Martin,1 Van T. Hoang,1 Steven Elliott,1 Melody Badoo,1 Theresa Phamduy,1 Aaron Buechlein,2 Douglas Rusch,2 Douglas Chrisey,1 Erik Flemington,1 Kenneth Nephew,2 Bridgette Collins-Burow,1 Matthew E. Burow1. 1 _Tulane University, New Orleans, LA;_ 2 _Indiana University Bloomington, Bloomington, IN_.

Breast cancer is the most commonly diagnosed cancer in women, with the second highest mortality rate. The overwhelming majority of breast cancer deaths are a direct result of distant metastatic spread to vital organs such as lung, bone, and brain. The zinc finger transcription factor ZEB2 has been implicated as a driver of cancer cell motility, tissue invasion and metastasis in a number of diverse malignancies, but its role in breast cancer is not completely understood. We chose to examine the effects of direct overexpression of ZEB2 in the MCF-7 cell line, a luminal and estrogen receptor positive (ER+) derived breast cancer line. MCF-7-ZEB2 cells demonstrated significantly increased migration and invasion as well as an altered morphology in vitro compared to that of vector. Additionally, MCF-7-ZEB2 cells exhibited increased lung metastasis in vivo when implanted in an orthotopic xenograft mouse model. ZEB2 function in metastasis has canonically been attributed to transcriptional repression of the cell junction protein E-Cadherin. Here we establish that in ER+ breast cancer cells, ZEB2 fails to repress E-cadherin and promotes cell motility and metastasis through the induction of an E-cadherin independent signaling cascade. Next generation RNA sequencing analysis of the MCF-7-ZEB2 cells compared to vector revealed alteration of the MAPK signaling cascade, evidenced by enhanced expression of key motility and MAPK associated genes, including PLAU, EGF, ACTA2, and MMP9. Pharmacological inhibition of MAPK pathway completely abrogated ZEB2 induced migration, cell morphology changes and expression of target motility genes, confirming a necessary role for MAPK signaling in ZEB2-driven cell motility in ER+ breast cancer. Together these results indicate that the ZEB2 transcription factor drives motility in breast cancer cells through a novel MAPK dependent pathway, warranting further investigation into the mechanisms involved in ZEB2 action. Elucidating the pathways involved in ZEB2 function which are specific to ER+ breast cancer is an important step in understanding the processes underlying metastasis and has the potential to yield new therapeutic targets.

#4411

Cadherin engagement induces a dramatic increase in tyr-705 phosphorylation of the signal transducer and activator of transcription-3 (Stat3α) but not the dominant-negative isoform Stat3β.

Zaid Taha,1 Stephanie Guy,1 Maximillian Niit,1 Leda Raptis,1 Adina Vultur,2 Rozanne Arulanandam3. 1 _Queen's University, Kingston, Ontario, Canada;_ 2 _The Wistar Institute, Ottawa, Ontario, Canada;_ 3 _Ottawa Hospital Research Institute, Ottawa, Ontario, Canada_.

The purpose of this study was to examine the role of Stat3α and Stat3β in survival and apoptosis of normal and tumor cells.

The Stat3 transcription factor is activated by cytokine receptors of the IL6 family, and receptor and non-receptor tyrosine kinases, plays an etiological role in neoplasia. Stat3 activation entails phosphorylation at tyr-705, Stat3 dimerization through a reciprocal, SH2 domain-phosphotyrosine interaction, and nuclear translocation. This triggers transcription of genes involved primarily in cellular survival such as survivin, MCl1 and Bcl-xL, as well as cell division such as myc. We previously demonstrated that engagement of E- or N-cadherin or cadherin-11 induces a dramatic increase in total protein levels and activity of the small GTPases, Rac and Cdc42, through inhibition of proteasomal degradation. Activated Rac leads to a surge in secretion of cytokines of the IL6 family through the transcription factor NFκB and Jak kinases, and this in turn, activates Stat3 in an autocrine manner. Stat3 inhibition in confluent, non-transformed cells induces apoptosis, pointing to a key survival role for Stat3.

Full-length Stat3 (termed Stat3α) is composed of an SH2 domain, tyr-705 and a COOH terminus encoding the transcription activation domain (TAD). Stat3β is a naturally-occurring splice variant which is lacking TAD. Therefore, Stat3β dimerizes with Stat3α but is defective in transcriptional activation, resulting in inhibition of Stat3α function. Since tyr-705 is present in both isoforms, we examined the phosphorylation pattern of Stat3α vs Stat3β. Our results demonstrate that cadherin engagement brought about through confluence of non-transformed mouse fibroblasts results in phosphorylation of Stat3α-tyr705, despite the fact that the sequence around tyr-705 is the same in both isoforms.

The Large Tumor antigen of Simian virus 40 oncogene (TAg) interacts with the p53 and pRb tumor-suppressors, and this leads to activation of the E2F family of transcription factors, targeting cell division genes. At the same time E2F is a potent apoptosis inducer, hence the high demand of transformed cells for antiapoptotic signals, such as Stat3. Interestingly, our data demonstrated that SVLT expression results in phosphorylation of Stat3β. Therefore, this feedback loop that reduces the activity of Stat3α, triggers apoptosis of transformed cells selectively, because of their high E2F levels. As a result, certain tumor cells which may naturally express high Stat3β levels would be very sensitive to pharmacological Stat3 inhibition, a finding which could have significant therapeutic implications.

#4412

c-Myc-driven lymphomas require calcium-modulating cyclophilin ligand (CAML) for survival and proliferation.

Jennifer C. Shing, Lonn D. Lindquist, Richard J. Bram. _Mayo Clinic, Rochester, MN_.

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions in a complex to mediate tail-anchored (TA) protein insertion into the ER membrane. CAML has also been implicated as a regulator of endocytic trafficking, modulator of intracellular calcium signaling, and essential factor for the survival and proliferation of specialized immune cells. To investigate the role of CAML in cell biology and cancer, we generated Eµ-Myc transgenic mice that carry a conditional deletion allele of CAML. From these mice, we derived B cell lymphoma cell lines driven by c-Myc and induced deletion of the CAML gene by treatment with tamoxifen to activate Cre-ERT2 in vitro. Homozygous loss of CAML in multiple independent B cell lymphoma lines activated apoptosis, while heterozygous deletion led to reduced cell proliferation. Apoptosis was inhibited by caspase inhibitor Q-VD-OPh or Bcl-2/xL overexpression; however, blocking apoptosis did not restore cell division. Loss of CAML did not activate the unfolded protein response, but did induce c-Jun and JNK phosphorylation. Structure-function studies indicated that lymphoma cell viability did not depend on interaction of CAML with TRC40, another component of TA protein insertion machinery. Conversely, expression of a minimal region of CAML that interacts with the TA insertion co-factor WRB was sufficient to rescue growth of CAML-deleted cells. In summary, our study demonstrates an essential pro-survival role for CAML in c-Myc-driven lymphomas. Future investigations will focus on the underlying mechanism of apoptosis in this system to advance our understanding of the biological functions of CAML, and consider CAML-dependent survival as a potential novel target for anticancer therapy.

#4413

Thrombospondin-1 is a master regulator of glioblastoma vascularization and infiltration.

Thomas Daubon,1 Celine Leon,2 Kim Clarke,3 Mathilde Poulet,2 Hrvoje Miletic,1 Francesco Falciani,3 Rolf Bjerkvig,1 Andreas Bikfalvi2. 1 _University of Bergen, Bergen, Norway;_ 2 _University of Bordeaux, Bordeaux, France;_ 3 _University of Liverpool, Liverpool, United Kingdom_.

Glioblastoma are heterogeneous tumours composed of different subpopulations of cells with different behavioral characteristics. Areas of highly angiogenic cells co-exist with populations of non-angiogenic but highly invasive and infiltrative cells. This heterogeneity is a key clinical challenge in glioma treatment.

We used a large scale RNA sequencing approach to investigate the molecular components which differentiate infiltrative from angiogenic cells, in a Patient Derived Xenograft (PDX) mouse model. Our bioinformatic analysis revealed TGFβ1 to be a master regulator of tumor development, and Thrombospondin-1 (Tsp1) to be up regulated in infiltrative areas as compared to angiogenic area. Thrombospondins are known as anti-angiogenic factors, however the full roles of these multi domain proteins in tumor development remain to be elucidated.

We found Tsp1 expression to be upregulated in grade IV-GBM and in silico in the GBM mesenchymal subclass. It is expressed not only in tumor cells, but also in tumor blood vessel endothelial cells (ECs). TGFβ1 transcriptionally regulated Tsp1 via Smad1 and Smad3. However, contrary to previous results, we found that Tsp1 was not involved in TGFβ1-activation in tumor cells. We showed that Tsp1 regulates cell migration and invasion both in vitro and in vivo. Inhibition of Tsp1 expression in vivo correlated with increased tumor vascularization in both the chick CAM assay and the PDX mouse model.

Anti-angiogenic treatments in the PDX mouse model leads to increased tumor hypoxia and invasive tumor cell behavior, as described in our previous work. In this context, both TGFβ1 and Tsp1 were upregulated in tumor cells. Downregulation of tumor-derived Tsp1 by shRNA in the presence of anti-angiogenic therapy led to reduced tumor growth and invasion in vivo. Finally, peptide-mediated inhibition of Tsp1 activity demonstrated that Tsp1/CD47 interaction is involved in the invasive capacity of GBM cells.

Taken together, our data suggest that Tsp1 inhibition may be a promising therapeutic approach to limit tumor infiltration induced by treatment with anti-angiogenic agents.

#4414

Insulin receptor substrate 1 is a key substrate for Pim protein kinases.

Jin H. Song, Andrew S. Kraft. _University of Arizona, Tucson, AZ_.

[BACKGROUND] Proviral integration site for Moloney murine leukemic virus (Pim) proteins are a family of serine/threonine kinases composed of three different isoforms (Pim1, Pim2 and Pim3) that are aberrantly expressed in a wide variety of tumor types, including hematopoietic malignancies. Overexpression of Pim kinases in cells exerts a pleiotropic effect on various signal transduction pathways, including cell cycle progression, cell proliferation and apoptosis. They are now being targeted in human clinical trials by small molecules inhibitors. However, the protein substrates and the mechanism of action of the Pim protein kinases are not known. To understand how Pim is regulating this diverse set of pathways, it is essential to define the substrates for this protein kinase.

[MATERIAL and METHOD] To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 serine 1101 (IRS1-S1101) as a potential Pim substrate. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation.

[RESULT] Our in vitro kinase assay showed that S1101 phosphorylation was markedly induced by incubation of active Pim1 kinase to IRS1 immunoprecipitants, demonstrating that Pim1 is capable of directly phosphorylating IRS1 protein on S1101. Mutation of this residue in IRS1 from serine to alanine (S1101A) completely abolished in vitro phosphorylation by Pim1. Similar results were obtained using the IRS2 protein that contains an identical Pim kinase phosphorylation consensus at 1149. To find whether one or all of the three Pim isoforms are regulating the phosphorylation IRS1, each isoform was transduced into Pim triple knockout (TKO) MEF cells using lentiviruses producing Pim1, Pim2 or Pim3. Each of the three isoforms was sufficient to induce the phosphorylation of IRS1 on S1101. Consistent with these results, the depletion of each Pim kinase isoform individually using siRNA in the various cancer cell lines did not decrease IRS1 phosphorylation, but the knockdown of all three isoforms abolished the phosphorylation of the IRS1 protein. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling.

[Conclusion] These results demonstrate that IRS1-S1101 is a novel substrate for the Pim kinases and provide the possibility of a novel marker for evaluation of Pim inhibitor therapy.

#4415

CPAP contributes to HCC malignancy through regulating inflammatory pathways.

Liang-Yi Hung. _National Cheng Kung Univ., Tainan, Taiwan_.

Hepatocellular carcinoma (HCC) is one of the most common malignancies, and the third most prevalent cause of cancer-related death worldwide. Its curative treatments are limited to liver resection and liver transplantation. In addition, HCC is characterized by hypervascular tumor which is described as a formation of many blood vessels during tumor growth. It has been shown that the transcription factors NF-κB and STAT3, both of which play an important role in the inflammatory pathway, are activated in HCC. Centrosomal P4.1-associated protein (CPAP) plays a vital role in centrosomal function and is identified as a transcriptional co-activator of NF-κB or STAT5. Our studies indicated that CPAP is overexpressed in HCC and involved in regulating the TNF-α-NF-κB and IL-6/STAT3 pathways. By reporter assay, Q-PCR, immunoblotting and ELISA analysis, we found that the activity of NF-κB or STAT3 is increased in HCC cells with CPAP overexpression upon TNF-α or IL-6 stimulation. Overexpressed GFP-CPAP significantly enhances NF-κB or STAT3-driven transcriptional activation and increases the expression of NF-κB or STAT3-targeted genes in response to TNF-α or IL-6 treatment. Furthermore, by immuoprecipitation and in situ PLA, CPAP could form a complex with NF-κB or STAT3. Expression of CPAP and NF-κB or STAT3 had a positive correlation in clinical HCC tissues. Interestingly, overexpression of GFP-CPAP augmented the expressions of pro-inflammatory cytokines and promoted tumor growth and metastasis in the orthotopic and splenic mouse models. The expression of CPAP was positively correlated with plasma proinflammatory cytokines expression in HCC patients. Our results suggest that CPAP participates in activating the TNF-α-NF-κB and IL-6/STAT3 pathways, which contributes to HCC malignancy.

#4416

Combination of HSP90 and proteasome inhibitor is effective in pancreatic cancer.

Ganji Purnachandra Nagaraju, Balney Rajitha, Leah Benton, Bassel F. El-Rayes. _Emory University, Atlanta, GA_.

Background: Heat shock protein (HSP90) and proteasome play an important role in cellular protein trafficking and degradation in pancreatic cancer (PC). Given the overlap in the mechanism of action, we investigated the effects of the combination of pharmacological inhibitors of HSP90 (Ganetespib) and proteasome (Carfilzomib) on PC cells in vitro and in vivo.

Methods: The combined effects of ganetespib and carfilzomib were evaluated in MIA PaCa-2 and HPAC cell lines using a cell proliferation

assay. Effects on the expression of survival (PI3K/AKT, and ERK), cell cycle (Cdc-2 and cyclin B1), angiogenesis (HIF-1α and VEGF) and autophagy (LC3 A/B, and Atg7) pathways were examined by Western blot. Cell cycle analysis was performed by FACS assay. HPAC cell lines were tested for efficacy to ganetespib, carfilzomib, alone and in combination, using an in vivo tumor xenograft model.

Results: The combination of ganetespib and carfilzomib significantly decreased cell proliferation, induced G2-M cell cycle arrest and induced autophagy in both MIA PaCa-2 and HPAC cell lines. Ganetespib and carfilzomib also decreased the activation of ERK, PI3K/AKT, and autophagy signaling molecules (LC3 A/B, and Atg7). In animal models, ganetespib potentiated the effects of carfilzomib, as measured by tumor

volume. Western blot analysis from tumors removed from animals confirmed the effects of ganetespib and carfilzomib on survival, cell cycle, autophagy and angiogenesis.

Conclusion: These observations provide preclinical proof-of-principle that combinatorial targeting of HSP90 and proteasome is a rational approach for development in PC clinical trials.

#4417

In vitro **efficacy of dasatinib alone or in combination with PARP inhibitor plus standard cytotoxic agent in triple-negative breast cancer cell lines.**

Yuliang Sun,1 Jennifer Carlson,1 Xiaoqian Lin,1 Pradip De,1 Casey Williams,1 Sally Hausman,2 Nandini Dey,1 Brian Leyland-Jones1. 1 _Avera, Sioux Falls, SD;_ 2 _NCI, Bethesda, MD_.

Background: Patients presenting with triple-negative breast cancer (TNBC) have the poorest prognosis of all breast cancer (BC) subtypes and there are limited treatment options, with no efficacious targeted therapies. Therefore, improved approaches to treatment of these cancers are an unmet need. Several molecular targets including: epidermal growth factor receptor (EGFR), poly ADP ribose polymerase (PARP), and Src family tyrosine kinases are under clinical investigation for the treatment of this disease (De et al., 2014; Kim et al., 2013). Src family tyrosine kinases play a critical role in signal transduction downstream of growth factor receptors and integrin family members. Preclinical studies have established a role for Src family kinases in proliferation, angiogenesis and invasion in prostate cancer model (Varcaris et al., 2014). Dasatinib is an rally-active ATP-competitive small molecule kinase inhibitor that potently inhibits Abl kinase, Src family kinases and other kinases (Lombardo et al., 2004), and has shown its anti-proliferative and anti-metastatic effectiveness against TNBC in both preclinical and clinical studies (Finn et al., 2011). Recently, PARP inhibitors in combination with chemotherapy have shown promising results in TNBC in clinical and preclinical studies (Tutt et al., 2010; De et al., 2014). We hypothesize that the inhibitor of Src family of kinases (dasatinib) in combination with PARP inhibitor (ABT888) plus standard cytotoxic agent (carboplatin) will attenuate the growth of TNBC cell lines. Methodology: BT-20 (PIK3CA mutated, H1047R), HCC70 (PTEN null), HCC1937 (PTEN null), MDA-MB-231 (KRAS/BRAF mutated), MDA-MB-468 (PTEN null) and SUM149PT (BRCA1 mutated, PTEN null) cells were used for this study. Growth inhibition, survival/proliferation, colony formation and apoptosis were examined using MTT assay, 2D proliferative/growth assay, 3D-ON-TOP assays, and annexinV staining respectively. Results: Our data showed that 1) High anti-proliferative activities were observed following the treatment of dasatinib along with ABT888 plus carboplatin in both 2D proliferation assay and 3D-ON TOP colony formation assay. 2) In the same line, dasatinib in combination with ABT888 plus carboplatin inducing early stage apoptosis was seen by Annexin V staining in all tested cell lines. 3) Our MTT data showed that anti-proliferative activity of dasatinib as a single agent is highly variable in different genetic background of TNBC cell lines. Conclusion: Our data suggest that the combination of Src inhibitor may enhance the antitumor activity of PARP inhibitor plus standard cytotoxic agent in TNBC models. Mechanistic studies are ongoing, the results of which will be presented in the meeting.

#4418

Identification and characterization of patient-derived PREX2 mutation in HCC.

Yen-Fu A. Chen,1 Kuo-Jui Lee,1 Yi-Hsiung Lin,1 Chung-Hsien Li,1 Chia-Hung Yen,1 Hui-Kuan Lin,2 Yi-Ming Chen1. 1 _Kaohsiung Medical University (KMU), Kaohsiung City, Taiwan;_ 2 _Wake Forest University, Winston-Salem, NC_.

Phosphatidylinositol 3,4,5-trisphosphate Rac exchange factor 2 (PREX2), a highly distributed GEF, is abundant in human cancer cells. Also, PREX2 is frequently mutated in melanoma, stomach, colorectal, and lung cancers. Recently, we identified HectH9 (homologous to E6AP carboxyl terminus homologous protein 9) is the E3 ligase of PREX2 and mediates its degradation. We found that PREX2 was overexpressed in HCC and associated with the following clinical characteristics: viral infection (P=0.004), tumor size (P=0.03) and AFP levels (P=0.04). Results from multivariate logistic regression analysis indicated that PREX2 expression profoundly correlated with hepatitis B virus infection (odds ratio=14.07, P=0.01). Moreover, high-expression level of PREX2 in the tumor tissues was associated with poor survival (P=0.02). In addition, we performed HaloPlex sequencing to analyze the PREX2 genome among tumorous, tumor-adjacent tissues and peripheral blood mononuclear cells from 68 HCC patients. Thirteen non-silent point mutations were detected in PREX2. Compared to wild-type PREX2, the S113R mutant displayed a weaker interaction with HectH9, a reduced ubiquitination level and prolonged half-life. Moreover, the S1113R mutant further enhanced AKT activation and cell proliferation. These results reveal a novel mechanism through which GNMT restrains AKT signaling and HCC tumorigenesis.

#4419

Exploration of alternative pathways mediated by IFNL4 and related to cell proliferation and death in a hepatoma cell line.

Fang Wang, Olusegun O. Onabajo, Nina Rao, Ludmila Prokunina-Olsson. _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

The ability to generate interferon lambda 4 (IFNL4), the most recently discovered member of the type-III interferon family, has been associated with impaired clearance of hepatitis C virus (HCV). IFNL4 signals through an IFNL receptor complex that serves all type-III interferons and consists of receptors IFNLR1 and IL10R2. Signaling through IFNL receptor complex leads to activation of the JAK-STAT pathway and transcriptional activation of interferon-stimulated genes (ISG). Intracellular expression of IFNL4 also causes reduced proliferation and increased cell death in human hepatic cells but it remains unclear if this is a result of JAK-STAT signaling through IFNL receptor complex or through some other alternative mechanisms. To address this question, we used CRISPR-CAS9 system to knock-out IFNLR1 in a hepatoma HepG2 cells. We designed and tested six guide RNAs (gRNAs) for IFNLR1 exons. CAS9 protein and gRNAs that most efficiently induced mutations in IFNLR1 were introduced into HepG2 cells to generate stable cell lines. No obvious defects were observed in IFNLR1-KO-HepG2 cells, indicating that loss of IFNLR1 had no deleterious effects on cell viability. We selected three stable clones which had sequencing-confirmed inactivating IFNLR1 mutations and Western blot-confirmed lack of IFNLR1 protein expression. Compared to the parental HepG2 cells the IFNLR1-KO-HepG2 cells were deficient in the ability to induce the luciferase-tagged interferon-stimulated response element (ISRE-Luc) reporter after treatment with recombinant IFNL4 and IFNL3 proteins, which signal through the same receptor complex. At the same time, response to IFNa treatment was not affected because IFNa signals through its own receptor complex. These results indicate that, as other type-III interferons, IFNL4 induces ISRE signaling through IFNL receptor complex. INFLR1-KO-HepG2 cells provide a highly efficient model for studies of factors involved in signaling of type-III interferons, including IFNL4. Next, we will investigate alternative pathways involved in anti-proliferative and cell death-related phenotypes induced by IFNL4 in HepG2 cell. We will perform RNA-seq on parental and IFNLR1-KO-HepG2 cells treated with or without IFNL4, IFNL3 and IFNa. These results will contribute to a better understanding of IFNL4 function, which can be relevant for viral infections and cancer.

#4420

Heparan sulfate proteoglycans mediate SW13 oncogenic potential and response to granulin.

McKale Davis, Elizabeth Hull. _Midwestern University, Glendale, AZ_.

The SW13 adrenal adenocarcinoma cell line exists as two epigenetically and phenotypically distinct subtypes. One is a highly proliferative epithelial subtype lacking the expression of key tumor suppressor proteins, Brm and Brg1, the intermediate filament protein vimentin, and the cell cycle check point protein CD44 (SW13-), while the other is a slow growing, mesenchymal-like subtype which expresses all of these proteins (SW13+). Evidence strongly suggests that subtype determination is epigenetic in nature, as HDAC inhibitors can induce an SW13- to SW13+ subtype switch, but the cellular and environmental factors that regulate cellular phenotype have yet to be characterized. We performed a comprehensive microarray analysis and identified heparan sulfate proteoglycan (HSPG) binding as a primary pathway affected between these two subtypes. HSPGs are glycoproteins present in the extracellular matrix that can have tumor-promoting or tumor-suppressing activities depending on the number and sulfation status of attached sugar chains known as glycosaminoglycans (GAGs). Quantitative RT-PCR was then used to confirm the increased expression of the HSPGs, serglycin and glypican-3, as well as the HSPG-binding growth factors granulin and fibroblast growth factors-1 and -2 in SW13+ cells. Furthermore, HDAC inhibition was also shown to increase the expression of these genes in a time-dependent manner. As granulin over-production is associated with cancer progression in a number of human cancers, we were interested in examining the role of this HSPG binding growth factor further. A pro-granulin ELISA showed that SW13+ cells secrete significantly more granulin than SW13- cells. Intriguingly however, treatment with exogenous granulin increases growth rates in the already highly proliferative SW13- subtype, but had no effect on SW13+ growth. We hypothesized that the pleiotropic response of SW13 cells to growth factor treatment may be the result of HSPG post-translational modifications, so we assessed total GAG sulfation was by dimethylmethylene blue assay, and indeed the SW13+ cells were found to have significantly higher levels of total GAG sulfation than SW13- cells. Thus, transcriptional and post-translational modifications of HSPGs may contribute to SW13 phenotype control, and ultimately may affect tumorigenesis.

#4421

Molecular features of hepatocellular carcinoma arising from non-alcoholic fatty liver.

Makoto Takeda, Takanori Sakaguchi, Ryouta Kiuchi, Takanori Hiraide, Yasushi Shibasaki, Yoshihumi Morita, Hirotoshi Kikuchi, Mitsutoshi Setou, Hiroyuki Konno. _Hamamatsu University School of Medicine, Hamamatsu, Japan_.

(Background)

The incidence of hepatocellular carcinoma (HCC) arising from non-alcoholic fatty liver (NAFL) has been recently increasing according to increase of patients having metabolic syndrome. HCC, which shows steatosis, cellular ballooning, Mallory-Denk bodies, inflammation and fibrosis in cancer tissue, are recognized as steatohepatitic HCC (SH-HCC). This HCC phenotype is more frequently found in NAFL patients than in viral-hepatitic patients.

(Purpose)

The purpose of this study is to elucidate molecular features of SH-HCC with NAFL background.

(Methods)

To profile SH-HCC-related genes, mRNA expression patterns in SH-HCCs were compared with those in viral hepatitis HCCs by RNA array. RNA expression of the identified SH-HCC related gene was analyzed in three human liver carcinoma cell lines (HepG2, Huh7, and HLE) in normal conditioned medium. By mimicking the lipid-rich environment, these cell lines were also cultured in the presence of saturated (palmitic or stearic acid, recognized as endoplasmic reticulum stressors) or unsaturated fatty acids (oleic acid: OA). To address the role of SH-HCC-related gene, cell proliferation and apoptosis were assessed in the presence or absence of fatty acids, by knock-down of the objective gene using siRNA.

(Results)

(1) Caveolin (cav)-1 and -2 were identified as SH-HCC related genes (1.63 and 1.58 fold expression of cav-1 and -2, respectively) by RNA microarray analysis.

(2) HLE and Huh7 showed high and low expression of both cav-1 and -2 mRNA, whereas HepG2 did not express either on qRT-PCR.

(3) In the presence of OA, HepG2 and Huh7 did not well proliferate, whereas HLE cell proliferation increased with concentration of OA and culture duration.

(4) Knock-down of cav-1 and -2 canceled OA-induced increase of cell proliferation. In the same condition, expression of cleaved caspase 3 was increased, suggesting the promotion of apoptosis.

(Consideration)

Cav-1 and -2, the important protein for endocytosis and cellular signaling, are highly expressed in SH-HCC. These proteins would increase cell proliferation and inhibit apoptosis in the presence of unsaturated fatty acids, presumably by using these fatty acids as energy source or by caveolae-related anti-apoptotic process.

#4422

Extracellularly secreted APE1/Ref-1 triggers apoptosis in triple-negative breast cancer cells via RAGE binding, which is mediated through acetylation.

Yu Ran Lee,1 Hee Kyoung Joo,1 Ki Mo Kim,2 Byeong Hwa Jeon,1 Sunga Choi1. 1 _School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Cancer Research Team, Korean Medicine Based Herbal Drug Research Group, Herbal Medicine Research Division, Korea Institute of Oriental Medicine, (KIOM),, Daejeon, Republic of Korea_.

The present study evaluated the mechanism of apoptosis caused by posttranslational modification, hyperacetylation in triple-negative breast cancer (TNBC) cells. We previously showed that trichostatin A (TSA) induced secretion of acetylated apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1). This is the first report showing that Ac-APE1/Ref-1 initiates apoptosis in TNBC cells by binding to the receptor for advanced glycation end products (RAGE). The functional significance of secreted Ac-APE1/Ref-1 was studied by induction of intracellular hyperacetylation through co-treatment with acetylsalicylic acid and TSA in MDA-MB-231 cells. In response to hyperacetylation, secretion of Ac-APE1/Ref-1 in vesicles was observed, resulting in significantly decreased cell viability and induction of apoptosis with increased expression of RAGE. The hyperacetylation-induced apoptosis was similar in two other TNBC cell lines: BT-459 and MDA-MB-468. Therefore, hyperacetylation may be a therapeutic target for treatment of TNBCs. This study introduces a novel paradigm whereby posttranslational modification induces apoptotic cell death in breast cancer cells resistant to standard chemotherapeutic agents through secretion of auto- or paracrine molecules such as Ac-APE1/Ref-1.

#4423

Methylation and acetylation are novel post-translational modifications identified for the pre-apoptotic protein kinase C (PKC) delta.

Noemi Kedei, Elliott L. Paine, Lisa M. Miller Jenkins, Peter M. Blumberg. _National Cancer Institute, Bethesda, MD_.

Members of the PKC family of diacylglycerol (DAG) receptors play a central role in cellular signaling, affecting cell proliferation, differentiation, apoptosis, etc. Their expression and/or function are often altered in cancer. Posttranslational modifications play an important role in the regulation and intracellular localization of PKCs. For example, phosphorylation of the hydrophobic and turn motifs are required for the maturation, stability and proper folding of most isoforms, and phosphorylation of S299, Y311, and Y334 in the hinge region are well characterized agonist (phorbol ester or H2O2) induced modifications of PKC delta. We have previously reported that PMA and bryostatin 1, two PKC activators having very different biology in multiple cell lines, induced a relatively complex and different pattern of PKC delta modification in LNCaP cells as detected by charge-based separation. Moreover, bryostatin 1 failed to induce Y311 phosphorylation whereas it induced S299 and T507 phosphorylation similarly to PMA. In this study, we explored by mass spectrometry the posttranslational modifications of PKC delta induced by the two drugs. GFP-tagged PKCdelta was overexpressed in LNCaP and HEK cells, and PKC delta was immunoprecipitated from treated cells (in total lysates or in cytoplasmic and nuclear fractions) using anti-GFP antibody. Using this approach, we were able to detect the previously described phosphorylations of S302/S304, S506, S645, S664 in all samples and the agonist-induced phosphorylation of S299, while detection of phosphorylated tyrosine residues was less efficient. Novel findings include detection of agonist-induced phosphorylation of S130, S359, and S647, and acetylation or methylation of multiple K or N sites, respectively. Acetylation of K275 in the C1b domain, of K319 in the hinge region, of K475 in the catalytic domain and of K649 at the C terminus was detected in almost all samples while methylation of N278, N283 and N292 in the C1b domain was detected in control LNCaP cells only. Agonist treatment induced acetylation and methylation at additional sites in the hinge region and in the C terminus of the protein. The modifications were not identical between the cytoplasmic and nuclear PKC delta after PMA treatment: phosphorylation of T50, methylation of N292 and acetylation of K382 was detected only in the cytoplasmic fraction while phosphorylation of S359 was present in the nuclear fraction only. The newly identified acetylation and methylation add to the complexity of posttranslational modifications of PKC delta that might have regulatory roles in the localization and/or activity of the protein.

#4424

Acetylation of Fascin on the lysine 471 residue produces a change in growth and mobility phenotype of esophageal cancer cells by inducing F-actin rearrangement.

Fa-Min Zeng, Xiao-Ning Wang, Ying-Li Zhang, Lian-Di Liao, En-Min Li, Li-Yan Xu. _Shantou University Medical College, Shantou, China_.

Elevated expressions of fascin mRNA and protein in cancer cells have been correlated with aggressive clinical course, poor prognosis and shorter survival. Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is reported that the lysine 471 residue (K471) of fascin can be acetylated and also involves in its actin binding, but the roles of K471 acetylation in filopodia formation are still uncertain. In this paper, we produced point mutants mimicking the active deacetylated (Lys471Arg, K471R) or inactive acetylated (Lys471Gln, K471Q) states of fascin. The actin-binding assay showed that the fascin K471Q mutation decreased the actin binding activity of fascin, compared with wild-type fascin (WT) and K471R mutant fascin in the esophageal cancer cells. Moreover, live imaging revealed that K471Q mutant fascin reduced the number and length of filopodia, whereas the K471R mutant increased filopodia frequency. The fascin K471Q mutation changed the straight filopodia became forniciform filopodia. The protrusion presented abnormal morphology when cells spread. Further functional experiments showed that the K471Q mutant fascin rendered the tumor cell migration, invasion and tumorigenesis decrease significantly, whereas K471R mutant fascin like WT fascin still maintained a strong ability. Taken together, we propose that the lysine K471 residue may be a key specific actin crosslinking site and also involved in the tumor biological function of fascin.

#4425

Comprehensive study of TGF-β pathway-driven functional molecular characterization of human hepatocellular cancer.

Jian Chen,1 Jiun-Sheng Chen,1 Jianping Zhang,1 YoungJin Gi,1 Lior Katz,2 Ji-Hyun Shin,1 YunSeong Jeong,1 Mitch Belkin,3 Wilma Jogunoori,3 Bibhuti Mishra,3 Jon White,3 Shoujun Gu,4 Milind Javle,1 Xiaoping Su,1 John Stroehlein,1 Marta Davila,1 Xuemei Wang,1 Jeffrey Morris,1 Patrizia Farci,5 Rehan Akbani,1 Lopa Mishra4. 1 _The University of Texas MD Anderson Cancer Center, Houston, TX;_ 2 _Sheeba Medical Center, Ramat Gan, Israel;_ 3 _Institute of Clinical Research, Washington, DC;_ 4 _George Washington University, Washington DC, DC;_ 5 _National Institutes of Health, Bethesda, MD_.

Background: TGF-β plays a complex role in cancer- from tumor suppression to immune modulation to tumor promotion that are unclearly defined to date. Recent clinical studies show that targeting TGF-β improves survival up to 21 months, yet prognostic significances are undefined. Moreover, the relationships between patterns of mutations and transcriptomic phenotypes for the TGF-β pathway are unclear. Methods: 1. We analyzed the transcriptome of 488 hepatocellular cancers and screened for mutations in the TGF-b pathway in 202 HCCs from The Cancer Genome Atlas (TCGA). 2. Next we correlated mouse models of HCC with a functional analysis of drivers. Results: 1. Transcriptomic analyses revealed aberrant TGF-β superfamily profiles in 72% of hepatocellular cancers, with mutations in 38% of patients. 2. Significantly, HCCs characterized by the "inactivated" TGF-β signature were associated with a significantly poorer survival particularly in early stage HCCs, compared to HCCs with the "activated" TGF-β signature (p=0.0027). 3. We observed the greatest number of functional mutations in the SPTBN1 gene (6%), which encodes a tumor suppressor TGF-β/Smad3 adaptor protein. 4. We found a strong association between DNA damage response genes and the TGF-β pathway at both transcriptomic and genomic levels. 5. We also observed a strong correlation between VD related genes and TGF-β pathway genes in the TCGA genomic analysis. 6. subsequent VD deprivation synergistically with TGF-β inactivation promotes liver tumor development. 7. Through a transcriptomic and functional analysis we observed that TLR7 mRNA levels are increased 4-fold in liver tissues from Smad3+/- mice, and TLR7 is a direct target of Smad3. Conclusions: The TGF-β pathway plays a pivotal role in liver tumorigenesis and the molecular signatures we characterize here have prognostic significance. In specific populations, VD deficiency and TGF-β disruption synergistically promote liver tumor growth, possibly through regulating TLR7 expression. The additional association with the DNA repair pathway supports new approaches to biomarker driven targeting of TGF-β, improving survival of liver cancer.

#4426

The PEAK1/ZEB1/ITGA1 pathway mediates survival, stemness and TGFbeta-induced EMT in pancreatic cancer.

Armen Gharibi, Sa La Kim, Daniel Brambilla, Yvess Adamian, Malachia Hoover, Joy Lin, Megan Agajanian, Laurelin Wolfenden, Jonathan A. Kelber. _California State University, Northridge - Biology, Northridge, CA_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States with a five-year survival rate of approximately 7%. Its highly invasive characteristics, lack of biomarkers for early detection, limited therapeutic targets and inherent resistance to chemotherapy are the central factors that lead to an overall poor prognosis for this malignancy. Since most cancer-related deaths are a result of metastatic tumors, we reasoned that our previously published proteomic signature of the cell pseudopodium (PD) may be a good pool from which to identify novel protein biomarkers or therapeutic targets in pancreatic cancer. We cross-referenced PD enriched proteins with Oncomine and identified 37 genes that are upregulated in pancreatic cancer. The function of integrin alpha 1 (ITGA1), a cell surface receptor for collagen and regulator of cell adhesion, has not been previously characterized in the context of PDAC. Our results indicate that ITGA1 is required for PDAC cell viability, promoting cell survival and tumor growth. We further analyzed ITGA1 cell-surface levels and discovered that ITGA1hi cells represent a subpopulation of Aldehyde Dehyrogenase high (ALDH1hi) cells, and that ITGA1 knockdown reduces the ALDH1hi cell population. One mechanism by which PDAC cells acquire metastatic potential is via epithelial-mesenchymal transition (EMT). Thus, we searched for prominent EMT genes among the top ITGA1 co-expressors using RNA-Seq data from the Cancer Bio Portal. Interestingly, ZEB1 (a transforming growth factor beta, TGFbeta, response gene and central regulator of EMT) and PEAK1 kinase (a protein we've shown to be required for pancreatic cancer progression and TGFbeta/ZEB1-induced EMT) co-expresses with ITGA1. We tested the function of ITGA1 on TGFbeta-induced EMT and found that ITGA1 is upregulated by TGFbeta during EMT, and required for metastasis and a complete EMT response to TGFbeta. We also demonstrate that PEAK1 is required for TGFbeta-induced ITGA1 in PDAC cells. Finally, we demonstrate that ITGA1 is responsible for collagen-induced spreading and an EMT-like shift in PDAC cell morphology. Taken together, these results point to a novel TGFbeta/PEAK1/ZEB1/ITGA1 signaling cascade that when targeted can abrogate PDAC cell survival, stemness, EMT and tumor growth/metastasis.

#4427

Role of Smurf2 in regulation of Smad anchor necessary for receptor activation (SARA) in TGFβ-receptor signaling.

Sanghyun Lee, John M. Di Guglielmo. _Western University, London, Ontario, Canada_.

Non-small cell lung cancer (NSCLC) is a major form of lung cancer and is the leading cause of cancer mortality in the western world. Transforming growth factor β (TGFβ) deregulation leads to many human diseases: TGFβ is a tumor suppressor in normal lung epithelium; however, it switches roles and promotes lung cancer metastasis in cancer cells. TGFβ induces cell proliferation, migration and invasion in NSCLC cells.

The canonical pathway of TGFβ is initiated through binding of TGFβ ligand to transmembrane Ser/Thr kinase receptors, which propagate their signaling via receptor regulated R-Smads; proteins that function as intracellular effectors in the TGFβ pathway. Once activated, R-Smads form a complex with common (Co)-Smads that translocates into the nucleus to regulate transcriptional responses. Access of R-Smads to the activated receptor complex is regulated by an adaptor protein called Smad anchor for receptor activation (SARA). SARA facilitates the activation of Smads and allow efficient Smad signalling.

In addition to R-Smads and Co-Smads, inhibitory Smads (I-Smads) regulate TGFβ signalling. I-Smads block the signalling by competing against R-Smads for the association with TβR complex or by targeting receptors for ubiquitin-mediate degradation. I-Smads recruit the E3 ubiquitin ligases, Smad ubiquitination regulatory factors (Smurfs), to catalyze degradation of the receptor complex. The overall aim of the project is to characterize SARA in TGFβ receptor signalling and study the interaction of SARA with different proteins involved in the pathway, such as Smurf2 and Smad7.

We first examined how SARA, Smurf2 and Smad7 influence each other by performing a multi-combination transfection study. We also examined the interaction between SARA and Smurf2 through co-immunoprecipitation. We observed that the level of SARA decrease in the presence of Smurf2 and Smad7. In the presence of Smurf2 ligase inactive mutant and Smad7, the level of SARA is somewhat recovered. However, SARA does not directly interact with Smurf2. These data together suggest that SARA and Smurf2 influence each other in a very close manner. It also suggest that it could be a transient interaction. Characterizing SARA in TGFβ receptor signalling will provide us with possible drug targets for NSCLC.

#4428

Nestin suppression attenuates invasive potential of endometrial cancer cells by down regulating TGF-β signaling pathway.

Amber A. Bokhari,1 Batsukh Dorjbal,1 Tabari M. Baker,1 Sana Waheed,1 Christopher M. Zahn,2 Chad A. Hamilton,3 George L. Maxwell,4 Viqar Syed1. 1 _Uniformed Services University of the Health Sci., Bethesda, MD;_ 2 _American College of Obstetricians and Gynecologists, Washington, DC;_ 3 _Walter Reed National Military Medical Center, Bethesda, MD;_ 4 _Women's Health Integrated Research Center at Inova Health System, Annandale, VA_.

Nestin, an intermediate filament protein and a stem cell marker is expressed in several cancers. Little is known about the expression level and role of Nestin in endometrial cancer. Compared to immortalized endometrial epithelial cell line EM-E6/E7/TERT, endometrial cancer lines express high to moderate levels of Nestin. Endometrial tumors and tumor cell lines have a cancer stem-like cells population expressing CD133. The AN3CA and KLE cells showed more CD133+ cells and expressed Nestin at higher levels than Ishikawa cells. Knockdown of Nestin in AN3CA and KLE, and overexpression in Ishikawa cells was associated respectively with attenuation and enhancement of CD133+ cells. Knockdown of Nestin increased cells in G0/G1 phase and decreased in S phase, whereas overexpression decreased cells in G0/G1 phase and increased in S phase of cell cycle. Nestin knockdown cells showed increased expression of p21, p27 and PNCA and decreased expression of cyclin-D1 and D3. Nestin overexpression revealed an inverse expression pattern of cell cycle regulatory proteins. Nestin knockdown inhibited cancer cell growth and invasive potential by down regulating TGF-β signaling components, MMP-2, MMP-9, vimentin, SNAIL, SLUG, Twist, N-cadherin, and upregulating E-cadherin. Conversely, Nestin overexpression enhanced cell invasiveness by upregulating TGF-β signaling components, MMP-2, MMP-9, mesenchymal markers and downregulaing epithelial marker, E-cadherin. Nestin knockdown inhibited and overexpression promoted invadopodia formation and pFAK expression. Knockdown of Nestin significantly reduced tumor volume in vivo. Finally, progesterone and a nitric oxide donor inhibited Nestin expression in endometrial cancer cells. These results suggest that Nestin can be a therapeutic target for cancer treatment.

#4429

CCL20 regulation via TGFB /BMP signaling pathway in murine immortalized colonocytes and colonic epithelium.

Tasia D. Brown, Anna Means, Tanner Freeman, Connie Weaver, Keith Wilson, R. Daniel Beauchamp. _Vanderbilt University Medical Center, Nashville, TN_.

TGFβ and BMP signaling through receptor-mediated activation of SMAD proteins is crucial for normal homeostasis in intestinal epithelium. Alterations in this pathway contribute to the development of colorectal cancers. To understand the role of these pathways in homeostasis of the colon we conditionally deleted loxP-flanked alleles of Smad4 in adult mouse colon. We used Lrig1CreERT allele to achieve greater than 80% SMAD4 gene deletion and a CK19CreERT allele for mosaic deletion in colonic epithelium. Our data indicates that SMAD4 activity regulates multiple interactions between intestinal epithelium cells and surrounding stroma and that deletion of SMAD4 in adult mouse colonic epithelial cells alters expression of cytokines, increases immune cell infiltration and predisposes mice to tumorigenesis. Increases in the chemokine CCL20 have been reported in CRC tumors beginning at the adenoma stage and it may regulate both Tregs and Th-17 cells in an active immune response. Luminex cytokine/chemokine array analysis revealed increased levels of CCL20 protein in colonic mucosa and RNAseq data revealed that when SMAD4 is knocked out in the epithelium there is an upregulation of CCL20 and CCR6 (receptor) mRNA in the epithelium and stroma, respectively. To further explore the regulation of CCL20, we used immortalized mouse colonocytes (IMC cells) to further determine how the TGFβ and BMP signaling pathways may be involved in CCL20 expression. We found that CCL20 mRNA expression was suppressed by TGFβ1 and BMP2 and increased by inhibitors of these pathways. The inhibition of CCL20 by the TGFβ and BMP2 pathways occurs even in the presence of a known strong inducer of CCL20, such as TNFα. We have further observed in mice induced to undergo deletion of SMAD4 in the intestinal epithelium that treatment with DSS results in 100% of mice developing mucinous adenocarcinomas in the distal colon. We conclude that ligand-dependent Smad4-mediated signaling provides important negative homeostatic regulation of CCL20 expression in colonic epithelium. Loss of Smad4 results in increased CCL20 expression and we are currently testing the hypothesis that CCL20 overexpression contributes to the inflammation induced carcinogenesis in this mouse model.

#4430

Implication of the primary cilium in the Hedgehog pathway in colorectal cancer cells.

Blanche Senicourt. _Université de Sherbrooke, Sherbrooke, Quebec, Canada_.

Background: The Hedgehog (HH) pathway is involved in the maintenance of numerous cell types both during development and in the adult. Often deregulated in cancers, its involvement in colorectal cancer has come into view during the last few years, although its role remains poorly defined. In most tissues, the HH pathway is highly connected to the primary cilium (PC), an organelle not expressed in the normal colonic epithelium which recruits the functional components and regulates the pathway.

Aims: Since the intestinal epithelium is known to be a non-ciliated tissue, the HH pathway as related to the PC has not been explored. We investigated the presence of PC in colon cancer tumors and cell lines. Our hypothesis is that PC formation controls the HH activation that has been reported in numerous colon cancer studies.

Materials and methods: We used cellular models to look at HH activation, focusing on the final effector Gli1. The link between PC and the HH pathway was shown by the recruitment of the Smo receptor in the PC. We looked for PC in a subset of 63 tumors from a Tissue Micro Array and in 4 colorectal cell lines by immunofluorescence using two well-known markers, acetylated α-tubulin and polyglutamylated tubulin. 3D deconvoluted pictures were obtained to characterize the shape and size of PC. Using cellular models we investigated HH pathway expression by qPCR. We assessed the functional link between HH pathway and PC through localization of the Smo receptor in the PC using immunofluorescence.

Results: We observed the presence of the PC in the epithelium of primary colorectal tumors at all stages but not in their normal counterparts. Using human colorectal cancer cell lines we found a clear correlation between the presence of the PC and the expression of the final HH effector, GLI1, and provide evidence of a functional link between the two by demonstrating the recruitment of the SMO receptor to the PC membrane.

Conclusion: We conclude that the PC directly participates in the HH pathway in colorectal cancer cells which is the first observation of PC expression in tumors arising from a non-ciliated tissue. Functional studies are required to determine the mode of action of this organelle in carcinogenesis.

### DNA Methylation 2

#4431

Tandem profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma identifies tumor-specific distribution of 5-hydroxymethylcytosine and associations with patient survival.

Kevin C. Johnson,1 E. Andres Houseman,2 Jessica E. King,1 Camilo E. Fadul,3 Brock C. Christensen1. 1 _Geisel School of Medicine at Dartmouth, Hanover, NH;_ 2 _Oregon State University, Corvallis, OR;_ 3 _University of Virginia, Charlottesville, VA_.

Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Genome-wide assessment of epigenetic alterations to cytosines in glioblastoma has largely been limited to 5-methylcytosine (5mC). 5-hydroxymethylcytosine (5hmC) is an additional cytosine modification that is deregulated in cancer, though the extent and context of these alterations at single cytosine resolution remain unclear. Through parallel processing of DNA with bisulfite and oxidative bisulfite treatments on Illumina 450K arrays we resolved both 5mC and 5hmC in 30 glioblastomas. We developed and applied a novel technique for estimating 5mC, 5hmC, and unmethylated proportions from array data to glioblastoma tissues and compare with normal brain tissue. Genomic distribution of 5hmC was associated with features of transcription despite the glioblastoma genome being relatively depleted of 5hmC. When integrating 5mC and 5hmC data using a Gaussian finite mixture model approach, we observed significant associations between 5hmC levels and gene-sets involved in immune and RNA regulatory processes while high 5mC classes were associated with receptor-mediated processes and cancer gene-sets. Significant differences in patient survival were observed among classes of 5hmC obtained from a recursively partitioned mixture model. Gene expression across epigenetic enzymes including methyltransferases and TETs was also investigated for relation with patterns and levels of 5hmC and 5mC. Analyses of elevated 5hmC with alternative splicing events that may promote disease progression are underway. Together, our results provide a genome-wide map of 5hmC in glioblastoma, indicate that transcription patterns as well as disrupted processes are associated with 5hmC patterns, and highlight the potential clinical prognostic utility of 5hmC in cancer.

#4432

Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation.

Shinichi Fukushige, Yasuhiko Mizuguchi, Kanchan Chakma, Yuriko Saiki, Akira Horii. _Tohoku Univ. Graduate School of Medicine, Sendai, Japan_.

DNA demethylating agents are useful therapeutic drugs for human cancer; 5-aza-2'-deoxycytidine and 5-azacytidine for myelodysplastic syndrome are good examples. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects. However, precise mechanisms for anticancer activity of these demethylating reagents have yet to be established, mainly due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulates more genes than ones expressing TET1-CDwt without MBD (TET1-CDwt) or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site (TSS). Interestingly, 65% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with 5-azacytidine, DNA demethylating agent. These results suggested a fact that gene reactivation by both methods was primarily based on DNA demethylation. In order to examine growth inhibitory effects of DNA demethylation to cancer cells, we utilized a tetracycline inducible system to regulate the expression of MBD-TET1-CDwt using a prostate cancer cell line. Induction of MBD-TET1-CDwt demethylated and upregulated the glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells. Flow cytometry analysis showed decreased cells in S phase and increased cells in G1 phase by MBD-TET1-CDwt expression. Our present results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation, and that our methods allow us to analyze the effects of global DNA demethylation in a wide variety of cancer cells.

#4433

Genome-wide CpG island methylation profiling analysis identifies an epigenetically dysregulated necroptosis mediator in osteosarcoma.

Aditya Sharma, Elaine Notis, Christine Mella, Steven J. Kuerbitz. _Akron children's hospital, Akron, OH_.

BACKGROUND

Aberrant CpG island (CGI) methylation with dysregulation of gene expression is a hallmark of cancer. To identify biologically-relevant targets of aberrant epigenetic silencing in osteosarcomas, we analyzed CGI genes exhibiting disproportionate high-frequency gain of methylation.

METHODS

Infinium HumanMethylation 450 Beadchip profiling quantitated methylation at 485,578 CpG sites in bisulfite-modified DNA of 16 primary human osteosarcomas (OS) samples. Diff scores (D), reflecting difference in prevalence of site-specific methylation for OS samples compared to pooled non-neoplastic controls, were analyzed for gene-associated CGI sites. Genes exhibiting disproportionate high frequency of gain of methylation (+D), were identified utilizing a program to rank order gene CGI loci along an observed/expected (Obs/Exp) hit distribution where "hits" were defined as DAverage > DThreshold.

RIPK3 CGI hypermethylation was quantitated by Combined Bisulfite Restriction Analysis (COBRA) and by bisulfite sequencing in 5 OS cell lines (HOS, MNNGHOS, MG-63, G-292, U2OS) and in human mesenchymal cells (hMSC). RIPK3 expression was analyzed by q-RT PCR. To evaluate the effect of RIPK3 expression on cisplatin-associated cytotoxicity, G-292 cells were stably transfected with RIPK3 or the corresponding empty vector (EV). Cisplatin cytotoxicity was assayed in RIPK3 transfectants versus vector-only controls.

RESULTS

At DTh = 50, >2500/15,000 CGI genes in the OS samples exhibited gain of CGI methylation in excess of what would be expected based on a uniform distribution of methylation (i.e., Obs/Exp > 1). 169 genes exhibited hypermethylation "hits" at all CGI sites assayed (Obs/Exp =11.95) including the receptor interacting serine-threonine kinase 3 gene (RIPK3). RIPK3 mediates necroptotic cell death and has been associated with cisplatin sensitivity. COBRA analysis of RIPK3 CGI showed extensive methylation in MNNGHOS, MG63 and G292 cells and partial methylation in HOS and U2OS. hMSCs were minimally methylated. COBRA data were congruent with the prevalence of methylation at 18 CpG sites within the CGI determined by bisulfite sequencing. Quantitation of RIPK3 mRNA expression by q-RT PCR confirmed transcriptional silencing in MNNGHOS, MG63, U2OS and G292 cells. G292 stable transfectants expressing RIPK3 were more sensitive (lower LD50) when cultured in cisplatin compared to vector-only controls.

CONCLUSIONS

Analysis of genome-wide methylation profiling data to identify CGI genes exhibiting a disproportionate gain of methylation in OS identified RIPK3 hypermethylation. Extensive RIPK3 CGI hypermethylation is associated with transcriptional silencing in OS cells. We further show that expression of RIPK3 is associated with enhanced cisplatin cytotoxicity in G-292 cells. These results suggest that methylation of RIPK3 may contribute to cisplatin resistance in OS.

#4434

CTCF acts as a master regulator to direct epigenetic events important for tumor development and progression.

Nathan A. Damaschke,1 Bing Yang,1 Anastasia Montgomery,1 John Svaren,1 Avtar Roopra,1 Jianhua Luo,2 Sunduz Keles,1 David Jarrard1. 1 _University of Wisconsin, Madison, WI;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Intro

Epigenetic changes, including altered DNA methylation, underlie the development and progression of prostate cancer (PCa). DNA methylation events are frequently seen in PCa, however, how these methylation changes are directed locally and regionally remain an important question. CTCF is a widely expressed chromatin insulator protein which plays important roles in transcriptional regulation, chromatin architecture, and the conservation of epigenetic marks. Previous work has shown that CTCF expression declines in the aging mouse prostate and in human metastatic prostate samples. It is our hypothesis that CTCF loss directs DNA methylation increases at specific loci resulting in gene expression and phenotypic advantages in cancer cells.

Methods

Cell lines with inducible shRNA expression targeting CTCF were created using 9E6/E7 human prostate epithelial cells and PPC1 and LNCaP cancer lines. Knockdowns were confirmed by qPCR and western blotting. DNA methylation was analyzed using MeDIP-chip, utilizing the Affymetrix Cytoscan HD Array and RNA expression analyzed using Affymetrix Human Transcriptome 2.0 Array. Bioinformatic analysis was performed in R 3.2.1 and Bioconductor 3.0. Motif analysis was performed using HOMER. Cell stress included exposure to H2O2 and CoCl2.

Results

We detected 9811 probes with differential methylation after CTCF knockdown (with P<0.01), of which 5,968 probes (60.1%) were hypermethylated. Of the hypermethylated probes, 417 (7%) were near gene transcription start sites and 3,361 (56.3%) were located within transcribed loci. Motif analysis indicated that CTCF target sequences were enriched in regions developing hypermethylation (P<0.01). Pyrosequencing confirmed methylation increases. RNA expression analysis with CTCF knockdown identified 1,394 transcribed loci with significantly altered expression (FDR<0.1). One-third of genes with altered transcriptional levels also contained a differentially methylated probe in its promoter or transcribed region (459/1394; 32.9%). We confirmed numerous genes with promoter hypermethylation corresponding to decreased RNA expression including CASP14, EDN1, EL, FSTL1, and WIPI1. Gene ontology indicates enrichment for anti-apoptotic genes. CTCF knockdown in epithelial and cancer cell lines led to a significantly increased survival of cells faced with hypoxia or oxidative stress.

Conclusion

Reduction of CTCF, an alteration that occurs in the development of advanced PCa, directs the hypermethylation of loci containing CTCF binding sites. These hypermethylated regions correspond to the transcriptional downregulation of genes involved in metabolism, ER stress, and apoptosis. CTCF knockdown results in resistance to hypoxia or oxidative stress. This study demonstrates, for the first time that CTCF acts as a master regulator to direct epigenetic events important for tumor development and progression and is a target for therapy.

#4435

Genome-scale DNA methylation changes delineate uterine leiomyoma subgroups.

Eevi Kaasinen,1 Amjad Alkodsi,2 Miika Mehine,2 Hanna-Riikka Heinonen,2 Netta Mäkinen,2 Mervi Aavikko,2 Kati Kampjärvi,2 Minna Taipale,1 Pia Vahteristo,2 Rainer Lehtonen,2 Lauri A. Aaltonen2. 1 _Karolinska Institutet, Huddinge, Sweden;_ 2 _University of Helsinki, Helsinki, Finland_.

Symptomatic uterine leiomyomas (ULs) occur in as many as 20% of women. These bening lesions are a major burden to women's health by causing health complications such as abnormal bleeding, pain and infertility, and they are a common cause of hysterectomy that is at present the only curative treatment. Similar to other solid tumors, genetic and epigenetic changes are likely to have value as predictors of UL clinical outcome, as well as response to treatment. Recent findings from us and others have provided evidence for existence of at least three distinct molecular UL subgroups, each displaying a characteristic genetic driver aberration; MED12 mutation, HMGA2 activation or FH mutation.

We conducted DNA methylation analysis of bisulfite sequencing data from 59 tumors and 38 normal myometrium samples on targeted genomic regions (84.5Mb) such as CpG islands, shores and shelves, GENCODE promoters and DNaseI hypersensitive sites from ENCODE data using Agilent SureSelect methyl-seq target enrichment system. The studied samples consisted of 17 MED12-mutated, 11 HMGA2-upregulated and 6 FH-mutated tumors. Whole-genome sequencing and expression array profiling had been performed previously from the same specimens (Mehine, Kaasinen, et al. 2014).

We developed a data processing pipeline that makes use of: (i) Trim Galore tool for quality control of sequencing data (ii) Bismark for alignment and DNA methylation calling (Krueger & Andrews 2011).

Hierarchical clustering of all detected CpG sites revealed that MED12- and FH-mutated tumors cluster according to the driver changes, and HMGA2-upregulated tumors cluster in two separate branches. Global hypermethylation profile was detected in FH-mutated tumors, which is compatible with previous reports in paragangliomas (Letouzé et al. 2013). Four tumors with COL4A5/COL4A6 aberration did not display uniform DNA methylation pattern and four tumors without known genetic driver changes clustered among normal myometrium samples; one of these tumors harbors a somatic TP53 rearrangement created through massive chromothripsis rearrangement event. As myometrium samples showed uniform global DNA methylation by clustering clearly separate from majority of the tumors, statistically powerful comparison of UL subgroups with different genetic background to myometrium samples was enabled. Furthermore, integration of differentially methylated regions with significant expression changes allowed us to characterize genes which are epigenetically activated or silenced in different UL subgroups. All ULs displayed increased methylation of genes enriched in developmentally related biological processes.

Our results provide strong evidence that the known UL subgroups are distinguishable by global DNA methylation profiling. The role of the identified epigenetically activated or silenced genes should be studied further to allow better understanding of the biological processes related to UL development and clinical outcome.

#4436

Synergistic effects of promoter associated DNA methylation and genetic alterations to better understand oncogenic gene expression profiles.

Claire Olson,1 Fang Yin Lo,1 Kerry Deutsch,1 Sharon Austin,1 Kellie Howard,1 Amanda Leonti,1 Lindsey Maassel,1 Christopher Subia,1 Tuuli Saloranta,1 Nicole Christopherson,1 Kathryn Shiji,1 Shradha Patil,1 Steven Anderson,2 Anup Madan1. 1 _Covance, Seattle, WA;_ 2 _Covance, Durham, NC_.

The emergence of Next Generation Sequencing (NGS) along with computational biology has broadened the scope in which diverse cellular processes can be interrogated. While there has been considerable progress in understanding the impact of genetic and epigenetic mechanisms in tumorigenesis using whole genomic, epigenomic and transcriptional analysis by NGS, there has been little consideration of the importance of interplay between these processes. We performed a comparative analysis of array and NGS technologies to identify differentially methylated CpG sites in colorectal cancer cell lines. NGS had more specificity in addition to profiling more CpG sites relative to Illumina 450K arrays.

Base-level resolution of sequencing data can identify any strand specific methylation bias. Our analysis shows that methylation frequency between the sense and antisense strand are highly correlated (average R2 ~ 0.81), and coefficient of variance (CV) between the strands is generally low (about half of observed sites have <10% CV). However, a small percentage of bases had strand specific biases. Using a minimum of 100% CV and difference in methylation frequency greater than 50% as filtering criteria, we found 1210, 569, 638, and 1484 CpG sites have strand specific biases with 7 overlapping bases among the samples tested. Further investigation will identify whether these bases are random or reside within a particular region, where biases occur, and what genes are potentially affected.

We also used NGS and publically available gene expression datasets in colorectal cancer cell lines-HCT116 and HCT116 DKO (cell line with genetic knockouts of both DNA methyltransferases DNMT1 and DMNT3b) to identify roles of differential methylation in regulating gene expression. A majority of genes were down-regulated between HCT116 and HCT116 DKO cell lines including those involved in chromatin, nucleic acid, and nucleotide binding and cell cycle regulation. Interestingly, many differentially expressed genes are also involved in immune response. We then used bisulfite treated genomic data to evaluate genetic regulation of gene expression. For this, we converted bisulfite treated data into genomic space using custom in-house developed bio-informatics tools that were first tested using DNA isolated from NA12878 cell line. Our analysis showed that 65% of the known variants detected in NA12878 cell line by the genome in a bottle consortium can be identified by bisulfite sequencing of promoter associated CpG islands. One limitation of this analysis is the inability to identify C>T genomic variants. This data is being analyzed to evaluate effects of genetic mutations in promoter binding sites on gene expression in colorectal cancers. Comparative analysis of genetic and epigenetic regulation of gene expression will allow better understanding of gene regulatory networks in colorectal cancer.

#4437

DNA methylation analysis in cisplatin resistant ovarian cancer cells and recurrent ovarian cancer patient samples.

Michaela L. Granados,1 Laurie G. Hudson,1 Li Luo,2 Sabrina L. Samudio-Ruiz1. 1 _University of New Mexico College of Pharmacy - Albuquerque, Albuquerque, NM;_ 2 _University of New Mexico, Albuquerque, NM_.

Acquisition of platinum resistance following first line platinum therapy is commonly observed in ovarian cancer patients and is a major obstacle to clinical effectiveness. Currently there are no options available to prevent platinum resistance; however, studies show that demethylating agents may resensitize patients to platinum therapy thereby demonstrating DNA methylation as a critical contributor to the development of platinum resistance. Using a cellular model of platinum resistance we have previously shown that resistant cells have increased DNA methyltransferase (DNMT) activity as well as increased global DNA methylation. These observations were dependent on cisplatin induced activation of the Epidermal Growth Factor Receptor (EGFR) which we previously identified as a novel a regulator of DNMT activity and DNA methylation. Inhibition of the EGFR conferred during a platinum resistance paradigm decreased DNMT activity, decreased global methylation and attenuated resistance in our platinum resistant model. We hypothesized that further genome wide analysis of DNA methylation in resistant, parental and EGFR inhibited cell lines at specific CG sites would reveal targets of methylation important for the development of platinum resistance. Using the Illumina 450K methylation array, we found few significant alterations in DNA methylation for given genes between control and cisplatin resistant (CPR) cells. However, additional DNA methylation analysis with the 450K methylation array of patient tumor samples at initial diagnosis as well as after relapse, while not significant, showed similar trends in the data for certain genes namely CSRNP3, LOC400904, LYPD6, MTMR9L, NIPAL4 and RBP7. We are still evaluating the effects of EGFR inhibition in vitro on DNA methylation patterns. We suggest that while the in vitro model of platinum resistance corresponds to patient samples in some aspects, ovarian cancer cells display a great deal of heterogeneity in DNA methylation as described by others and a much larger sample size is necessary to draw concrete conclusions. Current studies are evaluating gene expression in control and resistant cells to validate whether alterations in DNA methylation coincide with changes in expression.

#4438

Promoter hypermethylation status of Fanconi Anemia (FA) pathway genes FANCF, FANCL and FANCS in non-small cell lung cancer (NSCLC).

Andrew Fink, Arjun Kalvala, Li Gao, Kathleen Dotts, Brittany Aguila, Shirley Tang, Gregory A. Otterson, Miguel A. Villalona-Calero, Wenrui Duan. _Ohio State University, Columbus, OH_.

Gene promoter methylation is an epigenetic mechanism used by cells to control gene expression. Over recent decades, scientists have made various discoveries linking DNA methylation to several adverse outcomes, including human cancers. The Fanconi Anemia (FA) pathway is involved in homologous recombination, one of the major mechanisms of DNA repair. This pathway is essential for human cells to maintain integrity following DNA damage. Cancers with defective FA pathways are expected to be more sensitive to cross-link based therapy, or to treatments in which additional repair mechanisms are targeted. The FA pathway contains at least 19 genes, and some of the members have been implicated in susceptibility to a number of cancers by genetic or epigenetic alterations. Promoter methylation in FA genes is thought to play a role in the occurrence of cancer.

Recently we screened 139 non-small cell lung cancer (NSCLC) formalin-fixed, paraffin-embedded (FFPE) tumors for FANCD2 foci formation by FA triple stain immunofluorescence (FATSI) analysis. Among the 104 evaluable tumors, 23 (22%) were FANCD2 foci negative. Since epigenetic inactivation can be one of the mechanisms for FA functional deficiency in these tumors, we evaluated 39 NSCLC samples (21 foci positive and 18 foci negative; 21 adenocarcinomas, 17 squamous cell carcinomas, 1 large cell carcinoma) for FANCF, FANCL and FANCS (BRCA1) promoter methylation.

Human lung tumor tissue samples were obtained from The Tissue Procurement Shared Resources of the Ohio State University after IRB approval. Genomic DNA and total RNA samples were isolated from frozen lung tumor and matching non-tumor tissues. The promoter methylation status of FANCF, FANCL and FANCS was evaluated using methylation-specific PCR (MS-PCR).

Among the 18 FATSI negative tumors, promoter methylation was found in FANCF (1 adenocarcinoma), FANCL (1 adenocarcinoma) and FANCS (1 adenocarcinoma). Among the 21 FATSI positive tumors, no promoter methylation was detected in FANCF or FANCL. Promoter methylation in FANCS was found in 1 (squamous cell carcinoma) of 21 FATSI positive tumors.

The above observations suggest that epigenetic alterations, specifically methylation, can be one of the factors that contribute to FA functional deficiency in NSCLC patients. These findings may have clinical implications, since these tumors may be more sensitive to cross-link based therapy. However, an important caveat is that these changes may not be stable and could revert during treatment. Further studies in FA gene expression are needed to determine the impact of FA gene promoter methylation on FA repair foci formation.

#4439

Relationship between DNA methylation of TET genes and levels of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in hepatocellular carcinoma.

Lissette Delgado-Cruzata,1 Hui-Chen Wu,2 Jin Shen,2 Tiffany Thomas,3 Abby B. Siegel,4 Yu-Jing Zhang,2 Abhishek Goyal,2 Christine C. Hsu,5 Helen E. Remotti,3 Regina M. Santella2. 1 _City University of New York, John Jay College, New York, NY;_ 2 _Mailman School of Public Health, Columbia University, New York, NY;_ 3 _Department of Pathology and Cell Biology, Columbia University, New York, NY;_ 4 _Dept of Med Hematology & Onc, Columbia University, New York, NY; _5 _Dept of Medicine, Columbia University, New York, NY_.

Long-term survival after hepatocellular carcinoma (HCC) diagnosis has clearly improved as a result of treatment by means of a liver transplant. However, a large number of cases do not meet the transplant criteria and undergo resection. In these patients, loss of global DNA hydroxymethylation has been linked to worse prognosis, but no association has been found in transplant patients. Differential epigenetic processing is a possible reason for this, and has not been previously explored. The process of DNA demethylation is mediated by the family of ten-eleven translocation (TET) proteins, that catalyze the conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). There are three different TET protein variants: TET1, TET2 and TET3. Studies have shown that expression of at least TET1 is epigenetically regulated. In this study we investigated the association between DNA methylation of TET genes and global DNA methylation and hydroxymethylation in 66 tumor (T) and non-tumor (NT) paired samples of individuals treated at Columbia Presbyterian Medical Center in New York. Levels of 5mC and 5hmC were determined by UPLC/MS/MS, and DNA methylation of the TET promoters was obtained from Illumina Infinium 450k CpG array data. Mean levels of 5mC (T=3.15±0.49% vs. NT=3.80±0.12% (p<0.0001) and 5hmC (T=0.11±0.04% vs. NT=0.23±0.07%, p<0.0001) in tumor tissues were statistically significantly lower than in non-tumor tissues. In contrast, average DNA methylation levels of TET1 (T=0.40±0.06% vs. NT=0.35±0.02%, p<0.0001) were statistically significantly lower in non-tumor tissues than in tumor tissues. However, this was not the case for TET2 or TET3. TET1 DNA methylation levels negatively correlate with global DNA 5mC levels in tumor and non-tumor tissue (Spearman correlation coefficient: T= -0.47 (p=0.002) and NT= -0.60 (p<0.0001)). In contrast, associations between gene DNA methylation and 5hmC DNA levels were different for the tumor and non-tumor tissue samples. 5hmC levels positively and statistically significantly correlated with TET1 and TET2 DNA methylation (Spearman corr. coef TET1 NT= 0.57 (p<0.0001), and TET2 NT= 0.56 (p<0.0001)) in non-tumor tissue while there was a statistically significant negative correlation with TET3 (Spearman corr. coef. TET3 T= -0.37 (p=0.005)). No other associations were found between 5mC and 5hmC levels and TET gene DNA methylation levels. These findings suggest higher expression of TET1 correlates with a hypomethylated state and that the accumulation of 5hmC possibly results from a downregulation of TET1 and TET2 in non-tumor tissue. HCC carcinogenesis alters this process, and while further studies need to be conducted, TET3 seems to play a larger role in tumor cells. Our sample size was limited, but these results suggest that future research of 5hmC might help elucidate HCC underlying molecular events and aid in the design of prognostic markers.

#4440

Diffuse intrinsic pontine glioma epigenetic marks change depending on cell culture conditions: Implications for investigating epigenetically directed therapy.

Sama Ahsan, Michael Haffner, Charles Eberhart, Eric Raabe. _Johns Hopkins Hospital, Baltimore, MD_.

Methylation of cytosine is viewed as repressive of transcription. 5-methylcytosine (5mC) can be removed from DNA by activity of ten-eleven translocation (TET) methylcytosine dioxygenase. The first step in erasing the repressive 5mC mark is conversion to 5-hydroxymethylcytosine (5hmC) by TET. Subsequent steps lead to replacement of 5hmC by cytosine, providing a mechanism by which 5mC-mediated repression can be erased. We have previously identified 5hmC and 5mC as being imbalanced in primary tumor tissue samples from the universally fatal pediatric malignancy diffuse intrinsic pontine glioma (DIPG) compared normal brain and glioblastoma (GBM). The aberration in DNA methylation along with known aberrant histone methylation in DIPGs, likely creates an active transcriptional/translational state and potentiates tumor growth. We hypothesized that the elevation of 5hmC in DIPG is secondary to increased TET activity. We found two different epigenetic signatures for DIPG cells depending on their culture conditions. When cultured in serum-free media as neurospheres, to our surprise, we found cells had low 5hmC, in contrast to what we observed in primary tumor and in orthotopic xenograft tumors. We hypothesized that 5hmC status may vary depending on differentiation state, and found that when DIPG cells were differentiated on matrigel for 14 days in the presence of serum, 5hmC levels increased and were similar to that observed in primary tissue and xenograft tumors. Differentiation was confirmed by upregulation of GFAP (glial marker) and MAP2 (neuronal marker) expression and the elaboration of long processes. We also found that DIPG cells in the serum/matrigel/differentiated state had increased TET1 and TET2 mRNA expression relative to undifferentiated neurospheres (2.6 and 2.1 fold increase respectively for TET1 and TET2 mRNA expression). As a control for adherent versus nonadherent growth, we also plated DIPG cells on laminin in EGF/FGF media, and we found that the serum-differentiated cells upregulated TET1 and TET2 mRNA 6.7 and 3.3 fold respectively, compared to laminin-plated cells, providing further support that differentiation affects epigenetic marks in DIPG. In contrast to DIPG, GBM cells did not exhibit similar changes in TET mRNA expression when differentiated, emphasizing the uniqueness of the DIPG epigenetic signature. Our data shows that neurosphere cell culture models of DIPG may not be true epigenetic representations of DIPG tumor in vivo and the predicted response to potential therapeutic options may differ based on the epigenetic state used for preclinical testing.

#4441

**Non-pathogenic bacteria change host DNA methylation** in vivo **.**

Ang Sun,1 Jaroslav Jelinek,1 Shinji Maegawa,1 Christian Jobin,2 Jean-Pierre Issa1. 1 _Fels Institute for Cancer Research and Molecular Biology,School of Medicine, Temple University, Philadelphia, PA;_ 2 _Department of Medicine, Division of Gastroenterology, College of Medicine, University of Florida, Gainesville, FL_.

Introduction: More than a thousand species of bacteria live in a normal person's gastrointestinal tract. Epigenetic effects on the colon directly caused by the commensal bacteria remain elusive. In the current study, we explore the bacterial effects on DNA methylation of the host colon. Methods: Eight non-pathogenic bacterial species were introduced to the gut of germ-free (GF) mice to make specific-pathogen free (SPF) mice. Our study consisted of the following groups (n=6 for each): wildtype (WT) GF, WT SPF, IL-10 knockout (KO) GF, IL-10 KO SPF, and IL-10 KO GF or SPF treated with azoxymethane (AOM). We analyzed DNA methylation in genomic DNA extracted from the proximal colon using Digital Restriction Enzyme Analysis of Methylation (DREAM) and compared methylation levels at CpG sites among different groups. Results: 1) Principal component analysis (PCA) performed on methylomes of all the samples separated the mouse samples based on presence or absence of bacteria, IL-10 KO status or AOM treatment into 6 distinct groups. 2) We analyzed CpG sites differentially methylated between GF and SPF mice at p<0.05 significance level. In WT mice, there were 1871 CpG sites (11% out of 17475 analyzed sites), in IL10 KO mice, there were 5986 CpG sites (33% out of 18254 analyzed sites) and in IL10 KO AOM-treated mice there were 6755 CpG sites (31% out of 21697 analyzed sites) differentially methylated. 3) There were 756 common CpG sites differentially methylated in all SPF vs GF comparisons. Interestingly, these sites were enriched 2.6-fold for aging-related CpG sites. 4) WT SPF mice showed increased methylation at CpG sites hypomethylated in WT GF mice and, conversely, decreased methylation at CpG sites hypermethylated in WT GF mice. This pattern of DNA methylation change was also observed by comparing GF with SPF in IL-10 KO mice with or without AOM treatment. 5) Both in GF and SPF mice the DNA methylation changes were more pronounced in IL10 KO while AOM had little additional effect. 6) IL10 KO resulted in genome-wide increase of methylation. However, the introduction of bacteria in SPF mice resulted in the methylation change mainly at CpG islands. Conclusions: Non-pathogenic bacteria introduced in the colon cause methylation gains at CpG sites with low DNA methylation and methylation losses at sites with high DNA methylation in a pattern similar to the changes observed in aging and cancer.

#4442

Very low methylation is a novel epigenetic regulatory mechanism in normal tissues and predisposes to hypermethylation in cancer.

priyanka madireddi, Jaroslav Jelinek, Justin Lee, Takahiro Sato, Jean-Pierre Issa. _Temple University Medical School, Philadelphia, PA_.

High level DNA methylation of promoter CpG Island (CGI) represses gene expression. However, low-level CGI methylation is usually not distinguished from complete absence of methylation. Here we show that Very Low Methylated Regions (VLMRs 1-20%) on promoter CGIs are present in up to 20% of human and mouse genes and negatively correlate with gene expression in all tissues examined. In vitro, low-level methylation directly represses reporter gene expression; an effect mediated by methyl-CpG-binding proteins as transient knockdown of MBD4 reverses this repression. In vivo VLMR genes are enriched for polycomb occupancy but can also occur in H3K4me3 occupied promoters, where it correlates with gene repression independent of polycomb complex binding. VLMRs in promoter CGIs in healthy WBCs are 19 fold and 65 fold more likely to gain hypermethylation in Acute Myelogenous Leukemia and Myelodysplastic Syndrome patients respectively compared to unmethylated CGIs (<1%). Our study reveals a novel epigenetic control mechanism that has strong effects on gene expression in normal tissues and accounts for most of the gene specificity of hypermethylation in cancer.

#4443

Investigation of the promoter methylation status of the FHIT (fragile histidine triad) gene in larynx cancer.

Semra Demokan,1 Kubra Akalin,1 Cansu Ozkoklesen,1 Murat Ulusan,2 Necati Enver,2 Nejat Dalay1. 1 _Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey;_ 2 _Department of Otorhinolaryngology, Faculty of Medicine, Istanbul University, Istanbul, Turkey_.

Purpose: Development of carcinoma is associated with epigenetic modifications. Methylation of CpG islands in the promoter regions can participate in gene silencing and promote tumor aggressiveness. The Fragile histidine triad (FHIT) gene, localized on chromosome 3, is a tumor suppressor gene and the abnormal methylation of FHIT. The FHIT regulates the G2/M cell cycle check point and apoptotic pathwaysin various types of cancer. Inactivation of FHIT due to methylation has been shown in breast, cervical, gastric and colon carcinomas. In our study, we evaluated the FHIT gene promoter methylation in patients with larynx carcinoma (LC).

Experimental Design: Methylation of FHIT was investigated by bisulfite modification/methylation-specific polymerase chain reaction in tumors and matched normal tissue samples from 57 Turkish patients with LC.

Results: The promoter region of the FHIT was methylated in 96.49% and 29.82% of the primary tumors and the corresponding normal tissue samples, respectively [p=0.0001, chi-square=14.2105, OR(95%CI)= 64.7(14.4-296.07)]. A methylation rate (less than 10%) was observed in 3.51% (2/57) and 70.18% (40/57) of tumor and normal samples, respectively. Moderate methylation levels (10-25%) were found in 3.51% (2/57) of the tumors and 1.75% (1/57) of the adjacent normal tissues. High methylation levels (>25%) were observed in 92.98% (53/57) and 28.07% (16/57) in the tumor and normal tissues, respectively.

Conclusion: Methylation may play an important role in LC carcinogenesis. Although we found that there was significant association between the methylation levels of tumor and adjacent normal tissues, high methylation levels of the FHIT was observed in the tumors when compared to the levels in normal tissue. These results suggest that the methylation rates of FHIT gene may provide a suitable biomarker in LC carcinogenesis. Our study is still in progress to include a larger cohort of patients.

#4444

DNA methylation age is accelerated in breast tissue of healthy women donors.

Mary E. Sehl, Patricia A. Ganz, Steve Horvath. _UCLA David Geffen School of Medicine, Los Angeles, CA_.

Background: Advanced age and cumulative estrogen exposure are well known risk factors for breast cancer. However, the mechanisms underlying the link between cellular senescence, chronic estrogen stimulation, increased cell cycling, and breast carcinogenesis are poorly understood. We examined epigenetic alterations that occur in the female breast with advancing age, and estrogen exposure.

Methods: We utilized specimens from 49 women healthy tissue donors from the Komen Tissue Bank who donated both breast tissue and peripheral blood at two time points spaced at least 2 years apart. Ages of the donors ranged from 18-65 years and they were grouped by menopausal status (27 pre- and and 22 post-menopausal) and parity (21 nulliparous). We assessed epigenome-wide DNA methylation levels (Illumina 450K platform) from DNA extracted from breast and blood tissue, and calculated tissue "DNA methylation age (DNAm age)" based on methylation levels at 353 CpGs that have been shown to strongly correlate with chronologic age across tissues. We examined the relationship between this outcome and age and variables related to cumulative estrogen exposure, including age at menarche, gravidity, parity, and menopausal status.

Results: DNAm age was highly correlated with chronological age in our sample (cor=0.8, p<0.001), within both peripheral blood (cor=0.94, p<0.001) and breast tissues (cor=0.88, p<0.001). When we examine the residuals for each sample from our regression analysis of DNAm age versus chronological age, we find that age acceleration is significantly increased in breast relative to peripheral blood tissue (p<0.001). In a multivariate regression analysis, we found that earlier age at menarche (β=-0.62, p=0.04) and higher gravidity (β=0.95, p=0.03) were associated with higher DNAm age.

Conclusions: We find that DNAm age is accelerated in breast tissue compared with peripheral blood cells in a population of healthy donors to the Komen Tissue Bank. We further note a significant association between DNAm age and early menarche, and rising number of pregnancies, suggesting that cumulative cell cycling with menstrual cycles and the tissue plasticity occurring with pregnancy may contribute to accelerated epigenetic age in the female breast.

#4445

The estrogen related receptor alpha regulates one carbon metabolism and DNA methylation.

Mathieu Vernier,1 Etienne Audet-Walsh,1 Ingrid Tam,1 Geneviève Deblois,2 Vincent Giguère1. 1 _Mcgill University, Montréal, Quebec, Canada;_ 2 _University of Toronto, Toronto, Ontario, Canada_.

Besides genetic alterations, epigenetic mechanisms such as DNA methylation are implicated in the acquisition of malignant phenotype, and thus, the use of epigenetic drugs is a promising strategy for anti-cancer therapy. DNA methylation is the process by which DNA methyltransferase enzymes (DNMT) add methyl groups to cytosines, using S-adenosine methionine (SAM) as a methyl donor.Therefore, this process relies both on the expression of the DNMTs and on the availability of SAM, which is a component of the one carbon metabolism pathway. In the context of breast cancer, cell metabolism is tightly regulated by the orphan nuclear receptor estrogen-related receptor alpha (ERRa) and thus, we wondered whether ERRa could regulate SAM levels and, by extension, DNA methylation.

By analyzing chromatin immunoprecipitation experiments followed by DNA sequencing (ChIP-seq) of ERRa conducted in different breast cancer cell lines, we discovered that ERRa is located on the promoter of many genes implicated in one carbon metabolism. Inhibition of ERRa by RNA interference or with the chemical inhibitor C29 modified the mRNA levels of these genes and induced changes in SAM levels. Interestingly, we also observed ERRa binding on the promoter of DNMT1 and genetic and pharmacological inhibition of ERRa reduced DNMT1 expression at the mRNA and protein levels. Altogether, these changes ultimately decreased global DNA methylation in breast cancer cell lines, suggesting that ERRa is a major driver of this epigenetic mechanism. Surprisingly, genetic inhibition of DNMT1 or treatment with the DNMT inhibitor (DNMTi) SGI-1027 reduced the protein levels of ERRa in these cells, unraveling the existence of a feedback loop between these two pathways. Therefore, we explored whether treating breast cancer cells with a combination of C29 and SGI-1027 would represent a good therapeutic approach. We treated breast cancer cells with a given concentration of C29 and increased concentrations of SGI-1027 for 24h and observed that C29 highly sensitized these cells to the DNMTi. Further investigations on xenograft models will be conducted to validate these results in vivo and next-generation sequencing will be performed in the context of ERRa inhibition to study the global DNA methylation alterations and the consequences on gene expression.

In conclusion, we have uncovered a novel crosstalk between cell metabolism and epigenetics that lead to a better understanding of the regulation of these events and their influence on cancer cell biology and drug sensitivity. We propose that targeting these two pathways with a combination therapy possesses great therapeutic potential for breast cancer and may be efficacious in other cancers as well.

#4446

Compound gene mutations in the methylation pathway contribute to the chronic health problems and colorectal cancer: A family-based case-control study.

Pamela Shiao,1 Brandi Wasek,2 Teodoro Bottiglieri,2 Hongyan Xu1. 1 _Augusta University, Augusta, GA;_ 2 _Baylor University, Dallas, TX_.

The purpose of this project is to examine compound gene mutations in the methylation pathway for their contributions to the development of chronic health conditions and colorectal cancer (CRC) in a family-based case control study.

Fifty four cancer patients and matched 54 family controls were recruited from southern California areas by accessing California Cancer Registry data, with racial ethnic groups representing the proportions of population distributions in the communities. Home visits were conducted for interviews and collection of biological samples (buccal and/or blood) for genotyping. Blood plasma samples were processed within 30 minutes of collection, transported on ice, and stored in -80ᵒC for metabolite analyses. Family-Based Association Tests using the FBAT-toolkit, taking account of familial correlations due to shared genes, were performed. The test p-values and beta coefficients were reported.

New Data Findings

Plasma total homocysteine (tHcy) levels were highest in the advanced cancer group than the early stage cancer group, and control groups with chronic health problems and healthy control (p = 0.0066). For SNP analyses on five selected genes including methylenetetrahydro-folate reductase (MTHFR) 677 and 1298, methionine synthase (MTR) 2756, methionine synthase reductase (MTRR) 66, and dihydrofolate reductase (DHFR) 19 bp deletion, the advanced cancer group had the highest mutation rates for these genes combined, than other groups (p = 0.032), and the most MTHFR mutations (p = 0.016). Among the ethnic groups, the methylation status represented by the SAM/SAH ratio, were lower in African American (AA) and Asian than in Hispanics and Caucasian (p = 0.0199). Caucasian and Hispanics had higher MTHFR mutations than Asian, and AA had the lowest MTHFR mutations (p < 0.001). Hispanics had the lowest mutations (8.7%) on MTR gene (p = 0.069) than Asian (29.3%), Caucasian (41.2%), and AA (50%); and Hispanics had the lowest mutations (26.1%) on MTRR gene (p < 0.001) than Asian (50%), AA (70%), and Caucasian (79.4%). Hispanic samples had the lowest mutations (47.8%) for DHFR 19bp deletion than Caucasian (55.9%), AA (80%), and Asian (85.4%) (p < 0.001). FBAT analyses revealed that SAH levels were associated with MTRR mutation (p = 0.077), but betaine and choline levels were associated with MTR mutations (p < 0.05).

Conclusion

Based on the findings from these analyses, tHcy levels and common gene mutations in the OCM methylation pathways are associated with the development of chronic health problems and advanced stages of CRC. Ethnic groups present different mutation patterns on the genes in the methylation pathways for population health. These findings warrant personalized healthcare based on gene mutations and enzyme deficiency in the OCM methylation pathways.

#4447

EZH2 contributes to breast cancer metastasis.

Shira Yomtoubian,1 Seongho Ryu,2 Sharrell Lee,1 Lauren Havel,1 Dingcheng Gao,1 Vivek Mittal1. 1 _Weill Cornell Medical School, New York, NY;_ 2 _Soonchunhyang Institute of Med-Bio Sciences, Republic of Korea_.

While the 5-year relative survival rate of localized stage breast cancer is 99%, once the cancer spreads to distant lymph nodes and organs the survival rate falls to 25%. In order to develop strategies to combat metastasis, we have been studying epigenetic factors that contribute to the aggressive nature of breast cancer. We have focused on enhancer of zeste homolog 2 (EZH2), a histone methyl transferase, because breast cancer patients expressing increased EZH2 exhibit highest metastatic recurrence. We have found through in vivo studies that blocking EZH2 activity not only prevents metastasis formation, but also targets established metastases. Genome wide ChIP-seq analysis revealed that EZH2 may impact breast cancer metastasis through regulating the expression of EMT marker, E-cadherin, and stem cell markers, GATA3 and DKK2. Mammosphere assays from tumors treated with pharmacological blockade of EZH2 and limiting dilution analysis validates that EZH2 inhibition impacts the tumor-initiating cell population. Our data suggests that inhibition of EZH2 activity may have anti-metastatic therapeutic potential and may complement current standards of breast cancer therapy.

#4448

Development of DNA methylation biomarkers for pancreatic cancer diagnosis.

Keiko Shinjo,1 Keisuke Katsushima,1 Genta Nagae,2 Hiroyuki Aburatani,2 Kenji Yamao,3 Yutaka Kondo1. 1 _Nagoya City University, Nagoya, Japan;_ 2 _The University of Tokyo, Tokyo, Japan;_ 3 _Aichi Cancer Center Hospital, Nagoya, Japan_.

Pancreatic cancer is highly lethal malignancy despite the recent clinical advancement. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop minimally invasive biomarkers for detection. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancer. The presence of cell-free DNA (cfDNA) in blood stream has been known and cfDNA possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system might be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens of pancreatic cancer with clinical stage III-IV using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database, TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. In addition, analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We validated these methylation markers in different FNA sample set (n=19) by bisulfite pyrosequencing or quantitative methylation specific PCR and found that 18/19 (95%) cases were methylated in at least one marker (AUC=0.95). Finally, we performed methylation analysis in cfDNA from pancreatic cancer patients. From our preliminary data, we could detect aberrant DNA methylation in cfDNA from pancreatic cancer patients.

We identified potential DNA methylation biomarkers for the diagnosis of pancreatic cancers and these markers might be applicable for detecting cancer in cfDNA.

#4449

TET1 inhibits gastric cancer metastasis by demethylation and re-expression of PTEN.

Bingya Liu, Yao-fei Pei, Jian-fang Li, Liping Su, Ran Tao, Min Yan, Zhang-gang Zhu. _Shanghai Jiao Tong Univ. School of Medicine, Shanghai, China_.

TET1 is a member of ten eleven translocation enzymes, which can convert 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), eventually to promote CpG islands demethylation of specific gene.. In this study, we found that TET1 expression and 5-hmC content were low in tumor tissues compared to its adjacent non-tumor tissues of gastric cancer. Cell proliferation, migration and invasion were enhanced when TET1 knocked down in gastric cancer cell in vitro and in vivo, while it was suppressed when TET1 over-expressed. We also found that PTENis one of the target genes of TET1. TET1 directly binds to the promoter region of PTEN, activates its transcription through demethylating its CpG islands. Knockdown of TET1 results in hypermethylation of PTEN promoter and low expression of PTEN. PTEN knockdown activates AKT and FAK pathway, which are suppressed by PTEN. The activation of PTEN downstream AKT and FAK facilitates tumor migration, invasion and accelerates cell growth. In conclution, we found a novel mechanism that TET1 suppresses tumor cell growth, migration and invasion through conversion PTEN promoter CpG island methylation to demehylation and then re-expression PTEN.

#4450

Silencing NKD2 by promoter region hypermethylation promotes esophageal cancer progression by activating Wnt signaling.

Mingzhou Guo,1 Baoping Cao,1 Weili Yang,1 Yongshuai Jin,1 Meiying Zhang,1 Tao He,1 Qimin Zhan,2 James G. Herman,3 Guanglin Zhong1. 1 _Chinese PLA General Hospital, Beijing, China;_ 2 _Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China; _3 _University of Pittsburgh Cancer Institute, Pittsburgh, PA_.

Background: Despite the use of surgery or chemoradiotherapy to treat, esophageal cancer, its prognosis still remains poor, with a 5-year survival below 15%. Recent insights into the epigenetic mechanisms associated with multi-step carcinogenesis provide new opportunities to develop novel effective targeted therapies for esophageal squamous cell carcinoma. Naked cuticle homolog 2 (NKD2) was found frequently methylated in human breast cancer. The epigenetic changes and mechanisms of NKD2 in human esophageal cancer remain unclear. Methods: Nine esophageal cancer cell lines and 154 cases of primary esophageal cancer samples were analyzed using methylation specific PCR, immunohistochemistry, western blot, flow cytometry techniques and a xenograft mouse model. Results: Loss of NKD2 expression and complete methylation were found in KYSE150 and TE1 cells. Reduced expression and partial methylation were observed in KYSE30, KYSE70, KYSE410, KYSE140 and COLO680 cells. High level expression and unmethylation were detected in KYSE450 and TE8 cells. Re-expression of NKD2 was induced by 5-aza-2'-deoxycytidine in NKD2 unexpressed or reduced cells. NKD2 was methylated in 53.2% (82/154) of human primary esophageal cancer samples, and promoter region hypermethylation was associated with reduced expression of NKD2 significantly (p<0.01). NKD2 methylation was associated with TNM stage and lymph node metastasis (p<0.01). The results suggest that NKD2 is regulated by promoter region methylation and methylation of NKD2 may serve as a prognostic marker in esophageal cancer. Our further studies demonstrate that NKD2 suppresses cell proliferation, colony formation, cell invasion and migration, as well as induces G1/S check point arrest in esophageal cancer cells. NKD2 suppressed xenograft tumor growth and inhibited Wnt signaling in human esophageal cancer cells. Conclusions: NKD2 is frequently methylated in human esophageal cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses esophageal cancer progression by inhibiting Wnt signaling both in vitro and in vivo.

#4451

Different patterns of SIRT1, KLF4, DAPK1 and SPG20 methylation in B lymphocytes correlate with the clinical parameters of non-Hodgkin lymphoma subtypes.

Raffaele Frazzi,1 Eleonora Zanetti,1 Mariaelena Pistoni,1 Ione Tamagnini,2 Riccardo Valli,2 Francesco Merli3. 1 _Translational Research Laboratory, Arcispedale S. Maria Nuova IRCCS, Reggio Emilia, Italy;_ 2 _Pathology Division, Arcispedale S. Maria Nuova IRCCS, Reggio Emilia, Italy;_ 3 _Hematology Division, Arcispedale S. Maria Nuova IRCCS, Reggio Emilia, Italy_.

Background. DNA methylation is one of the best studied epigenetic modifications and one major constituent of the epigenome of a cell. It contributes to normal development as well to carcinogenesis. Nowadays, many efforts are being made in order to use DNA methylation as a biomarker. The aim of our work is to characterize the expression and methylation of SIRT1, HIC1, BCL6, KLF4 and other genes relevant for Non-Hodgkin lymphomas (NHL) pathogenesis. Methods. Immunohistochemistry (IHC) on 72 formalin-fixed paraffin embedded tissue sections (FFPE). B-lymphocytes were purified from 36 biopsies of follicular hyperplasias (non-malignant B-lymphocytes), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL). Gene expression were analysed by quantitative retrotranscribedPCR (qRTPCR). Quantitative CpG promoter methylation analysis was performed by pyrosequencing after bisulfite conversion or by Methyl II array qPCR on genomic DNA. Results. In a total of 72 FFPE samples of follicular hyperplasias (n=17), FL (n=36) and DLBCL (n=19), SIRT1 staining is localized in the germinal center of the majority of follicular hyperplasias and FL samples. SIRT1 localizes preferentially in the centroblasts of the GC of the follicles where it correlates with Ki67. BCL6 is uniformly positive in follicular hyperplasias and FL, but heterogeneously distributed in DLBCL. Interestingly, SIRT1 and BCL6 expression correlate in FL.

By quantitative pyrosequencing we analyzed 3 CpG sites for the SIRT1 promoter (corresponding to the binding sites for CREB, ARID and PPARG transcription factors). Follicular hyperplasias display higher methylation levels (52.88%) than FL (38.36%) and DLBCL (32.65%) on SIRT1 promoter suggesting a possible inverse correlation between tumor aggressiveness and SIRT1 methylation.

Next, we selected a panel of genes whose expression is linked to lymphoma pathogenesis. By Methyl II array qPCR, we show that BCL6 methylation does not vary among samples. KLF4, DAPK1 and SPG20, show statistically significant methylation increases in FL and DLBCL compared to follicular hyperplasias, indicating a possible role of these proteins in lymphoma pathogenesis. On the contrary, no significant differences are observed for the other markers MZB1, MGMT, LMO2 and ASXL1. Notably, KLF4, DAPK1 and SPG20 mRNA expression levels anti-correlate with their promoter methylation in FL.

Conclusions. Epigenetic changes in SIRT1 methylation inversely correlate with NHL aggressiveness (decreasing in the order: follicular hyperplasias - FL - DLBCL), while KLF4, DAPK1 and SPG20 show a methylation increase that correlates with tumor aggressiveness. These data suggest that different patterns of methylation correlate with the clinical and prognostic parameters of these NHL subtypes.

#4452

Oxdative stress suppresses the expression of 15-hydroxyprostaglandin dehydrogenase via upregulation of DNMT3a/Snail in human colon epithelial cells.

Ja-Young Lee, Jeong-Eun Lee, Hye Kyung Na. _Sungshin Women's University, Seoul, Republic of Korea_.

Overproduction of prostaglandin E2 (PGE2) is implicated in pathogenesis of inflammation-associated carcinogenesis. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme that catalyzes the conversion of PGE2 to biologically inactive 15-keto-PGE2 metabolite. 15-PGDH has been considered to be a tumor suppressor as its expression is frequently repressed in human malignancies via hypermethylation of its promoter. In our previous study, oxidative stress caused by Helicobacter pylori infection suppressed the expression of 15-PGDH. Reactive oxygen species (ROS) can induce methylation as well as mutation of genes including those that encode tumor suppressor proteins, thereby contributing to carcinogenesis. This prompted us to investigate whether 15-PGDH expression could be down-regulated by oxidative stress in normal colon epithelial CCD841 cells. When CCD841 cells were treated with H2O2, the expression level of 15-PGDH was significantly reduced in both concentration and time dependent manners. Hydrogen peroxide (300 μM) induced the expression of DNA methyltransferase (DNMT) 3a, which was blocked by an inhibitor of DNMT, 5-Aza-2′-deoxycytidine (5-Aza). The 5-Aza abrogated the down-regulation of 15-PGDH expression induced by H2O2. The antioxidant, N-acetylcysteine (NAC) abrogated the down-regulation of 15-PGDH and upregulation of DNMT 3a expression induced by H2O2. The transcriptional repressor Snail plays a central role in the suppression of 15-PGDH expression. H2O2 treatment also elevated expression of Snail, which was abrogated by NAC and 5-Aza. Taken together, these finding suggest that ROS suppresses the expression of 15-PGDH through hypermethylation of CpG island in the 15-PGDH promoter.

#4453

Molecular hallmarks of drug resistance to DNA methylation inhibitors and alternative therapeutic regimen for overcoming resistance.

Khushboo Agrawal,1 Petr Vojta,1 Dusan Holub,1 Rastislav Slavkovsky,1 Ivo Frydrych,1 Petr Dzubak,1 Marcela Krecmerova,2 Marian Hajduch1. 1 _Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic;_ 2 _Institute of Organic Chemistry and Biochemistry, ASCR v.v.i, Prague, Czech Republic_.

Aberrant DNA methylation perturbing the epigenetic machinery has been continuously underlined as the major hallmark involved in all types of cancer. The archetypal DNA methylation inhibitor, 2'-deoxy-5-azacytidine (5-AZA-CdR) have been approved by FDA as the effective epigenetic drug for the treatment of blood malignancies, over a decade. However, the successful epigenetic therapy remains impeded owing to its chemotherapeutic resistance. To investigate the molecular mechanism of resistance to 5-AZA-CdR, we developed several HCT116 p53 wild-type cell clones, resistant towards 5-AZA-CdR, and principally studied the molecular alterations during the development of resistance, using high throughput RNA sequencing based transcriptomics and mass spectrometry based proteomics. The molecular profiling of HCT116 cells sensitive to 5-AZA-CdR treatment versus 5-AZA-CdR resistant cell clones distinguished the differential expression of various genes and the proteins coded by the genes, compared with untreated HCT116 wild-type cells (ANOVA p<0.05). The major affected cellular pathways were (i) Cell cycle: role of 14-3-3 proteins in cell cycle regulation, G1/S transition and initiation of mitosis (ii) Apoptosis and survival: granzyme A signaling, BAD phosphorylation, p53 dependent apoptosis (iii) Transcription: role of heterochromatin protein I family in transcriptional silencing, role of AP1 in regulation of cellular metabolism (iv) DNA damage: role of SUMO in p53 regulation. Further, we used MTT cytotoxicity assay to determine the cross-resistance or sensitivity of the 5-AZA-CdR resistant cell clones towards the inhibitors of epigenetic "Readers-Writers-Erasers". The study revealed that 5-AZA-CdR resistant cell clones exhibited cross-resistance towards all the tested epigenetic inhibitors, however, significant sensitivity was exceptionally observed for bromodomain inhibitors. Additionally, the well-known bromodomain inhibitor, (+)-JQ1, showed significant anti-tumor activity sensitizing 5-AZA-CdR resistant HCT116 xenografts, suggesting bromodomain inhibition as the alternative therapeutic regimen for overcoming resistance to DNA methylation inhibitors. Validation of relevant genes and/or proteins as biomarkers for predicting clinical response to 5-AZA-CdR, including mutation analysis and methylome sequencing, and studies validating bromodomains as alternative therapeutic target for re-sensitizing the cancer patients, resistant to DNA methylation inhibitors is currently ongoing.

#4454

Genome-wide analysis of DNA methylation induced by environmental carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice.

Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. _Penn State University College of Medicine, Hershey, PA_.

We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n=3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1X50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value < 0.01 and percent methylation difference > 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465.

#4455

DNA methylation as biomarkers to predict early recurrence of T1-2N0 lung cancer.

Chen Chen,1 Andrew Yang,1 Devlin Danielle,1 Kristen Rodgers,1 Peng Huang,1 Zhihao Lu,1 Candace Griffin,1 Beverly Lee,1 Richard Battafarano,1 Fenglei Yu,2 Tza-Huei Wang,1 Stephen Baylin,1 James Herman,3 Alicia Hulbert,1 Malcolm Brock1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _The Second Xiangya Hospital of Central South University, Changsha, China;_ 3 _University of Pittsburgh, Pittsburgh, PA_.

Purpose: Many patients die of recurrent NSCLC. Current clinical and molecular tests fail to predict patients at high risk for recurrence. We investigated the relationship between gene promoter methylation and recurrence of NSCLC and its prediction accuracy.

Materials and Methods: We prospectively enrolled patients who underwent surgery for T1-2N0 NSCLC. We collected primary tumors, regional lymph nodes (RLN) and mediastinal lymph nodes (MLN). A total of 112 patients were included; cases (n=39) and controls (n=73) were defined based on cancer recurrence within 40 months of surgery. Promoter methylation of eight cancer-related genes (CDO1, TAC1, SOX17, p16, CDH13, RASSF1A, APC and MGMT) was obtained by using nanoparticle-based DNA extraction followed by qMSP. Logistic regression analysis adjusted by age, gender, race, tumor size and pack year was used to study the relationship between the gene promoter methylation and the risk of recurrence of NSCLC; machine learning assessed its prediction accuracy.

Results: Cases had significantly higher p16 methylation levels than controls in all three tissue types by univariate and multivariate analysis after adjusting for confounders (age, gender, race, tumor size, smoking history, and other gene methylation levels). SOX17 was significantly methylated among cases in both RLN and MLN samples, and remained significant in MLN after adjusting for confounders. Table 1 shows the prediction accuracy of p16 and SOX17 using tumor tissue alone and combined with RLN and/or MLN. Prediction accuracies were increased after adding biomarkers from lymph nodes.

Conclusion: Increased methylation status in the promoter region of p16 and SOX17 is associated with an increased risk of cancer recurrence among patients with T1-2N0 NSCLC treated with curative resection. Adding molecular analyses of lymph nodes to primary tumor samples significantly increased the diagnostic accuracy of predicting recurrence in these patients.

Table 1. Prediction accuracy using tumor and combined with RLN and/or MLN.

---

Tissue Samples | Sensitivity | Specificity | PPV | NPV

Tumor | 0.41 | 0.84 | 0.52 | 0.76

Tumor+RLN | 0.68 | 0.96 | 0.90 | 0.84

Tumor+MLN | 0.56 | 0.88 | 0.71 | 0.79

Tumor+RLN+MLN | 0.69 | 0.90 | 0.79 | 0.85

#4456

Preliminary investigation of the promoter methylation status of BRCA1, BRCA2, TP53 genes of some breast cancer patients.

Titilola A. Samuel, babatunde ayo james, Muhammad HABEEB, temitope oshodi, olubunmi magbagbeola. _university of Lagos, Lagos, Nigeria_.

Epigenetic alterations in BRCA1, BRCA2 and TP53, such as DNA methylation, may be an effective approach in diagnosis, prognosis and therapy in breast cancer. This study examines the DNA methylation signature of BRCA1, BRCA2 and TP53 in some breast cancer patients attending a Clinic in Lagos, Nigeria. In this study, 14 blood specimens with Invasive Ductal Carcinoma (IDC) were investigated. The samples were modified by Bisulfite modification, and then assessed by Methylation-Specific PCR (MSP). The methylation analysis indicates that 7(50%) of the 14 patients had a segment of the promoter region of BRCA1 gene methylated, 8 (57.14%) of BRCA2 methylated and 11(78.57%) of TP53 promoter region. This study provides preliminary evidence for the gene expression inactivation of BRCA 1, BRCA 2, and TP53 Genes in over 50% of the 14 patients with Stage 2 invasive ductal carcinoma who participated in this study.

### Epigenetic Biomarkers and Therapies

#4457

Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy.

Joana G. Marques,1 Berkley Gryder,2 Maria Boehm,1 Marco Wachtel,1 Young Song,2 Hsien-Chao Chou,2 Rajesh Patidar,2 Hongling Liao,2 Javed Khan,2 Beat W. Schaefer1. 1 _University Children's Hospital, Zurich, Switzerland;_ 2 _National Cancer Institute - NIH, Bethesda, MD_.

Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric tumor driven by an oncogenic fusion protein, PAX3-FOXO1, which acts as a transcription factor. Conventional chemotherapy is effective for low risk patients who have a 5-year overall survival greater than 65%, while high risk patients, including those with metastatic disease, have less than 40% survival. Consequently, we hypothesize that targeting the fusion protein or its collaborators in transcription regulation will provide novel therapies for this aggressive subtype of RMS. To identify new druggable PAX3-FOXO1 interactors, we performed a combined proteomic and genetic screen which led to the discovery of the NuRD complex (Nucleosome Remodelling and Deacetylase) as a major PAX3-FOXO1 co-regulator. The NuRD complex is unique among the chromatin remodelling complexes due to its dual enzymatic activity. It can act by histone deacetylation through HDAC1/2 (histone deacetylases) or influence nucleosome positioning through CHD4 (chromodomain-DNA-binding protein 4). Intriguingly, it has been associated with both activating and repressive activities in gene expression and its role in cancer development is not fully understood yet. We found that in FP-RMS, silencing of CHD4 affected the expression of approximately 50% of PAX3-FOXO1 regulated target genes. These were mainly genes which are usually upregulated, suggesting an activating role for NuRD. Consistent with CHD4 activation activity, ChIP-seq experiments demonstrated that CHD4 and HDAC2 co-localize with the fusion protein in cis-regulatory sites of a subset of its target genes. Interestingly, gene expression analysis showed that both CHD4 and HDAC2 are highly expressed in tumor tissue and myoblasts when compared to normal skeletal muscle, inferring a potential role of the NuRD complex in maintaining the undifferentiated phenotype observed in FP-RMS. Importantly, CHD4 silencing had no effect on myoblasts proliferation whereas a profound growth reduction was seen in FP-RMS cell lines, suggesting a unique tumour dependency on this chromatin remodeler. In addition, depletion of CHD4 caused a complete regression of xenograft tumours in mice.In summary, we have identified the NuRD complex as an essential positive co-regulator of PAX3-FOXO1 transcriptional activity. Our data propose a critival role of one of the NuRD core component CHD4 in FP-RMS cell viability, making CHD4 an attractive new target for therapy. To our knowledge, CHD4 is the first chromatin remodeler identified to associate with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy.Ongoing work is currently underway to identify first-in-class small molecules to inhibit CHD4 protein.

#4458

Discovery of novel epigenetic biomarkers in premalignant/malignant oral cavity lesions by methylation arrays.

Semra Demokan,1 Sevde Comert,1 Cansu Ozkoklesen,1 Murat Ulusan,2 Gulsum Ak,3 Canan Alatli,4 Nejat Dalay1. 1 _Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey;_ 2 _Department of Otorhinolaryngology, Faculty of Medicine, Istanbul University, Istanbul, Turkey;_ 3 _Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Istanbul University, Istanbul, Turkey;_ 4 _Department of Tumor Pathology and Oncology Cytology, Istanbul University, Istanbul, Turkey_.

Purpose: The incidence of the oral squamous cell carcinoma (OSCC) is around 50% of all head and neck cancer (HNC), the sixth most common type of cancer in the world. Aberrant hyper/hypo-methylation in the promoter region of some known or putative tumor suppressor/protoonco-genes occurs frequently during the development of various cancers including OSCC. Both of genetic and environmental factors play roles in the carcinogenesis of malignant lesions. Etiological factors predisposing to OSCC are smoking, alcohol and tobacco consumption, snuffuse, viral factors, chronic irritation, iron and various vitamin deficiencies, poor oral hygiene and diseases such as syphilis. Despite the advances in cancer diagnosis and treatment, the 5-year survival rate of OSCC patients is poor and because of advanced stage of disease, it has high mortality and morbidity. Another the most important ethiological factor of OSCC is oral premalignant lesions (OPML), since OPML is related with malignant transformation. In our study, we included three different OPML associated with different risks of leading to malignancy: Oral leukoplakia has a high risk of conversion into malignancy, whereas oral lichen planus and oral lichenoid dysplasia are associated with lower risk. We aimed to investigate the epigenetic characterization of OSCC and OPML relative to the epigenetic changes in these groups.

Experimental Design: Tumor and corresponding healthy tissues from 12 and 10 Turkish patients with OPML and OSCC, respectively were collected. The samples were analyzed histopatologically and after DNA extraction and bisulfite modification, methylation patterns of the samples were investigated by using the Illumina Infinium HumanMethylation450 (450K) BeadChip array.

Results: According to our preliminary results obtained from the methylation array experiments, CpG regions in the UOX, ABCC12, FCRLB, ABL2, LGR6 and SLC22A18 genes were found unmethylated while CpGs located in YPEL-3 were highly methylated (p<0.005).

Conclusion: We concluded the UOX, ABCC12, FCRLB, ABL2, LGR6, SLC22A18 and YPEL-3 may be potential biomarker candidate genes playing role in the transformation mechanism of OPML/OSCC. In the further studies, we will compare the methylation patterns between OPML subgroups to discriminate the potential candidates.

*This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK-SBAG-114S497).

#4459

Altered signal transduction in Isocitrate Dehydrogenase (IDH) mutated glioblastomas and implication on precision medicine.

Jie Li, Zachary J. Taich, Johnny Akers, Scott Vandenberg, Clark C. Chen. _UCSD Moores Cancer Center, La Jolla, CA_.

Glioblastoma (GBM) is the most common form of primary brain cancer and remains one of the most devastating of human diseases. One recurrent physiologic states in glioblastoma is defined by mutation of the Isocitrate Dehydrogenase (IDH) genes. IDH genes encode enzymes that catalyze oxidative decarboxylation of isocitrate. Nearly all IDH mutations in GBM involve substitution of arginine 132 of IDH1 with histidine (IDH1-R132H). The mutant protein loses the capacity to carry out its innate function and instead produces 2-hydroxyglutarate (2HG). The aberrant production of 2HG, in turn, alters the epigenetic landscape in these GBM cells. While it is clear that IDH1 mutated GBMs harbor a distinct cell state, the signaling cascades contributing to this biology remain poorly understood.

Using published mRNA signatures associated with EGFR activation, we demonstrate that IDH mutated GBMs harbored decreased EGFR signaling using four different GBM cohorts, including the Chinese Glioma Genome Atlas(CGGA; n=155), the REMBRANDT dataset (n=288), The Cancer Genome Atlas (TCGA; n=406), and the UCSD cohort (n=25). Further analysis revealed that IDH mutated clinical specimens and cell lines harbored lowered mRNA levels for EGFR and H-Ras. These suppressions were associated with increased deposition of the repressive histone markers, H3K9me3 and H3K27me3, in the EGFR and H-Ras promoter regions. Finally, the exogenous expression of IDH1-R132H conferred resistance to the EGFR inhibitor, Gefitinib, suggesting IDH1 mutation as a predictive biomarker for EGFR inhibitor response in GBM patients.

We further analyzed mRNA signatures associated with p53 activation and DNA damage accumulation in IDH mutated GBMs. We found that that IDH mutated specimens exhibited lowered level of signatures associated with p53 activation, suggesting decreased DNA damage accumulation. We subsequently determined whether this state of lowered DNA damage accumulation is associated with altered sensitivity to DNA damaging agents. Exogenous expression of IDH1-R132H in multiple glioblastoma and glial lines conferred increased resistance to temozolomide (TMZ). This resistance is associated with decreased accumulation of γ-H2AX. Consistently, we found that in a prospective glioblastoma registry of 274 GBM patients, IDH mutation is associated with poor response to TMZ (p<0.001).

In contrast to EGFR and p53 signaling, where IDH mutation is associated with pathway suppression, we found an association between elevated MYC expression and IDH mutation in both clinical specimens and cell-line models. Moreover, exogenous IDH1-R132H expression is associated with increased sensitivity to agents that suppress MYC expression, including LSD1 inhibitors. These results suggest potential therapeutic opportunities to target IDH mutated GBMs through epigenetic modulation of MYC expression.

#4460

Precision cancer risk diagnosis by accumulation of epigenetic alterations.

Toshikazu Ushijima, Kiyoshi Asada, Masahiro Maeda, Takeshi Nakajima, Taichi Shimazu. _National Cancer Ctr. Research Inst., Tokyo, Japan_.

Background and Aim: Aberrant DNA methylation is induced by chronic inflammation, and can be accumulated in normal-appearing tissues [reviewed in Ushijima and Hattori, Clin Cancer Res, 2:143, 2012]. Cross-sectional studies have shown that the degree of accumulation of aberrant DNA methylation was correlated with cancer risk, especially inflammation-associated cancers. In this study, we aimed to demonstrate that cancer risk can be predicted by an epigenetic cancer risk marker by a multicenter prospective cohort study.

Methods: We enrolled 826 patients with early gastric cancer, aged 40-80 years, who had undergone endoscopic resection. If a patient had active Helicobacter pylori (H. pylori) infection, the patient was enrolled after eradication of H. pylori. At the time of enrollment, one gastric biopsy was taken, and methylation levels of three preselected genes, miR-124a-3, EMX1 and NKX6-1, were measured by quantitative methylation-specific PCR. The patients were followed up annually by endoscopy to detect metachronous gastric cancer. To exclude cancers undetected at the time of enrollment, "authentic" metachronous gastric cancers were defined as those developed 1 year after enrollment.

Results: With a median follow-up of 2.97 years, 782 of 826 patients had at least one follow-up. Authentic metachronous gastric cancers developed in 66 patients: 29, 16 and 21 patients at 1-2, 2-3 and ≥3 years after the enrollment, respectively. The highest quartile of the miR-124a-3 methylation level had a significantly high HR in univariate analysis (95% CI) [2.17 (1.07 - 4.41); p = 0.032] and adjusted HR in multivariate analysis [2.30 (1.03 - 5.10); p = 0.042] [Asada et al, Gut, 64:388, 2015]. More importantly, with a median follow-up of 4.84 years, a multivariate analysis yielded a larger HR [2.79 (1.34 - 5.81)] with a smaller p value (0.0061).

Conclusions: This study, for the first time, demonstrated the usefulness of an epigenetic cancer risk marker by a multicenter prospective cohort study. DNA methylation accumulated in normal-appearing tissues was considered to be highly useful for precision cancer risk diagnosis.

#4461

Epigenetics in doxorubicin and docetaxel-resistant triple negative breast cancer.

Terra Sztain, Helena Chang. _UCLA Breast Ctr., Los Angeles, CA_.

Triple negative breast cancers (TNBC) do not express estrogen receptor, progesterone receptor, or Her2/neu hence there are no targeted therapies for this type of breast cancer. Chemotherapy is the systemic treatment option with doxorubicin and docetaxel being the two most commonly used drugs. It is well known drug resistance can lead to treatment failure in these patients. It is therefore essential to uncover information on the mechanism of these resistance pathways, and identify novel molecular markers for developing potential pathway driven treatment. Our previous studies have shown that genes, ataxia telangiectasia mutated (ATM), TGF-β, Bcl-2, NDRG1 and NDRG2 are closely related to cancer stem cell marker CD24 expression, and drug resistance toward doxorubicin and docetaxel in TNBC. In this study, two TNBC cell lines HCC1806 and HCC1937 were epigenetically examined by ChIP-qPCR. The gene promoters of the above five genes, within a 1kb range were analyzed. Results showed that both drugs significantly decreased total Histone 3 quantity, which is representative of total nucleosome density, at ATM and TGFB1 promoters, and additionally doxorubicin at NDRG1 promoter. Doxorubicin also decreased histone acetylation and methylation of H3K9 significantly at NDRG1 and consistently at the other gene promoters, while docetaxel showed consistent increase of methylation at H3K9. Results suggest that treatment with the two drugs affects an epigenetic pathway involving one or several epigenetic cofactors (methyltransferases, acetyltransferases, nucleosome remodeling complexes, etc.). Elucidating this pathway may provide valuable information for the treatment of TNBC.

#4462

Cytotoxic effects of a novel BRD4 inhibitor in uveal melanoma cells with Gnaq/11 mutations.

Grazia Ambrosini, Gary K. Schwartz. _Columbia University, New York, NY_.

Uveal melanoma (UM) is an aggressive intraocular malignancy with high tendency to metastasize to the liver. The majority of UM harbor activating mutations in the G proteins Gnaq/11, and amplification of the oncogene c-Myc. Currently available drugs have shown limited response in patients with UM and there is an urgent need for new effective therapies. Recent findings from our laboratory demonstrated that UM cells with Gnaq/11 mutations are dependent on BRD4 activity. BET proteins are epigenetic readers involved in chromatin remodeling and transcriptional regulation. BRD4 inhibitors consist of small molecules that competitively displace BRD4 from acetylated histones, resulting in the suppression of the MYC transcriptome and other BRD4-dependent genes. Here, we report that a novel clinical BRD4 inhibitor from Plexxikon (Berkeley, CA), PLX51107, induces high cytotoxic activity in UM cell lines at nanomolar concentrations. In cells with Gnaq/11 mutations, PLX51107 showed an IC50 of 250nM, similar to the pre-clinical compound JQ1. In contrast, PLX51107 did not have a significant effect (IC50>3000nM) in cells without the mutations. PLX51107 induced marked apoptosis in Gnaq/11 mutant cells, as detected by Annexin V staining and induction of cleaved PARP. It also suppressed the expression of BRD4 targets, i.e c-Myc, Rad51, and induced the pro-apoptotic protein Bim. In mouse xenograft models of UM PLX51107 significantly inhibited tumor growth. Since the MAPK pathway plays a major role in UM, we explored the combination of PLX51107 with a MEK inhibitor, and found increased activity in vitro and in vivo. All together, these observations support the idea that this clinical BRD4 inhibitor represents a novel therapeutic intervention against UM with Gnaq/11 mutations, and clinical development is planned.

#4463

Epigenetic regulation of gene expression in high-risk B-cell acute lymphoblastic leukemia by Casein Kinase II.

Morgann Loaec,1 Jonathon Payne,1 Elanora Dovat,1 Chunhua Song,1 Kimberly J. Payne,2 Sinisa Dovat1. 1 _Penn State University College of Medicine, Hershey, PA;_ 2 _Loma Linda University, Loma Linda, CA_.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Although advances in the treatment of ALL have resulted in a high cure rate for this disease, high-risk ALL is characterized by both resistance to conventional chemotherapy and a poor prognosis. The pathogenesis of high-risk ALL is still not understood. Casein Kinase II (CK2) is an oncogenic kinase that is overexpressed in both B-cell ALL (B-ALL) and T-cell ALL (T-ALL) and is associated with poor outcome. Inhibition of CK2 results in a strong therapeutic effect in a preclinical model of leukemia. However, the mechanism by which CK2 promotes oncogenesis in leukemia is unknown. Here, we studied how CK2 regulates expression of histone demethylase KDM5B in ALL. The KDM5B gene encodes a histone demethylase that regulates levels of histone modification H3K4me3 in leukemia. Molecular inhibition of CK2 using shRNA that targets the CK2 catalytic subunit resulted in transcriptional repression of KDM5B in ALL as evidenced by qRT-PCR. A similar effect was observed when leukemia cells were treated with the CK2 inhibitor CX-4945. Inhibition of CK2 resulted in reduced expression of KDM5B with an increase in the global cellular level of H3K4me3 as evidenced by Western blot. The use of quantitative chromatin immunoprecipitation (qChIP) showed that CK2 inhibition enhances DNA binding of the Ikaros tumor suppressor to the promoter of the KDM5B gene. Ikaros is a DNA-binding protein that regulates transcription of its target genes via chromatin remodeling. Loss of Ikaros function results in high-risk ALL. Serial qChIP analysis demonstrated that the increased Ikaros binding to the KDM5B promoter following CK2 inhibition is associated with an alteration of the epigenetic signature at the DNA region that surrounds the Ikaros binding site. Specifically, enhanced Ikaros binding results in increased occupancy of the H3K27me3 histone modification, along with a reduced occupancy of the H3K9ac histone modification at the KDM5B promoter. These results are consistent with the formation of heterochromatin and transcriptional repression. We tested the effect of CK2 inhibitors on Ikaros-mediated repression of KDM5B in primary, high-risk B-ALL cells that have a deletion of one Ikaros allele. Results showed that CK2 inhibition in high-risk B-ALL restores Ikaros binding to KDM5B promoter and represses KDM5B transcription. These data suggest that the inhibition of CK2 controls expression of KDM5B and the global H3K4me3 level in ALL by regulating the function of Ikaros as a transcriptional repressor of KDM5B. This presented data demonstrates the role of the CK2-Ikaros signaling axis in the regulation of both gene expression and the global epigenetic signature in ALL, and provide a mechanistic insight into the role of CK2 in the pathogenesis of ALL.

#4464

The role of KMT5A in prostate cancer.

Mahsa Azizyan, Kelly Coffey, Craig N. Robson. _Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom_.

Castrate resistant prostate cancer (CRPC) remains a significant clinical problem with no currently available cure. The Androgen receptor (AR) continues to function in CRPC through a plethora of mechanisms and the AR remains the primary target for therapeutic intervention in CRPC. Targeting AR co-regulating proteins to indirectly target AR signaling may prove beneficial. Recently, our group identified KMT5A as a potential regulator of AR from selective siRNA library screening.

KMT5A is a methyltransferase which monomethylates histone 4 on lysine 20. The establishment of this epigenetic mark directly impacts chromatin structure, genome stability and cell cycle progression, all hallmarks of cancer. In addition, it monomethylates non-histone proteins such as p53. It is therefore important that KMT5A activity and its cellular localization are under tight regulation. Using a relevant in vitro CRPC model we have shown that KMT5A acts as an AR co-activator while in androgen sensitive models KMT5A restricts AR activity. This highlights the importance of studying how KMT5A itself is regulated. KMT5A is primarily regulated through post-translational modifications in a cell cycle dependent manner. KMT5A polyubiquitination by E3 ligases CUL4-Cdt2 and APC-Cdh1 causes its proteasomal mediated degradation in S and late mitosis cell cycle phases, respectively. Moreover, the Skp2 E3 ligase has been suggested to play a role in KMT5A ubiquitination and degradation; but there is no direct evidence to show this to date. Therefore, the role of Skp2 in regulating KMT5A is understudied and remains elusive. It is also not known whether KMT5A could be modified directly by ubiquitination without leading to its degradation. As such, we aimed to investigate KMT5A monoubiquitination and the role of Skp2 in regulating KMT5A. We demonstrated monoubiquitinated KMT5A is present in a panel of PC cell lines. This was further confirmed by performing ubiquitination assays in COS7 cells. We determined that the KMT5A C-terminal SET domain is targeted for monoubiquitination and identification of the site(s) is ongoing. Furthermore, monoubiquintinated KMT5A was found to be exclusively cytoplasmic. In addition, Skp2 markedly enhances KMT5A monoubiquitination in a concentration dependent manner. We were also able to show that KMT5A and Skp2 interact. Skp2 mediated monoubiquitination of KMT5A however is not associated with protein turn over as addition of MG132 (proteasome inhibitor) does not further stabilize KMT5A. Interestingly, Skp2 mediated monoubiquitination of KMT5A downregulates MG132 mediated stabilization of KMT5A, possibly indicating this monoubiquitination impacts the accessibility of KMT5A for other ligases which promote KMT5A polyubiquitination and degradation. The newly characterized modification of KMT5A is a prevalent form of total KMT5A which can affect its function. Thus, insight into its physiological significance may provide a novel therapeutic target to indirectly target the AR.

#4465

Targeted disruption of HDAC3 induces terminal myogenic differentiation of embryonal rhabdomyosarcoma.

Michael Phelps, Jenna Bailey, Terra Vleeshouwer-Neumann, Eleanor Chen. _University of Washington, Seattle, WA_.

Embryonal rhabdomyosarcoma (ERMS), one of the most common and lethal pediatric sarcomas, is characterized by an arrest in myogenic differentiation. We have previously shown that treatment of ERMS cells with pan histone deacetylase (HDAC) inhibitors promotes myogenic differentiation. However, the underlying mechanism specific to each HDAC in ERMS remains unknown. We have completed a high-efficiency CRISPR/Cas9 gene knockout screen of class I and II HDAC genes, revealing HDAC3 as a major driver of myogenic suppression in ERMS. HDAC3-deficient ERMS cells showed robust terminal myogenic differentiation with the formation of multinucleated myotubes. Expanding on our in vitro studies, we developed a novel tamoxifen-inducible CRISPR gene-targeting system in ERMS xenografts established in immune-compromised mice. Disruption of HDAC3 in vivo significantly suppressed tumor growth by inducing myogenic differentiation of ERMS cells. By combining inducible CRISPR gene targeting with HDAC3 structure-function analysis, we showed that HDAC3 functions in concert with the nuclear receptor co-repressor (NCOR1 and NCOR2) complex to regulate gene transcription. HDAC3/NCOR binding and HDAC3 deacetylase activity are both required for suppressing myogenic differentiation in ERMS. RNA sequencing analysis of differentially expressed genes in HDAC3-deficient ERMS cells highlighted key pathways dysregulated in ERMS. Our results suggest that epigenetic silencing of the myogenic program is a major mechanism required for ERMS tumor growth. Identification of the essential interaction between HDAC3 and the NCOR transcriptional repressor complex as well as downstream genes and pathways provides important insights into the biology of ERMS tumorigenesis. Our findings highlight potential new drug targets specific to the activity of HDAC3 in order to improve treatment of ERMS patients.

#4466

GULP1 is a potential tumor suppressor that modulates chemo-resistance through regulation of Nrf2-Keap1 signaling axis in urothelial carcinoma.

Elisa Guida,1 Masamichi Hayashi,1 Alexander Baras,1 Leonel Maldonado,2 Mariana Brait,1 Leonardo Reis,1 akira oki,1 Evgeny Izumchenko,1 trinity bivalaqua,1 george J. Netto,1 Wayne Koch,1 David Sidransky,1 mohammad O. Hoque1. 1 _Johns Hopkins University, BALTIMORE, MD;_ 2 _University of South Alabama, Mobile, AL_.

By an integrated genomic approach, we recently identified engulfment adaptor PTB-domain-containing 1 (GULP1) as a potential tumor suppressor gene (TSG), selectively silenced in different types of solid tumors by promoter methylation (PM). Here, we aimed to test GULP1 PM in urothelial carcinoma (UC) and to functionally characterize GULP1 during UC initiation, progression and acquisition of chemo-resistance.

Initially, using a panel of cell lines, we observed that silencing of GULP1 expression is mediated by PM in UC cell lines. Furthermore, to test if this phenomenon is clinically relevant, we first employed quantitative methylation specific PCR on 20 primary tumors and matched normal samples. We observed higher frequency of GULP1 PM in tumors (35.0%) than in matched normal tissues (10.0%). Secondly, additional 76 tumors of different stages and grades of UC were tested and the methylation frequency were similar in UCs (27.1%) and significantly higher than in normal urothelial tissues (9.5%). Interestingly, UC cases with GULP1 PM had a tendency of lymphatic/venous invasion (P=0.066) when compared with cases without GULP1 PM. Moreover, to explore the correlation of GULP1 expression with different clinicopathological parameters, we performed immunohistochemical analyses on 2 different UC Tissue-Micro-Arrays. Overall GULP1 silencing was observed in 85% of UC cases and silencing is significantly more in muscle invasive UC (MIUCs) than in non-muscle invasive UC (NMIUCs) (P<0.001, Fisher's exact test).

Numerous cell based assays revealed that GULP1 silencing confers growth advantage to tumor cells. Further mechanistic analysis revealed that GULP1 has a crucial role in the regulation of Nrf2-KEAP1 axis, maintaining actin cytoskeleton architecture and assisting KEAP1 to prevent Nrf2 nuclear translocation. By using RT2 pathway expression array analysis of ShGULP1 T24 cells and T24 cells, we discovered that GULP1 silencing induces activation of Nrf2 targets, such as heme oxygenase 1 (HMOX1). We further confirmed the inverse correlation between GULP1 and HMOX1 expression in numerous UC cell lines by forcefully modulating GULP1 expression. Since constitutive activation of Nrf2 signature has been defined as responsible for chemoresistance, we analyzed GULP1 PM and expression in cisplatin based therapy responsive and resistant primary UC samples; and in isogenic cisplatin sensitive and resistant T24 cell lines. Interestingly, GULP1 expression is lower both in resistant primary UC samples and in resistant T24 cell line.

Altogether, our findings determined that GULP1 is an epigenetically silenced potential TSG in UC and its inactivation lead to malignant progression by constitutive activation of NRF2 pathway in UC carcinogenesis. Furthermore, GULP1 expression and/or PM may guide in selecting candidate patients for cisplatin based neo-adjuvant therapy.

#4467

PRSS8 methylation and its significance in esophageal squamous cell carcinoma.

Yonghua Bao,1 Qian Wang,2 Wancai Yang3. 1 _Jining Medical University, Jining, China;_ 2 _Xinxiang Medical University, Xinxiang, China;_ 3 _Jining Medical University, University of Illinois at Chicago, Chicago, Chicago, IL_.

Esophageal cancer is one of the most common cancers worldwide, and the incidence and mortality is increasing rapidly in recent years in China, but the underlying mechanisms are largely unclear. Herein we used immunohistochemical staining on an esophageal squamous cell carcinoma (ESCC) tissue microarray (TMA) containing 362 cases of ESCC and found that the expression of PRSS8, serine protease prostasin, was significantly decreased in ESCC. Interestingly, the reduction of PRSS8 was well correlated with poor differentiation, less stromal lymphocyte infiltration, and shorter survival time. PRSS8 mRNA levels were also significantly reduced in ESCC compared to the adjacent normal esophagus assayed by quantitative real-time PCR. Methylation specific PCR showed that PRSS8 promoter region was hypermethylated in ESCC tissues and cell lines, which was associated with downregulation of PRSS8 expression and decreased activities of PRSS8 promoter. Promoter deletion and truncation approaches showed that the CpG island in PRSS8 promoter region played an important role on the determination of promoter activities. De-methylation agent decitabine was able to restrore PRSS8 expression in esophageal cancer cell lines, leading to the inhibition of cancer cell proliferation, motility, migration and cell cycle arrest. However, the restoration of PRSS8 and its tumor inhibition could be reversed by small interfering RNA targeting PRSS8. Immunoblotting results showed that tumor inhibition of PRSS8 may be associated with proliferation- and epithelial-mesenchymal transition - related proteins (e.g. upregulation of E-cadherin and downregulation of Twist and Snail) in ESCC cells. The findings from current study strongly suggested that PRSS8 acted as a tumor suppressor and PRSS8 promoter region hypermethylation has clinical significance of in ESCC.

#4468

Targeted deep sequencing of plasma circulating cell-free DNA shows VIM and FBLN1 methylation as potential biomarkers for hepatocellular carcinoma.

Reetta Holmila,1 Athena Sklias,1 David C. Muller,1 Davide Degli Esposti,1 Paule Guilloreau,2 James Mckay,1 Suleeporn Sangrajrang,3 Petcharin Srivatanakul,3 Pierre Hainaut,4 Philippe Merle,5 Andre Nogueira da Costa,1 Zdenko Herceg1. 1 _International Agency for Research on Cancer, Lyon, France;_ 2 _Croix-Rousse Hospital, Lyon, France;_ 3 _National Cancer Institute, Bangkok, Thailand;_ 4 _Institut Albert Bonniot, INSERM Unité 823, La Tronche, France;_ 5 _UMR INSERM 1052, Lyon, France_.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, but is still lacking sensitive and specific biomarkers for diagnosis and prognosis. HCC carcinogenesis is a multi-step process that usually arises in background chronic metabolic, inflammatory and/or infectious liver disease and it exhibits a wide range of epigenetic alterations that could potentially be used as biomarkers. In this study, we applied the targeted massively parallel semiconductor sequencing to assess in plasma circulating cell-free DNA (cfDNA) the methylation of a panel of genes (FBLN1, HINT2, LAMC1, LTBP1, LTBP2, PSMA2, PSMA7, PXDN, TGFB1, UBE2L3, VIM and YWHAZ) and to evaluate the potential of these genes as novel HCC biomarkers in two different series, one from France and one from Thailand. Methylation in cfDNA was detected for FBLN1, PSMA7, PXDN and VIM, with substantial differences in methylation patterns between cases and controls for FBLN1 and VIM. Further, the average methylation level across analyzed CpG-sites was associated with higher odds of HCC for VIM (1.48 [1.02, 2.16] for French cases, and 2.18 [1.28, 3.72] for Thai cases), and lower odds of HCC for FBLN1 (0.89 [0.76, 1.03] for French cases and 0.75 [0.63, 0.88] for Thai cases). We also analyzed a set of HCC and adjacent tissues as well as The Cancer Genome Atlas' (TCGA) data to further investigate the origin of methylation pattern in cfDNA and noted that the analysis of VIM could be improved based on the methylation pattern detected in TCGA data. Overall, our study provides evidence that changes in methylation levels of cfDNA could be used as potential cancer biomarkers and that VIM and FBLN1 methylation in cfDNA are associated with HCC and may represent useful plasma-based biomarkers for improved detection and patient surveillance.

#4469

Twelve epigenetic marker panel for circulating DNA in plasma can detect early breast cancer.

Natsue Uehiro,1 Fumiaki Sato,1 Shigehira Saji,2 Masakazu Toi1. 1 _Kyoto University, Kyoto City, Japan;_ 2 _Fukushima Medical University, Fukushima City, Japan_.

Introduction: Breast cancer is usually divided into several subtypes, and clinical courses are different by subtypes. To manage patients adequately, many biomarker studies are conducted. Circulating DNA (ciDNA) is one of the attracted biomarkers for cancer management, but there are little reports that ciDNA is useful for early detection of breast cancer. DNA methylation pattern is also different among subtypes, it has possibilities to be a biomarker. To detect early breast cancer, we developed epigenetic marker panel for ciDNA by using the subtype-specific methylation markers and common methylation markers among subtypes.

Methods: Twelve novel methylation markers containing four common methylation markers and eight subtype-specific methylation markers were selected by methylation analysis. DNA samples for methylation analysis were obtained from 56 samples of microdissected cancer cells, 34 samples of cell lines and 29 samples of blood from healthy volunteers. These markers might detect circulating tumor DNA. We also selected four novel internal controls for methylation specific PCR (MSP) to estimate amount of ciDNA. Digital droplet MSP was used in this panel for estimation. Algorithms for discrimination between healthy volunteers and cancer patients were built by using ciDNA in plasma from 80 healthy volunteers and 87 cancer patient as a training set. All combination of 12 methylation markers and three parameters (geometric mean of internal controls, geometric mean of methylation markers, number of positive methylation markers) was estimated with Support Vector Machine (SVM). The best algorithm was selected by leave-one-out cross-validation method. We evaluated the algorithm with ciDNA in plasma from 53 healthy volunteers and 58 breast cancer patients as an independent validation set. The result of each samples was output as a diagnostic index.

Results: The best algorithm adopted four methylation markers and two parameters. The area under the curve of receiver operating characteristic curve by SVM was 0.92 in the training set and 0.88 in the validation set. Sensitivity and specificity were 0.91 and 0.83 in the training set, 0.84 and 0.79 in the validation set, respectively. Sensitivity of stage0-I, IIA, IIB-III, and metastatic breast cancer were 0.86, 0.87, 0.84, and 0.97, respectively. This panel realized high sensitivity even in early breast cancer. There was statistically significant trend that higher stage category showed higher diagnostic index. Sensitivity of triple negative, luminal HER2 and HER2 positive breast cancer were all 1.0 whereas sensitivity of luminal breast cancer was 0.83. This panel also realized high sensitivity regardless of subtype.

Conclusions: Our epigenetic marker panel could detect early breast cancer. It had high accuracy in every subtype, it could be a strong tool for blood-based screening of breast cancer.

#4470

ST3GAL1-associated transcriptomic program portends poor prognosis in glioblastoma.

Yuk Kien Chong,1 Edwin Sandanaraj,1 Lynnette Koh,1 Moogaambikai Thangaveloo,1 Melanie SY Tan,1 Geraldene Koh,1 Tan Boon Toh,1 Grace GY Lim,1 Joanna Holbrook,2 Oi Lian Kon,3 Mahendran Nadarajah,1 Ivan Ng,1 Wai Hoe Ng,1 Nguan Soon Tan,1 Kah Leong Lim,1 Carol S. Tang,1 Beng Ti Ang1. 1 _National Neuroscience Institute, Singapore, Singapore;_ 2 _Singapore Institute for Clinical Sciences, Singapore, Singapore;_ 3 _National Cancer Centre, Singapore, Singapore_.

Cell surface sialylation has been associated with tumor cell invasiveness in several cancers. Patients with grade IV glioblastoma multiforme (GBM) often show a median survival period of fifteen months, even with the current standard-of-care treatment. Among the reasons for this poor prognosis lies in the cellular and molecular heterogeneity of tumor cells. The Cancer Genome Atlas efforts have shown that histologically identical GBM tumors can be divided into four molecular subtypes based on gene expression, with each subtype corresponding to unique genomic aberrations and clinical outcome. These findings highlight the limitation of relying solely on morphological approaches to diagnose and subsequently treat patients. We conducted a lectin screen with the goal of identifying candidates that stained normal and tumor cells differentially. We identified a sialyltransferase, ST3Gal1 as a mediator of the binding pattern of one such lectin, Peanut Agglutinin. We demonstrate that ST3Gal1 promotes tumor cell invasiveness and enriches for self-renewal potential in the mesenchymal tumor subclass of patients, typified by highly aggressive and recurrent profiles. Depletion of ST3Gal1 extends tumor latency and prolongs survival in mice. Importantly, an enrichment of the ST3GAL1 transcriptomic program portends poor prognosis in glioma patients. Moving forward, since there are no mutations or changes in copy number of ST3GAL1, we explored epigenetic regulation as a possible mechanism. By tapping into data from the NIH Roadmap Epigenome, and transcriptomic analysis of our patient tumor-derived cells, we identified a histone clustering signature that correlates with survival outcome and disease progression. We are currently investigating one such histone modifier, lysine (K)-specific demethylase 1A (LSD1) and its role at modifying ST3GAL1 transcription and activity.

#4471

Overcoming the resistance to the anti CD20 treatments in chronic lymphocytic leukaemia.

Annarita Scialdone, Jesper Kofoed Damm, Urban Gullberg, Kristina Drott. _BMC - Lund University, Lund, Sweden_.

Chronic lymphocytic leukaemia is the most common leukaemia in the Western countries. Treatment of CLL and other lymphomas is based on cytostatic drugs in combination with rituximab or other monoclonal antibodies targeting the B-cell surface marker CD20. Although the CD20-antibodies have revolutionized the therapeutic perspectives of indolent B-cell lymphomas, the treatment of CLL with CD20-antibodies is often inefficient, a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown previously that CD20 is epigenetically regulated in some lymphoma cell lines and that some histone deacetylase inhibitors (HDACi) can increase CD20 expression in B-cell lymphomas both in vitro and in vivo. Therefore, we utilized the HDACi valproate to upregulate CD20 both in CLL cell lines and in patients.Two CLL patients with del13q were treated with valproate at serum levels around 800 µM for three days in three 21-day cycles. CD20 expression was analyzed by Real time-PCR and flowcytometry on the B-CLL cells isolated from peripheral blood.In addition, the valproate-induced histone modifications and changes in repressor/activator complexes of the CD20 promoter were investigated in the CLL cell lines and in patients.In the patient 1, CD20 expression was increased 2 times after 2 weeks of treatment, suggesting long-term effects by valproate. Neither the mRNA stability nor the protein stability seems to be affected by Valproate, suggesting transcriptional effects. However, no effects were observed in the patient 2. In contrast to patient results, Valproate increases CD20 transcript and protein levels by 2,5 times in the CLL-cell line I83-E95 after 48 hours. Here, the activating histone mark, acetylated H3K9, is enriched on the CD20 promoter suggesting valproate-related effects on the epigenome. Our study will continue the characterization of valproate-related effects on the epigenetic signatures of the CD20 promoter in CLL cells in vitro and in vivo, dissecting the mechanisms underling low CD20 expression in CLL.

#4472

Targeting the oncogenic transcription factor EWS-Fli1 by BET bromodomain inhibition in Ewing sarcoma.

Camille JACQUES,1 François LAMOUREUX,1 Marc BAUD'HUIN,1 Lidia RODRIGUEZ-CALLEJA,1 Thibaut QUILLARD,1 Pierre PERROT,1 Franck TIRODE,2 Françoise REDINI,1 James E. BRADNER,3 Dominique HEYMANN,1 Benjamin ORY1. 1 _Laboratoire de Physiopathologie de la resorption osseuse, NANTES cedex 1, France;_ 2 _Institut Curie, Paris cedex 05, France;_ 3 _Dana Farber Cancer Institute, Boston, MA 02115, France_.

Objective

Ewing Sarcoma is the second most common primitive malignant bone tumor after osteosarcoma. This childhood cancer is defined by a chromosomal translocation leading to the production of a chimerical transcription factor, EWS-Fli1, which is implicated in the progression of this malignancy. As current treatments improve its outcome, therapeutic resistances remain and reduce the survival rates. Despite protocols' optimization efforts, patients still relapse and it is important to develop new therapeutic approaches. The recently synthesized molecule JQ1, an inhibitor of the BET bromodomain proteins could be a new original weapon against this cancer. BET proteins interact with acetylated histones to regulate the chromatin accessibility to transcription factors and RNA polymerases. As JQ1 was recently shown to reduce the transcription of "super-enhancers"-dependent-genes such as oncogenes, which are highly sensitive to the BET proteins presence, it sounds relevant to hypothesize that EWS-Fli1's expression depends on the activity of these proteins.

Method

The cell viability, the cell proliferation and the cell's clonogenic potential consequent to a JQ1 treatment were studied in vitro. Using a Ewing Sarcoma nude mice model, the JQ1 effects on both the tumor growth and the animals overall survival were assessed. The EWS-Fli1 expression and ones of its transcriptional targets were evaluated by RT-qPCR and Western blotting and the direct regulation of EWS-Fli1 expression by BRD4 was checked by ChIP-qPCR.

Results

We demonstrated that JQ1 reduces the viability and the clonogenic capabilities of the cells as well as it leads to a G1-phase blockade, delays the burden of the tumor growth and improves the mice overall survival. Histological analysis shows that JQ1 reduces the vascularization and the cell proliferation within the tumors. Those effects are correlated to the associated silencing of EWS-Fli1 and the consequent modulation of some of its target-genes resulting from the depletion of BRD4 from the EWS-Fli1 promoter.

Conclusion

Our results shed light on the BET bromodomains' role as essential regulators of Ewing Sarcoma carcinogenesis. These proteins are indeed required to control EWS-Fli1's expression, consequently impacting its downstream signaling, which is functionally decisive for the maintenance of the tumorigenic features of these cancer-cells. Thus, the BET bromodomain proteins could be promising therapeutic targets in the Ewing tumors context.

#4473

The role of HBO1 in epithelial ovarian cancer.

Marcos Quintela. _Houston Methodist Research Institute, Houston, TX_.

Epithelial ovarian cancer (OC) is now the fourth leading cause of cancer death among women in the U.S and the leading cause of death in gynecological cancers. This lethality is due to both its asymptomatic nature and the lack of effective treatment strategies for advanced stage disease. OC comprises a variety of tumor types (histotypes) that differ in morphology, prognosis, etiology and molecular biology. Despite the knowledge of OC heterogeneity, current treatment strategies are very similar, and unfortunately, very ineffectual. New approaches, to inhibit OC growth and progression are therefore being sought, with few obvious targets for directed therapy. Currently, large-scale gene expression, DNA copy number and mutational screens have not been able to pinpoint new high frequency drugable targets. Epigenetic therapy conceives a whole genome approach, potentially circumventing the reliance on therapies that target infrequently mutated genes. So far in OC, most studies have focused on the inactivation of tumor suppressor genes (DNMTi, EZH2i, HDACi⋯) with mixed success, while little is known about the epigenetic activation of cancer-associated genes. Histone acetyl-transferases like GCN5, PCAF and p300 have been targeted in cancer previously, and the main purpose of this study is to assess the role of one such acetyl-transferase, Histone acetyl-transferase binding to origin of replication complex 1" (HBO1, also known as KAT7 and MYST2) in the progression of OC. HBO1 is a histone acetyltransferase (HAT) that plays important roles in diverse molecular processes including DNA replication, transcription and DNA repair. Importantly, HBO1 is overexpressed in primary cancers (breast, testis, ovarian), although the molecular basis of its role is still unclear. To better understand HBO1's role in OC, we characterized its expression in well-defined OC cell lines both at the mRNA and protein level. Observed in vitro patterns of HBO1 expression correlate well with previous observations; HBO1 is overexpressed in several OC cell lines. Interestingly, the serous histotype (both the most common and the most aggressive) generally shows the highest expression. Thus far, we have now analyzed HBO1's downstream regulatory pathways in several OC cell lines through use of siRNA knock-down coupled to microarray analysis. Comparisons of the various datasets have revealed pathways common to all cell lines studied, as well as distinction. Our approach aims to identify known epigenomic signatures that may be associated with HBO1 binding and downstream signaling which may drive differentiation toward the most advanced stages of the disease. Translating this new information through epigenetic targeting and structural inhibition may lead to novel, next-generation therapies for this most complex of disease presentations.

#4475

SMYD2 inhibitor BAY-598 reveals the giant scaffold AHNAK as a novel multi-methylated substrate.

Erik Eggert, Timo Stellfeld, Daniel Korr, Carlo Stresemann. _Bayer Pharma AG, Berlin, Germany_.

The SET and MYND domain-containing protein 2 (SMYD2) has been reported to mono-methylate several lysine residues on histone and non-histone proteins. Overexpression of SMYD2 has been reported in cancer cell lines as well as in esophageal squamous cell carcinoma (ESCC), bladder carcinoma, gastric cancer and pediatric acute lymphoblastic leukemia patients and correlates with lower survival rates. Although several studies uncovered important roles of SMYD2 in promoting tumorgenesis by protein methylation of key proteins (e.g. p53, PTEN, Rb, PARP), the biology of SMYD2 and its possible role in cancer biology is still far from being fully understood. Therefore, we employed the SMYD2 inhibitor BAY-598 (AACR-2015 abstract 2829, publication in preparation), and used proteomics to find novel substrates of SMYD2 in cancer cells.

We confirmed with both BAY-598 and by knockdown experiments that the giant scaffold protein AHNAK is a novel major substrate of SMYD2. Different studies have tried to decipher the function of AHNAK in normal and diseased tissues. AHNAK forms multi-protein complexes acting as a structural scaffold involved in different processes, including calcium channel regulation, cell contact signaling, migration, and tumor metastasis. Interestingly, we found AHNAK to be mono-methylated by SMYD2 on multiple sites in its central repeated domain as well as on its C-terminal domain. BAY-598 potently inhibited methylation at these sites on AHNAK. To further determine the prevalence of AHNAK methylation, we analyzed different cancer cell lines and tissues from mice. AHNAK methylation correlated with SMYD2 expression, but not with AHNAK expression, suggesting tissue-specific regulation of AHNAK methylation by SMYD2.

In summary, the novel finding of AHNAK regulation by methylation further complements the understanding of the roles of SMYD2 in cancer biology and underlines the utility of using probe inhibitors like BAY-598 to explore potential therapeutic targets in cancer.

#4476

Epigenetically altered circulating nucleosomes as blood biomarkers for early detection of cancer: clinical studies in NSCLC, CRC, PCA and PC.

Jake V. Micallef. _Belgian Volition SA, Namur, Belgium_.

BACKGROUND: The epigenetic structure of chromosomes in cancer tissue is altered with respect to histone modification, histone isoform and DNA modification composition as well as the composition of non-histone chromatin proteins - for example; the androgen receptor (AR) in prostate cancer.

Short (>180bp) fragments of tumor-derived cell free DNA circulate in the blood of cancer patients as mono-nucleosomes.

AIM: To evaluate whether epigenetically altered circulating nucleosomes provide a blood biomarker opportunity for early detection of cancers.

METHODS: We have developed more than 25 ELISA tests for circulating nucleosomes containing different epigenetic signals. These assays employ a solid phase anti-nucleosome antibody in combination with a labelled antibody directed to bind to a particular epigenetic signal - for example a histone modification, histone isoform, modified cytosine residue or a nucleosome-AR adduct. We have conducted clinical studies in collaboration with a number of centres to investigate the use of these ELISA tests for the detection of epigenetically altered circulating nucleosomes in serum samples taken from patients with lung cancer, colorectal cancer, pancreatic cancer and prostate cancer and from control subjects with no cancer and with or without concomitant disease of the same organ.

RESULTS: A collaborative study of 121 patients referred for colonoscopy at the CHU Dinant Godinne - UCL Namur, Belgium, who either showed potential symptoms of CRC or were high-risk subjects found that a panel test of five epigenetic nucleosome biomarker assays detected 87% of colorectal cancer cases and 67% of dysplastic polyps at 90% specificity.

A 59 subject collaborative study with Lund University, Sweden, including 25 patients with operable stage 2 pancreatic cancer, 10 patients with other pancreatic diseases and 24 healthy individuals found that a panel of 5 assays including 4 epigenetic nucleosome ELISA assays and CA19-9 detected 92% of early-stage pancreatic cancer cases, with 100% specificity among the healthy subjects and 2 false positives among patients with benign pancreatic disease.

A study of 73 patients conducted with the Liege University Hospital, Belgium, including 29 NSCLC cases, 22 COPD cases and 22 subjects healthy lungs showed that a panel of 4 epigenetic nucleosome assays combined with the smoking history detected 93% of NSCLC cases, with 91% specificity and also differentiated NSCLC and COPD cases.

Collaborative studies in prostate cancer have also shown excellent results and data from these studies will be presented at the conference.

CONCLUSION: Epigenetic profiling of circulating nucleosomes using simple ELISA methods offers a biomarker opportunity for the early detection of a variety cancers.

#4477

Loss of EZH2 accelerates STAT5 loss mediated fatty liver and cancer development through non-methyltransferase function.

Woo Kyun Bae, Hyun Jeong Shim, Sang Hee Cho, Ik-Joo Chung, Kyung Keun Kim. _Chonnam National Univ. Hwasun Hospital, Hwasun-Gun, Republic of Korea_.

Background: The molecular mechanisms underlying the development of fatty liver and hepatocellular carcinoma are not fully understood. Loss of STAT5 from liver tissue results in hepatosteatosis and hepatocellular carcinoma (HCC). Enhancer of zeste homolog 2 (EZH2) mediates epigenetic silencing of gene expression and is frequently up-regulated in human HCC. STAT5 recruits EZH2, which represses a substantial subset of genes regulated by STAT5 during B lymphopoiesis. However, it is not clear if EZH2 can affect STAT5-target genes in liver.

Methods: To investigate the role of STAT5 and EZH2 in liver, mice were generated that carried EZH2fl/fl alleles, STAT5fl/fl alleles, EZH2fl/flSTAT5fl/fl alleles and an Alb-Cre transgene. Lipid analysis in serum and liver, and RNA-seq were examined at 3 & 8 months of age. Mice were also examined by chronic 3 months injection by carbon tetrachloride (CCl4).

Results: The deletion of STAT5 in hepatocyte caused fatty liver and increased cholesterol in serum and liver. The deletion of EZH2 did not make a difference in H3K27me3 expression, and lipid in serum and liver. However, combined loss of EZH2 and STAT5 caused severe fatty liver, increase triglyceride and the ratio liver/body weight compare to STAT5 KO mice (the ratio; 0.097 vs. 0.058, p < 0.05). In response to 3 months treatment with CCl4, combined loss of EZH2 and STAT5 showed accelerated development of hepatocellular carcinoma at 12 months of age. Transcriptome analysis identified the expression of several genes was significantly increased (cd36, pparγ, cyp4a14, lipoprotein lipase, fasn) and decreased (nox4, bbc3, bcl2l1, cdkn2b) which were well known as STAT5 target genes in combined loss of EZH2 and STAT5.

Conclusions: This work demonstrates that loss of EZH2 in liver affects the expression of STAT5 target genes and accelerates STAT5 loss mediated fatty liver and hepatocellular carcinoma development through non-methyltransferase function.

#4478

EGFR tyrosine kinase inhibitors overcome resistance to chemotherapy in malignant pleural mesothelioma.

Bernard Staumont,1 Chrisostome Costa,1 Fabian Vandermeers,1 Sathya Neelature Sriramareddy,1 Céline Mascaux,2 Arnaud Scherpereel,3 Bernard Duysinx,4 Renaud Louis,4 Philippe Delvenne,5 Pascale Hubert,5 Luc Willems1. 1 _Molecular and Cellular Epigenetics (GIGA) and Molecular Biology (GxABT), University of Liège, Liège, Belgium;_ 2 _UMRS911, Faculty of Pharmacy, Aix-Marseille Université, Marseille, France;_ 3 _Calmette Hospital, CHRU of Lille and University of Lille II, Lille, France;_ 4 _Service of Pneumology, CHU of Liège, Liège, Belgium;_ 5 _Laboratory of Experimental Pathology (GIGA), ULg, Liège, Belgium_.

Background: Malignant pleural mesothelioma (MPM) is a cancer of the pleura mainly caused by exposure to asbestos fibers. Current treatments are unsatisfactory due to intrinsic chemoresistance of the tumor. We hypothesized that chemoresistance was due to epigenetic errors and evaluated the ability of HDAC inhibitors to improve treatment efficacy. We previously showed that valproic acid (VPA) improves the first line regimen of MPM both in vitro and in vivo (Vandermeers et al, 2009, Clinical Cancer Research 15: 2818). A clinical trial also demonstrated that VPA in combination with doxorubicin increases the response rate of second line patients (Scherpereel et al, 2011, European Respiratory Journal 37:129).

Methods: Transcriptomic profiling was performed by microarray analyses (Agilent). Gene expression was validated by quantitative RT-qPCR. Modulation of TGFα expression was performed by shRNA interference and transfection of a cDNA vector. Onset of apoptosis was assessed with the Annexin V assay.

Results: To evaluate the mechanisms associated with the response to chemotherapy, we compared two types of MPM cell lines (M14K and H28) characterized by a difference in sensitivity to doxorubicin+VPA. Microarray analyses and bioinformatic modeling of gene expression profiles revealed the most relevant candidate genes associated with sensitivity or resistance to this regimen. Among these, TGFα expression was associated with resistance to doxorubicin + VPA in a series of MPM cell lines. Silencing of TGFα by RNA interference in H28 cells correlated with a significant increase in apoptosis. On the other hand, overexpression of TGFα desensitized M14K cells to doxorubicin+VPA -induced apoptosis. Since TGFα interacts with the EGF receptor, we evaluated pharmacological inhibition using EGFR tyrosine kinase inhibitors (erlotinib and gefitinib) and the dual HDAC/EGFR inhibitor CUDC-101. As predicted, these TKI inhibitors improved efficacy of doxorubicin+VPA.

Conclusions: Our data demonstrate that TGFα is involved in resistance of MPM to chemotherapy and that TKI inhibitors overcome resistance to second line regimen.

#4479

Unveiling the relationship between the SWI/SNF chromatin remodeling complex and noncoding RNAs.

Paola Peinado Fernández,1 Isabel Fernández Coira,1 Eva E. Rufino Palomares,1 Octavio A. Romero,2 Chanatip Metheetrairut,3 Laura Boyero Corral,1 Julián Carretero,4 Esther Farez Vidal,1 Marta Cuadros Celorrio,1 Fernando Reyes Zurita,1 Victoria Sánchez Martín,1 Carlos Baliñas Gavira,1 Jose A. Lupiáñez Cara,1 Montse Sánchez Céspedes,2 Frank Slack,5 Pedro P. Medina1. 1 _University of Granada, Granada, Spain;_ 2 _Bellvitge Institute for Biomedical Research (IDIBELL), Barcelona, Spain;_ 3 _Siriraj Hospital, Bangkok, Thailand;_ 4 _University of Valencia, Valencia, Spain;_ 5 _Beth Israel Deaconess Medical Center, Boston, MA_.

Chromatin remodeling complexes are crucial for the viability of the cells due to their role in regulating interactions between DNA and histones and, therefore, modifying the accessibility of the genetic information to the transcriptional machinery. This relevance can also been seen in the SWI/SNF complex that has been associated with cancer in the last deep-sequencing efforts on tumoral genomes.

BRG1 is the helicase/ATPase catalytic subunit of the SWI/SNF complex and it is frequently lost in NSCLC cell lines with a high mutation rate. In primary tumors, the loss of expression of BRG1 is also frequent, however it cannot be explained by mutations or by promoter hypermethylation.

In this study we have focused on the regulation of the expression of BRG1 by microRNAs. These are small RNA molecules that do not code for any protein but they have key roles in gene expression regulation by binding to the 3'UTR of the transcripts. In addition, their levels have been found altered in several diseases including cancer. Our hypothesis is that the deregulation observed in BRG1 levels in primary tumors can be associated with changes in the microRNAs that control its expression.

We have studied the 3'UTR of BRG1 by performing a Rapid Amplification of 3' cDNA Ends (3' RACE) and we have cloned the resulting 3'UTRs after a luciferase coding sequence of a plasmid in other to use the luminescence signal as a method to analyze the binding of the microRNAs tested to the 3'UTR of BRG1. Previous bioinformatic analyses were performed to choose the best microRNAs that could potentially regulate BRG1.

Our preliminary results indicate that some microRNAs are able to bind to the 3'UTRs and successfully repress the expression of catalytic subunit of SWI/SNF. Importantly, one of these microRNAs is well known for developing oncogenic activities in lung cancer and it has been related with poor prognosis.

In conclusion, the activity of the SWI/SNF complex can be regulated by microRNAs and this regulation may be relevant during lung carcinogenesis. From the other perspective, SWI/SNF complex could also impact in lung carcinogenesis regulating microRNAs expression, an aspect that we are currently evaluating.

#4480

Blood epigenetic age may predict cancer incidence and mortality.

Yinan Zheng,1 Brian T. Joyce,1 Elena Colicino,2 Lei Liu,1 Wei Zhang,1 Qi Dai,3 Martha J. Shrubsole,3 Warren A. Kibbe,4 Tao Gao,1 Zhou Zhang,1 Nadereh Jafari,1 Joel Schwartz,2 Andrea A. Baccarelli,2 Lifang Hou1. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _Harvard T.H. Chan School of Public Health, Boston, MA;_ 3 _Vanderbilt University School of Medicine, Nashville, TN;_ 4 _National Cancer Institute, Rockville, MD_.

Biological measures of aging are important for understanding age-related cancers as the population ages. Since the epigenome is closely related to aging, epigenetics may help predict these and other age-related diseases. We aimed to prospectively examine whether blood Δage (the discrepancy between epigenetic and chronological age) can predict cancer incidence/mortality. In a prospective cohort (the Normative Aging Study), Δage and its rate of change over time were calculated using Illumina Human Methylation 450K array data in 834 blood leukocyte samples longitudinally collected from 442 participants free of cancer at the blood draw. About 3-5 years before cancer onset or death, Δage was associated with cancer risks in a dose-responsive manner (test for trend P=0.02) and time-dependent Cox models showed a one-year increase in Δage was associated with all-cancer incidence (HR (hazard ratio): 1.06, 95% CI: 1.02-1.10) and mortality (HR: 1.17, 95% CI: 1.07-1.28). Participants with smaller Δage and decelerated epigenetic aging over time had the lowest risks of cancer incidence (log-rank P=0.003) and mortality (log-rank P=0.02). Spline analysis suggested Δage was associated with cancer incidence in a 'J-shaped' manner, and with cancer mortality in a time-varying manner. We conclude that blood epigenetic age may mirror epigenetic abnormalities related to cancer early development or the immune response to it. Those with older epigenetic age relative to their chronological age have an elevated risk of cancer events within 3-5 years. Thus, epigenetic age could potentially serve as a novel, minimally invasive biomarker for cancer prediction.

#4481

Phosphorylation regulates EZH2 neoplastic functions in triple-negative breast cancer.

Talha Anwar, Caroline Arellano-Garcia, Boris Burman, Celina G. Kleer. _University of Michigan Medical School, Ann Arbor, MI_.

Background: Triple negative (estrogen receptor, progesterone receptor, HER2-neu negative) breast cancers (TNBC) comprise 15% of all breast cancers but are responsible for a disproportionately high number of deaths. Overexpression of the histone methyltransferase EZH2 (Enhancer of Zeste Homolog 2) is an independent prognostic biomarker significantly associated with poorly-differentiated TNBCs and poor patient outcome. We previously identified a novel link between EZH2 and the p38 mitogen-activated protein kinase, an important mediator of progression and metastasis of TNBC. We found that EZH2 binds to p38, and that EZH2 and activated p38 are concordantly expressed in the metastases of breast cancer patients. Based on these data and previous in vitro studies, we hypothesized that p38 MAPK may also regulate EZH2 through phosphorylation in breast cancer. We further hypothesized that this phosphorylation event may be important for EZH2 contribution to malignancy.

Methods: In order to test this hypothesis, we performed knockdown rescue experiments in triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-436. Stable knockdown of EZH2 was achieved using shRNA targeting the 3'UTR. Knockdown of EZH2 was rescued by reintroduction of myc-EZH2 (WT) or a T367A-EZH2 phosphorylation-deficient mutant. Cell lines were then used in functional assays of proliferation, migration, and invasion. In order to further interrogate the importance of this phosphorylation event, we developed a phospho-specific EZH2 T367 antibody.

Results: p38-mediated phosphorylation of EZH2 at T367 contributes to the migratory and invasive properties of TNBC. Mechanistically, phosphorylation by p38 does not affect binding to other PRC2 members but may affect EZH2 activity. We are currently investigating the relevance of pEZH2 as a biomarker of breast cancer survival with our new antibody.

Conclusions: We provide evidence that p38 phosphorylation of EZH2 at T367 contributes to malignancy of triple-negative breast cancers. Our data suggest a new mechanism by which EZH2 is regulated and may offer an additional mechanism by which EZH2 contributes to TNBC progression.

#4482

Expression profiling of SETD family genes in breast cancer patients.

Joao N. Matos Neto. _UNB, Brasilia, Brazil_.

Epigenetic alterations have recently been recognized to play an important role in the progression of many tumor types. Protein methyltrasferases are among the most relevant epigenetic effectors. Dysregulation in the expression or activity of these enzymes is among the major epigenetic alterations involved in carcinogenesis. Methyltransferases of the SETD family share a SET domain which is involved in the methylation of lysine residues on histones and non-histone proteins. So far, little is known about the relation of SETD family genes with mammary carcinogenesis. In the present study we evaluated the relative expression of 10 SETD family genes in 18 breast tumor samples and non-tumor breast tissues from patients with breast cancer by real time qPCR. The comparative expression profile of each gene was correlated with the most relevant clinicopathologic prognostic information available from patients. We found a significant increase in the expression of SETMAR in tumor samples compared with the normal breast tissues. In addition, triple negative tumor subtype (n = 7), when compared with a pool of normal tissue samples (n = 13) presented an increased relative expression of SETMAR. Interestingly, SETD8 was 10 times overexpressed when the angiolymphatic invasion (n = 8) was present compared to the absence of this feature (n = 10). Patients with strong axillary tumor burden defined as the presence of at least 10 axillary lymph nodes with cancer (n = 6), showed an increased relative expression of SETD5 and SETD8 genes when compared to the absence of this feature (n = 12). Evaluating the expression on paired samples (normal tissue and tumor tissue from the same patient (n = 7), we observed an increased relative expression of SETD1B. Also in the paired samples, we found a strong correlation among the expression of the genes SETD1B, SETD5, SETD8 and SETMAR in tumor samples with no correlation in normal tissues. Furthermore, immunohistochemical analysis revealed a stronger expression of SETD5 and SETMAR in carcinomas in situ when compared to normal tissues and invasive tumors. Taken together, these data points to a likely role of SETMAR and SETD1B genes in mammary carcinogenesis, with relevant clinical implications and with the possible involvement of SETD5 and SETD8 genes in more advanced stages of the disease. The correlation on the expression of these genes with relevant clinical prognostic factors in breast cancer opens the way to exploit them not only as therapeutic targets, but also as new prognostic markers.

Keywords: SETD gene family, expression profile, breast cancer

#4484

Differentially methylation between ER negative and ER positive breast cancer identifies master regulators to expose potential epigenetic drivers of aggressive disease.

Maria J. Worsham, Kang Mei Chen, Indrani Datta, Josena K. Stephen, Dhananjay Chitale, George Divine. _Henry Ford Health System, Detroit, MI_.

The majority of published studies investigating driver genes have focused primarily on genomic mutations which have led to novel study designs (basket trials) where patients with a rare mutation, regardless of tumor histology, are matched to a drug expected to work through the mutated pathway. This dominant focus on genomic mutations has yet to configure in epigenetics. There has been relatively little advancement in changing the management of women with ER negative BC, mainly due to a dearth of actionable therapeutic targets. Our drill-down approach to identified an ER negative-specific 16 gene methylation signature (AHNAK, ALPL, ANXA2R, CCND1, CIRBP, CPQ, DST, EGFR, ESR1, GPRC5B, HERC5, IL22RA2, MITF, OBSL1, POU3F3, RB1CC1) starting from a discovery approach (Illumina Infinium HumanMethylation450 BeadChip, 40 ER negative vs. 40 ER positive BC) followed by expression verification, significant rankings in biological pathways (Ingenuity Pathway Analysis), and confirmation by targeted sequencing using Illumina MiSeq.

Causal Networks are small hierarchical networks of regulators that control the expression/methylation of dataset targets. They can enhance understanding of the effect of master regulators on disease or function.

The objective of this study was to identify regu¬latory networks utilizing IPA's Causal Network Analysis (CNA) in order to illuminate possible causes and mechanisms underlying the biological activities of the 16 candidate gene signature differentiating ER negative from ER positive BC. To reflect expected gene expression direction implied by methylation changes for the 16 candidate genes, the inverse of the methylation ratio from ER negative vs. ER positive tissue was used for CNA.

CNA software identified 4 hierarchical networks and their corresponding master regulatory molecules, diethylstilbestrol, MSH2, 15-ketoprotaglandin E2, and transcription regulator SP1. Diethylstilbestrol and SP1 had direct regulatory influence (depth level 1) to the candidate molecules ALPL, CCND1, EGFR, ESR1 and CCND1, CIRBP, EGFR, ESR1, respectively. CNA raised the profile of ALPL, CCND1, CIRBP, EGFR, ESR1 (5/16 candidate genes) for further consideration as potential epigenetic drivers of ER negative BC.

Support: Komen Foundation: KG110218

#4485

Regulation of chronic lymphocytic leukemia (CLL) immunobiology by histone deacetylase 6 (HDAC6).

Eva Sahakian,1 Kamira Maharaj,1 John Powers,1 Renee M. Fonesca,1 Susan Deng,1 Javier Pinilla-Ibraz,1 Steven N. Quayle,2 Simon S. Jones2. 1 _Moffitt Cancer Center & Research Institute, Tampa, FL; _2 _Acetylon Pharmaceuticals, Inc., Boston, MA_.

In CLL, an immunosuppressive phenotype enables the malignant B cell to evade immune detection leading to immune suppression. In recent years, epigenetic changes prompted by proteins known as histone deacetylases (HDACs) have gained special attention predominantly because of their active role in the regulation of pathogenesis and immune-related pathways in CLL; though the precise mechanism by which these regulatory events take place has yet to be elucidated. In addition to the well-recognized role of histone deacetylase inhibitors (HDACi) in the control of cell cycle and apoptosis, HDACi have a potential role in modulating the immunobiology of CLL. Remarkably, HDACi alter the inflammatory status of immune cells and tumor cells themselves. Among the family of HDACs, we have recently demonstrated that HDAC6 has a regulatory role in transcriptional regulation of IL-10, and is responsible for T cell anergy in murine and human antigen presenting cells.

Activation of T-cells is predominantly dependent on both co-stimulatory and co-inhibitory molecules, including PD-1/PD-L1 as well as CTLA4-B7, OX40-OX40L, CD40-CD154, GAL9-TIM3, and 41BBL/41BB. Furthermore, most of these molecules have been identified to have epigenetically induced changes in expression.

Previously we had reported that expression of HDAC6 is increased in CLL patient samples. Recent studies from our lab show that selective HDAC6 inhibitors modify the expression of immunomodulatory molecules, which may ultimately lead to increases in the immunogenicity of CLL. Thus far, findings from our lab reveal that CLL cells treated with HDAC6i show 1) a dose dependent cell kill, 2) a reduction of IL-10—an important cytokine in the regulation of cell proliferation in CLL, and 3) synergistic reduction of viability when combined with the BTK inhibitor ibrutinib. Subsequently, we have also demonstrated that MEC2-HDAC6KD cells exhibit an increase in MHCII and a decrease in PD-L1 expression. Interestingly, we also observed a decrease in the expression of PD-L1 and other immune checkpoint markers in CLL cell lines treated with low-doses of HDAC6i. Additionally, malignant B cells isolated from euTCL1 mice and treated ex vivo with HDAC6i become more immunogenic and elicit greater type I allogeneic T cell immune response. Lastly, utilizing our in vivo CLL murine models (euTCL1, and euTCL1-HDAC6KO), we have been able to demonstrate a reduction in circulating lymphocytes in euTCL1-HDAC6KO, as well as euTCL1 mice receiving systemic administration of HDAC6i.

In conclusion, selective inhibition of HDAC6 in CLL results in the reduction of co-inhibitory molecules, dose dependent increases in cell killing as single treatment as well as strong synergy when combined with ibrutinib. These findings could provide a successful combination immunotherapeutic strategy for the treatment of CLL.

#4486

Epigenetic vulnerabilities in ovarian cancer.

Jessica A. Thomes Pepin, Horacio Cardenas, Cody Moore, Salvatore Condello, Jiang Guanglong, Liu Yunlong, Daniela Matei. _Indiana University School of Medicine, Indianapolis, IN_.

Background: Histone acetylation regulates gene expression in cancer and can be exploited for therapeutic targeting. Histone deacetylase inhibitors (HDACi) increase histone acetylation, promoting transcription of genes involved in growth arrest and apoptosis and have been used as anti-cancer agents. BRCA mutations are the best characterized genetic alteration associated with ovarian cancer and can be targeted therapeutically by recently approved PARP inhibitors. We hypothesized that the epigenetic landscape of ovarian cancer is altered by the presence of BRCA mutations and can be manipulated by HDACi for therapeutic benefit.

Materials and Methods: We utilized isogenic cell lines carrying mutated/functional BRCA 1 (UWB1.289; UWB1.289-BRCA), and respectively BRCA 2 genes (PE01/PEO4). We measured HDAC expression and activity by western blotting and an enzymatic assay and characterized globally chromatin marked by H3K9ac (active chromatin) and H3K27ac (poised chromatin) by using ChIP sequencing. To determine whether HDAC inhibitors increase susceptibility to PARP inhibitors, cellular proliferation was measured in ovarian cancer cells expressing mutated and wild type (WT) BRCA.

Results: HDAC1 expression and activity were decreased in cell lines carrying defective BRCA proteins. ChIP sequencing identified chromatin regions differentially marked by H3K9ac (178 promoters in BRCA1-null compared to BRCA WT cells) and H3K27ac (131 promoters in BRCA WT compared to BRCA null cells) suggesting differences in gene transcription dependent on histone acetylation in BRCA mutated vs BRCA functional cancer cells. A subset of genes differentially marked by H3K9ac were validated at mRNA level by RT PCR confirming BRCA dependent regulation (TGM2, Wnt7a, DKK1, MET, FLI1, AXL, TG2, PMEPA1, TWIST2 upregulated) and (E2F2, NID2 downregulated) in BRCA1-null ovarian cancer cells. HDAC1 inhibition with trichostatin A (TSA) and entinostat (MS-275) increased H3K9ac and was associated with an increase in TGM2, FLI1, DKK1 and E2F2, and a decrease in TWIST2 gene expression in both BRCA1-wt and -null cell lines confirming epigenetic regulation of this subset of genes. Functionally, HDACi synergized with PARP inhibitors in BRCA null ovarian cancer cells (combination index 0.63), consistent with the altered gene expression changes induced by differences in chromatin marks.

Conclusions: In conclusion, the chromatin landscape in BRCA null ovarian cancer cells is altered and can be targeted by HDAC inhibitors. Identifying pathways and specific genes altered by histone acetylation may enable future rational targeted combination therapies. 

### Germline/Somatic Genomics and Personalized Medicine

#4487

An INDEL variant confers melanoma risk through PARP1 expression regulation.

Jiyeon Choi,1 Matthew Makowski,2 Tongwu Zhang,1 Matthew Law,3 Wendy Kim,1 Michael Kovacs,1 Hemang Parikh,1 Lauren Aoude,3 Michael Gartside,3 Jeffrey Trent,4 Michiel Vermeulen,2 Stuart Macgregor,3 Nicholas Hayward,3 Mai Xu,1 Kevin Brown1. 1 _NCI, Bethesda, MD;_ 2 _Radboud University, Netherlands;_ 3 _Queensland Institute of Medical Research, Australia;_ 4 _Translational Genomics Research Institute, Phoenix, AZ_.

Recent genome wide association studies identified several new loci for melanoma susceptibility including chr1q42.1 locus encompassing Poly [ADP-ribose] polymerase 1 (PARP1). As an effort to identify effecter genes and functional risk variants from this locus we performed expression quantitative trait loci (eQTL) analysis in 62 melanoma cell lines. Among the genes in 1Mb area eQTL was identified for PARP1 and subsequently validated by qPCR, where increased PARP1 levels are significantly correlated with the risk allele (p=0.03, copy number adjusted). Further allele discrimination qPCR of PARP1 transcripts in 14 melanoma cell lines heterozygous and of neutral copy for the lead SNP indicated significantly higher proportion for the risk allele (p=.0001). Same allelic imbalance was also observed in 51 heterozygous melanomas with neutral copy numbers from The Cancer Genome Atlas (p=.028). To identify functional risk variants mediating these effects we annotated this locus using six melanoma relevant cell types available from ENCODE and Roadmap database. Based on recent fine mapping data suggesting single-SNP model for this locus we prioritized high LD variants for nomination. Among 65 SNPs of high LD with the lead SNP or imputed best SNP (r2>0.6 using 1000 Genomes phase3), four exhibited strong evidence as potential transcriptional enhancers. Electro Mobility Shift Assays and luciferase assays on these four variants demonstrated that one of them, a six-base pair INDEL (-/GGGCCC) in the first intron, displayed strong allelic functionality. Consistent with the expression data melanoma-associated deletion allele results in higher luciferase activity while protective insertion allele binds a group of proteins with higher affinity. Subsequent comparative mass-spectrometry for these insertion-binding proteins identified a striking collection of Guanine-quadruplex binding proteins including RECQ1 as potential inhibitors of PARP1 expression. Consistent with this hypothesis over-expression of RECQL results in more pronounced allelic difference in luciferase activities indicating that RECQ1 contributes to allelic PARP1 expression. Further interrogation of RECQ1 and PARP1 expression correlation analysis suggested that RECQ1 levels are inversely correlated with PARP1 in melanomas carrying insertion alleles. These data demonstrate that increased PARP1 expression is correlated with melanoma risk and an INDEL variant mediates differential PARP1 expression possibly through secondary DNA structure binding proteins including RECQ1.

#4488

Targeted sequencing revealed distinctive and pathogenic mutations in African Americans with colorectal cancer.

Hassan Ashktorab,1 Hamed Azimi,1 Mike Nickerson,2 Sara Bass,3 Joseph Boland,4 Meredith Yeager,2 Sudhir Varma,1 Mohamed Daremipouran,1 Zaki Sherif,1 Shima Ghavimi,1 Babak Shokrani,1 Edward Lee,1 Adeyinka Laiyemo,1 Hassan Brim1. 1 _Howard University, Washington, DC;_ 2 _National Cancer Institute, Bethesda, MD;_ 3 _National Institute of Health, Bethesda, MD;_ 4 _National Cancer Institue, Bethesda, MD_.

Background & Aim: We recently analyzed 12 pairs of African American CRC tumors and their matched normal tissue using whole exome sequencing and established a panel of novel mutations that are potentially useful for the detection of CRC in this population. In this study, we examined APC, MSH3, and MSH6 mutations using targeted exome sequencing (TES) to determine distinctive mutations frequencies and their chronology in the neoplastic transformation.

Materials & Methods: A total of 140 colon samples: (30 normal, 21 adenomas, 33 advanced adenomas & 56 tumors collected from African Americans were used as our discovery set on an Ion Torrent platform. A subset of 36 samples were used as validation set on an Illumina platform. Bioinformatic analysis was performed on both sets of data. Common mutations were considered validated.

Results: Two novel MSH6 mutations were validated. Four known mutations in MSH3 were validated and were located in nonsynonymous (exon 10) and 1synonomous mutation (exon 18). As for the APC, 20 mutations were validated include 4 novel mutations. The novel mutations were 3 stopgain and 1 nonsynonymous located at the EB1 binding site, in the mutational cluster region (MCR), and at the 15 Amino Acid repeat.

Conclusion: We here defined novel mutations that target DNA MMR and APC genes in African Americans with colorectal lesions. Greater frequency of mutations in cells defective for DNA repair and APC genes is an advantage in cell growth and genetic instability and relevant to the initiating events of colon tumorigenesis.

#4489

Using functional data from Roadmap Epigenomics to inform analysis of rare variants linked to gene expression in a large colorectal cancer study.

Stephanie A. Bien,1 Tabitha A. Harrison,1 Paul L. Auer,2 Flora Qu,1 Jeroen Huyghe,1 Barbara Banbury,1 Peyton Greenside,3 Goncalo R. Abecasis,4 Sonja I. Berndt,5 Stephane Bézieau,6 Hermann Brenner,7 Graham Casey,8 Andrew T. Chan,9 Jenny Chang-Claude,7 Sai Chen,4 Joshua D. Smith,10 Loic Le Marchand,11 Christopher Carlson,1 Polly A. Newcomb,1 Christian Fuchsberger,4 Marty L. Slattery,12 Hyun M. Kang,4 Emily White,1 John Potter,1 Steven J. Gallinger,13 Michael Hoffmeister,7 Stephen B. Gruber,8 Deborah A. Nickerson,10 Ulrike Peters,1 Anshul Kundaje,3 Li Hsu1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Wisconsin, Milwaukee, WI;_ 3 _Stanford University, Stanford, CA;_ 4 _University of Michigan, Ann Arbor, MI;_ 5 _National Cancer Institute, National Institutes of Health, Bethesda, MD;_ 6 _Service de Génétique Médicale, Nantes, France;_ 7 _German Cancer Research Center, Heidelberg, Germany;_ 8 _University of Southern California, Los Angeles, CA;_ 9 _Massachusetts General Hospital, Boston, MA;_ 10 _University of Washington, Seattle, WA;_ 11 _University of Hawaii Cancer Center, Honolulu, HI;_ 12 _University of Utah, Salt Lake City, UT;_ 13 _Mount Sinai Hospital, Toronto, Ontario, Canada_.

To investigate the role of low frequency and rare genetic variation in colorectal cancer (CRC) susceptibility, the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) and the Colorectal Cancer Family Registry (CCFR) conducted whole genome sequencing and imputed into genome-wide association studies (GWAS) of 14,718 CRC cases and 12,186 controls. These data provide a unique opportunity to investigate rare variants, which contribute to the majority of the variation in the genome. To improve power for discovering rare CRC susceptibility variants (<1% MAF), Roadmap Epigenomics data were used to construct biologically relevant testing sets of enhancers, promoters and exons for gene-based association testing across the genome. Since enhancers exert their effects by impacting expression of target genes, we defined enhancer-gene networks by linking enhancer(s) to target gene expression using Roadmap chromatin state maps and gene expression. Variants in linked enhancers from digestive and immune tissues were aggregated together with variants in the promoter and non-synonymous coding variants in the target gene. We tested 9,884 variant sets for association with CRC risk using the Mixed effects Score Test (MiST). Our most significant findings are for acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor-ACADS (p=1x10-4), AlkB homologs, including AlkB homolog 1-ALKBH1 (p=2x10-4), and SRA stem-loop interacting RNA binding protein-SLIRP (p=2x10-4). We will replicate these findings within the Colorectal Cancer Transdisciplinary Study (CORECT), as well as additional samples currently genotyped in CCFR and GECCO (over 25,000 CRC cases and controls). Although the top findings are statistically non-significant in this initial dataset, each of these genes linked to molecular pathways implicated in CRC carcinogenesis (fatty acid metabolism, DNA/RNA repair, and Nuclear Receptor signaling pathway, which interacts with the Wnt, beta-catenin pathways to result in a diverse array of cellular effects including altered cellular adhesion, tissue morphogenesis, and oncogenesis). Our current findings suggest that although functional insight can improve power for novel discovery, even larger sample sizes and/or pathway-based analyses are necessary to understand the role of rare variants in CRC carcinogenesis.

#4490

Comparisons of genetic alterations of breast cancer between East and West: Special emphases on young patients with ER+/HER2- tumors.

Ching-Hung Lin,1 Tzu-Pin Lu,2 Ruby Yun-Ju Huang,3 Yen-Shen Lu,1 Ko-Yun Lo,4 Kuan-Ting Kuo,1 Eric Y. Chuang,2 Pei-Fang Wu,1 I-Chun Chen,1 Jean Paul Thiery,5 Chiun-Sheng Huang,1 Ann-Lii Cheng1. 1 _National Taiwan Univ. Hospital, Taipei, Taiwan;_ 2 _National Taiwan Univ., Taipei, Taiwan;_ 3 _National University Hospital, Singapore, Singapore;_ 4 _Academia Sinica, Taipei, Taiwan;_ 5 _National University of Singapore, Taipei, Singapore_.

A rapid surge of female breast cancer has been observed in young East Asians. As a first step toward understanding this phenomenon, we compared the genetic alterations between breast cancer form Taiwan (East) and Western countries (West). Under the same array platforms, the differences in gene copy number alterations (n = 120 in East and 1,948 in West) and gene expressions (n = 327 in East and 423 in West) were analyzed as further stratified by age and estrogen receptor (ER)/ HER2 status. Gene-gene interaction networks were explored by analyzing the overlapping gene with ingenuity pathway analysis. The ingenuity pathway analysis revealed the common differences between East and West in NFkB, PI3K, Akt, MAPK, ERK, IFNα, and Jnk networks. Among the patients <50 years with ER+/ HER2- tumors, we identified a uniquely higher frequency of the APOA1/C3/A4/A5 (the lipid metabolizing genes) gene cluster loss (29% vs. 12%, P = .005) and low expressions of these four genes (East/ West ratios ranged from .07 to .39) in East than that in West. We conclude that NFkB, PI3K, Akt, MAPK, ERK, IFNα, and Jnk networks are common differences between East and West, while APOA1/C3/A4/A5 loss and low expressions are unique genetic alterations for young Asian patients with ER+/ HER2- tumors.

#4491

Pseudoexons may underlie unbalanced expression of APC alleles in familial adenomatous polyposis.

Taina Tuulikki Nieminen,1 Walter Pavicic,2 Noora Porkka,1 Heikki J. Jarvinen,3 Anna Lepisto,4 Paivi Peltomaki1. 1 _Univ. of Helsinki, Helsinki, Finland;_ 2 _Laboratorio de Citogenética y Mutagénesis, Instituto Multidisciplinario de Biología Celular (IMBICE-CONICET-CICPBA)., La Plata, Argentina;_ 3 _Helsinki University Central Hospital, Helsinki, Finland;_ 4 _Helsinki Unversity Central Hospital, Helsinki, Finland_.

Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC gene. Clinically, the disease can be subdivided into classical FAP with hundreds or thousands of polyps throughout the colon and rectum and Attenuated Familial Adenomatous Polyposis (AFAP) with a milder polyposis. Conventional screening protocols fail to detect any APC mutation in a considerable fraction of adenomatous polyposis families, especially those representing AFAP. We investigated 56 FAP and AFAP families from Finland that had remained APC mutation-negative after protein truncation test, exon-specific Sanger sequencing, and multiplex ligation-dependent probe amplification. Of particular interest were four families with constitutionally unbalanced mRNA expression of APC alleles by Single Nucleotide Primer Extension. These families were interrogated by next generation sequencing of the whole genomes (WGS) and RNA (RNA-seq); additionally, APC promoter methylation was addressed by methylation-specific multiplex ligation-dependent probe amplification. RNA-seq raised the suspicion of a pseudoexon (erroneous inclusion of intronic sequence in the mature mRNA) in three families. A pseudoexon between exons 5 and 6 of the APC gene was present in one family and a pseudoexon between exons 10 and 11 in two families. By WGS, the exon 5-6 pseudoexon consisted of c.646-1806 T>G, which generated a cryptic splice site leading to a 127-bp intronic insertion in the APC mRNA. This pseudoexon was associated with classical FAP. Two alternative genetic changes underlay the exon 10-11 pseudoexon: c.1408+729 A>G was present in one family (AFAP) and c.1408+731 C>T in another family (FAP); both created a new splice donor site leading to an identical 83-bp insertion in APC mRNA. The exon 10-11 pseudoexon resulting from c.1408+731 C>T has previously been described in two unrelated German patients with FAP; the remaining two pseudoexon-associated mutations are novel. All three pseudoexons were predicted to cause premature stop codons leading to APC protein truncation. Sanger sequencing was used to confirm the mutations and to test their presence in the entire series of 56 families; no additional mutation-positive cases were detected. Taken together, deep intronic mutations of the APC gene may explain a proportion of FAP and AFAP families that screen mutation-negative by traditional methods. Specifically, such mutations accounted for three out of four families with the unbalanced expression phenotype of APC in our study. We further conclude that RNA-seq is an effective method to reveal deep intronic mutations and is worth considering in apparent mutation-negative families.

#4492

Investigation of the germline mutational landscape of GBA in general cancer patients and its role in the tumorigenesis.

Junghoon Shin,1 Daeyoon Kim,2 Youngil Koh,1 Sungsoo Yoon1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Cancer Research Institute, Seoul National University, Seoul, Republic of Korea_.

Introduction

Gaucher disease (GD) is an autosomal recessive disease resulting from defective enzymatic activity of acid β-glucosidase (β-glucocerebrosidase). Approximately 400 point mutations in GBA, located in 1q21, have been described and genotype-phenotype correlation was revealed. Previous studies have shown the association of type 1 GD and development of various types of cancers. However, the mutational landscape of GBA and its clinical implication in general cancer patients have not been systematically explored.

Methods

We used publicly available data obtained from ICGC and TCGA data portal (sftp://dccsftp.nci.nih.gov) for the analysis of single nucleotide variants (SNVs) in GBA in general cancer patients. GenBank reference sequence NM_001005741 was used for SNP identification.

Results

A total of 2124 cancer patients comprising 29 cancer types were included. Mutations with allele frequency lower than 0.3 or coverage depth lower than 15 were filtered out from analysis. Overall, 7% (151 out of 2124) were GBA mutation carriers, including 1 nonsense (involving exon 1) and 153 missense mutations, including 5 homozygous alternate. The most frequently detected SNVs included rs2230288 (c.1093G>A:p.E365K, 3 homozygous alternate), rs75548401 (c.1223C>T:p.T408M), rs143187997 (c.58A>G:p.I20V, 1 homozygous alternate), rs150466109 (c.38A>G:p.K13R, 1 homozygous alternate), rs76763715 (c.A1226G:p.N409S), and rs421016 (c.1448T>C, p.L483P), accounting for 1.88%, 1.55%, 1.27%, 1.04%, 0.85%, and 0.28% of the entire cancer population, respectively. We compared our data with 1000 Genomes data of 2504 normal subjects (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502), which revealed that the prevalence of rs2230288, rs75548401, and rs76763715 carriers was significantly higher in the cancer group than 1000 Genomes population (p=0.01, <0.001, and <0.001 by chi-square test, respectively). Regarding somatic mutational profile of tumor samples of 151 germline GBA mutation carriers, the most frequently mutated genes included TP53, PIK3CA, and KRAS, accounting for 28.5%, 7.9%, and 7.9%, respectively.

Conclusion

Although overall prevalence of germline GBA mutation carriers was not significantly different between cancer patients and general population collected from 1000 Genomes data, SNVs in some specific positions showed statistically significant difference in prevalence between 2 groups, implying association between specific mutational loci of GBA and cancer-developing mechanism, even in heterozygous carriers.

#4493

Germline findings in targeted tumor sequencing using matched normal DNA.

Subha Krishnan,1 Gargi D. Basu,1 Laura Gonzalez-Malerva,1 Thomas Royce,1 Tracey White,1 Janine R. Lobello,1 Amy King,1 Kevin Lau,1 Ahmet Kurdoglu,1 Matthew Halbert,1 Jeff Kiefer,2 Irene Cherni,2 Jessica Aldrich,2 Tyler Izatt,2 Megan Russell,2 Sara Nasser,2 John D. Carpten,2 David W. Craig,2 Jeffrey Trent,2 Michael J. Demeure1. 1 _Ashion Analytics, Phoenix, AZ;_ 2 _The Translational Genomics Research Institute, Phoenix, AZ_.

Background: The promise of precision medicine is vast and efforts are needed to increase utilization of precision oncology tests. Studies have suggested that mutations in cancer predisposition are more common than previously thought. Herein, we investigated germline alterations in cancer patients undergoing tumor profiling to identify the potential genetic predisposing factors as well as therapeutically relevant germline variants.

Methods: Comprehensive genomic profiling was performed on tumor samples from 67 patients from 24 cancer types with ages ranging 23-77 years. We performed targeted exome sequencing of 562 genes in tumor and paired normal DNA which included 116 genes that have been associated with cancer predisposition syndromes. In addition to identifying the somatic changes, we characterized germline mutations in select 116 genes using public databases (ClinVar, Ensembl).

Results: Pathogenic germline mutations were found in 17.91% (12/67) of cases, which included 10 unique variants in 8 genes, the majority of which included DNA repair genes (ATM, BRCA2, ERCC4, NBN, PSM2, MUTYH, XPC and AR). Likely pathogenic mutations were found in 23% (16/67) of the cases and 12% (8/67) were predicted pathogenic by SIFT/PolyPhen. In select cases we found existence of a 2nd hit which may have an impact on certain targeted therapeutics such as PARP inhibitors. All of the cases profiled carried either benign or variants of unknown significance (VUS) mutations. Germline findings in cancer susceptibility genes that were concordant with the individual's cancer type included ATM (R2227C) in HER2+ breast cancer patient; BRCA2 missense mutation (K944X) in peritoneal cancer and HER2+ breast cancer; BRCA2 (W1692MfsX3, I2588YfsX60) in patients with pancreatic and breast cancer respectively and PMS2(L729QfsX6) in colorectal cancer (CRC). Germline pathogenic findings in cancer susceptibility genes that were dis-concordant with the individual's cancer type included ERCC4 (R799W) in endometrial cancer; XPC (P334H) in CRC; MUTYH (G393D) in esophageal and CRC; NBN (K219NfsX16) in HER2+ breast cancer and AR (A646D) mutation in CRC. Interestingly, FLT4 (R1324L) mutation which is associated with decreased response and toxicity to sunitinib in renal cell cancer was found across several tumor types including lung adenocarcinoma (n=3), pancreatic cancer (n=3), HER2+ breast cancer (n=2), CRC (n=2) and n=1 in gallbladder carcinoma, cholangiocarcinoma and endometrial adenocarcinoma. The comparison of somatic to germline mutation will be presented.

Conclusion: Germline data analysis of a small cohort of advanced cancer patients revealed that unselected cancer patients may benefit from germline evaluation which may influence the evaluation and care of the patient or the patient's family members. Even though majority of germline mutations were VUS, several mutations were identified which may be amenable to targeted therapeutic strategies.

#4494

Whole genome sequencing reveals different patterns of mutational mechanisms in breast tumors between women of African and European descent.

Jason J. Pitt,1 Toshio F. Yoshimatsu,1 Yonglan Zheng,1 Jason Grundstad,1 Jigyasa Tuteja,1 Jiebiao Wang,1 Abayomi Odetunde,2 Galina Khramtsova,1 Wendy Clayton,1 Adeyinka Ademola,2 Temidayo O. Ogundiran,2 Adenike T. Adeniji-Sofoluwe,2 Millicent Obajimi,2 Adewunmi Adeoye,2 Chinedum Babalola,2 Oladosu A. Ojengbede,2 Christopher O. Olopade,1 Oluwasola A. Olayiwola,2 Lin Chen,1 Dezheng Huo,1 Kevin P. White,1 Olufunmilayo I. Olopade1. 1 _University of Chicago, Chicago, IL;_ 2 _University of Ibadan, Ibadan, Nigeria_.

Across race/ethnicities, breast cancer incidence and mortality rates markedly differ. Numerous studies have demonstrated that individuals of African ancestry acquire aggressive, early-onset breast cancers more frequently than other populations for reasons that remain unexplained. The sources of these disparities are complex, and a comprehensive characterization of mutation landscapes amongst indigenous Africans, African Americans (AA), and Caucasian breast tumors has not been performed.

We generated high-depth whole genome sequencing on 31 Nigerian breast cancers (90x) along with matched blood (30x). Breast cancer whole genomes (tumor-normal pairs) from The Cancer Genome Atlas were harmonized with our samples, resulting in a cohort of 31 Nigerians (17 estrogen receptor negative, ER-), 31 AA (22 ER-), and 43 Caucasians (19 ER-). High confidence somatic mutations (substitutions and insertions/deletions) were obtained by using multiple variant callers. Regardless of race, ER- tumors carried similar numbers of mutations than their estrogen receptor positive (ER+) counterparts (Welch t-test p = 0.57 - 0.82). TP53 (64%) was the most frequently mutated gene in ER- disease, while canonical PIK3CA activating mutations were prevalent in ER+ cases (33.3%). Additionally, tumor suppressor genes RB1, NF1, and PTEN were disrupted via structural rearrangements in ~6 to 15% of samples. Rearrangements in the H3K27 methylation regulator EZH1 were identified in six Caucasians but only one individual with African ancest. Notably, within coding regions, no striking mutation rate differences amongst races were identified. However, global substitution patterns in ER+ and ER- cancers varied widely by race/ethnicity. In ER- cases, Nigerians carried the highest proportion of canonical APOBEC-associated substitutions, particularly C>T transitions. Conversely, Caucasians with ER+ disease showed a higher proportion of C>T than both Nigerians (Welch t-test p = 0.044) and AA (Welch t-test p = 0.011). Kataegis, or clustered mutations, was most prevalent in Nigerian samples, regardless of ER status. Evidence for kataegis was often corroborated by structural variant breakpoints and aberrant copy number states at the hypermutated locus. Mutation signature analyses highlighted multiple APOBEC signatures, with moderate contribution differences across race and ER status.

Overall, our data suggests potential mutation spectra differences in Caucasian, African American and indigenous African breast tumors. Identification of these molecular characteristics by ancestry and geography may help understand race-associated phenotypes and exposures that drive outcomes in breast cancer.

#4495

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

Rong Chen. _Icahn School of Medicine at Mount Sinai, New York, NY_.

We implemented a personalized cancer therapy (PCT) program in a clinical setting, applying an integrative genomics approach to maximally elucidate the complexity of each tumor. For patients in the study, we performed whole exome sequencing and SNP microarrays on tumor and patient-matched normal samples, as well as RNA sequencing on available frozen samples, to identify somatic mutations, copy number alterations, gene fusions, and gene expression alterations in the tumor. Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was used to ensure high sensitivity in cancer mutation hotspots. Genomics results were integrated with cancer knowledge databases and for each cancer type a specific workflow was developed to optimize data interpretation. We returned genomics findings to 46 patients and their physicians, characterizing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-, 6.9-, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91% of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in 4 cases. These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based personalized cancer therapy strategies.

#4496

Genomic profiling of late stage tumors powering precision medicine in Japan.

Masashi Kanai,1 Matthew Schu,2 Tomohiro Sakuma,3 Steven Abbott,2 Tadayuki Kou,1 Ross Gagnon,2 Thomas Halsey,2 Shigemi Matsumoto,1 Mayumi Kamada,4 Masahiko Nakatsui,4 Yasushi Okuno,4 Akinori Hiroshima,3 Hiroaki Mochizuki,3 Patrick Hurban,2 Manabu Muto,1 Victor J. Weigman2. 1 _Dept. of Clinical Oncology, Kyoto University Hospital, Kyoto, Japan;_ 2 _EA Genomics, A Q2 Solutions Company, Morrisville, NC;_ 3 _Biomedical Dept., Mitsui Knowledge Industry co.,ltd, Toyko, Japan;_ 4 _Department of Clinical System Onco-Informatics, Graduate School of Medicine, Kyoto University, Kyoto, Japan_.

Much evidence has shown that genomic profiling of tumors provides patients with treatment options specific to their tumor's biology in the US. Within the Kyoto University Hospital Cancer Center, we offer patients access to these tests through the OncoPrimeTM for those patients of cancer with unknown primary (CUP), rare tumor types or for which standard treatment options have failed. Our institution sees over 6,000 cancer patients each year with over 1,500 receiving chemotherapy and a progressive climate makes this kind of testing favorable. We aim to demonstrate the efficacy of genomic profiling along with successful execution of international specimen logistics as FFPE samples are shipped to EA Genomics and clinical report is provided within 3 weeks.

We officially launched the product publicly in March 2015 and the majority of patients have paid for testing out of pocket in lieu of insurance coverage. During this first year, we saw 30% of patients with CUPs, and 16% of both pancreas and biliary tract with the rest being other rare cancers. Patients going through the testing were generally successful (93.5%) with the specimen failures likely indicative with FFPE artifacts or severe tumor heterogeneity. Actionabilty for our population (84.8%) is consistent with previously published numbers for information provided in the clinical report.

Additional information was collected after the clinical reports to determine early indicators of follow up and see that the accepted term 'actionability' holds different meaning during the practice of patient care. While we had 17% of patients receive the suggested indication we have yet seen positive response from patients who have returned for follow-up. Selected tissues were tested by alternative profiling vendors, and in one case alternate action with Erlotinib was recommended by one vendor; this action has shown promising response. This suggests that heterogeneous interpretation is still a largely unsolved issue with genomic profiling. This may suggested that interpretation is still a largely unsolved issue with genomic profiling. While almost all other patients tested qualified for clinical trials (84.8%) we noted 3 main barriers to entry: geography, cost of intervention and deterioration of patient condition.

We show our results in line with previous studies but strive to increase adoption of results and are compiling information of pan cancer biomarker statuses and treatment success/failures to improve interpretation prior to wider adoption of this kind of testing.

#4497

NYGC glioblastoma clinical outcomes pilot study: Discovering therapeutic potential in glioblastoma through integrative genomics.

Kazimierz O. Wrzeszczynski, Nicolas Robine, Vladimir Vacic, Anne-Katrin Emde, Bo-Juen Chen, Will Liao, Kanika Arora, Minita Shah, Ewa A. Grabowska, Vanessa Felice, Esra Dikoglu, Catherine Reeves, Mayu Frank, Vaidehi Jobanputra, Michael C. Zody, Toby Bloom, Robert B. Darnell. _New York Genome Center, New York, NY_.

Current adjuvant therapeutic options for the treatment of Glioblastoma (GBM) are often determined by limited histological information. Additionally, most GBM clinical trials for targeted chemotherapeutic agents do not distinguish the genetic mutational tumor profiles of the patients recruited and have failed to reach successful treatment endpoints. The New York Genome Center (NYGC) has undertaken a glioblastoma clinical sequencing outcome pilot study to better determine personal treatment options for patients with GBM using integrated genomic data. During the initial phase of this study for 2015 NYGC has performed whole genome sequencing (WGS) on 10 primary GBM tumor-normal pairs, analyzed each patient's tumor for single nucleotide variants, structural variants and copy number alterations. In addition RNA sequencing and a DNA methylation assay were also performed on several of the patients. The patient's genomic profile was then compared to a database of known targeted therapeutic approaches. A final tumor board composed of NYGC scientists, GBM consortium scientists and treating oncologists then reviewed all data prior to identifying a final therapeutic strategy. Data from the first 10 patients revealed RB1 variants to be predominant in half of the patients. SNV's in NF1 or PIK3R1 were also discovered in 4 out of 10 samples. Remaining lower frequency variants occurred in TP53, PDGFRA, PTEN, PIK3CA, ERBB3, SMO, STAG2, ACVR1, NFKB1 and JAK3. Analysis of copy number alterations resulted in 8 of 10 patients containing the characteristic chromosome 7 amplification combined with chromosome 10 deletion, affecting EGFR and PTEN, respectively. Extreme amplification with potential double minute structural variation of EGFR containing the A289V mutation was observed in 2 of 10 samples. Two samples contained a potentially targetable over-amplification of the PDGFRA/KIT/KDR chromosome 4 locus. Predominant deletions resided in CDKN2A, ESR2, PTEN and FLT3. Hemizygous deletions of RB1 combined with RB1 nonsense or missense variants were observed in 4 samples. To date, RNA sequencing was performed on 5 patient samples. Most strikingly the combination of DNA and RNA sequencing revealed the presence of a putative activating MET exon skipping event in the extracellular domain. This MET variant was considered as a potential targetable variant. Therapeutic options resulting from WGS genomic profiles were the PI3K inhibitor BKM120 (60%), half of these had an additional aberration in MET and were recommended for the combinatorial trial NCT01870726. Drug recommendations for the treatment of GBM based on specific N=1 patient genomic profiles were also made for nilotinib, vismodegib and palbociclib. Here, we present the first phase of the NYGC GBM clinical outcome study demonstrating how patient WGS information can provide more precise therapeutic options in the treatment of glioblastoma.

#4498

RNA-Seq in the NYGC GBM Clinical Outcomes Pilot Study.

Bo-Juen R. Chen, Nicolas Robine, Kazimierz Wrzeszczynski, Vladimir Vacic, Anne Katrin Emde, Kanika Arora, Minita Shah, Ewa A. Grabowska, Vanessa Felice, Esra Dikoglu, Catherine Reeves, Mayu Frank, Vaidehi Jobanputra, Michael C. Zody, Toby Bloom, Robert Darnell. _New York Genome Center, New York, NY_.

Whole genome and targeted sequencing data have just started to revolutionize clinical care for cancer patients. Based on genetic variants in each tumor, clinicians can tailor treatment options for patients. While genomic sequencing data provide the landscapes of genetic variants, RNA-seq data can provide complementary information and allow us to interpret the functional effects of discovered variants. However, N-of-1 analysis with RNA-seq data poses several challenges, including integration with DNA data and normalization of gene expression. To tackle these challenges, we developed RNA analytics for integrative analysis for tumor samples in a clinical setting.

For variant analysis, we use RNA to validate somatic variants (both SNVs and indels) and to elucidate potential functional effects of the variants. Because RNA-seq has different coverage patterns from WGS, we developed tools to calculate and compare variant allele frequency in both DNA and RNA data, which allows us to validate variants and evaluate allelic expression. Additionally, leveraging on RNA-seq, our tools look for evidence of aberrant splicing events, such as exon skipping and intron retention that are caused by genetic variants. These analyses not only provide an integrative view of the data, but also prioritize variants and potentially reveal variant effects.

Because many cancers are driven by fusion genes and some fusion genes are recurrent in GBM, we use our data to detect gene fusion supported by RNA-Seq, and if possible by WGS. We use FusionCatcher and STAR-Fusion, coupled with OncoFuse to discover and annotate fusions in RNA-Seq. We subsequently look for supporting evidence by any of the structural variant callers we use.

Gene expression quantification is most valuable as a comparative measurement. However, this is challenging for N-of-1 analysis, since such assessment requires proper normalization and comparison to a proper cohort. Batch-effects due to library preparation and other covariates exacerbate the issue of normalization. We tackle this issue with various normalization and correction strategies. Utilizing GC-bias correction, batch-effect adjustment, and public datasets, our analytic provides gene expression measurement that allows researcher to interpret whether the gene is up- or down-regulated compared to a cohort (such as TCGA).

Our RNA analytics comprehensively utilize transcriptomic data and provides solutions to integrative analysis. We have applied our tools to sequence data from five glioblastoma patients and showed how RNA-seq can compliment WGS. Our integrative analysis helps identify an exon-skipping event in MET in one patient, which was validated with Sanger sequencing. The expression analysis also helps identify potential aberrant activity of the SMO pathway, possibly driven by a SMO missense mutation and loss of SUFU.

#4499

Integration of targeted RNA sequencing to targeted DNA sequencing for the characterization of clinically relevant variants in a population of thousands of patients treated on clinical trial.

Woochang Lee, Heejoon Jo, Xiaoying Yin, David A. Eberhard, Nirali M. Patel, Michele C. Hayward, Ashley H. Salazar, Joel S. Parker, William Y. Kim, Henry S. Earp, Norman E. Sharpless, David N. Hayes. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Background: Targeted sequencing has become common in the care of some patients with cancer, both for the detection of standard of care clinical mutations as well as in the care of patients with advanced disease who are looking for clinical trials or other non-standard therapies. Many patients have mutations detected, although many variants are of unknown significance, and many patients have no mutations at all. We added RNA targeted sequencing along with DNA targeted sequencing to add utility of targeted sequencing strategy in cancer genomics and assessed the performances of RNA sequencing analysis.

Methods: Genomic sequencing of investigative biomarkers was prospectively offered to selected patients. DNA and RNA libraries were prepared separately from a retrieved archival FFPE tumor sample or a fresh frozen tumor sample from each patient. Relevant targets were enriched by custom designed Agilent SureSelect hybrid capture baits using standard protocols. Samples were sequenced on Illumina HiSeq 2000/2500 platforms. We compared somatic variants detected by DNA alone to those detected by DNA plus RNA using the UNCeqR algorithm and software.

Results: From a population of 2200 patients consented as part of LCCC1108 (NCT01457196), a subset of 300 patients was selected at random for RNA profiling. Selected patients were 7 to 83 years old (median, 54) and the cases included more than 20 cancer types including uterus, breast, ovary, and thyroid etc. And stages of cancers were as follows; I, 34.6%, II, 16.8%, III, 25.2%, and IV, 23.4% respectively. Sequencing of RNA was successful in 90% of specimens using 2.5 ug of RNA. RNA sequencing could detect 97.5% of sequence variants called by DNA sequencing and detect 20% more variants than DNA sequencing. Also 97.9 % of variants showed higher mutant allele fraction (MAF) in RNA sequencing than DNA sequencing. We further interrogated the impact of certain classes of mutations on transcript structure and abundance. Specifically, we observed that splice site mutations and indels were associated with detectable alterations in the full length transcripts of their respective genes as well as overall gene abundance, with nonsense and frameshift mutations associated with decreased relative expression of the transcript relative to controls. We also observed that gene amplification of EGFR and ERBB2 was reflected in the RNA sequencing as increased gene expression with statistical significance by Fishers' exact test (P < 0.05), which suggests that RNA can be used as a surrogate for known protein and nucleic quantitative assays.

Conclusion: Using clinical samples, including relatively small quantities, we were able to confirm that nucleotide variants detected at DNA level leads to significant alteration at the transcription level and to have additional information potentially helpful for better management of cancer patients.

#4500

Normal tissue and data resources for cancer research from the GTEx program.

Ping Guan,1 Abhi Rao,1 Simona Volpi,2 Susan Koester,3 Helen M. Moore,1 GTEx consortium. 1 _National Cancer Institute, Bethesda, MD;_ 2 _National Human Genome Research Institute, Bethesda, MD;_ 3 _National Institute of Mental Health, Bethesda, MD_.

The NIH Common Fund's Genotype-Tissue Expression (GTEx) project aims to study gene expression and regulation across multiple human tissues from approximately 1000 healthy normal postmortem donors. GTEx will provide valuable insights into gene regulation and its tissue specificity, to identify correlations between genetic variations and variations in gene expression levels as expression quantitative trait loci (eQTLs), and to help understand inherited susceptibility to disease.

Initiated in 2010, the GTEx program has generated a large volume of data associated with each donor, including clinical and histopathological, as well as genotyping and gene expression data from whole genome sequencing, whole exome sequencing, expression array, and RNAseq data. The program has published research results in multiple scientific journals over the past year.

The following public resources are available from the GTEx program:

1. GTEx Portal: an open access database of GTEx expression data and analysis results: http://www.gtexportal.org

2. Database of Genotypes and Phenotypes (dbGaP): controlled access of comprehensive GTEx clinical data and raw sequencing data: http://www.ncbi.nlm.nih.gov/gap

3. Access to residual GTEx biospecimens for research: http://www.gtexportal.org/home/samplesPage

4. SOPs and best practices from GTEx biospecimen collections: http://biospecimens.cancer.gov/resources/sops/library.asp

5. GTEx histological image viewer: http://biospecimens.cancer.gov/resources/tissue_image_library.asp

6. GTEx donors' families website: a lay description of the GTEx project tailored to the GTEx donors' families: http://www.genome.gov/gtex

GTEx data can be used in combination with other data sets such as The Cancer Genome Atlas (TCGA) and genome-wide association studies (GWAS) to further explore the genetic causes and biology of cancer. The eQTL data from multiple tissues will help prioritize candidate genes within GWAS-associated loci; allow evaluation of tissue specificity of associated loci, and pinpoint target tissues for disease studies. Researchers are using GTEx resources to: study cancer heterogeneity by subtracting normal tissue expression; identify mutations in protein-truncating variants which may cause cancer; and explore gene activity from multiple donors across multiple tissue types to better understand the age/gender bias in cancer and other diseases.

#4501

Immunophenotypic identities of clinical samples have the potential to correlate with overall survival in cytogenetically normal AML patients.

Pawel Gaj,1 Dominika Nowis,2 Stefano Volinia3. 1 _Center of New Technologies, University of Warsaw, Warsaw, Poland;_ 2 _Medical University of Warsaw, Center of New Technologies, University of Warsaw, Warsaw, Poland;_ 3 _University of Ferrara, Ferrara, Italy_.

Tumor infiltrating lymphocytes (TILs) have been described to have great potential to modulate course of the disease. Capacity to eliminate cancer cells primarily depends on the interplay between tumor and its microenvironment. Despite the great interest which this topic gathered over last decades the role of immune cells interaction with cancer has hardly been studied in acute myeloid leukemia patients.

Multitude of patient-specific factors, as well as a great repertoire of different kinds of tumor-infiltrating immune cells, potentially affecting disease progression should be the motivation to investigate implication of tumor immune cells interplay in possibly maximized cohorts of patients. Due to technical reasons the traditional microscopy-based techniques do not allow for the desired high-throughput approach.

In the present study we demonstrate an inter-platform-compatible method which allowed us to predict the gene expression component involvement of 37 cell populations representing different stages of hematopoietic linage differentiation. The predictions were done for an extensive set of peripheral blood mononuclear cells (PBMC) and bone marrow aspirate material sampled from cytogenetically normal (CN) AML cases.

Our results nicely confirmed poor survival prognosis observed for the patients demonstrating high contribution of the CD34+, CD38- hematopoietic stem cells in their bone marrow samples HR=2.05, 95%CI=1.22-3.43, p-value=6.73E-03. We also show that a specific Mature NK cell_CD56+ CD16+ CD3- component found in gene expression signatures of bone marrow samples correlated independently with significantly worse survival in CN AML patients HR=2.56, 95%CI=1.49-4.42, p-value=7.03E-04.

Same analysis carried for the CN AML PBMC samples indicated independent positive effects for the predicted involvement of the Erythroid_CD34+ CD71+ GlyA- and Hematopoietic stem cell_CD133+ CD34dim cell populations in The Cancer Genome Atlas (TCGA) CN AML cohort.

Acknowledgements

The research was supported by a grant from European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION, a grant from the Ministry of Science and Higher Education of Poland [IP2012 048172 (DN)], and a grant from the National Science Centre Poland [2013/10/E/NZ5/00778 (DN)].

#4502

Molecular impact of in vitro exposure to electronic cigarette vapor in human bronchial epithelium.

Elizabeth Moses,1 Teresa Wang,1 George R. Jackson,2 Sean Corbett,1 Eduard Drizik,1 Daniel Brooks,1 George O'Connor,1 Catalina Perdomo,1 Steven Dubinett,3 Patrick Hayden,2 Marc E. Lenburg,1 Avrum Spira1. 1 _Boston University, Boston, MA;_ 2 _MatTek Corp, Ashland, MA;_ 3 _University of California Los Angeles, Los Angeles, CA_.

Electronic cigarettes (ECIGs) are an emerging alternative tobacco product thought by some to potentially be safer than traditional tobacco cigarettes (TCIGs). Despite the increasing prevalence of ECIG use, few studies have evaluated the potential physiological effects of ECIG exposure. In this study we aimed to determine the global gene expression effects of ECIG exposure on bronchial epithelium in vitro. Human bronchial epithelial cells (HBECs) grown at Air Liquid Interface (ALI) were exposed to TCIG smoke and ECIG vapor derived from tobacco or menthol flavored products with and without nicotine. We identified a number of gene expression alterations that were induced by both ECIG and TCIG exposure as well as a novel set of changes uniquely induced by ECIG exposure. ECIG exposure induced the expression of genes involved in oxidative and xenobiotic stress pathways and increased the production of reactive oxygen species, similar to, but generally lower in magnitude than, the effects of TCIGs. Furthermore, TCIG and ECIG exposure both decreased the expression of genes involved in cilia assembly and movement, suggesting that the integrity of the bronchial epithelium is concordantly impaired by both exposures. We additionally identified a number of ECIG-specific cell cycle and cell division pathway changes. Finally, we observed that ECIG-induced changes were dependent on both flavor and nicotine content. Together, these results indicate that ECIG vapor can induce cellular stress and molecular alterations within airway epithelium that share similarities with the effects of TCIG smoke. Based on these findings, further studies are warranted to determine whether ECIG use will lead to similar deleterious health outcomes as those caused by TCIGs.

#4503

Actionable mutations and mutational burden in renal cell carcinoma.

Susan M. Mockus,1 Sara E. Patterson,1 Jason R. Pettus,2 Gregory J. Tsongalis2. 1 _The Jackson Laboratory, Farmington, CT;_ 2 _2The Geisel School of Medicine at Dartmouth and Dartmouth Hitchcock Medical Center, Lebanon, NH_.

Clear cell renal cell carcinoma (CCRCC), the most common subtype of renal cell carcinoma, is a heterogeneous cancer with variable outcomes and molecular aberrations. Further elucidation of mutational profiles and actionable mutations may improve patient outcomes in CCRCC. Somatic mutational profiling via next-generation sequencing was conducted on 30 CCRCC whole section FFPE patient samples to determine the mutational burden as compared to other solid tumor types. The sequencing failure rate for these samples was 20% (6/30). A 358-gene panel was also run on an additional 57 non-CCRCC solid tumor FFPE patient samples (81 samples in total). FASTQ files generated from Illumina's CASAVA software were submitted to the JAX Clinical Genome Analytics (CGA) data analysis pipeline to perform automated read quality assessment, alignment, and variant calling. Identified variants were then submitted for clinical curation using the JAX Clinical Knowledgebase (CKB) for actionable mutational analysis and overall mutational burden. Of the CCRCC sample set, 33% had one or more actionable mutations. This was low compared to actionable mutations in colon (16/16, 100% actionable), TNBC (18/20, 90% actionable), squamous lung (8/9, 89% actionable), and pancreatic (8/12, 67% actionable). Furthermore, the CCRCC set had a low overall mutational burden with 22 ± 6.7 nonsynonymous SNV's as compared to colon (29 ± 11), squamous lung (43 ± 17), and TNBC (31.5 ± 12). However, the pancreatic set had even lower SNV's at 12.7 ± 3.4. The number of indels was comparable across the five sets, except for colon, which averaged 6.9 ± 8.3. Significant copy number variations (CNV's; amplification validated at greater than or equal to 6 copies) also had a wide range with squamous lung at the highest (11 ± 11), followed by TNBC (5.6 ± 5.0), and then colon (2.3 ± 3.0). Both the CCRCC and pancreatic sets had few CNV's at 0.8 ± 1.5 and 0.17 ± 0.40, respectively. Common mutations identified in the CCRCC set included EPHB6 duplications near S166, which were identified in 25% of samples. Gene specific mutations in CCRCC were also identified in VHL (54%) and SETD2 (21%). These three genes, EPHB6, VHL, and SETD2 have been previously implicated in CCRCC pathogenesis. Six variants identified in VHL were C77*, P86R, Q132fs, A149D, R177fs, and L184fs. Due to the low mutational burden and/or actionable mutations in CCRCC, even large NGS panels may not be adequate to profile somatic mutations and whole exome or whole genome may be more conducive for molecular subtyping of renal cell carcinoma.

#4504

Developing a PTEN-ETS signature to improve molecular risk stratification in prostate cancer.

Luigi Marchionni, Tamara L. Lotan. _Johns Hopkins University School of Medicine, Baltimore, MD_.

Prostate cancer (PCa) is a heterogeneous disease and the development of a molecular classification is critical to distinguish lethal from indolent tumors and reduce overtreatment. Genomic alterations of the PTEN and ETS genes (e.g., ERG) are among the most common in PCa and there is a huge interest in exploiting these alterations for risk assessment. We have found that PTEN loss is associated with PCa death most strongly in patients whose tumors do not carry an ERG rearrangement, contradicting findings from mouse models.

Despite technological advances have enabled extraordinary insight into PCa molecular alterations, our understanding of the interaction between PTEN and ETS genes during PCa progression remains very limited. Hence, we have analyzed a large collection of multi-omics datasets to characterize the molecular underpinnings of PTEN loss in the context of ETS-negative versus ETS-positive tumors and further investigate their role in driving PCa prognosis.

First we have developed a classification approach for PCa stratification based on dichotomized gene expression of PTEN and ETS family members. We have then validated this approach using TCGA DNA copy number variation and rearrangement status information. Our results confirmed that transcriptome analysis can be used for detecting PTEN and ETS alterations, and that in the absence of ERG rearrangements, tumors with PTEN loss frequently overexpress alternative ETS genes.

Next, we examined whether somatic mutation profiles might differ by PTEN/ETS status, providing an potential explanation for the lethal behavior of ERG-negative/PTEN-negative tumors. As expected SPOP mutation was found to be mutually exclusive with ERG expression, but it did not vary significantly by PTEN status. Most interestingly, TP53 mutation - the most frequently altered gene in lethal metastatic PCa - was significantly enriched among PTEN-negative tumors irrespective to ERG- status. Collectively these findings suggest that known prognosis-driving somatic mutations do not explain the aggressive behavior of ERG-negative/PTEN-negative tumors.

Finally, we analyzed differential gene expression associated with PTEN loss in the presence or absence of ERG gene expression and identified the associated biological processes and signaling pathways. This analysis failed to confirm the correlation between ERG/ETV1 expression and higher androgen signaling output previously reported in murine models. Furthermore, our findings revealed that MYC targets and the genes involved in cell cycle progression are the most up-regulated by PTEN loss in ETS-negative tumors, while genes involved in cell differentiation, response to DNA damage, and TNF signaling are the most induced by PTEN loss in ERG-positive tumors.

Overall, our analysis clarified some important aspects of the role of PTEN/ETS network in PCa progression, suggesting a number of promising targets for the development of novel therapeutic interventions.

#4505

Pan-cancer analysis of fusion gene druggability and neoantigen potential.

Henrik Edgren, Anja Ruusulehto, Kalle Ojala, Maria Laaksonen. _MediSapiens Ltd, Helsinki, Finland_.

Fusion genes are a well known type of oncogenic mutation, in which a genomic rearrangement, such as e.g. a translocation, inversion or deletion, joins together parts of two genes, which normally are not located adjacent to each other. In the context of cancer driver mutations, the result of this rearrangement is a novel chimeric gene with oncogenic potential. While fusion genes have been known longest in hematological malignancies, over the last years they have been shown to occur in nearly all types of cancers studied. Notable examples include ETS-family fusions, such as TMPRSS2-ERG, in prostate cancer.

In previous work, we have used our FusionSCOUT fusion gene detection pipeline to analyze over 8000 cancer samples from 28 different TCGA cancer types. In this study, we extend this target discovery to further characterize the genomic alteration contexts in which gene fusions occur, both by examining the copy number states at inferred rearrangement breakpoints, as well as by correlating the numbers of fusion genes identified in tumors with the level of genomic instability in the tumors. We also analyze the frequency at which different druggable protein domains occur in fusion genes, comparing these to the expected random distribution. Finally, we predict, for each tumor, the neoantigen potential of the identified fusion proteins and compare this to the same predictions for simple somatic mutations, in order to characterize the contribution of fusion proteins to neoantigen load.

In summary, we extend our pan-cancer fusion gene identification analysis with a detailed characterization of the genomic alteration contexts at which fusion genes occur. We also report on the drug treatment potential of fusion genes, both in terms of small molecule drugs, as well as highlight the degree to which fusion gene derived chimeric proteins should be taken into account when estimating the neoantigen load of tumors.

#4506

Identification of druggable transcriptional outliers in urothelial carcinoma.

Bishoy S. M. Faltas, Rohan Bareja, Ethan Shelkey, Rebecca Mayer, Andrea Sboner, Mark A. Rubin, Olivier Elemento. _Weill-Cornell Medical College of Cornell University, New York, NY_.

Background: Urothelial carcinoma (UC) is a major public health problem affecting new 75,000 patients and resulting in 15,000 deaths annually. Despite a variety of genetic and epigenetic alterations described in UC, few therapeutically exploitable drivers have been identified and validated. We hypothesized that transcriptional outliers from primary tumor biopsies could reveal such therapeutically exploitable weaknesses. Identification of these druggable outliers would be critical for individualized therapy of patients with UC. Methods: In order to identify transcriptional outliers we calculated Z-scores for a set of 72 druggable cancer-related genes from RNA sequencing data from our Institute of Precision Medicine (IPM) cohort of advanced platinum-resistant UC. We performed similar outlier analyses on The Cancer Genome Atlas (TCGA) cohort of UC and the Cancer Cell Line Encyclopedia (CCLE) database of urothelial cell lines. Common outliers between IPM and CCLE datasets or between TCGA and CCLE datasets were nominated for further in-vitro testing. Sensitivity scores of cell lines for 130 drugs were extracted from the publicly available cell line browser database and the variance these sensitivity scores across (640) cell lines was calculated. The drugs targeting the outlier genes (real sensitivity z-score) were compared to the mean z-scores of randomly chosen drugs associated with those genes (control sensitive z-score). To investigate the mechanisms underlying comparison of outliers methylation patterns of the promoter regions of outlier genes were examined. Results: 6 druggable outlier genes were common between IPM and CCLE cohorts, and 15 outlier genes between CCLE and TCGA cohorts. Common outliers included FGFR3, KIT, BCL2, ABL1, and SMO. The presence of outlier genes in cell lines correlated with higher sensitivity to drugs predicted to target these genes in comparison to randomly selected drugs (p<0.05). In vitro cell viability assays confirmed sensitivity of RT112 and RT4 cell lines to Dovitinib, an FGFR3 inhibitor as predicted by our algorithm. Outlier genes were frequently involved in gene fusions, amplification or DNA hypomethylation (p<0.02). Conclusions: Identification of druggable transcriptional outliers can elucidate novel oncogenic dependencies and aid in selecting precision therapies for UC patients.

#4507

New JAK2 heterozygous loss: A role in overall survival in acute myeloid leukemia patients.

Viviana Guadagnuolo,1 Maria Chiara Fontana,1 Cristina Papayannidis,2 Marco Manfrini,1 Antonella Padella,1 Giorgia Simonetti,1 Anna Ferrari,1 Giovanni Marconi,1 Andrea Ghelli Luserna di Rorà,1 Stefania Paolini,1 MariaChiara Abbenante,1 Sarah Parisi,1 Chiara Sartor,1 Emanuela Ottaviani,1 Giovanni Martinelli1. 1 _L. & A. Seragnoli, bologna, Italy; _2 _L. & A. Seragnoli, Bologna, Italy_.

Acute Myeloid Leukemia (AML) is a clonal hematopoietic disorder characterized by an abnormal proliferation and differentiation of immature blast cells in the bone marrow.

SNP microarray approach has resulted in genome-wide screening for genomic alterations with information not previously achievable and also it allows to map all the genes involved in these alterations which may plays a role in oncogenesis.

Our objective is to evaluate the prognostic impact of these genetic alterations on clinical outcome.

We analyzed 285 AML samples at diagnosis of which 221 by SNP Array 6.0 (Affymetrix) (Affymetrix), while 64 by by CytoScan HD Array (Affymetrix). The results obtained were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 softwares (BioDiscovery).

The 285 AML patients (pts) analyzed are equally distributed between both sexes, they have a median age of 60 years, include miscellaneous cytogenetic abnormalities and normal karyotype. The 44% of pts are treated with chemotherapy (CHT), the 16% of pts with low-dose cytosine arabinoside, the 20% of pts with 5-azacytidine and the 19% of pts are not treated or had only supportive therapy. The ratio between responders and non responders pts was 57/43. Each pt presented an average of 1448 events so distributed: 31% of homozygous deletions, 32% of homozygous amplifications, while irrelevant events are related to heterozygous deletions and amplifications. We also found 36% of uniparental disomy (UPD)events.

Among all these macroscopic and submicroscopic alterations, we focused on JAK2 gene, which is located on human chromosome 9 at p24.1 locus. It is composed of 25 exons and encodes a 1132 amino-acid protein of 130.7 kDa.

This gene was amplified in 12/285 pts (4%) and deleted in 13/285 (5%). All these pts are treated with CHT but only those characterized by JAK2 deletion are responsive in first line. We showed that the group of pts which present this deletion had a better overall survival rate than the group with the amplification of this gene (p-value < 0,01).

The deletion event of JAK2 gene involved two different regions: the first region had a length of 7.429 bp involving the intron 4 and was found in the database genome variant (DGV); the second deletion presented a variable length between different pts: the smallest deleted region had a length of 3342 bp and included the exons 17, 18, 19, while the largest one had a length of 76903 bp which included the portion from exon 9 to exon 19. This second deletion has not been described in DGV and it involved the pseudokinase and kinase domain of JAK2 protein.

We have identified Copy Number Variation involving important cancer genes in AML. We have identified a new JAK2 deletion involving its Pseudokinase and Kinase domain and we observed that this deletion of JAK2 correleted with overall survival.

ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project

#4508

Genomic profiling of ER+ breast cancers treated with prolonged neoadjuvant letrozole reveal a high frequency of NOTCH2 mutations in clinically resistant tumors.

Angel Guerrero-Zotano,1 Thomas Stricker,1 Katherine E. Hutchinson,1 Luigi Formisano,1 Jennifer Giltnane,1 Amparo Ruiz,2 Carlos L. Arteaga1. 1 _Vanderbilt University, Nashville, TN;_ 2 _Instituto Valenciano de Oncologia, Valencia, Spain_.

Hypothesis: Approximately 20% of patients with early ER+ breast cancer (BC) treated with adjuvant antiestrogen therapy eventually relapse with endocrine-resistant metastatic disease. We hypothesized that profiling newly diagnosed ER+ breast tumors that persist following prolonged estradiol deprivation with letrozole would identify genomic alterations causally associated with endocrine resistance.

Methods: We treated 57 postmenopausal women (median 77 years; range 60-86) with ER+/HER2- early BC with neoadjuvant letrozole (median 7.5 months; range 3-36) followed by surgery and adjuvant endocrine therapy. Patients were followed with serial ultrasounds and defined as non-responders if they developed recurrent locally or metastatic disease, or had a preoperative endocrine response index (PEPI) ≥4 (composite score of post-treatment ER, Ki67, T and N status). DNA extracted from post-treatment specimens was profiled using targeted exome sequencing of 300 cancer-related genes. We screened for variants with a high probability of disrupting protein function and excluded variants likely to be germline by filtering out every alteration not present in the Catalogue of Somatic Mutations in Cancer (COSMIC), if the variant had an allele frequency >0.2% as per the Exome Aggregation Consortium (ExAC) dataset.

Results: Ten (10) patients had a PEPI 0 score, 32 patients were PEPI 1-3, and 15 were PEPI ≥4. After a median follow-up of 50 months (range 12-100), 9 patients have recurred with metastatic disease (4 with PEPI1-3, 5 with PEPI≥4). We identified 535 variants with a median coverage >200x (387 nonsynonymous, 23 stop-gain, 124 indels). The median number of mutations was similar in responder vs non responders (8.03 vs 8.6). Recurrently mutated genes (>5%) included PIK3CA (38%), NOTCH2 (31%), KMT2C (22%), VEZF1 (21%), TP53 (12%), NF1 (9 %), MTOR (9%), ESR1 (7%) and ERBB2 (5%). Recurrent amplifications were detected in CCND1 (15%), ERBB2 (12%), FGF19 (12%) and FGFR1 (8%). The only mutations enriched in the non-responders and that correlated with long-term outcome were those in NOTCH2. The relapse-free survival rate at 40 months for patients with NOTCH2 mutations was 89% vs 41% for patients with wild-type NOTCH2 (p=0.001). NOTCH2 mutations clustered as a frameshift deletion (P6fs) in exon 1, with a 20% average allele frequency. This mutation has been reported in TCGA and COSMIC at a mutation rate of 6%, across all tumor types and is predicted to produce an N-terminally truncated NOTCH2 protein (39 amino acids shorter) lacking the signal peptide.

Conclusions: Genomic profiling of residual ER+ breast cancers treated with prolonged neoadjuvant letrozole revealed a high frequency of NOTCH2 mutations. These alterations in NOTCH2 may cause hormone independence in ER+ breast cancer and are worthy of further mechanistic and clinical investigation.

#4509

Clinical genomic profiling of 1000 metastatic breast cancer patients: actionable targets, novel alterations, and clinical correlations.

Pedram Razavi, Matthew T. Chang, Sumit Middha, Dara S. Ross, Ahmet Zehir, Tracy A. Proverbs-Singh, Cyriac Kandoth, Sarat Chandarlapaty, Maura N. Dickler, Jorge S. Reis-Filho, Sujata Patil, Venkatraman Seshan, Lillian Smyth, Neil M. Iyengar, Komal Jhaveri, Shanu Modi, Chau T. Dang, Mark E. Robson, Larry Norton, Clifford A. Hudis, Marc Ladanyi, Maurizio Scaltriti, Nikolaus Schultz, David Hyman, Michael F. Berger, Barry S. Taylor, David B. Solit, José Baselga. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Most large genomic profiling efforts in breast cancer have focused on primary breast tumors. Profiling of metastatic breast cancer (MBC) could more accurately define actionable genomic alterations and reveal novel alterations that arise under the selective pressure and clonal evolution of prior therapy. Utilizing a 410-gene targeted capture-based sequencing platform (MSK-IMPACT), we analyzed 920 tumors (62% metastatic, 38% primary) from 874 MBC patients for somatic mutations, DNA copy number alterations, and structural rearrangements (planned final analysis 1000 patients). Detailed clinical data including treatment outcomes was collected for all patients. The cohort was representative of the well characterized clinical subtypes of breast cancer with 71% ER+ or PR+ and HER2-, 17% HER2+, and 12% triple negative breast cancer (TNBC).

Our analysis revealed recurrent alterations in multiple pathways including PI3K/AKT/mTOR (56%), cell cycle regulation (42%), RTK signaling (40%), epigenetic regulation (33%) and MAPK/ERK (19%) pathways. The most frequent actionable alterations include: PIK3CA mutation (36%), ERBB2 amplification (15%), FGFR1 amplification (12%), ESR1 mutation (11%), PTEN mutation/deletion (10%), AKT1 mutation (6%), and ERBB2 mutation (5%). The genomic landscape was significantly different across breast cancer subtypes. For example, the PI3K/AKT/mTOR pathway was altered in ER+ MBC mainly through activating PIK3CA mutations whereas PTEN deletion/mutations were most common in TNBC. We also identified significant differences in the genomic profiles of metastatic and primary tumor samples. Gene enrichment analyses revealed a subset of genes more frequently altered in metastatic tumors (STK11: 13 vs 2; ROS1: 14 vs 2; FGFR4: 15 vs 1) suggesting that mutations in these genes may play a role in breast tumor metastasis and/or therapy resistance. ESR1 mutations were predominantly present in metastatic tumors (88%) and were significantly associated with duration of prior hormonal therapy (P<0.0001). ESR1 mutations were also associated with a poor response to the ER degrader fulvestrant (median PFS: 4.8 vs 13.7 months, mutated vs wild type; P=0.01). Analysis for potentially novel hotspot mutations revealed recurrent RHOA G17 mutations in 6 MBC patients, nominating RHOA, a GTPase transducer of membrane receptors, as a candidate driver in a subset of breast cancers. While identified previously in other tumors, RHOA G17 has never been implicated in breast cancer.

In summary, our genomic analyses of this large cohort of MBC patients revealed actionable alterations in over 60% of the patients. 29% of patients with these alterations were enrolled in clinical trials of matched targeted therapies to date (PIK3CA 27%, AKT1 30%, ESR1 23%, ERBB2 39%) suggesting that prospective genomic characterization can accelerate enrollment of patients with MBC onto therapeutic clinical trials.

#4510

KIT melanomas are developmentally racially biased.

Margaret I. Sanchez, James M. Grichnik. _Sylvester Comprehensive Cancer Center - University of Miami Leonard M. Miller School of Medicine, Miami, FL_.

KIT is a critical pathway for melanocytic homeostasis and it is often down-regulated in cutaneous melanoma. However, KIT activation plays a critical role in a subpopulation of melanomas especially those in acral locations and melanomas on chronically sun damaged skin. We analyzed exome and RNAseq data from The Cancer Genome Atlas (TCGA) Network (321 cutaneous melanomas in the data set) to identify patterns unique to KIT activation. The melanomas were divided based on known mutually exclusive mutations (BRAF, NRAS and KIT) vs those without (WT). We compared RNA data between each group. KIT group had significantly higher expression of KIT than BRAF, NRAS and WT (p<0.01, FDR<0.01) and isoform 2 expression was higher than 1. KIT melanomas were found to overexpress Wnt pathway, pigmentation and genes involved epithelial-mesenchymal transition. From the exome data, germline SNPs with incidence in ethnic groups (Caucasian, Asian and African) and more likely to be present in a specific melanoma groups were selected. Comparing the 3 ethnicity groups across the 4 tumor groups Asian SNPs were significantly enhanced in KIT tumors, African in WT and Caucasian in BRAF and NRAS melanomas. We evaluated the tumors for the primary type of genetic damage. The three signatures that appeared most divergent were CC>TT for UV, (G/A)C(G)>(G/A)T(G) for deamination, and G>T for oxidative damage. UV and deamination appeared inversely proportional, while oxidative damage appeared to be independent of those other two features. KIT and WT had a greater % of non UV high deamination tumoral environments. A fraction of these tumors also had very high oxidative signatures. We then compared 2 KIT subgroups UV vs non UV (absence of CC>TT mutations) for germline ethnicity differences. Asian SNPs were highly increased in non UV subgroup whereas Caucasian SNPs were in UV subgroup. Further, we divided KIT non UV subgroup into deamination and oxidative damage subgroups and compared ethnicity differences. Deamination was significantly higher in Asians whereas oxidative damage was higher in Caucasians. A similar analysis was done to the WT group, where African SNPs were significantly increased in the non UV subgroup and were primarily associated with oxidative damage. This data implies that KIT mutant melanomas develop in a unique genetic and mutational environments and makes this an ideal system to in which study racial disparities at the molecular level.

#4511

Characterization of tumor neoantigen repertoire in patients with germline BRCA1/2 mutations.

Adam A. Kraya, Kara N. Maxwell, Brandon M. Wenz, Bradley Wubbenhorst, Daniel DeSloover, Nicole Lunceford, Jennifer D. Morrissette, Michael Feldman, Robert H. Vonderheide, Susan M. Domchek, Katherine L. Nathanson. _University of Pennsylvania, Philadelphia, PA_.

Tumor neoantigens are peptides unique to the cancer genome that can induce endogenous T-cell activity and contribute to the efficacy of immunotherapy. We sought to characterize the neoantigen repertoire in 20 breast and 19 ovarian tumors from patients with germline BRCA1/2 mutations. Loss of BRCA1/2 function leads to genomic instability and increased mutational burden, a key correlate with clinical benefit from immunotherapy. To determine patient class I Human Leukocyte Antigen (HLA) haplotypes, whole exome sequencing data were analyzed using OptiType. Class I HLA alleles were typed to four-digit accuracy and were used in the prediction of tumor neoantigens using NetCTLpan 1.1. Briefly, non-synonymous variants from tumor exome data were translated to peptides of length 15, 17, and 19 amino acids centered on the variant of interest, where NetCTLpan analyzed all 8-, 9-, and 10-mer amino acid substrings, respectively, within the peptide sequences to predict HLA allele-specific binding affinities. IC50 values, predicted probability of binding to the TAP transporter, and probability of C-terminal cleavage were used to prioritize candidate neoepitopes of interest. When considering peptides with a predicted affinity less than 500nM, the minimum criterion for a likely binding occurrence in vivo, neoantigen loads varied extensively across both breast and ovarian patients, ranging from a minimum of 10 predicted binders in patients with the lowest neo-antigen load to greater than 300 predicted binders in patients with the highest load (mean 87, std. dev. 71). 10 of 20 breast cancer patients and 13 of 19 ovarian cancer patients harbored a moderate neoantigen load of 50-150 predicted neoantigens. In comparison, tumor types with low neoantigen load (e.g. acute myeloid leukemia) and high neoantigen load (e.g. melanoma) exhibit 8 and 480 predicted neoantigens on average, respectively, consistent with their missense mutational burden. Positive correlates of neoantigen load in our studies included mutational burden (p<0.01), genome gain/loss, tumor ploidy, and BRCA1/2 loss of heterozygosity (p<0.05), all related markers of genomic instability. These relationships will be further validated in an independent sample set of germline BRCA1/2 mutation carriers from the TCGA, and expression levels of these predicted neoantigens will be investigated by RNASeq. Further, additional neoepitope prediction tools will be used to generate a consensus list of top-ranking candidate neoepitopes with high predicted immunogenic potential, which will be functionally evaluated for T-cell reactivity. Our work demonstrates that breast and ovarian cancer patients with BRCA1/2 mutations vary substantially in terms of predicted immunogenicity and suggests that a subset of such patients may be responsive to immunotherapy.

#4512

High-throughput identification of neoepitopes for the development of patient-specific cancer immunotherapies.

Andrew Nguyen,1 J Zachary Sanborn,1 Charles J. Vaske,1 Shahrooz Rabizadeh,2 Kayvan Niazi,2 Patrick Soon-Shiong,3 Steven C. Benz1. 1 _NantOmics LLC, Santa Cruz, CA;_ 2 _NantWorks LLC, Culver City, CA;_ 3 _NantWorks LLC; CSS Institute of Molecular Medicine, Culver City, CA_.

Introduction: Immunotherapies such as checkpoint inhibitors, CAR T cells, NK cells, and therapeutic vaccines are revolutionizing cancer medicine. Although these agents have resulted in remarkable responses in some patients, many fail to respond, suggesting that a patient-specific approach is needed for immunotherapies to realize their full potential. Here we report the analysis of whole genome sequencing (WGS) and RNA sequencing (RNAseq) data from The Cancer Genome Atlas (TCGA) to identify neoepitopes that may serve as viable immunotherapeutic targets and that could be used to develop next-generation, patient-specific cancer immunotherapies.

Methods: HLA-A compatible neoepitopes were identified by creating all permutations of 9-mer amino acid strings derived from identified single nucleotide variants (SNVs) occurring in expressed genes. All neoepitopes were filtered against a database of 9-mers created using every known human protein along with common variations obtained through dbSNP to reduce off-target effects. HLA typing was determined from WGS data using the HLA forest algorithm. NetMHC 3.4 was used to obtain the predicted binding affinities of neoepitopes to HLA-A alleles

Results: We analyzed 750 patients across 23 cancer types using WGS data and RNAseq data, when available. Mutational and neoepitope loads varied across cancer types, with skin cutaneous melanoma and thyroid carcinoma having the highest and lowest mutational and neoepitope loads, respectively. Neoepitope counts identified by WGS correlated with neoepitope expression identified by RNAseq across a wide variety of cancers (Pearson's r=0.99 for all cancers combined). The distribution of HLA-A and HLA-DRB1 alleles determined from TCGA were generally comparable to that in the US population; however, the frequency of HLA-DRB1*15:01 (associated with several diseases) was ~2-fold higher than the US population. Among patients who expressed HLA-A*02:01 (n=143), the numbers of predicted neoepitopes identified by WGS, neoepitopes expressed per RNAseq, and neoepitopes having binding affinity to HLA-A*02:01 were 23272, 9619, and 138, respectively. Almost all neoepitopes were unique to each patient, with a maximum of only 2-shared neoepitopes among any cancer type. Among 26 triple negative breast cancer (TNBC) samples, the numbers of predicted neoepitopes, expressed neoepitopes, and neoepitopes with affinity to HLA-A*02:01 were 17925, 8184, and 228, respectively. There were no shared neoepitopes among TNBC samples. Across all cancers, ~6% of neoepitopes occurred in cancer driver genes.

Conclusions: Neoepitopes are rarely shared among cancers, and almost all are unique to each patient. For patients with limited treatment options and poor outcomes, such as TNBC, high-throughput identification of neoepitopes could serve as the basis for the development of next-generation, patient-specific immunotherapies.

#4513

A prognostic model for pulmonary carcinoid tumors based on large chromosomal alterations.

Konstantinos Leventakos, Aaron S. Mansfield, Stephen J. Murphy, Sarah H. Johnson, Sarah E. Kerr, Marie Christine Aubry, George Vasmatzis, Julian R. Molina. _Mayo Clinic, Rochester, MN_.

PURPOSE: Previous mutational analyses have failed to identify a predictive or prognostic marker for pulmonary carcinoid tumors (PCT). The heterogeneity of PCTs coupled with a lack of detailed information about the tumor biology, impedes patient stratification into groups based on tumor phenotypes or treatment response. We sought to use high-throughput next-generation sequencing to improve prognostication.

METHODS: We stratified 37 patients with resected PCTs as low risk (n=17) and high risk (n=20) based on a 5 year long follow up for recurrence. Patients with no recurrence within 5 years from initial treatment were deemed low risk. Macrodissection was followed by genomic DNA isolation and next-generation sequencing was performed using an Illumina Mate Pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction.

RESULTS:

Carcinoids commonly harbor rearrangements (median 3.5, range 1-167). Large chromosomal gains or losses were found in 24 of the cases (64.8%). We developed a prognostic score that included the total amount of genetic alterations (deletions and translocations). ROC analysis revealed an AUC of 0.76. High risk patients had a mean score of 20 and low risk patients had a mean score of 5.45 (p value= 0.03, Welch Two Sample t-test). Further analysis of the specific genetic alterations harbored by high and low risk PCTs and correlation with the pathologic and immunohistochemical information is currently underway.

CONCLUSION:

Large chromosomal rearrangements and aneuploidy portend a poor prognosis for patients with PCTs. Mate Pair sequencing of PCTs provides valuable prognostic information for this highly heterogeneous group of cancers.

### Histone Modifications and Chromatin Dynamics

#4514

Transcription down-regulation of c-Myc through nucleic acid clamp-mediated stabilization of the promoter G-quadruplex.

Taisen Hao, Tracy Brooks. _University of Mississippi, Oxford, MS_.

MYC overexpression occurs in up to 80% of all cancers. Down-regulating MYC's expression is an effective therapeutic strategy for the treatment of many of these cancers, and particularly for non-Hodgkin's lymphoma (NHL). NHL is the fifth most common cancer type, accounting for 5% of all cancers and 4% of all cancer deaths. Developing MYC-targeting therapeutics will be very impactful to NHL patients. In the current study, we have developed a nucleic acid clamp to transcriptionally down-regulate MYC expression by stabilizing the G-quadruplex (G4) formed in the nuclease hypersensitivity element III1 promoter region. The clamps complement nucleotides on the 5'- and 3'- regions flanking the G4-forming sequence, linked together with an 18 angstrom abasic moiety. To date, the binding affinity and specificity, G4 structure stabilizing ability, MYC down-regulation capability, and the selective cytotoxicity on cancerous vs non-cancerous cell lines of these clamps have been confirmed by EMSA and electronic circular dichroism, DMS footprinting, luciferase reporter assay, and MTS viability assay respectively. Recently, various replacements have been explored to replace the abasic moiety with non-complementary nucleic acids and to optimize the phosphate backbone with peptide nucleic acids. In addition, complexation with block co-polymers is under investigation to develop a functionalizable nanotherapeutic for NHL. The highly selective G4 stabilizing ability of the clamps used in the current approach offers an advantage over traditional G4 stabilizing small molecules. This novel approach is a promising therapeutic for the treatment of MYC related malignancies, and provides a framework for future G4-therapeutic development for other high-value genes, such as kRAS, VEGF, c-KIT and many more.

#4516

Evaluating the chromatin state as a predictive biomarker for BET inhibitor sensitivity in hematological malignancies.

Joseph Castillo,1 Ryan Raisner,1 Karen Gascoigne,1 Gary Dos Santos,2 Emer Clarke,2 Alex Huang,1 Mark Lackner,1 Zineb Mounir1. 1 _Genentech, South San Francisco, CA;_ 2 _ReachBio, WA_.

The BET (bromodomain extra-terminal) family of proteins are a group of epigenetic regulators that control gene transcription by reading acetylated lysines on histone tails and recruiting transcription complexes. The various BET inhibitors developed show strong anti-tumor effects against a broad range of hematologic malignancies. The BET inhibitor JQ1 shows strong anti-proliferative activity in sub-sets of multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and lymphoma cell line models. At present, however, predictive biomarkers of BET inhibitor sensitivity have not been identified. We hypothesize that the chromatin state characterized by various histone post-translational modifications (acetylation, methylation, etc.), can drive sensitivity to BET inhibition. By examining basal expression of epigenetic marks in panels of sensitive and resistant heme cell line models and further validating these histone modification trends in primary AML and multiple myeloma cultures, we may be able to identify predictive biomarkers of BET inhibitor sensitivity in hematological malignancies.

#4517

Epigenome landscape of human normal purified hepatocytes: analysis by the International Human Epigenome Consortium (IHEC).

Eri Arai,1 Fumihito Miura,2 Yasushi Totoki,3 Satoshi Yamashita,3 Ying Tian,1 Masahiro Gotoh,3 Hidenori Ojima,1 Hiroyuki Nakagawa,4 Yoriko Takahashi,4 Hiromi Nakamura,3 Natsuko Hama,3 Mamoru Kato,3 Hiroshi Kimura,5 Yutaka Suzuki,6 Takashi Ito,2 Tatsuhiro Shibata,3 Yae Kanai1. 1 _Keio University School of Medicine, Tokyo, Japan;_ 2 _Kyushu University Graduate School of Medical Sciences, Japan;_ 3 _National Cancer Center Research Institute, Tokyo, Japan;_ 4 _Mitsui Knowledge Industry Co., Ltd., Tokyo, Japan;_ 5 _Tokyo Institute of Technology, Yokohama, Japan;_ 6 _The University of Tokyo, Japan_.

As a research group participating in the International Human Epigenome Consortium, we have performed whole-genome bisulfite sequencing (WGBS) using post-bisulfite adaptor tagging, chromatin immunoprecipitation (ChIP)-sequencing (seq), RNA-seq and whole-genome sequencing (WGS) using normal hepatocytes purified from partial hepatectomy specimens of 6 Japanese patients without hepatitis virus infection, chronic liver disease or hepatocellular carcinoma.

DNA methylation profiles such as low CpG methylation levels in the region 200 bp upstream from the transcription start site (TSS200), the first coding exon and CpG island were obtained. CHH methylation was observed more frequently on the anti-sense strands than on the sense strands in the 5' untranslated region (UTR), first intron, gene body and 3' UTR. non-CpG methylation was inversely correlated with gene expression levels.

Personal differentially methylated regions (pDMRs) were observed less frequently in TSS200, the first coding exon, TSS1500, and CpG island where CpG methylation levels were low, indicating that the regions important for expression regulation may be protected from personal variations of CpG methylation. Histone modification profiles of pDMRs differed considerably among samples. pDMRs were observed around the TSSs of genes whose expression levels are reportedly regulated by CpG methylation, such as LRP1B, RASGRF2 and TFF1. pDMRs were located more frequently in the vicinity of loci showing genetic variation (single-nucleotide variations [SNVs] and/or insertions/deletions [indels]) than on all autosomes. Although further study is needed to clarify the molecular background of such genome-epigenome interaction, we speculate that SNVs and indels may affect the binding of non-coding RNAs and/or protein complexes that induce histone modification or CpG methylation alterations around the SNV and indel loci. MetaCore pathway analysis revealed that genes showing both genetic (SNVs and indels) and epigenetic (pDMRs) variations in multiple samples were significantly accumulated in signaling pathways participating in hepatocyte function and disease susceptibility.

Our data suggest that genetic variations may induce epigenetic variations and generate individual differences in the phenotypes of normal hepatocytes and/or determine disease susceptibility through variations in expression. After accumulation of numerous reference epigenome profile data in the IHEC database, comparison between IHEC data for normal cells and cancer cells may facilitate the accurate identification of cancer-specific epigenome profiles, which would be indispensable to the development of biomarkers and molecular targeted therapies.

#4518

4CSeq analysis of a breast cancer susceptibility locus on 6q25.1.

Sofie van Huffel,1 Robert C. Day,1 Jisha Antony,1 Judith Marsman,1 Julia A. Horsfield,2 Anita K. Dunbier1. 1 _University of Otago, Dunedin, New Zealand;_ 2 _University of Otago, Dunedin, OR_.

Single nucleotide polymorphisms within the genomic region immediately upstream of ESR1, a gene on 6q25.1 encoding the estrogen receptor α (ER), have been found to be associated with increased risk of breast cancer1,2. Three genes within this region, CCDC170, RMND1 and ARMT1 are co-expressed with ESR1 in ER+ve breast cancers and CCDC170 expression is inversely associated with proliferation and recurrence free survival in a tamoxifen-treated patient cohort3. In addition, gene fusions between ESR1 and CCDC170 have been associated with aggressive and endocrine-resistant luminal B tumours4. This study aimed to investigate chromatin structure within the 6q25.1 region using targeted chromatin confirmation capture (3C) and circularised chromatin confirmation capture analysis sequencing (4Cseq) to explore whether the spatial organisation of the chromatin might be associated with the regulation of genes and mutational patterns at this locus.

To carry out 4Cseq analysis, chromatin from MCF7 cells was cross-linked, enzymatically digested, ligated, re-digested, circularised and then amplified before the purified PCR products were sequenced on an Illumina HiSeq2500. After the sequence reads were trimmed and processed, FourSig software was used to identify cis and trans interactions from the nine anchor points used in the assay. For 3C analysis, chromatin was cross-linked, digested, ligated, and analysed by quantitative real-time PCR. Data was normalised to a synthetically generated fragment.

Analyses revealed that the ESR1 promoter region contacted a number of different upstream regions including the promoters of RMND1 and CCDC170. A high frequency of interactions was observed in the intergenic region between CCDC170 and ESR1 where a number of SNPs associated with breast cancer risk have been found. In addition, contact between the 3' end of the ESR1 gene and the 3' end of CCDC170 suggests that there may be a regulatory interaction in this region. Contacts were also observed within CCDC170, immediately adjacent to the point at which gene fusions between ESR1 and CCDC170 have previously been observed4. Overall, these data support the hypothesis that chromatin structure may drive the co-expression of the genes at this locus and suggests that some interactions within the region occur in areas where genomic rearrangements in metastatic breast cancers have been observed.

References:

1.

Turnbull C et al. Nature Genetics 2010;42:504-7.

2.

Zheng W et al. Nature Genetics 2009;41:324-8.

3.

Dunbier A et al. PLoS Genetics 2011;7:e1001382.

4.

Veeraraghavan J. et al., Nature Communications 2013;5:4577.

#4519

Nanoscale ChIP-seq analysis in primary hepatocellular carcinoma tissues reveals tumor-specific chromatin deregulation for oncogene activation.

Feng Wu,1 Yingying Lee,2 Sau Dan Lee,3 Patrick Tan,4 Nathalie Wong,5 Kevin Yuk-Lap Yip,3 Kai F To,6 Alfred Sze Lok Cheng2. 1 _Departments of Anatomical and Cellular Pathology and School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong;_ 2 _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong;_ 3 _Dept. of Computer Science and Engineering, The Chinese University of Hong Kong, Hong Kong;_ 4 _Duke-NUS Graduate Medical School Singapore, Singapore;_ 5 _Departments of Anatomical and Cellular Pathology,The Chinese University of Hong Kong, Hong Kong;_ 6 _Departments of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong_.

Introduction: Emerging evidence that epigenetics converts alterations in nutrient and metabolism into heritable pattern of gene expression has profound implications in understanding human physiology and diseases. Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of metabolic syndrome including obesity and diabetes which elevate the risk of hepatocellular carcinoma (HCC). The metabolites derived from excessive insulin, glucose and lipid may perturb epigenetic gene regulation through DNA methylation, histone modifications, and RNA interference, leading to activation of pro-inflammatory signaling and oncogenic pathways[1]. Transcriptional and chromatin patterns are mostly interrogated by chromatin immunoprecipitation (ChIP). However, conventional ChIP protocols necessitate the use of large numbers of cells and usually limit to the studies in cell lines, which may display epigenetic patterns distinct from primary tumors.

Aims and methods: To study chromatin deregulation in primary human NAFLD-related HCC tumor and adjacent non-tumor tissues, we performed nanoscale ChIP sequencing (nano ChIP-seq)[2], which allows genome-wide location of histone modifications for scanty materials, of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac) for active promoters and enhancers, respectively. We also integrated the nano ChIP-seq profiles with transcriptomic data from TCGA and in-house cohorts.

Results and summary: We found that primary human HCC tumors were associated with increased H3K4me3 occupancy, which occurred predominantly nearby the transcription start sites. H3K4me3-occupied promoters were enriched with signal transducer and activator of transcription (STAT) binding sites, which is in accordance with the crucial role of interleukin 6/STAT3 signaling in NAFLD-HCC initiation and progression. Moreover, tumor-specific H3K4me3 and H3K27ac profiles correlated with gene over-expression, including novel STAT3-driven oncogenes e.g. UBE2C and PLK1 in HCC. In summary, our findings demonstrated the feasibility of profiling chromatin using limited HCC tissues and identified aberrant promoter and enhancer regulatory elements associated with NAFLD-related hepatocarcinogenesis.

Acknowledgements: This project was supported by the University Grants Committee through the Collaborative Research Fund C4017-14G, Theme Based Research Scheme T12-403/11-1 and General Research Fund 14102914.

References:

1. Tian, Y., et al., Epigenetic regulation of hepatocellular carcinoma in non-alcoholic fatty liver disease. Semin Cancer Biol, 2013. 23(6 Pt B): p. 471-82.

2. Muratani, M., et al., Nanoscale chromatin profiling of gastric adenocarcinoma reveals cancer-associated cryptic promoters and somatically acquired regulatory elements. Nat Commun, 2014. 5: p. 4361.

#4520

The effect of EPZ011989, an enhancer of Zeste homolog 2 inhibitor, in acute myeloid leukemia.

Sydney Fobare,1 Cecelia Miller,2 Shelley J. Orwick,2 Heike Keilhack,3 Erin Hertlein,2 John C. Byrd2. 1 _Hendrix College, Conway, AR;_ 2 _The Ohio State University, Columbus, OH;_ 3 _Epizyme, Inc, Cambridge, MA_.

Enhancer of Zeste Homolog 2 (EZH2) is a catalytic core subunit of the Polycomb repressive complex 2, PRC2. EZH2 catalyzes the tri-methylation of lysine 27 on histone H3 (H3K27me3) which often results in the silencing of associated gene promoters. The over-expression of EZH2 is associated with cancer progression and poor prognosis in solid tumors and hematological malignances. EZH2 mutations in lymphoma generally are activating whereas those in myelodysplasia and acute myeloid leukemia are typically loss of function. Additionally, EZH2 mutations are not isolated to a hotspot and therefore discerning phenotype is challenging. The purpose of this study was to determine if the inhibition of EZH2 induces cell differentiation or apoptosis in AML, and to determine the significance of EZH2 mutations in AML patients. As H3K27me3 deposition is catalyzed by EZH2, we hypothesized that analysis of H3K27me3 levels could differentiate an inactivating EZH2 mutation. We analyzed H3K27me3 levels in AML patient samples with wild type (N=5) and mutant (N=3) EZH2, and found that overall H3K27me3 is reduced in the mutant patient samples. Furthermore, the sample with the highest variant allele frequency (VAF) of the EZH2 mutation had noticeably lower levels of H3K27me3 compared to those that had a lower VAF. This finding demonstrates that diminished H3K27me3 levels can predict the presence of an inactivating EZH2 mutation in AML cells. We next assessed if the EZH2 tool molecule EPZ011989 produced a similar phenotype in wild-type EZH2 AML cell lines (Kasumi-1, MOLM-13, and MV4-11). EPZ011989 inhibited H3K27me3 in all of the cell lines at a concentration of 0.625 μM after only four days of treatment. At these concentrations and this time point we demonstrated no change in viability among these three cell lines and only minimal proliferation inhibition in MV4-11 and MOLM-13 cells. Longer time points are currently being investigated as EZH2 inhibitors often induce anti-proliferative effects only after longer incubation periods.

Therefore, our results support that inactivation of EZH2 with EZP011989 in AML cell lines induces the same cell-biochemical consequence (i.e. H3K27me3 loss) associated with EZH2 mutations in AML patients. This potentially will allow diverse pharmacologic studies in cell lines relative to gene targets and also synergistic lethality studies utilizing genetic and pharmacologic screening techniques to potentially develop therapeutic agents to treat this subtype of AML.

#4521

Substrate profiling for SETD8 reveals novel protein targets involved in DNA replication and DNA damage response.

Fabio Pittella Silva, Gil Blum, Chamara Senevirathne, Minkui Luo. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Protein lysine methyltransferases utilize S-adenosyl-L-methionine (SAM) to catalyze the addition of methyl groups to specific lysine residues in both histones and non-histone proteins. Recent evidence indicates that their dysregulation is closely linked to the development and progression of cancer, making them potential therapeutic targets. SETD8 is the sole methyltransferase responsible for mono-methylation of histone H4 lysine 20 (H4K20me1). Its activity is essential for cell viability and has been associated with many essential cellular processes including transcriptional regulation, heterochromatin formation, genomic stability and cell-cycle progression. SETD8 is overexpressed in several types of cancer and may play important roles on malignancy through its trans-methylation activity or by direct interaction with its binding partners. It has been reported that the association between SETD8 and TWIST, a regulator of epithelial-mesenchymal transition, enhances the invasiveness of breast cancer cells. In addition, it was shown that SETD8-mediated methylation of p53 suppresses p53 dependent transcription activation in cancer cells. Despite the identification of a few non-histone proteins such as p53, PCNA and Numb as SETD8's substrates, a comprehensive investigation of new potential substrates is lacking to date. Here we used Bioorthogonal Profiling of Protein Methylation (BPPM) with engineered SETD8 and synthetic SAM analogues to profile novel substrates for SETD8. We identified SETD8 mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this distinct chemical moiety into target proteins for subsequent pulldown and identification of the modified substrates. The BBPM technology applied here revealed previously known substrates of SETD8 as well as novel SETD8 targets. Among the main targets of our interest are those involved in DNA replication and DNA damage response. Our finding is expected to bring new perspectives on the biological importance of SETD8 as well as on its role in carcinogenesis.

#4522

Epstein-Barr virus infection suppresses the DNA repair mechanisms in nasopharyngeal epithelial cells via reduction of the H3K4me3 mark.

Maria L. Lung, Arthur KL Cheung, Wei Dai, Merrin ML Leong, George SW Tsao. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

Introduction: Epstein-Barr Virus (EBV) is an oncovirus, which contributes to development of various cancers, including nasopharyngeal carcinoma (NPC). EBV latent proteins play critical roles in modulation host cell histone modifications in order to regulate its signaling pathways. However, the role of EBV in regulation histone modifications in epithelia cell system is still not very clear. Further studies are necessary to characterize the functional role of EBV in regulating epithelial cell modifications.

Aim: in this current study, we aim to investigate the role of EBV infection in regulating a promoter and transcription activation histone marker, H3K4me3, in the host cell genome.

Methodologies: Chromatin immunoprecipitation sequencing (ChIPseq) was performed by using the next-generation sequencing approach. The ChIP reactions were prepared by utilizing the antibody targeting the H3K4me3 mark in two pairs of immortalized non-tumorigenic nasopharyngeal epithelial (NPE) cell lines, which were artificially infected with (550, 550-EBV, 361, and 361-EBV). The ChIPseq results were validated by the ChIP-QPCR and RT-QPCR.

Results: A total of 1747 genes show losses of H3K4me3 in both sets of EBV-infected NPE cell lines. Among them, 628 (36%) genes show losses of H3K4me3 in promoter regions. Interestingly, a total of 18 DNA damage repair signaling members in the base excision repair (BER), homologous recombination, non-homologous end-joining, and the mismatch repair pathways showed significant losses of H3K4me3 in EBV-infected NPE cells. Based on utilizing the DAVID annotation tool for pathway analysis, members in the BER pathway were significantly enriched (FDR= 0.0709, cut-off<0.1). The ChIPseq results were validated by ChIP- and RT-QPCR and showed significant losses of the H3K4me3 and expression of the BER members in both sets of EBV-infected NPE cell lines. The clinical significances were further confirmed by detection of significant down-regulation of the BER members in NPC paired biopsies.

Conclusion: EBV infection induces changes of host cell H3K4me3 levels and results in down-regulation of the BER members.

Acknowledgement: This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, People's Republic of China: Grant number AoE/M-06/08 to MLL and the Seed Funding Programme for Basic Research of the University of Hong Kong: Grant number 201308159003 to AKLC.

#4523

Decreased H2B monoubiquitination is associated with enhanced proliferation and resistance to apoptosis in human lung cancer.

Keqiang Zhang,1 Jinhui Wang,2 Xiwei Wu,2 Theresa A. Reno,1 Tommy R. Tong,3 Jae Kim,1 Dan Raz1. 1 _Division of Thoracic Surgery, City of Hope National Medical Center, Duarte, CA;_ 2 _The Integrative Genomics Core lab of Department of Molecular Medicine, City of Hope National Medical Center, Duarte, CA;_ 3 _Department of pathology, City of Hope National Medical Center, Duarte, CA_.

Monoubiquitination of histone H2B (H2Bub1) is an important chromatin modification that plays multiple roles in transcription, mRNA processing, DNA repair, cell differentiation, and maintenance of stemness. Recent studies have indicated that dysregulation of H2Bub1 and the resulting alterations of global gene expression are key contributors in cellular transformation and metastasis. However, the importance of H2B ubiquitination in lung cancer is unknown. Here, by using immunohistochemical analysis (IHC), we have demonstrated that the protein level of H2Bub1 is frequently decreased in a cohort of human lung cancer tissues in comparison to paired adjacent normal tissues. We also found that decrease in H2Bub1 is associated with increased cellular proliferation, as measured by Ki67, and is associated with decreased overall survival. Furthermore, using RNA-seq and transcriptional profiling analysis, we identified that reduction of H2Bub1 via attenuating the expression of histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) leads to significant changes of global gene expression, modulating multiple malignancy-promoting genes, including c-Met, E2F2, and BMF. Signaling pathways including p53, DNA replication, DNA repair, and cellular proliferation, and regulation of apoptosis in lung cancer cells are also altered. Additionally, we have further shown that decrease of H2Bub1 by RNF20 knockdown significantly promotes growth, migration/invasion, and strongly enhances resistance to cytotoxic chemotherapy-induced apoptosis in lung cancer cells. Our findings suggest that dysregulation of H2Bub1 signaling may play an important role in growth and drug resistance of lung cancers, and may potentially serve as a prognostic biomarker and therapeutic target for lung cancer patients.

#4524

A rational approach for the discovery of inhibitors of NSD2 for the treatment of multiple myeloma.

Claudia Fromond, Xavier Espanel, Severine Estevez, Vanessa Adarbes, Stephanie Bocart, Bruno Loillier, Anne Soude, Nathalie Urban, Frederic Bell, Philippe Masson, Benaïssa Boubia, Pierre Broqua, Christian Montalbetti. _Inventiva, Daix, France_.

Multiple myeloma (MM) is a plasma cell malignancy which accounts for approximately 10% of hematologic malignancies. Despite the introduction of new therapeutic agents, MM remains incurable and nearly all patients ultimately relapse. About 20% of MM are due to a chromosomal translocation t(4;14) leading to overexpression of the NSD2 histone methyltransferase. NSD2 catalyzes dimethylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Several studies have shown that in MM harboring the translocation t(4;14), oncogenic programming is dependent on the methyltransferase activity of NSD2. Thus, NSD2 inhibitors are potential therapeutics for this devastating cancer

Using the AlphaLisa™ technology, we screened 240,000 compounds coming from our proprietary library. The assay was based on the detection of H3K36me2 on nucleosome by a specific antibody. False positives hits were removed by a technological counterscreen, and compounds with redox or metal contamination activity were excluded. For confirmation purposes, an orthogonal counter-screen based on 3H SAM incorporation was performed. The IC50 obtained for one of the best compounds across the AlphaLisa and 3H SAM incorporation are very similar, showing values of 0.15 µM and 0.29 µM respectively for compound 1.

Binding affinities measured by MST and SPR for compound 1 were consistent with the IC50 observed in the biochemical assays KD= 0.19 µM for MST and 1.7 µM by SPR. This compound was also shown to be a good binder by NMR (STD), and further experiments are planned to elucidate the selectivity and MOA of this series.

To our knowledge, no NSD2 inhibitor have been identified to date despite several screening efforts performed by other groups. We believe that our hits are promising starting points to generate potent and selective NSD2 inhibitors.

#4525

Distinctive chromatin organization of p53 binding sites in normal and cancer cells.

Feng Cui, Peter LoVerso, Feifei Bao. _Rochester Institute of Technology, Rochester, NY_.

The tumor suppressor p53 is arguably one of best known proteins in humans, which induces or represses hundreds of genes to prevent tumor development. Now, it becomes clear where p53 binds across the human genome and how it regulates gene expression. Recent studies have established that p53 can access its target sites on the nucleosomal DNA and the rotational positioning of a nucleosome is critical to determine the accessibility of p53 sites. We have found that p53 response elements (REs) associate with cell cycle arrest such as p21 REs are likely to be exposed on the nuclesomal surface. However, it remains unclear if other chromatin elements such as histone modifications and DNA methylation affect p53 in vivo binding and if such chromatin organization differs in normal cells versus cancer cells.

We analyzed the pre-existing chromatin landscape of thousands of p53 binding sites identified so far in normal (GM12878) and cancer (K562) cells. The p53 sites derived from the normal cells are often found in the vicinity of the transcription start sites (TSSs) of nearby genes, characterized with relative low nucleosome occupancy, high levels of transcriptionally active histone marks such as H3K4me3 and low levels of DNA methylation. By contrast, the p53 sites found in the cancer cells are located remotely from TSSs, with relatively high nucleosome occupancy, low levels of H3K4me3 and DNA methylation. The shift in p53 genomic binding patterns in the normal and cancer cells reflects the cancer-associated epigenetic dysregulation. Overall, in contrast to most of DNA-binding transcriptional factors, p53 binding sites occur in nucleosome-enriched regions. These findings indicate that p53 belong to a set of transcription factors including the pluripotency factors Oct4, Sox2 and Klf4 that can directly interact with nucleosomes in the context of chromatin.

#4526

Proscillaridin A effects on chromatin modifications and oncogene degradation in acute lymphoblastic leukemia.

Gregory Armaos, Simon Jacques-Ricard, Elodie Da Costa, Annie Beaudry, Noël J-M Raynal. _Université de Montréal, Montréal, Quebec, Canada_.

Acute lymphoblastic leukemia (ALL) represents approximately 25% of all pediatric cancers diagnosed every year. In about 80% of cases, pediatric patients will attain an event-free 5-year survival. Unfortunately, patients who are resistant to treatment or who relapse have a poor prognosis. Hence, novel therapeutic approaches are necessary to increase survival rates. Epigenetic alterations, such as DNA methylation and histone modifications, are involved in disease development, progression, and in particular, resistance to treatment. These reversible alterations represent additional targets in ALL. Recently, we discovered candidate epigenetic drugs in FDA-approved drug libraries. We hypothesize that one drug in particular, the cardiac glycoside proscillaridin A, has both epigenetic and anti-cancerous properties in preclinical models of ALL and can therefore be repositioned for the treatment of the disease. To test our hypothesis, we treated two ALL cell lines Nalm-6 (pre-B ALL) and Molt-4 (T-ALL) in vitro with clinically relevant concentrations of proscillaridin A and analyzed cell growth, cell cycle, gene expression and chromatin modifications. We observed dose-dependent growth inhibition in all cell lines, with IC50 values of 3.0 and 2.3 nM in Nalm-6 and Molt-4, respectively. Our results using BrdU staining indicate a block in the G2/M phase of the cell cycle. By western blot, we detected a reduction in histone acetylation levels and in histone modifying enzymes CBP and Tip60, as well as the c-myc oncogene. These promising results illustrate the perspective of using the cardiac glycoside proscillaridin A as a novel epigenetic drug for the treatment of relapsed or refractory ALL.

#4527

Cellular stress response 1 down-regulates the expression of epidermal growth factor receptor and platelet-derived growth factor receptor through inactivation of splicing factor 3A3.

Jian-Hua Luo, Ze-Hua Zuo, Yan P. Yu. _University of Pittsburgh School of Medicine, Pittsburgh, PA_.

Cellular stress response 1 (CSR1) is a tumor suppressor gene that plays an important role in regulating cell death. In this report, we showed that the N-terminus of CSR1 interacts with splicing factor 3A, subunit 3 (SF3A3). The SF3A3 binding motif was identified in the region of amino acids 62-91 of CSR1 through cell-free binding analyses. The interaction between CSR1 and SF3A3 led to migration of SF3A3 from nucleus to cytoplasm. The cytoplasmic redistribution of SF3A3 significantly reduced the splicing efficiency of epidermal growth factor receptor and platelet-derived growth factor receptor. Induction of CSR1 or down-regulation of SF3A3 also significantly reduced the splicing activity of oxytocin reporter gene both in vivo and in vitro. Mutant CSR1 that lacks the SF3A3 binding motif contained no RNA splicing regulatory activity, while the peptide corresponding to the SF3A3 binding motif in CSR1 interfered with the wild type CSR1 mediated inhibition of RNA splicing. Interaction of CSR1 and SF3A3 is essential for CSR1 mediated cell death. To our knowledge, this is the first report demonstrating that RNA splicing is negatively regulated by redistribution of a splicing factor.

#4528

Chromatin regulation by ING3 leads to tumor suppressive effects in pancreatic cancer through distinct signaling pathways.

Marina Koutsioumpa,1 Maria Hatziapostolou,2 Christos Polytarchou,3 Angelos Oikonomopoulos,4 Swapna Mahurkar-Joshi,1 Sara Huerta-Yepez,5 Belen Tirado-Rodriguez,5 Dimitrios Karavias,6 Helen Kourea,6 George A. Poultsides,7 David W. Dawson,8 Timothy R. Donahue,9 Dimitrios Iliopoulos1. 1 _Center for Systems Biomedicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA;_ 2 _Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom;_ 3 _Interdisciplinary Biomedical Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom;_ 4 _Center for Inflammatory Bowel Diseases, Melvin and Bren Simon Digestive Diseases Center, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA;_ 5 _Unidad de Investigacion en Enfermedades Oncologicas, Hospital Infantil de Mexico, Mexico City, Mexico;_ 6 _Department of Pathology, School of Medicine, University of Patras, Patras, Greece;_ 7 _Department of Surgery, Stanford University School of Medicine, Stanford, CA;_ 8 _Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA;_ 9 _Department of Surgery, Division of General Surgery, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA_.

Background: Despite our increased knowledge of the molecular events underlying the multi-step development of pancreatic cancer (PanCa), clinically meaningful improvement in survival rates has not yet been achieved. A growing body of evidence supports that pancreatic tumorigenesis is not only led by genetic alterations but also aberrant epigenetic regulation. Identification of epigenetic drivers of the clinical disease is of major importance, as it could potentially offer a means to interfere with novel targets in PanCa therapy.

Methods: Gene expression microarrays were employed in PanCa tissues and adjacent uninvolved tissues. mRNA and protein levels were assessed by qRT-PCR and immunohistochemical analysis of tissue microarrays in extended cohorts of patients. RNAi interference assays or lentiviral expression vector systems were applied for knockdown/overexpression experiments, as evidenced by qRT-PCR and Western Blot Analysis. Molecular analysis, cell proliferation, invasion and colony formation assays were conducted in genetically modified cells. Subcutaneous xenograft mouse models were used to evaluate tumor growth in vivo. Immunoprecipitation followed by mass-spectroscopy analysis was used to evaluate protein-protein interactions. Histone H4 at lysine 16 acetylation (H4K16ac) levels were assessed by Western Blot analysis, while chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) was performed for mapping protein-DNA interactions.

Results: Differential expression analysis of chromatin regulators in PanCa versus normal tissues shows that 27 epigenetic molecules are significantly deregulated (>1.5 fold, P<0.05). The reduced expression of inhibitor of growth family member 3 (ING3) was further validated by qRT-PCR and immunohistochemical analysis in three different cohorts of patients. Importantly, transient or stable depletion of ING3 promotes cell proliferation, anchorage-independent growth, invasiveness and in vivo tumor growth, while its overexpression has opposite effects. Cells lacking or overexpressing ING3 were utilized to reveal ING3-related gene signature. Dual-specificity phosphatase 5 (DUSP5) and c-Met were identified as ING3-downstream targets that mediate different ING3-regulated PanCa cellular properties. Interestingly, genetic manipulation of ING3 leads to profound effects in H4K16ac. Moreover, ING3 expression seems to affect p53 binding to gene promoters, possibly due to the formation of histone H1-ING3 complex that antagonizes for H1-p53 interaction.

Conclusions: Conclusively, we provide evidence that PanCa is characterized by loss of the chromatin regulator ING3 and we elucidated the tumor suppressive role of the latter in PanCa cell growth and invasion in vitro and in vivo.

#4529

Tailoring approaches for global epigenome analysis from archival formalin-fixed paraffin-embedded tissue samples.

Sudipto Das,1 Bruce Moran,2 Dominiek Smeets,3 Gillian Peuteman,3 Rut Klinger,2 Bozena Fender,4 Kate Connor,1 Matthias Ebert,5 Timo Gaiser,5 JHM Prehn,6 Orna Bacon,7 Elaine Kay,7 Bryan Hennessy,7 Verena Murphy,8 William Gallagher,9 Annette Byrne,6 Diether Lambrechts,3 Darran O'Connor1. 1 _Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 3 _Department of Translational Genetics, VIB, K.UK, Leuven, Belgium;_ 4 _OncoMark Ltd, NOVA UCD, Dublin, Ireland;_ 5 _University of Heidelberg, Mannheim, Germany;_ 6 _Department of Physiology, Royal College of Surgeons in Ireland, Dublin, Ireland;_ 7 _Beaumont Hospital, Dublin, Ireland;_ 8 _Irish Clinical Oncology Research Group, Dublin, Ireland;_ 9 _Conway Institute, University College Dublin, Dublin, Ireland_.

Novel DNA extraction methodologies allow the use of archival material from formalin fixed paraffin embedded (FFPE) tissue samples in genomic studies. A major limitation of this source of DNA is the fragmented nature and low overall yield generally obtained from clinical materials, making downstream applications such as epigenetic analysis challenging. Previous published attempts have focussed on smaller regions of CpG islands, such as the use of Illumina's 450k arrays (which measure methylation at approximately 485,000 sites). The objective of this study was to optimize experimental and analytical workflows that allow effective interrogation of global DNA methylation profiles from FFPE samples.

Methylation capture was conducted on DNA from matched FFPE and fresh frozen samples from the same metastatic colorectal cancer patient as well as two colorectal cancer cell lines, using the SeqCap Epi (Roche) methyl capture system. The custom capture designed includes 5.5 million CpG sites across the genome, a greater than 10-fold increase compared to previously published studies. The wet-lab protocol was robustly optimized for several parameters, such as overall yield and bisulphite conversion efficiency (measured by shallow-read next generation sequencing). A data analysis pipeline composed of the bisulfite-converted DNA aligner BWA-meth, as well as in-house Perl and R scripts, was used to generate detailed methylation maps for individual sample types in order to identify differentially methylated regions (DMRs), which were further validated using targeted bisulphite sequencing for selected loci.

Preliminary analysis of the data revealed 98% bisulphite conversion efficiency and low PCR duplicate rate (4-5%) across both sample types. Intriguingly, we observe an 80% concordance between the overall DNA methylation profiles between FFPE and fresh frozen samples, with a 60% overlap between the FFPE and fresh frozen samples in terms of direction of methylation i.e. hyper or hypomethylation. Mapping overall methylation levels to CpG 'resorts' (+/- 4kb of CpG islands) indicated ~10% of methylation occurred in these regions totalling 245MB. Known genomic features, including exons, promoters of coding/non-coding genes and enhancers, contained ~30% (1.5 million) of the methylated positions. In addition, this methodology also allowed us to identify C to T transitions across all samples, a common artefact of formalin fixed samples. The analysis revealed that a significantly low proportion of C to T transitions were detected across all samples, the levels of the transitions being consistent between the two sample types. Taken together, these results demonstrate a robust and novel approach to generate DNA methylation profiles from difficult-to-handle, but frequently available material, thus establishing a suitable platform for a whole methylome profiling from archival samples.

#4530

EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing.

Miljana Tanic,1 Simon Rodney,1 James Barrett,1 Hannah Parker,2 Andrew Feber,1 Stephan Beck1. 1 _UCL Cancer Institute, University College London, London, United Kingdom;_ 2 _Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland_.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes necessary for cellular differentiation and plays a crucial role in gene regulation of many oncogenes and tumor suppressor genes. Aberrant DNA methylation at the level of both individual gene promoters and on a genome-wide scale has been heavily implicated in cancer initiation and progression.

Whole genome bisulfite sequencing (WGBS) is the gold standard method for studying alterations in DNA methylation at a single base pare resolution. However, the high cost of WGBS is still prohibitive for high-throughput observational studies on large sample cohorts. Moreover, the WGBS data analysis requires substantial IT resources and a complex bioinformatics analysis. Targeted bisulfite sequencing enables a cost-effective and focused analysis of genomic regions of interest where most of DNA methylation alterations occur, such as promoters, CpG islands and shores, and enhancer regions. Currently, there are 2 main approaches for targeted bisulfite sequencing of medium-to-large genomic regions, reduced representation bisulfite sequencing (RRBS) and hybridization-based enrichment.

We have performed a comparison of 5 commercially available platforms for RRBS and in-solution hybridization-based enrichment panels. We analyzed a panel of two cancer cell lines (Hela and Coriell NA12878) and a normal blood reference gDNA sample, in duplicate on each platform using recommended sample inputs, and compared the results to WGBS data. Given that sample input requirements differ significantly between RRBS (~100ng or less), and hybridization based methods (500ng - 1 µg), we have analyzed the normal blood reference gDNA sample at 500ng input in duplicate across all platforms. All samples were sequenced to a ~30x depth on an Illumina HiSeq 2500 sequencer. We compared the genomic coverage, precision and off-target effects and overall concordance in methylome profiles for each method.

In summary, here we provide a comprehensive comparison of the performance of 5 different commercially available methods for medium to large scale targeted bisulfite sequencing, facilitating information to researchers for selection of appropriate method depending on required application.

#4531

Dynamic long-range chromatin interaction controls expression of Il-21 in CD4+ T cells.

Yeeun Choi. _Yonsei University, Seoul, Republic of Korea_.

IL-21, a member of four-α-helical bundle type I cytokines, causes many pleiotropic effects, including terminal differentiation of B cells, immunoglobulin production, and the development of follicular helper T cells. IL-21 also promotes differentiation of Th17 cells and is implicated in the development of autoimmune disease. IL-21 can be strongly induced in CD4+ T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il-21 gene at the chromatin level. Here, we identified insulator elements in the Il-21 locus with enhancer-blocking activity, which constitutively bind CTCF and cohesin. In naïve CD4+ T cells, these upstream and downstream CTCF-binding sites interact with each other to make a DNA loop; however, the Il-21 promoter does not interact with any cis-elements in the Il-21 locus. In contrast, stimulation of CD4+ T cells with IL-6 in the presence of αCD3 and αCD28 leads to recruitment of STAT3 and NFAT transcription factors to the promoter, as well as to novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity, facilitating IL-21 expression. The long-range interaction between the promoter and distal enhancer region was dependent on JAK/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. In addition, we demonstrated that CTCF was required for optimal IL-21 expression and formation of chromatin looping in the Il-21 locus. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of Il-21 gene.

#4532

Deregulation of retinoic acid (RA) epigenetic control of sphingolipid signaling.

Stefano Rossetti, Vincenzo Gagliostro, Nicoletta Sacchi. _Roswell Park Cancer Institute, Buffalo, NY_.

All-trans retinoic acid (RA), the bioactive derivative of vitamin A, can paradoxically exert both anti-cancer and cancer-promoting actions. According to our studies, RA promotes breast cancer cell growth and invasion whenever it fails to exert its genome-wide epigenetic control of transcription via the RA receptor alpha (RARA). Factors that negatively affect RARA genomic function lead to genome-wide deregulation of the transcriptome epigenetically regulated by RA, including RARA-target genes controlling critical sphingolipid functions. We show here that functional inhibition of genomic RARA concomitantly leads to epigenetic repression of neutral sphingomyelinase 2 (nSMase2/SMDP3), a critical enzyme involved in the synthesis of pro-apoptotic ceramide, and upregulation of S1PR1, a key receptor of sphingosine-1-phosphate (S1P), a sphingolipid with pro-proliferative and pro-invasive activity. Selective activation of S1PR1 recapitulates RA pro-proliferative/pro-invasive action, while selective inhibition of S1PR1 counteracts it. Apparently, activation of the S1P-S1PR1 axis, combined with lack of RARA-mediated epigenetic control of SMPD3-ceramide axis, contributes to determine RA tumorigenic action.

This study was supported by the NCI R01 CA127614 grant (NS).

#4533

Disruption of intrachromosomal interactions by deficient cohesin initiate epithelial-mesenchymal transition and stemness in cancer.

Jiyeon Yun. _Seoul National Univ. Cancer Research Inst., Seoul, Republic of Korea_.

PURPOSE

Although EMT-MET process is a crucial reversible event in development of human tumorigenesis controlled by complex networks, very little is known about how EMT-MET occurs reversibly and timely in cancer. Recently, different levels of Rad21 protein, a subunit of cohesin high-order-chromatin-architectural complex, between mesenchymal and epithelial cancers have been described.

METHODS

To elucidate how Rad21 determines EMT states, we silenced Rad21 in epithelial cancers using lentiviral shRNAs. We performed microarray to compare transcriptional changes by Rad21-KD. Additionally, we measured cell cycles and mitotic chromosome segregations to rule out the role of Rad21 as a sister chromatid segregation molecule. We observed cell morphology through confocal microscopy and checked high-order-chromatin structures using Chromosome-Conformation-Capture (3C) and Chromatin immunoprecipitation (ChIP) assay.

RESULTS

We identified high-order-chromatin structures mediated by Rad21 plays critical regulatory roles in transcription of EMT related genes for the first time. Rad21-KD in epithelial cancers caused reduction of Rad21 enrichment on TGFB1 and ITGA5 genes, leading to disruption of the-gene-specific-chromatin-looping structures. This dramatically recruited PolII and AcH3, active transcription marks, causing rapid induction of the gene transcriptions. Indeed, induced EMT-related genes directly led to morphological changes with EMT traits. We ultimately found that cancer stem cell-like cells showed same results with mesenchymal cancer and Rad21-KD epithelial cells.

These findings indicate that the initiation of EMT in cancer might crucially require changes in high-order-chromatin structures of EMT-related genes mediated by cohesin, contributing to rapid and precise reversible changes in EMT or MET state to adapt to phenotypes of human cancer development.

#4534

Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer.

Mohammad Imran Khan, Abid Hamid, Vaqar Mustafa Adhami, Chloe Lang, Hasan Mukhtar. _University of Wisconsin-Madison, Madison, WI_.

Prostate cancer (PCa) is the most common cancer and a major cause of cancer related deaths in men in North America. The phosphatase and tensin homolog gene (PTEN) is frequently inactivated in PCa. Approximately 40% of primary and 70% of metastatic PCa's have genomic alterations in the PI3K-signaling, mostly through the loss of PTEN. Majority of metastatic PCa associated with PTEN loss are treated with docetaxel; an FDA approved therapy for recurrent PCa. However, in PTEN null PCa docetaxel treatment enhances senescence and does not significantly impact tumor volume. These findings were further confirmed in PCa patients with decreased levels of PTEN. In order to understand the global impact of docetaxel in PTEN null PCa we performed an unbiased RNA-seq of prostate tissues harvested from docetaxel treated prostate specific PTEN null mice. Data analysis showed strong enrichment of chromatin modifying enzymes reactome, mainly PRC2 as one of the top ranked categories. Among them EZH2, an oncogenic methyltransferase was found to be significantly upregulated. Quantitative PCR analysis confirmed statistically significant upregulation of EZH2 in docetaxel treated mice compared to vehicle control. EZH2 upregulation was independent of androgen levels as both androgen dependent and independent cells showed striking upregulation of EZH2 when treated with docetaxel. Next, we confirmed the nature of the upregulated EZH2 (catalytic or non-catalytic) by docetaxel. EZH2 methyltransferase activity assay data showed significant induction of methyltransferase activity by docetaxel in both androgen dependent and independent conditions and in PTEN null mouse tissues. Further, we measured other methyltransferase marks i.e. H3K27me1&2, H3K4me3, H3K9me3 in the presence of docetaxel and found only specific upregulation of H3K27me3, confirming upregulation of only catalytic EZH2 by docetaxel. These data suggested a need for co-targeting EZH2 during docetaxel treatment in PTEN null PCa conditions. In order to provide a proof-of-principle we performed a tumor xenograft study in nude mice implanted with PTEN null PC3 cells. Combination of DZNeP (an EZH2 inhibitor) and docetaxel significantly inhibited tumor growth compared to each agent alone. Results obtained from the xenograft studies prompted us to perform a preclinical study in prostate specific PTEN null mice. Data obtained from this study clearly showed a significant reduction in number of high grade lesions in PTEN null tumors in combination group when compared to either docetaxel or DZNeP treated mice. We also observed clearance of senescent cells and accumulation of CD8+ T cells and classical dendritic (CD8+ Clec9a+ positive) cells. Our data suggests the necessity of designing a combination therapy of targeting EZH2 during docetaxel treatment specifically, which will likely increase the efficacy of docetaxel in PTEN null PCa.

#4535

SUV420H1 enhances the phosphorylation and transcription of ERK1 in cancer cells.

Theodore Vougiouklakis, Vassiliki Saloura, Yusuke Nakamura, Ryuji Hamamoto. _University of Chicago, Chicago, IL_.

The oncogenic protein ERK1, a member of the extracellular signal-regulated kinase (ERK) cascade, is a signaling molecule involved in various types of human cancer. Functional regulation by phosphorylation of kinases in the ERK pathway has been extensively studied, however methylation of the ERK1 protein has not been reported to date. Over the past decade, a number of protein methyltransferases have been identified and reported to methylate histone proteins as well as non-histone substrates related to various molecular functions that have pivotal roles in tumorigenesis. To identify candidate enzymes that may methylate ERK1, we performed an in vitro methyltransferase assay using various recombinant methyltransferases and found that SUV420H1 methylates ERK1. Mass spectrometry analysis validated our findings and identified ERK1 to be tri-methylated by SUV420H1 at lysines 302 and 361. Given that some protein methylation is known to affect protein phosphorylation, we then investigated the biological importance of the individual K302 and K361 methylated lysine residues on the phosphorylation status of ERK1. Substitution of methylation sites (lysine to arginine) diminished phosphorylation levels of ERK1 (Thr202 and Tyr204), supporting that methylation of these respective residues enhances ERK1 phosphorylation levels. Concordantly, siRNA-mediated SUV420H1 knockdown reduced phosphorylated ERK1 protein levels as visualized on immunoblot, while it interestingly also suppressed ERK1 at the transcript level. SUV420H1 knockdown caused a significant growth-suppressive effect in two squamous cell carcinoma head and neck (SCCHN) cell lines, and flow cytometric cell-cycle analysis showed a significant reduction in the proportion of cells at the S phase, and an increase of those at the G0/G1. Our results indicate that overexpression of SUV420H1 may result in activation of the ERK signaling pathway through enhancement of ERK phosphorylation and transcription, thereby providing new insights in the regulation of the ERK cascade in human cancer.

#4536

Polyunsaturated fatty acids alter the epigenetic landscape in triple-negative breast cancer (TNBC).

Amy M. Chattin, Mykelti O'Brien, Ronald S. Pardini. _University of Nevada, Reno, Reno, NV_.

Breast cancer is the second leading cause of cancer-related deaths in US women. The lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor II (HER2) in basal-like breast cancer subtypes, commonly referred to as triple-negative breast cancer (TNBC), account for 10-20% of cases and are associated with the poorest short-term prognosis due to a lack of targeted therapies. Epigenetic therapies are currently of interest to target TNBC, as many theorize epigenetic reprogramming is required for malignant transformation and invasion.

Research has shown omega-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA; C22:6, n-3) and eicosapentaenoic acid (EPA; C20:5, n-3) inhibit tumorigenesis, whereas linoleic acid (LA; C18:2, n-6) has been shown to promote neoplastic growth; supporting the association of increased cancer incidence with omega-6 PUFA dietary intake. Arachidonic acid (AA; C20:4, n-6) is associated with pro-inflammatory signaling processes, and therefore tumorigenesis; however, conflicting results exist in the literature across tumor types. PUFAs are bioactive nutrients known to have epigenetic effects within normal cells and recently, the ability of PUFA to alter DNA methylation and histone acetylation was reported in HCT-116 colon carcinoma (Cho, Y. et al, 2014).

Current research focuses on the potential of dietary intervention in TNBC as an epigenetic therapy. This study monitors epigenetic alterations in MDA-MB-231 TNBC in response to DHA, LA, EPA and AA. DHA, EPA and AA all elicit similar levels of apoptosis in MDA-MB-231, while LA promotes tumorigenesis. Initial results demonstrate promoter methylation changes (p<0.05) with a decrease in PTGS2 (prostaglandin-endoperoxidase synthase 2), and SOCS1(suppressor of cytokine signaling 1) and an increase in CDX2 (caudal type homeobox 2), HIC1 (hypermethylated in cancer 1), SPARC (Secreted Protein, Acidic, Cysteine-Rich (Osteonectin)), TSC1 (tuberous sclerosis 1), and WIF1 (WNT inhibitory factor 1) post DHA treatment. Studies are on-going to assess if the promoter methylation changes result in transcriptome and proteome level fluctuations. We are also investigating global gDNA methylation alterations, including 5-methylcytosine, in response to treatment. DHA, EPA, LA and AA exhibit dose-dependent effects on histone acetylation and HDAC class I/II activity. There is an increase in HDAC class I/II activity with the EC75, but not with the EC50 treatment for all PUFAs. Histone 3 global acetylation is decreased for all PUFA treatments at the EC75 versus EC50 dose. However, only LA and DHA have decreased H3 acetylation with an EC75 dose whereas EPA and AA have increased acetylation levels compared to the control. Research is ongoing to define a possible mechanism, but current data suggests PUFA may be useful as epigenetically-targeted cancer therapeutic agent or adjuvant.

#4537

Hexavalent chromium induces H4K16 hypoacetylation by NUPR1-mediated inactivation of histone acetyltransferase hMOF.

Qiao Yi Chen, Xiaoru Zhang, Thomas Kluz, Max Costa, Hong Sun. _New York University School of Medicine, Tuxedo Park, NY_.

Hexavalent chromium [Cr(VI)] is a well-established carcinogen linked to respiratory cancer and has been found at half of US toxic waste sites. In spite of considerable research efforts, the mechanism of Cr(VI)-induced carcinogenesis remains largely unknown. A growing body of evidence suggests that Cr is capable of inducing epigenetic changes, such as DNA methylation, histone modification, and microRNA. Acetylation of histone lysine residues neutralizes the positive charge from their side chains and decreases the affinity between histone tails and DNA, which subsequently leads to increased DNA accessibility and transcriptional activation in the gene promoter region. However, the effect of Cr(VI) on histone acetylation is still not clear. Here, we report that BEAS-2B cells exposed to 5 or 10 uM of Cr(VI) for 24 hours resulted in a striking and dose dependent decrease in H4K16ac. Co-treatment with Cr(VI) and Benzo(a)pyrene diolepoxide (BPDE) induced further decrease in H4K16ac. Cr (VI) transformed cells showed considerable decrease in H4K16ac compared to the control cells, which further indicates the correlation between Cr(VI) exposure and hypoacetylation of H4K16. H4K16 acetylation is known as an important determinant in higher-order chromatin structure and active gene expression. Male Absent on the First gene (MOF) is a H4K16 specific acetyltransferase. Our results indicate that Cr(VI) exposure significantly reduced MOF activity while its expression levels remained the same, thus suggesting that Cr(VI) may affect MOF activity rather than its expression. Moreover, Nuclear protein 1 (NUPR1), a MOF inhibitor, was increased in Cr(VI)-exposed cells. Depletion of NUPR1 in cells by small RNA interference rescued Cr(VI)- reduced H4K16ac. Together, our results indicate that Cr(VI) inhibits MOF activity through inducing NUPR1 expression which subsequently leads to H4K16 hypoacetylation. Since hypoacetylation of H4K16 is a known hallmark of human cancer, the results from our study presents strong evidence supporting the role of H4K16 hypoacetylation in chromium carcinogenesis.

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#4538

Heterochromatin protein 1 alpha modulates the invasive potential of malignant cells.

Xiaoyan Cui,1 Lance Stimpson,2 Tracy Hale,2 Alejandro Contreras1. 1 _M.D. Anderson Cancer Center, Houston, TX;_ 2 _Massey University, Palmerston North, New Zealand_.

Altered chromatin patterning is observed in neoplastic cells, with loss of heterochromatin associated with aggressive malignancies. Heterochromatin is the tightly packed form of chromatin characterized by limited transcription and is identified by the localization of Heterochromatin Protein 1 alpha (HP1 alpha) and Heterochromatin Protein 1 beta (HP1 beta). HP1 alpha and HP1 beta help to maintain chromatin organization during the cell cycle and regulate gene expression. We have previously shown that HP1 alpha and HP1 beta are both downregulated in papillary thyroid carcinomas and that both proteins are lost to a greater degree in 3 out of 4 metastatic lesions of papillary thyroid carcinomas. In addition, others have shown a loss of HP1 alphais associated with increased invasive potential and metastatic lesions in a range of human tumors.

By modulating the level of HP1 alpha in breast cancer cell lines we demonstrate a causal role for HP1 alpha in regulating invasive potential. To identify the molecular pathways through which HP1 alpha regulates this phenotype, RNA-seq was used to measure changes in both miRNA and mRNA expression after knock-down of HP1 alpha in the poorly invasive breast cancer cell line, MCF7. Reduction of HP1 alpha levels increased their invasive potential by 44% and this corresponded with differential expression of genes that promote an epithelial to mesenchymal transition (EMT). In addition, a tissue microarray containing 56 invasive breast carcinomas was immunohistochemically stained for HP1 alpha and HP1 beta . A majority of the breast carcinomas showed decreased expression of HP1 alpha when compared to HP1 beta (32/56 tumors). Taken together, these data suggest that loss of HP1 alpha results in a phenotype that is an intermediate of the EMT, and is supported by their altered cytoskeleton, cell-cell and cell-matrix interactions. HP1 alpha loss may confer a more aggressive, invasive potential in breast carcinomas.

By exploring these HP1 alpha δεπενδεντ pathways, we seek to further elucidate the regulatory signals that allow a malignant epithelial cell to undergo EMT and begin to invade surrounding tissue.

#4539

ATAD2 mediates DNA replication in cancer.

Seong Joo Koo,1 Amaury Ernesto Fernandez-Montalvan,1 Simon Holton,1 Oliver von Ahsen,1 Volker Badock,1 Sarah Vittori,2 Christopher J. Ott,2 James E. Bradner,2 Matyas Gorjanacz1. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _Broad Institute, Cambridge, MA_.

ATAD2 (ATPase family AAA domain-containing protein 2) is an epigenetic regulator which associates with chromatin through its Bromodomain specialized in Acetyl-Lys binding of histones. ATAD2 was also shown to directly associate with multiple transcription factors such as ERα, AR, E2F and MYC, and is believed to function as an oncogenic transcription factor in breast cancer.

Here, we propose that ATAD2 facilitates DNA replication. ATAD2 is specifically expressed in S and G2 phase during which it co-localizes with newly synthesized DNA. We found ATAD2 on nascent chromatin together with newly synthesized histone H4 acetylated on K12 and Proliferating Cell Nuclear Antigen (PCNA), a central protein coupling replication with chromatin restoration, but not on post-replicative chromatin. In line with these observations depletion of ATAD2 by siRNA led to reduced DNA replication, perturbed loading of PCNA onto chromatin and inhibition of cell proliferation. Interestingly, a brief cycloheximide treatment of the cells to prevent the deposition of newly synthesized histones (e.g. H4K5,12diac) abrogated the recruitment of ATAD2 to nascent chromatin suggesting that ATAD2 might recognize and interact with these histone marks. Indeed, extensive biochemical and biophysical analyses involving TR-FRET, MST (MicroScale Thermophresis), Biocore, and NMR revealed that the bromodomain of ATAD2 preferentially interacts with these marks characteristic of newly synthesized histones. Consequently, overexpression of ATAD2 mutants unable to interact with these marks impaired DNA replication and recruitment of PCNA onto chromatin. Taken together, our data suggest that ATAD2 is essential for DNA replication and thus predicts that it is expressed in cells undergoing S phase. To further strengthen this hypothesis we compared the expression of ATAD2 with the proliferation marker Ki67, and the late S and G2/M marker TOP2A, in various cancer types such as colorectal, gastric, lung, prostate and breast cancers by immunohistochemistry. Indeed ATAD2 expression was restricted to Ki67 and TOP2A expressing areas of tumors, independent of cancer type. Moreover, aggressive tumors, such as triple negative breast cancer and metastatic castration-resistant prostate cancer, showed more intense and abundant expression of ATAD2 whereas slow-growing tumors showed low expression of ATAD2. This research identifies a role for ATAD2 in replication, providing mechanistic and translational support for therapeutic development in cancer.

### Protein Modification and Transcriptional Regulation

#4540

Use of SIAH E3 ubiquitin ligase as a biomarker in pancreatic adenocarcinoma.

Elizaevta Svyatova. _Eastern Virginia Medical School, Virginia Beach, VA_.

The dismal prognosis of patients diagnosed with pancreatic cancer points to our limited arsenal of effective anticancer therapies. Oncogenic K-RAS pathway activation is commonly detected in pancreatic cancer. These activations confer drug resistance, drive aggressive tumor growth and metastasis, and ultimately contribute to poor clinical outcome. Currently, there is no curative treatments for pancreatic cancers. Thus, early detection, biomarker discovery, and identifying novel means of countervailing hyperactive K-RAS signals are urgently needed.

Seven-In-Absentia Homolog (SIAH), E3 ubiquitin ligase, is an essential downstream component of the RAS signal transduction pathway. SIAH is a member of a highly evolutionarily conserved family of RING finger E3 ubiquitin ligases.

Our previous immunohistochemistry (IHC) studies have shown SIAH is a tumor-specific biomarker and SIAH expression is progressively increased as pancreatic tumor progresses from pancreatic intraepithelial neoplasia (PanIN) to pancreatic adenocarcinoma (PDCA) at late stages. In this study, we expand our observations by comparing the expression patterns of SIAH and several newly identified SIAH-interacting proteins using Mayo clinic pancreatic cancer SPORE tissue microarrays (TMA) in a hope of identifying new vulnerability and novel prognostic biomarkers in pancreatic cancer. We hypothesized that SIAH and SIAH-interacting proteins may serve as useful biomarkers and will help to assess effectiveness of anticancer treatment, disease progress, predict clinical outcome and patient survival in the clinical settings. Moreover, we are evaluating SIAH's role in the regulation of the SIAH-interacting proteins and validating the potential mechanisms of these dynamic interactions identified in vitro using pancreatic cancer patients TMA. Ultimately, we aim to explore the new cancer vulnerability, develop new therapeutic strategies, and validate novel prognostic biomarkers for pancreatic cancer patients.

This study is performed in a double blind control settings and we are currently awaiting the disclosure of the clinical information from the pathologist.

#4541

Utilization of K48 poly-ubiquitin modulation as a biomarker of target engagement for a protein homeostasis inhibitor in the clinic: Preclinical validation with CB-5083, a first-in-class inhibitor of the AAA ATPase p97.

Mary-Kamala MENON, Ferdie SORIANO, Steve WONG, Eduardo VALLE, Stevan Djakovic, Brajesh KUMAR, Bing YAO, Antonett MADRIAGA, Tony WU, Julie RICE, Jinhai WANG, Alessandra CESANO, Laura SHAWVER, Han-Jie ZHOU, David WUSTROW, Daniel ANDERSON, Mark ROLFE, Ronan LE MOIGNE. _Cleave Biosciences, Burlingame, CA_.

Background:

Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is unknown. We recently discovered CB-5083, a novel small molecule inhibitor of p97, a protein involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. Accumulation of K48 poly-ubiquitinated proteins is a hallmark of protein degradation inhibition, and we used this proximal biomarker to follow CB-5083 target engagement on p97 in various pre-clinical models.

Results:

CB-5083 is a potent inhibitor of p97, with a biochemical IC50 of 11 nM. When cancer cells are exposed to CB-5083, biological consequences linked to p97 inhibition are detected, including ERAD inhibition, ER (endoplasmic reticulum) stress, ER stress-mediated cell death and accumulation of poly-ubiquitinated proteins. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of K48 poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in K48 poly-ubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Accumulation of K48 poly-ubiquitin also occurs in other tissues in the body and we developed a quantitative method to detect accumulation of K48 poly-ubiquitin as a marker of target engagement in whole blood. With this method, we were able to compare the kinetics and level of K48 poly ubiquitin accumulation following CB-5083 administration in both tumor and whole blood. We also performed a dose escalation of CB-5083 in cynomolgus monkeys and defined the minimal plasma AUC and Cmax required to see accumulation of K48 poly-ubiquitin in monkey whole blood. This approach allowed us to predict human exposures that should lead to target engagement and consequent biological activity in our ongoing phase 1 studies where a flow cytometry based assay is being used to monitor K48 poly-ubiquitin levels in patient blood samples.

Conclusion:

K48 poly-ubiquitin is a target engagement biomarker of p97 inhibition. CB-5083, a p97 inhibitor, can induce sustained induction of K48 poly-ubiquitin, not only in tumor but also in surrogate tissues such as whole blood. K48 poly-ubiquitin accumulation is currently being assessed to follow target engagement in the blood of patients in our ongoing phase 1 dose escalation studies of CB-5083.

#4542

Ube2d family members, Ube2e family members and Ube2w modulate the ubiquitination and degradation of EGFR by Cbl.

Ke Ma,1 Philip Ryan,1 Rachel Klevit,2 Stanley Lipkowitz1. 1 _NIH, Bethesda, MD;_ 2 _University of Washington, Seattle, WA_.

Ubiquitin ligases (E3s) are critical component of ubiquitination. In collaboration with ubiquitin-conjugating enzymes (E2s), E3s confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins. Cbl proteins are RING finger E3s that play a significant role in regulating activity of many tyrosine kinases (e.g., EGFR, MET) by ubiquitination. In our study, we used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that the three Ube2e family members and Ube2w interact with the RF domain of Cbl. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases ubiquitination of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can monoubiquitinate Cbl and increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl.

#4543

Substrate trapping proteomics reveals novel mechanism for regulation of mTORC1 signaling by βTrCP-FNIP1/2-FLCN axis.

Tai Young Kim,1 Jee Yun Chang,1 Jin Mo Ku,1 Se Hyang Hong,1 ji Hye Kim,1 Hyeong Sim Choi,1 Kangwook Lee,1 Myeong-Sun Kim,1 Sang Mi Woo,1 Michael B. Major,2 Seong-Gyu Ko1. 1 _College of Korean Medicine, Kyung Hee University, Seoul, Republic of Korea;_ 2 _Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between βTrCP2 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to "trap" ubiquitylated substrates on the SCF (βTrCP2) E3 complex. Comparative mass spectrometry analysis of immunopurified βTrCP2 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. Interestingly, many novel substrates for βTrCP2, including TBC1D4, HCFC1, DENND4C, FNIP1, and FLCN are related to cell metabolism. TBC1D4 encodes a GTPase activating protein for the small GTPase Rab that controls insulin-dependent trafficking of the GLUT4 glucose transporter in adipocytes. DENND4C acts as a guanine nucleotide exchange factor for Rab10 and its activity is required for insulin-stimulated GLUT4 translocation to plasma membrane in adipocytes. HCFC1 is a member of the host cell factor family, affecting gluconeogenesis by modulating PGC-1α stability. These suggest βTrCP2 might play important roles in glucose homeostasis by regulating stability of several different target proteins. Here, in focused study, we found that βTrCP1/2 bound, polyubiquitylated, and destabilized FNIP1, FLCN interacting protein 1. We further demonstrated that FNIP1 degradation was promoted by AMPK activation after glucose depletion and expression of a degradation-resistant FNIP1 mutant results in sustained activation of mTORC1 signaling. Hence, our findings reveal that βTrCP1/2 is involved in nutrient sensing through the AMPK-FLCN-FNIP1 and mTORC1 signaling pathways.

#4544

FBW7 induces S-phase arrest caused by DNA double strand breaks through targeting SOX9 for proteasomal degradation.

Xuehui Hong,1 Wenyu Liu,1 Ruipeng Song,1 Hiroyuki Inuzuka,2 Hatem E. Sabaawy,1 Katherine M. Morgan,1 Jamie J. Shah,1 Samuel F. Bunting,1 Xing Feng,1 Chi-Kwan Tsang,1 Zhiyuan Shen,1 X. F. Steven Zheng,1 LianXin Liu,3 Sharon R. Pine1. 1 _Rutgers-The Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _The First Affiliated Hospital of Harbin Medical University, Harbin, China_.

SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis or progression of many human tumors through increasing cell proliferation and epithelial-mesenchymal transition. We observed that, in response to UV irradiation or certain chemotherapeutic agents, SOX9 is actively and rapidly degraded by a ubiquitin pathway dependent mechanism across several different tumor types including lung cancer, colon cancer, osteosarcoma and melanoma, as well as normal human bronchial epithelial cells. We found that SOX9 is phosphorylated by GSK3β at Ser-236, facilitating the direct binding and degradation of SOX9 via the F box protein, FBW7α. We also determined that the de-ubiquitinase, USP28, stabilizes SOX9 under normal conditions by sequestering FBW7, but is released from FBW7 after UV irradiation, allowing FBW7 to bind SOX9 and target it for destruction. DNA damage-induced SOX9 degradation was independent of p53, ATM, ATR and MAPK pathways. Failure to deplete SOX9 attenuated the DNA damage-induced intra-S-phase checkpoint and increased long-term cell survival. Moreover, mutations within the FBW7 phosphodegron binding site of SOX9 prevented SOX9 degradation after DNA damage, and incurred resistance of non-small cell lung cancer (NSCLC) cells to cisplatin in vivo. We found that cancer patients with tumors expressing high Sox9 and low Fbw7 levels exhibit inferior survival. Our discovery reveals a novel function of SOX9 in the cellular response to DNA damage. Induced degradation of SOX9 may be part of the protection mechanisms to maintain genomic stability. This new regulatory mechanism of the FBW7-SOX9 axis in cancer could have diagnostic and therapeutic implications.

#4545

The E3 ubiquitin ligase COP1 controls STAT3 turnover and its loss leads to increased STAT3 stabilization and activation in prostate cancer.

Cecilia Dallavalle,1 George Thalmann,2 Carlo V. Catapano,1 Giuseppina M. R. Carbone1. 1 _Institute of Oncology Research (IOR), Bellinzona, Switzerland;_ 2 _University of Bern, Inselspital, Bern, Switzerland_.

The E3 ubiquitin ligase COP1 acts as a tumor suppressor and is deleted in a small percentage of prostate cancers. We reported that COP1 was repressed in prostate tumors through miRNA-mediated silencing, which represents an alternative and more frequent event than genetic deletion. COP1 controls ubiquitination and turnover of c-Jun and ETV1 and its loss has been associated with over-expression of these transcription factors. In this study, we identify STAT3 as a novel substrate of COP1 and report that concomitant deregulation of COP1 and STAT3 leads to prostate cancer progression. In a panel of prostate cancer cell lines, low expression of COP1 was associated with increased level of STAT3 protein and a more aggressive phenotype. Knockdown of COP1 in normal prostate epithelial RWPE-1 cells increased total (tSTAT3) and phosphorylated STAT3 (pSTAT3) and promoted tumorigenic properties. Conversely, over-expression of COP1 in DU145 cells, expressing low level of COP1, reversed the transformed phenotype and reduced tSTAT3 and pSTAT3 level and activation. COP1/STAT3 anti-correlation suggested that STAT3 was a substrate of COP1 for ubiquitination and degradation by the ubiquitin-proteasome system. Consistently, the proteasome inhibitor PS-341 prevented down-regulation of STAT3 in response to COP1 in DU145 cells. Furthermore, COP1 knockdown delayed significantly STAT3 protein turnover in RWPE-1 cells, indicating that COP1 regulated STAT3 degradation in these cells. Co-IP showed that COP1 and STAT3 directly interacted in RWPE-1 and DU145 cells. Interestingly, co-IP with the wild type and phosphorylation defective Y705F mutant STAT3 showed that the interaction with COP1 did not depend on STAT3 phosphorylation. Furthermore, the level of ubiquitinated STAT3 in RWPE1 cells was reduced after COP1 knockdown, whereas increased in DU145 cells after COP1 over-expression. These data provided evidence of COP1-dependent ubiquitination and degradation of STAT3 in normal prostate epithelial cells and, conversely, STAT3 accumulation and activation in prostate cancer cells with loss of COP1. To assess the clinical relevance of these findings, we examined the level of COP1, tSTAT3 and pSTAT3 by IHC in primary prostate tumors (n=136) from patients with long-term clinical follow up. Low COP1 levels were significantly associated with high tSTAT3 expression (Fisher test p<0.001). Furthermore, pSTAT3 was prevalently observed in the group of COP1 negative/STAT3 high tumors indicative of STAT3 activation. The combination of low COP1 and high STAT3 expression was associated with significantly higher risk of disease recurrence after prostatectomy (p0.01) marking patients at risk of poor clinical outcome. Collectively, this study identifies STAT3 as a substrate of COP1 and provides evidence of a novel pathway leading to STAT3 activation in human tumors.

#4546

Ubc9 sumoylation is required for its interaction with CDK6 through SUMO-interacting motif (SIM) and regulates CDK6 sumoylation in glioblastoma.

Anita Bellail, Chunhai Hao. _Henry Ford Health System, Detroit, MI_.

Small ubiquitin-like modifier-1 (SUMO1) is a conserved member of the ubiquitin-related protein family. Recently we have shown that the cell cycle G1 phase cyclin-dependent kinase-6 (CDK6) is modified by SUMO1 in glioblastoma (Bellail et al., Nat Commun. 2014). CDK6 sumoylation stabilizes CDK6 protein and its kinase activity and drives cell cycle progression for the cancer development and progression. SUMO1 is covalently attached to the Lysine 216 of the CDK6 protein through catalytic reactions by an E1 (SUMO-activating enzyme 1/2) and an E2-conjugating enzyme (UBC9). In this study, we further showed that UBC9 is phosphorylated by CDK1 kinase during mitosis and UBC9 phosphorylation increases the interaction of UBC9 with CDK6 and enhances SUMO1-CDK6 conjugation. Knockdown of SUMO1 by small hairpin RNA (shRNA) abolishes the interaction of UBC9 and CDK6 and SUMO1-CDK6 conjugation. UBC9 is conjugated by SUMO1 and recognizes its substrate protein through interaction of SUMO1 and SUMO-Interacting motif (SIMs) on the substrate protein. Indeed, the survey of CDK6 amino acid sequence and structure identified four SIMs on CDK6 protein. Each SIM was mutated to abrogate its binding property. Of the 4 SIMs, the CDK6-SIM4 is responsible for UBC9-CDK6 interaction as demonstrated by the finding that mutation of the first two amino-acids of the SIM sequence abolishes UBC9-CDK6 interaction and SUMO1-CDK6 conjugation. Our data show that UBC9 sumoylation is required for its interaction with CDK6 protein through SIM and thus involved in glioblastoma progression.

#4547

Rebalancing of Bcl-2 family proteins mediate the vulnerability of pevonedistat-treated acute lymphoblastic leukemia cells towards MEK/ERK pathway inhibition.

Shuhua Zheng, Gilles M. Leclerc, Guy J. Leclerc, Joanna DeSalvo, Ronan T. Swords, Julio C. Barredo. _University of Miami Miller School of Medicine, Miami, FL_.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children and the relapse rate in adult ALL patients is about 50%, highlighting the need for new therapeutic strategies. Data from our laboratory and others showed that ALL cells are sensitive to drugs that induce endoplasmic reticulum (ER) stress/unfolded protein response (UPR). In search for novel strategies to target the ER stress/UPR in ALL, we tested the efficacy of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924, pevo) in ALL. We found ALL cells exhibited significant in vitro and in vivo sensitivity to pevo-induced ER stress/UPR. Specifically, proteotoxic/ER stress was observed in pevo-treated ALL cells secondary to their inability to halt protein translation following pevo-induced activation of the mTOR pathway and concomitant de-phosphorylation of p-eIF2α (S51). In addition, aberrant activation of MEK/ERK has been correlated with resistance/relapse in pediatric ALL (Blood 2014; 124: 3420-3430). In our Bp- and T-ALL cell line models, we found consistent induction of p-ERK1/2 (T202/Y204) following pevo treatment, suggesting phosphorylation of ERK1/2 as a compensatory survival mechanism in response to pevo's cytotoxicity. Supporting this hypothesis, we observed significant in vitro synergy between the MEK inhibitor selumetinib (SEL) and pevo (CI = 0.017). On this basis, we tested the in vivo efficacy of pevo + SEL in NSG mice injected with NALM6 cells expressing the luciferase gene (NALM6/LUC). Engrafted NSG mice were treated with pevo (s.c., 66 mg/kg) and SEL (p.o., 50 mg/kg) twice daily on weekdays and once per day on weekends. Bioluminescence analysis of animals 21 days post ALL injection revealed significant reduction of tumor burden in mice treated with pevo alone or pevo + SEL (p<0.05 for both vs. control). Kaplan-Meier curves showed a significant survival advantage for mice treated with pevo + SEL compared with the control group (p<0.05). Mechanistic studies showed that pevo led to induction of NOXA and BIM whereas Mcl-1 levels were stabilized, suggesting sequestration of the pro-survival activity of Mcl-1 by NOXA/BIM. Indeed, co-IP analysis demonstrated that binding between NOXA and/or BIM with Mcl-1 was enhanced in pevo-treated ALL cells. Further, significant downregulation of Mcl-1 was observed in ALL cells co-treated with pevo + SEL. We found that the synergy of this combination was prevented by co-treatment with the pan-caspase inhibitor Z-VAD, but observed persistence of Mcl-1downregulation whereas BIM expression remained unchanged. We conclude that MEK/ERK pathway inhibition rebalances the Bcl-2 family proteins in favor of synergistic apoptotic death in ALL cells treated in vitro and in vivo with the NAE inhibitor pevonedistat. Our data supports further investigations of agents targeting NAE and the MEK/ERK pathway in relapsed/refractory ALL.

#4548

High throughput siRNA screens uncover a high rate of USP8 and ESCRT pathway dependency in squamous carcinoma cell lines.

Wendy Zhong,1 Elissa Cosgrove,1 Elissa Swearingen,1 Mike Ollmann,1 Jayee Banerjee,1 Vivienne Watson,1 Peter Jaeckel,2 Mariana Pfreimer,2 Silvia Materna-Reichelt,2 Holger Beckmann,2 Paul Kassner,1 Astrid Ruefli-Brasse,1 Olivier Nolan-Stevaux1. 1 _Amgen, Inc., South San Francisco, CA;_ 2 _Amgen, Inc., Regensburg, Germany_.

USP8 and the ESCRT pathway (endosomal sorting complex required for transport) are required for cellular homeostasis through key functions in shuttling ubiquitinated proteins towards lysosomal degradation, and through regulating the maturation of autophagosomal structures. In large-scale siRNA screens, the vast majority of cancer cell lines do not demonstrate dependency on USP8 or ESCRT pathway components for viability. However, we identified three cell lines (HCC70, HCC1954 and PFSK1) that are acutely dependent on USP8 and other ESCRT components (HGS, TSG101) for viability. Using a transcriptome-wide association approach, we found that these three USP8-dependent cell lines also had in common the expression of SerpinB3, also known as SCCA1 (Squamous Cell Carcinoma Antigen 1). After selecting additional Squamous Cell Carcinoma cell lines for further validation of USP8-dependency by siRNA screening, we found that Squamous Carcinoma cell lines, irrespective of their SerpinB3 expression status, were far more likely to be dependent on USP8 for viability (23%; N=22) than cancer cell lines of non-Squamous epithelial origin (4.5%; N=67). To unravel possible molecular mechanisms underpinning the requirement of USP8 for the viability of USP8-dependent Squamous Carcinoma cell lines (including SCABER, BICR22, CALU1 and EBC1), we conducted additional transcriptome association studies and genetic interaction screens to uncover genes that are synthetic lethal with USP8 knock-down.

#4549

Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy.

Silvia A a Batista, Rahul Samant, Paul A. Clarke, Paul Workman. _The Institute of Cancer Research, Sutton, United Kingdom_.

HSP90 has emerged as an important target in cancer therapy as inhibition of HSP90 can result in the degradation of many oncogenic client proteins. The two major cellular protein degradation pathways are the ubiquitin-proteasome system and autophagy. The role of the ubiquitin-proteasome pathway in client protein degradation following HSP90 inhibition is well established. We recently described how this process is controlled by an E3-ubiquitin ligase, cullin 5 (CUL5). We sought to further understand how both major protein degradation pathways are regulated in response to HSP90 inhibition. To do this, we performed a proteomic screen on the ubiquitination status of proteins extracted from HT29 cells following HSP90 inhibition by 17-AAG and / or siRNA silencing of CUL5 expression. We found the level of ubiquitination of 125 proteins increased by more than 2 fold in response to 17-AAG treatment. For 66 of these proteins, CUL5 silencing eliminated the 17-AAG-induced increase in ubiquitination; for 30 proteins CUL5 silencing partially reduced the 17-AAG-induced increase in ubiquitination; and for 29 proteins CUL5 silencing had no effect. Importantly, we found some known regulators of autophagy to be among those proteins ubiquitinated in response to 17-AAG treatment and rescued by CUL5 silencing. There was a correlation between levels of ubiquitination and levels of protein degradation. We measured markers of autophagic flux, LC3B and p62, and found clear evidence for altered pathway flux in response to HSP90 inhibition and CUL5 silencing. This study indicates that the interplay between HSP90 inhibition and CUL5 can control both proteasomal degradation and autophagy.

#4550

High-level nuclear GSK3β promotes glioma tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22 **.**

Aidong Zhou, Kangyu Lin, Sicong Zhang, Suyun Huang. _MD Anderson Cancer Center, Houston, TX_.

Glioblastoma multiforme (GBM) is the most lethal brain tumors and is resistant to most therapeutic endeavors. Glioma stem cells (GSCs) in GBM are responsible for glioma propagation and resistance to conventional therapies. GSK3β is a multifunctional serine/threonine kinase. Growing evidences have supported the oncogenic roles of GSK3β in diverse cancers, including in GBM; however, the functional mechanism of GSK3β in tumor initiation and propagation remains elusive. Our study showed that nuclear GSK3β is responsible for overexpression of the histone demethylase KDM1A and critically regulates histone H3K4 methylation during glioma tumorigenesis. GSK3β phosphorylates KDM1A, which induces its binding and deubiquitination by USP22, leading to KDM1A stabilization. GSK3β and USP22-dependent KDM1A stabilization is required for the demethylation of histone H3K4, thereby repression of BMP2, CDKN1A, and GATA6 transcription, and result in GSCs self-renewal and brain tumorigenesis. Moreover, introduction of a GSK3β-phosphorylation-mimic mutant KDM1A rescues the effect of GSK3β depletion on the inhibition of glioma tumorigenesis. In human glioblastoma specimens, KDM1A levels are correlated with nuclear GSK3β and USP22 levels. Furthermore, a GSK3 inhibitor tideglusib in a GBM mouse model inhibits the tumor initiating ability of GSCs, sensitizes tumors to temozolomide,

and prolongs mice survivals via KDM1A down-regulation. Our findings demonstrate that nuclear GSK3β and USP22-mediated KDM1A stabilization is essential for glioma tumorigenesis and highlights that targeting GSK3β as a therapeutic target for GBM.

#4551

Regulation of replicative stress by deacetylation and dephosphorylation.

Anja Göder,1 Claudia Schäfer,2 Teodora Nikolova,1 Günter Schneider,3 Oliver H. Krämer1. 1 _Institut für Toxikologie, Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany;_ 2 _CMB Jena, Jena, Germany;_ 3 _II. Medizinische Klinik und Poliklinik Klinikum rechts der Isar der Technischen Universität München, München, Germany_.

Over 3 billion base pairs of a mammalian cell have to be replicated with every cell division. Limitations in the supply of nucleotides and DNA lesions slow down replication fork speed and trigger a complex replicative stress response to prevent genomic instability and cancer development. The checkpoint kinases ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), checkpoint kinase-1 (CHK1), and checkpoint kinase-2 (CHK2) are at the heart of this response. These factors catalyze processes that slow down the cell cycle as well as mechanisms that stabilize the replication fork and processes that initiate DNA repair. Such pathways ensure the faithful transmission of DNA without epigenetic alterations and DNA mutations. As expected for such a pivotal mechanism, checkpoint kinase signaling is highly regulated. Posttranslational modifications including phosphorylation and acetylation regulate checkpoint kinases at multiple levels. Recent data show that class I histone deacetylases (HDACs) can affect checkpoint kinase signaling and genomic stability. How these HDACs control checkpoint kinases exactly and if they show specificity for certain checkpoint kinases and DNA repair pathways has not been resolved. Moreover, although it is known that phosphorylation triggers checkpoint kinase activation and that the trimeric phosphatase PP2A attenuates checkpoint kinase phosphorylation it is unknown how this activity of PP2A is modulated. PP2A consists of the subunits A (structural component, PPP2R1A/B), C (catalytic activity, PPP2CA/B), and B (discriminates between substrates to allow specificity of the PP2A holoenzyme). Our data illustrate that class I HDACs are required for checkpoint kinase phosphorylation in human and murine cells. We show that these enzymes suppress the expression of certain PP2A subunits and we reveal that one of the B type subunits specifically targets checkpoint kinases for dephosphorylation by the PP2A holoenzyme. With genetic and biochemical approaches, including RNAi, CRISPR-Cas9, DNA fiber assays, confocal microscopy, and DNA and protein analyses, we demonstrate how these mechanisms maintain S phase arrest and the survival of cells undergoing replicative stress.

#4552

Profiling signalling protein expression, modifications and interactions with multi-dimensional antibody microarrays.

Steven Pelech, Lambert Yue, Jeffrey White, Ryan Hounjet, Dirk Winkler. _University of British Columbia, Vancouver, British Columbia, Canada_.

Antibody microarrays permit sensitive and semi-quantitative analysis of the expression, covalent modification and interactions of proteins in lysates of cells and tissues. At Kinexus, we have developed high content Kinex KAM microarrays that feature nearly 900 pan- and phosphosite-specific antibodies for monitoring protein kinases, phosphatases and other low abundance cell signalling proteins with combinations of different detection systems. One method involved capture of in vitro dye-labeled proteins (e.g. with Cy3) from lysates from cells subjected to diverse treatments. Another method involved the detection of changes in their total phosphorylation with biotinylated pIMAGO stain and an anti-biotin antibody that is labeled with a different dye (e.g. Cy5). Alteration in protein-tyrosine phosphorylation were monitored with a dye-labelled, generic phosphotyrosine-specific PYK antibody in a sandwich antibody microarray (SAM) format. The SAM technique was also used to explore the interactions of adapter, scaffolding and chaperone proteins with hundreds of potential target signal transduction proteins with dye-labeled reporter antibodies for these highly interactive proteins.

We used several human cancer cell lines (e.g. A431, HeLa, Jurkat, MCF7) subjected to diverse treatments (e.g. growth factors) to identify biomarkers for the actions of these agents. Reproducible results were obtained with as little as 25 µg of lysate protein, with a dynamic range of detection exceeding 6000-fold, and a median error range for duplicates measurements of ±12%. Typically 10-15% of the proteins tracked with these arrays demonstrated perturbations exceeding 50%. More than a third of the leads from our antibody microarrays were confirmed by immunoblotting studies. The major limitation associated with validation by Western blotting was the much lower sensitivity with immunoblotting compared with antibody microarrays. We also explored the specific interactions of heat shock proteins, adapter proteins, 14-3-3 and calcium-binding proteins with the antibody microarray captured lysate proteins from cancer cell lines. By combining these detection strategies, it was feasible to obtain over 7000 data points from use of a single antibody microarray slide with two lysate samples and duplicate measurements.

The goal of our proteomics and bioinformatics studies is to use the experimental results from the application of these microarrays to map the architecture of signalling networks in a cell- or tissue-specific manner. Such multi-tiered microarray-based analyses permit target-directed identification of diverse regulatory protein changes in different experimental model systems with greater sensitivity, breadth, selectivity and economy when compared to any other competing proteomics methodologies.

#4553

Identification of CD30/TNFRSF8 as a primary target of the Myc oncoprotein and potential biomarker of Myc-driven cancer.

Victoria Gennaro,1 Xiao-yong Zhang,1 Lauren DeSalle,1 Duonan Yu,2 Andrei Thomas-Tikhonenko,2 Christopher Vakoc,3 Steven B. McMahon1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA;_ 3 _Cold Spring Harbor Laboratory, Cold Spring Harbor, NY_.

Pathological activation of the transcription factor Myc induces a multitude of human malignancies. In Myc-driven tumor progression, there is a robust dependence on Myc activity; however, strategies developed to explicitly modulate Myc function in cancer have yielded little clinical success. Patients harboring Myc-dependent tumors would benefit from a therapy that circumvents molecular targeting of Myc expression and instead directs against a distinct downstream effector.

Our preliminary data from an unbiased expression-profiling screen identify CD30 as a primary target of the Myc oncoprotein. CD30 is a member of the tumor necrosis factor superfamily and resides on the extracellular membrane; it is highly expressed in several lymphomas while acutely restricted in normal cells. Within the last few decades, CD30 has emerged as a diagnostic marker and therapeutic target of many lymphoproliferative diseases. However, the underlying mechanisms that regulate CD30 expression remain unclear. We report that CD30 is regulated at the transcriptional level by Myc. Through a genetic and biochemical approach, we define CD30 induction in a MYC-dependent manner across a variety of human cell lines of both lymphomagenic and non-lymphomagenic origin. Furthermore, deliberate modulation of Myc-activity demonstrates a positive correlation with CD30 surface protein expression in multiple malignancy models. With CD30 induction as a potential mechanism by which to identify and target Myc-driven oncogenesis, we can reshape detection and treatment of Myc-regulated cancer.

#4554

Toll-like receptor expression and ligation promotes in vitro invasion in pancreatic cancer.

Ibtehaj A. Naqvi,1 Jessica McDade,1 Jaewoo Lee,2 Rebekah White2. 1 _Duke University Medical School, Durham, NC;_ 2 _Duke University Medical Center, Durham, NC_.

Introduction: Pancreatic cancer (PC) has the worst prognosis of all the major cancers. The role of inflammation in tumor progression and metastasis, particularly in PC, is widely accepted. Our lab has recently shown that levels of circulating cell-free DNA (cfDNA) and associated proteins (histones, HMGB-1, etc.) are elevated in PC patients at baseline and increase in response to standard therapies. It is also known that components of the innate immune system respond to cfDNA and associated proteins. Specifically, the toll-like receptors (TLRs) have been shown to be activated by cfDNA and associated proteins. These TLRs, specifically TLR-4 and TLR-9, have been shown to promote tumor progression and metastasis in other cancers such as breast, colorectal, and skin cancers. We wanted to investigate the role of TLRs in PC by characterizing receptor expression and downstream function. We also attempted to inhibit the downstream effects of TLR signaling using nucleic acid binding polymers (NABPs) that our lab has previously utilized in other disease models such as systemic lupus erythematosus.

Methods: Three human (MiaPaCa-2, BxPC-3, PANC-1) and one murine (KPC-4580P) PC cell line were characterized for TLR-4 and TLR-9 expression by western blot. Briefly, cells were plated and lysed using RIPA buffer. Total protein concentration was quantified using bicinchoninic acid (BCA) assay and normalized prior to running the cell lysates on SDS-PAGE. The protein bands were then transferred to immunoblotting membrane and probed with specific primary antibodies, followed by fluorescent secondary antibodies. Blots were exposed and quantified via LI-COR. Downstream function of TLR expression was tested using Transwell-matrigel invasion assays wherein cells were plated in the Transwell system in the presence of TLR-4 and TLR-9 specific agonists (LPS and CpG or mtDNA, respectively). These cells were also treated with NABPs to inhibit TLR activation.

Results: All PC cell lines expressed TLR-4 and TLR-9. The invasion assays indicated that in the presence of TLR-4 and TLR-9 specific agonists, PC cells exhibited increased invasion. Moreover, PC cells that invaded in response to TLR-9 agonists were inhibited in the presence of NABPs. Our lab has also prepared conditioned media (CM) from cells treated with clinically relevant doses of radiation. Our PC cell lines displayed increased invasiveness in response to CM and decreased with NABP treatment.

Conclusion: We have previously shown that there are elevated levels of cfDNA and associated proteins in PC patient serum and have now shown that these factors can have direct effects on PC cells via TLRs that are expressed by the cancer cells. Treating cells with NABPs in the presence of TLR agonists can abrogate TLR mediated invasion in vitro. Our goal is to further characterize the effects of TLR signaling on cancer cells including RNA transcription and protein translation profiling.

#4555

Identification of bone morphogenetic protein 2 splice variant in pancreatic cancer reveals cross-talk between endocytic and autophagy pathways.

Eric Cruz, Surinder K. Batra. _Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE_.

The inherent heterogeneity in pancreatic cancer (PC) confers complex transcription patterns that require a subset approach to identifying novel gene targets. Thus, we performed meta-analysis on 11 COPA (Cancer Outlier Profile Analysis) transformed datasets (www.oncomine.org) depicting the expression profiles of 416 pancreatic tumors. This analysis revealed Bone Morphogenetic Protein 2 (BMP-2), a pivotal regulator of cellular proliferation, motility, & differentiation, to be increased in a subset of PC tumors. Canonical BMP-2 signaling is mediated via type 1 & type 2 serine/threonine kinase receptors, which upon ligand binding, phosphorylate SMAD proteins leading to initiation or attenuation of gene expression programs. Receptor mediated endocytosis has been shown to be an integral regulator of BMP signaling intensity and duration whereby endocytic internalization of activated receptors leads to their subsequent inactivation and recycling back to the plasma membrane or degradation via the lysosomal pathway. Our investigation of BMP-2 in PC has revealed a novel splice variant (BMP-2 SV), which due to exclusion of its signal peptide bearing exon, escapes the secretory pathway and is endowed with an intracellular pattern of expression. Furthermore, this BMP-2 isoform undergoes preferential degradation via the autophagy lysosomal pathway (ALP). Endosomal and autophagosomal trafficking has been previously shown to converge, through endosome and autophagosome vesicular fusion, to form a hybrid structure described as an amphisome. We hypothesize that endocytosed bone morphogenetic receptors and autophagocytosed BMP-2 SV converge in the amphisome thereby promoting signal transduction. Indeed, immunofluorescence analysis has revealed colocalization of the exogenous BMP-2 SV with components of both the endocytic and autophagic pathways. Furthermore, overexpression of BMP-2 SV in HEK-293 and pancreatic cancer cell lines was performed, which led to a corresponding increased Smad 1/5/8 phosphorylation. In contrast, biochemical inhibition of receptor mediated endocytosis with PitStop ® 2 or disruption of microtubule-dependent vesicular transport with vinblastine was found to diminish the BMP-SV dependent increase in Smad 1/5/8 phosphorylation. Likewise, increasing autophagy through Rapamycin treatment led to a corresponding increase in Smad 1/5/8 phosphorylation. Altogether, our results suggest a novel amphisome-mediated mechanism of BMP-2 signaling due to convergence of endocytosed receptors and autophagocytosed BMP-2 SV. Consequently, this intracellular mode of signaling may endow pancreatic tumor cells with a cell-autonomous means of driving its own growth and metastasis.

#4556

Activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1.

Mingnan Cao,1 Limei Guo,2 Juan Li,2 Chun Liu,1 Yiguo Zhang,3 Siwang Yu,1 Tony A-N. Kong4. 1 _Peking University School of Pharmaceutical Sciences, Beijing, China;_ 2 _Peking University School of Basic Medicine, Beijing, China;_ 3 _Bioengineering College of Chongqing University, Chongqing, China;_ 4 _Rutgers University School of Pharmacy, Piscataway, NJ_.

Liver-specific inactivation of NF-E2 p45-related factor 1 (Nrf1) results in sequential development of non-alcoholic steatohepatitis (NASH), fibrosis, and finally hepatocellular carcinoma (HCC). However, the detailed mechanisms responsible for this phenotype remain uncertain. In the present study, a β-naphthoflavone (β-NF)-inducible liver-specific Nrf1 knockout mouse line which harbors an Nrf1flox/flox allele and a rat CYP1A1-Cre transgene was employed to investigate potential mechanism underlying Nrf1 deficiency-induced NASH.

Β-NF-induced liver-specific knockout Nrf1 resulted in a reversible typical NASH and mild fibrosis phenotype. β-NF treatment almost abolished Nrf1 expression after 2 weeks, and induced dramatic elevation of serum transaminase, inflammation, necrosis, ballooning degeneration and HSC activation, but these phenotype diminished after 16 weeks, accompanied by restoration of Nrf1 expression. These observations clearly demonstrated the dependency of NASH and fibrosis on loss of Nrf1, and provide a novel model of reversible non-alcoholic liver diseases.

Notably, disruption of Nrf1 caused a sustained activation of sonic hedgehog (shh) signaling pathway accompanied by pronounced endoplasmic reticulum (ER) stress. More importantly, vismodegib, a hedgehog signaling inhibitor, dramatically reversed the deleterious effects caused by loss of Nrf1, such as elevation of serum transaminase activities, inflammatory cells infiltration and hepatocyte necrosis. It has been reported that hepatocytes undergoing endoplasmic reticulum stress and showed elevated expression of hedgehog ligands. However, the mRNA level of hedgehog ligand was not changed in Nrf1 deficiency mice, while the protein levels of both shh and Gli2 were significantly increased. Furthermore, chemical chaperone 4-PBA which can promote protein degradation partially reversed the effects of Nrf1 deficiency on shh signaling. Collectively, these data demonstrate that activation of sonic hedgehog signaling is essential for non-alcoholic steatohepatitis induced by liver-specific disruption of Nrf1, possibly through post-translational modification/procession of shh, and hedgehog signaling could be targeted to treat non-alcoholic liver diseases.

The present works were supported by the National Natural Science Foundation of China (No. 91429305, 81272468, and 81372266)

#4557

Tip110-mediated regulation of NF-κB activity is key to tumorigenesis.

Khalid Amine Timani, Amanda Whitmill, Ying Liu, Johnny J. He. _UNTHSC, Fort Worth, TX_.

HIV-1 Tat-interacting protein (Tip110), also known as squamous cell carcinoma antigen recognized by T cells 3 (SART3), is a multifaceted nuclear protein and has been shown to function in tumor antigenicity. Tip110 protein expression is very low in normal tissues and non-proliferating cells but becomes highly elevated in a number of malignant tumor cell lines and cancerous tissues, pointing to its possible role in tumorigenesis. Our previous studies have shown that Tip110 specifically interacted with oncogenic deubiquitinating enzyme, USP15, and that ectopic expression of the Tip110 protein led to re-distribution of USP15 from the cytoplasm to the nucleus. USP15 is known to inhibit TNF-α-induced NF-κB activity by modulation of IκB-α ubiquitination. In the current study, we demonstrated that Tip110 augmented TNF-α-induced transcription activity of NF-κB, and that deletion of the nuclear localization domain in Tip110 obliterated the observed enhancement of TNF-α-induced NF-κB activity, providing the very first evidence to support the specific role of Tip110 in NF-κB activation. In addition, we found that Tip110 expression modulated IκB-α phosphorylation and that activation of NF-κB by Tip110 was mediated through the p38/MAPK pathway. Moreover, we showed that ectopic expression of Tip110 induced translocation of NF-κB p65 from the cytoplasm to the nucleus, reminiscent of the aforementioned translocation of USP15, in the absence of direct interaction of Tip110 with either p65 or IκB-α. Taken together, these results indicate that opposing regulation of TNFα-induced NF-κB activity by Tip110 and USP15 could be pivotal for modulation of tumorigenesis. Further delineation of the underlying signaling cascades will likely provide new insights into development of anti-tumor agents.

#4558

PIN1 expression level is maintained by a negative feedback loop through regulation of Myc and miRNAs.

Ka Wai Leong, Chi Wai Cheng, Yok Lam Kwong, Wai Choi Eric Tse. _The University of Hong Kong, Hong Kong, Hong Kong_.

Introduction: PIN1 is a peptidyl-prolyl-isomerase (PPIase) that binds specific motifs (serine/threonine preceding a proline) in proteins, thereby catalyzing cis/trans isomerization of the peptide bond between the phosphorylated serine/threonine and proline. As a result of such conformational changes, stability, sub-cellular localization, functional activity as well as protein-protein interactions of the PIN1-bound proteins may be altered. PIN1 has been demonstrated to play an important role in controlling various cellular processes such as cell proliferation, apoptosis and migration. Consequently, its expression has to be tightly controlled. Previously, we found that two miRNAs, miR-296-5p and miR-874-3p, served as negative regulators of PIN1. However, the mechanism regulating these two miRNAs remains to be defined.

Methods: The online database-UCSC Genome Bioinformatics (NCBI/hg18) was used to search for the upstream regulator of miR-296-5p and miR-874-3p. Luciferase reporter assay was conducted to examine the effect of the identified regulator on the promoter regions of miR-296 and miR-874-3p. Quantitative polymerase chain reaction (Q-PCR) was used to detect the expression levels of miR-296-5p and miR-874-3p. Protein expression levels of the identified regulator and PIN1 were examined by western immunoblotting.

Results: By screening the online database-UCSC, Myc was identified as a potential transcription factor for the miRNAs, miR-296-5p and miR-874-3p. As demonstrated by luciferase reporter assay, expression of Myc activated the promoters of miR-296-5p and miR-874-3p. Myc over-expression also increased the expression levels of these two miRNAs, which in turn led to down-regulation of PIN1. Interestingly, we found that over-expression of PIN1 increased the expression of Myc, miR-296-5p and miR-874-3p. We therefore hypothesized that PIN1 expression is maintained via a negative feedback loop of Myc-miRNAs-PIN1. Consistent with this hypothesis, we demonstrated that over-expression of exogenous EGFP-tagged PIN1 led to the enhancement of Myc, miR-296-5p and miR-874-3p expression, and thereby subsequently decrease the endogenous PIN1 level.

Conclusion: Taken together, our results suggested that the expression level of PIN1 is maintained by a negative feedback loop through the regulation of Myc, miR-296-5p and miR-874-3p.

#4559

Sprouty2 differentially regulates signaling and phenotypic responses of glioblastoma cells to DNA damaging agents and receptor kinase inhibitors.

Nisha G. Sosale, Sally Shin, Matthew J. Lazzara. _University of Pennsylvania, Philadelphia, PA_.

Key to improving glioblastoma patient survival is the exploration of the signaling mechanisms that regulate cellular response to approved and investigational therapeutics, including DNA damaging agents and targeted therapeutics such as EGFR and MET kinase inhibitors. Previous work from our group demonstrated that Sprouty2 (SPRY2) surprisingly acted more as a tumor promoter than tumor suppressor in glioblastoma cell lines and tumor xenografts, with SPRY2 knockdown reducing proliferation of human glioblastoma cells and antagonizing the growth of subcutaneous tumor xenografts in mice. We hypothesized here that these effects may result from a perturbation in the cell cycle and that such effects may augment cell cycle shifts induced by DNA damaging agents or kinase inhibitors. Flow cytometry measurements indicated shifts in DNA content distribution in glioblastoma cell lines consistent with cell cycle arrest in response to SPRY2 knockdown alone, and that these shifts were substantially augmented by carboplatin or to a lesser degree by a combination of EGFR and MET inhibitors, but not by temozolomide. Western blotting indicated that SPRY2 knockdown resulted in increased phosphorylation of Ataxia Telangiectasia Mutated (ATM), a kinase involved in the DNA damage response pathway, at a serine residue (1981) that regulates the dissociation of inactivate ATM homodimers into activated monomers. In response to carboplatin, but not the other therapeutics tested, a robust increase was observed in Checkpoint kinase 1 (CHK1) phosphorylation at serine 345, which has been reported to cause cell cycle arrest. This effect was further enhanced by SPRY2 knockdown, which may help explain why the largest cell cycle perturbations were observed in SPRY2-deficient cells treated with carboplatin. Increased phosphorylation of p38 was also observed in response to carboplatin or EGFR and MET inhibitors. This may have contributed to cell cycle arrest, but may also have cooperated with the inhibition of AKT phosphorylation that occurred only in response to EGFR and MET inhibition to drive cell death, an effect that was also augmented by SPRY2 knockdown. Overall, this work has identified a small network of SPRY2-regulated signaling processes that control phenotypic responses of glioblastoma cells to different clinically relevant therapeutics and provides motivation for further study of the role of SPRY2 in glioblastoma.

#4560

Induced Sprouty4 expression in breast invasive ductal carcinoma cells downregulates ERK/MAPK activity and restores an epithelial-like phenotype.

Ethan J. Brock, Ryan Jackson, Seema Shah, Quanwen Li, Mansoureh Sameni, Stephen A. Krawetz, Shihong Mao, Bonnie F. Sloane, Raymond R. Mattingly. _Wayne State University, Detroit, MI_.

Breast cancer is one of the most common cancers in U.S. women, with approximately 25% of these cases being in situ disease. Sprouty proteins are currently recognized as important regulators of ERK/MAPK signaling, and have been studied in various cancer types. By employing RNA-seq, previous studies from our laboratories determined specific gene expression changes common to three models of ductal carcinoma in situ (DCIS)—MCF10.DCIS, SUM 102, and SUM 225—when compared to MCF10A cells (a model of non-transformed breast epithelium). All cell lines were grown in three-dimensional (3D) reconstituted basement membrane overlay culture because research has shown that the behavior of cancer cells in 3D matrices is more reflective of an in vivo response when exposed to drugs and radiotherapy than if they are cultured on plastic. Among the three models of DCIS, 63 genes were consistently upregulated. We identified 244 promoters associated with these 63 genes and performed bioinformatic data mining that revealed a high level of enrichment for a promoter framework shared by three of these genes: one of which encoded for the protein Sprouty4. We hypothesize that Sprouty4 is an endogenous inhibitor of ERK/MAPK signaling in DCIS, and its loss or reduced expression is a mechanism by which triple-negative lesions progress toward invasive ductal carcinoma (IDC). Using immunohistochemistry we found that Sprouty4 was highly expressed in certain human premalignant breast tissue samples, and that this expression was reduced in malignant triple-negative samples. These results correspond with immunoblot data from our 3D culture model of breast cancer progression in which Sprouty4 expression was higher during DCIS than in the IDC stage. Efficient over-expression of Sprouty4 reduced both ERK/MAPK activity as well as the aggressive phenotype of MCF10.CA1d IDC cells. Immunofluorescence experiments revealed data consistent with the relocation of E-cadherin back to the cell surface and the restoration of adherens. To determine if these effects were due to changes in ERK/MAPK signaling IDC cells were treated with MEK162, an allosteric MEK inhibitor. Nanomolar concentrations of drug produced a restoration of an epithelial-like phenotype similar to Sprouty4 over-expression. From these data we conclude that Sprouty4 may act to control ERK/MAPK signaling in a subset of DCIS, thus limiting the progression of these premalignant breast cancers.

#4561

Dissecting mechanisms of CART signalling through the estrogen receptor in ER+ breast cancer.

Brian Mooney,1 Sudipto Das,1 Rut Klinger,1 William Gallagher,2 Darran O'Connor1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _University College Dublin, Dublin, Ireland_.

The cocaine- and amphetamine-regulated transcript (CART) was first discovered as a peptide up-regulated by the administration of cocaine and amphetamine to rats. CART peptides are involved in regulating physiological processes, such as feeding, reward and neuro-protection. However, recent studies have associated high CART expression with worse overall survival in patients with small-bowel carcinoid tumours [1] and estrogen receptor-positive (ER+), lymph node-negative breast cancer [2]. Interestingly, in ER+ breast cancer, CART was also shown to be associated with poor patient response to tamoxifen, suggesting CART may play a role in conferring tamoxifen resistance [2].

The aim of this study was to elucidate the mechanism(s) by which CART signals in ER+ breast cancer. In order to test whether CART could mediate the ligand-independent activation of ER-alpha, MAP-Kinase pathway activation and levels of downstream gene-targets of ER-alpha were assessed post CART stimulation. Additionally, the ability of CART to activate ER-alpha using three LXD motifs (nuclear receptor co-activator recognition motifs) present within the CART sequence was also assessed. This was achieved through selectively mutating these motifs and testing whether CART still possessed the ability to activate ER-alpha using an ERE-Dual luciferase reporter assay.

Treatment of cells with CART demonstrated an increase in MAP-Kinase activity through the detection of increasing phosphorylated ERK levels. An increase in the phosphorylation of ER-alpha at serine 118 (a phosphorylation site thought to be involved in tamoxifen resistance) was also evident following CART stimulation. CART stimulation also resulted in an increase in levels of the progesterone receptor, a known ER-alpha gene target. Mutagenesis of each LXD motif within CART resulted in significant decreases in ER-alpha activity, suggesting a potential structure-function relationship between CART and ER-alpha.

In conclusion, we suggest that CART can activate ER-alpha in a ligand-independent manner through the MAP-Kinase pathway, and also potentially through specific LXD motifs within its sequence. Further investigation into the relationship between CART and ER-alpha will help us gain a better understanding of, not only the potential structure-function relationship between CART and ER-alpha, but also the role CART plays with regards to tamoxifen resistance in ER+ breast cancer.

[1] Landerholm K et al., Expression of cocaine- and amphetamine-regulated transcript is associated with worse survival in small bowel carcinoid tumors. Clin Cancer Res. 2012 Jul 1;18(13):3668-76

[2] DJ Brennan, DP O'Connor et al., The cocaine- and amphetamine-regulated transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer. Oncogene 2011 Dec 5. Doi: 10.1038/onc.2011.519

#4562

Inhibition of STAT3 signaling in human liver cancer cells using Evista.

Yina Wang,1 Chongqiang Zhao,2 Haiyan Ma,1 Huameng Li,3 Jiagao Lu,1 Chenglong Li,3 Jiayuh Lin,4 Li Lin1. 1 _Division of Cardiology, Department of Internal Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China;_ 2 _Division of Cardiology, Tianjin First Center Hospital, Tianjin, China;_ 3 _Ohio State Univ. College of Pharmacy, Columbus, OH;_ 4 _Center for Childhood Cancer, The Research Institute at Nationwide Children's Hospital, Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, OH_.

Liver cancer is one of the major causes of morbidity and mortality in China and also around the World. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in human cancer including liver cancer and has emerged as a viable molecular target for liver cancer treatment. Novel therapeutic approaches of more effective treatments are much needed. To date, few STAT3 inhibitors are available for the therapy of liver cancer yet. Evista (Raloxifene HCI) is used for the prevention and treatment of osteoporosis and was approved for reducing the risk of invasive breast cancer in postmenopausal women with osteoporosis and in postmenopausal women at high risk for invasive breast cancer. We reported the discovery of Raloxifene as novel inhibitors of IL-6/GP130 proteinprotein interactions (PPIs) using multiple ligand simultaneous docking (MLSD) and drug repositioning. The possible effect of Evista in STAT3 signaling or liver cancer cells is still unclear. The ability of Evista to inhibit STAT3 was tested. Evista inhibited the P-STAT3 stimulated by IL-6 but not the induction of STAT1, STAT2, and STAT4 phosphorylation by IFN-α and IL-4. Evista inhibited STAT3 phosphorylation and resulted in the induction apoptosis on human liver cancer cell lines HEPG2, 7721, and HUH-7 as evidenced by caspase-3 cleavage. Evista also inhibited the cell viability, cell migration and colony formation in liver cancer cells. Furthermore, administration daily of Evista suppressed the HEPG2 tumor growth in mice in vivo. In summary, our study is the first report indicating that Evista also exhibits biology activity against persistent STAT3 signaling in human liver cancer cells. These results suggest that Evista may also be a chemoprevention agent for liver cancer by targeting persistent STAT3 signaling.

#4563

Role of ezrin in PKA dependent colorectal cancer cell fate.

Premila Leiphrakpam, Michael G. Brattain. _UNMC Eppley Cancer Center, Omaha, NE_.

Introduction: Metastasis is the main cause of cancer related death in CRC. However, the molecular basis of CRC metastasis is poorly understood. Ezrin is a known cAMP dependent AKAP. We have found ezrin hyperphosphorylation at T567 is critical for CRC cell survival. In this study we evaluated the dependency of PKA activation with ezrin phosphorylation at T567 on its downstream cell survival mechanisms.

Methods: Small molecule ezrin kinase inhibitor NSC668394 was used to test the effect of inhibition of ezrinT567 phosphorylation on PKA activation and its downstream cell survival signaling. A genetic knockdown approach was utilized to further confirm the result. In another approach, growth factor mediated activation of ezrin T567 phosphorylation was performed and ezrin/PKA signaling was then analyzed for its downstream cell survival effects. To further confirm the findings ezrin phospho-mimetic (T567D) and phospho-deficient (T567A) mutants were generated through site directed mutation approaches.

Results: We demonstrated that dephosphorylation of ezrin at T567 activates PKA in a cAMP independent manner and leads to CRC cell death through inhibition of XIAP and survivin. Moreover, the stable knockdown of ezrin showed similar result. Interestingly, ligand mediated ezrin hyperphosphorylation at T567 has the opposite effect in which there is cAMP dependent PKA activation leading to CRC survival through upregulation of XIAP and survivin. This was further confirmed with ezrin T567 phospho-mimetic and phospho-deficient mutants which demonstrate PKA activation by cAMP dependent and independent mechanisms respectively leading to different CRC cell fates. Therefore, ezrin signaling demonstrated two opposing pathways in which both requires activation of PKA and yet result in different CRC cell fates by regulating XIAP and survivin expression.

Conclusions: Two different ezrin signaling dependent pathways were identified which are dependent on the same enzymatic activity but with opposite effects on CRC cell fates. Therefore, ezrin might be a novel target that could be utilized for the development of new therapeutic strategies and further understanding the mechanism of ezrin/cAMP-PKA activation and its role in CRC cell survival could have potential impact for treatment of distant metastasis in CRC.

#4564

Elucidating the mechanism of the STAT3/NF-κB/EP4 signaling axis in pancreatic cancer.

Amanda R. Munoz, James W. Freeman, Addanki P. Kumar. _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Despite recent advances to pancreatic cancer (PanCA) management, the 5 year survival rate remains only 7%. This poor survival rate highlights the importance of increasing our molecular understanding of PanCA pathogenesis to develop effective therapies to improve patient outcomes. We previously demonstrated that targeting the STAT3/NF-κB signaling axis with Nexrutine® (a Phellodendron amurense bark extract) was able to inhibit autophagy, ROS production, and proliferation in multiple PanCA cell lines. Our data also indicated the potential involvement of prostaglandin receptor EP4 in mediating these effects. To our surprise, we observed decreased levels of EP4 in human pancreatic tumors implicating a possible protective role. However, the mechanism behind STAT3/NF-κB signaling and its link to EP4 are still unknown. Using genetic and chemical inhibition of STAT3 and EP4, we have sought to define the STAT3/NF-κB/EP4 signaling axis. Our results indicate that inhibition of STAT3 via knockdown (STAT3 KD) using shRNA does not affect the proliferative ability or viability of PanCA cell lines when compared to non-transfected control (NTC) cells. Even though proliferation and viability were un-affected, STAT3 KD increased Cox-2 levels in a KRAS mutant cell line (Capan-2) and inhibited Cox-2 in a KRAS WT cell line (BxPC-3). Inhibition of EP4 increased STAT3 and NF-κB activity in KRAS mutant cell lines while decreasing activity in the KRAS WT cell line. Furthermore, EP4 inhibition increased protein levels of IL-6 and dual inhibition of STAT3 and EP4 yielded increased NF-κB activity and decreased Cox-2 levels. Converse experiments in cells treated with IL-6 revealed inhibited EP4 expression and decreased NF-κB activity. This may be the reason behind the increased STAT3 activity noted in EP4 inhibited KRAS mutant cells and supports the notion that EP4 may be protective against PanCA development. Taken together, these data suggests a feedback loop in regulating the STAT3/NF-κB/EP4 signaling cascade and that STAT3/NF-κB signaling axis members are differentially regulated upon STAT3 or EP4 inhibition possibly depending on KRAS status. Supported by Initiative for Maximizing Student Development (IMSD) Training Grant (R25 GM095480; NMJB), NCCIH (R01 AT007448; APK), and VA-MERIT Award (I01 BX 000766; APK)

#4565

Antizyme inhibitor regulates the expression of proteins involved in mitosis and affects prostate cancer cell growth.

James M. Rice,1 Amanda Kusztos,2 Bruce R. Zetter1. 1 _Boston Children's Hospital, Boston, MA;_ 2 _Boston College, Boston, MA_.

Upregulation of polyamine biosynthesis is a requirement for cell growth and proliferation. Increases in polyamine levels are mediated by the enzyme ornithine decarboxylase (ODC), which is inhibited by antizyme (AZ). Rescue of ODC activity is accomplished by expression of antizyme inhibitor (AZI), which prevents binding of AZ to ODC. Increases in AZI gene copy number, mRNA transcripts, protein expression and AZI nuclear localization are all associated with prostate cancer. Furthermore, knockdown of AZI expression prevents prostate cancer cell growth in vitro and in vivo. To determine if AZI regulation affects prostate cancer cell growth via mechanisms that are independent of polyamide levels, we conducted a proteomic analysis of the AZI interactome by co-IP and MS/MS. This resulted in the identification of 29 high-confidence interacting proteins (HCIPs), 10 of which are associated with microtubules or with the mitotic spindle including TPX2 and KIFC1. Constitutive overexpression of AZI in PC3M-LN4 prostate cancer cells leads to a decrease in expression of the microtubule associated protein KIFC1, further supporting an interaction between AZI and KIFC1. Silencing of AZI gene expression using siRNA nanoparticles results in a decrease in prostate cancer cell proliferation and altered expression of proteins involved in mitosis. Taken together, this data suggests that AZI may influence prostate cancer cell proliferation via mechanisms that are independent of polyamine regulation.

#4566

GRK3 is a direct target of CREB activation and promotes neuroendocrine differentiation of prostate cancer cells.

Mohit Hulsurkar,1 Meixiang Sang,1 Yan Zhang,1 Dayong Zheng,1 Songlin Zhang,1 Michael Ittmann,2 Jianming Xu,3 Wenliang Li1. 1 _University of Texas Health Science Center at Houston, Houston, TX;_ 2 _Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX;_ 3 _Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX_.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer that can arise de novo, but more commonly arises through neuroendocrine differentiation (NED) of prostate adenocarcinoma (PCA) after androgen deprivation therapy (ADT). The molecular mechanisms of NEPC development and NED in PCA cells remain poorly understood and no effective therapeutic is available for NEPC. We have reported that G protein-coupled receptor kinases 3 (GRK3, also called ADRBK2) promotes prostate cancer progression. In this study, we demonstrate that GRK3 is induced by ADT, is more highly expressed in NEPC than in PCA cell lines and mouse models. While studying its regulation, we found that GRK3 is a direct target of cAMP response element binding protein (CREB) that is activated by ADT. GRK3 expression positively correlates with the expression/activity of CREB in human prostate cancer cell lines and tissues. Notably, overexpression of GRK3 in PCA cells increased the expression of NE markers in a kinase activity dependent manner, while silencing GRK3 blocked CREB-induced NED in PCA cells, inhibited NE phenotypes and proliferation of NEPC cells. Taken together, these results indicate that GRK3 is a new critical regulator of NE phenotypes and mediator of CREB activation in NED of prostate cancer cells.

#4567

Functional characterization of P58IPK-PERK pathway in prostate cancer cells.

Xia Sheng, Margrethe Storm, Yang Jin, Fahri Saatcioglu. _University of Oslo, Oslo, Norway_.

Androgen signaling is important for the normal development and function of the prostate as well as for prostate cancer (PCa). We recently found that androgens activated the IRE1α-XBP1 arm of the canonical unfolded protein response (UPR) pathways and simultaneously inhibited PERK-eIF2α signaling. Activation of the IRE1α-XBP1 arm was mediated by direct binding of the androgen receptor (AR) in the vicinity of IRE1, as well as XBP1s target genes, and increase in their expression. Consistently, AR and IRE1α pathway gene expression are correlated in human PCa samples and XBP1s protein expression is significantly increased in cancer compared to normal prostate. However, the mechanisms behind androgen mediated inhibition of PERK-eIF2α signaling are not clear at present. One possible mediator in this regard is P58IPK, an XBP1s target gene that can interact with and inhibit PERK and eIF2α phosphorylation. Here, we show that P58IPK expression is increased by androgens in a time-dependent manner and that it plays a pro-survival role in PCa cells. P58IPK knockdown activated PERK expression as well as subsequent eIF2α phosphorylation when induced with the UPR activator thapsigargin. These findings suggest that P58IPK mediates, at least in part, the differential androgen effects between the IRE1α and PERK signaling in PCa and may be a potential therapeutic target.

#4568

14-3-3z-mediated oncogenic signaling downstream of HER2 in breast cancer.

Tsz Yin Chan, Courtney Banks, Bennet Peterson, Joshua Andersen. _Brigham Young University, Provo, UT_.

The goal of this study is to identify mechanisms by which the oncogenic tyrosine kinase receptor HER2 promotes the growth and metastasis of breast cancer. Previously published work suggests that the phospho-binding protein 14-3-3ζ, which is highly expressed in a variety of breast cancer subtypes, promotes HER2-mediated oncogenic signaling. In this study, we hypothesized that 14-3-3ζ sustains HER2 signaling by binding to and modulating a network of proteins that are phosphorylated downstream of HER2 activation. Using proteomics as a tool to identify 14-3-3ζ interacting proteins in HER2+ breast cancer cells treated with or without Lapatinib (HER2 inhibitor), we identified a subset HER2-dependent 14-3-3ζ interacting proteins. We have focused our current effort on one of these proteins, SH3BP4, which plays a critical role in controlling mTOR activation. We have identified a nutrient-sensitive phosphorylation within a putative 14-3-3ζ binding site on SH3BP4 and we are currently working to understand its mechanism of regulation and impact on mTOR-mediated growth signaling downstream of HER2.

#4569

Use of ACPD and ICA-1 as inhibitors of atypical proteinkinase C-zeta (ζ) and iota (ι) in metastasized melanoma cells.

Wishrawana S. Ratnayake, Mildred Acevedo-Duncan. _University of South Florida, Tampa, FL_.

The number of melanoma cases report is increased every year. Malignant melanoma is very common among all Caucasian populations worldwide. New cases in these populations are expected to be doubled every 10-20 years. According to the NIH Surveillance, Epidemiology and End Results (SEER) program, 73,870 new cases and 9,940 deaths were reported in 2015 in USA [http://seer.cancer.gov/statfacts/html/melan.html (11/10/2015)]. It is not totally understood which intracellular chemicals are involved in certain signaling pathways and which governs the metastasis of melanoma cancer cells. Even though PKC- iota (ι) was not reported in normal melanocytes, it was detected in high amounts in both transformed melanocytes and melanoma metastases. PKC- zeta (ζ) was also reported in both normal melanocytes and melanoma metastases [Melanoma Res. 12:201-209 (2002)]. We believe these atypical PKCs play an important role in cell motility of melanoma therefore we tested two inhibitors for them. The objective of this study was to test the inhibition effectiveness of ACPD [Diabetes. 63:2690-2701 (2014)] on both PKC-ι and PKC-ζ and ICA-1 [Int. J. Biochem. Cell Biol.43:784-794(2011)] as an inhibitor of PKC-ι. SK-MEL-2 metastasis melanoma cells and PCS-200-013 normal melanocyte cells were cultivated and treated with ACPD and ICA-1 in separate sets of flasks for three consecutive days while taking the cell count for every 24 hrs. Preliminary results of this experiment shows statistically significant decrease in cell number in SK-MEL-2 cells while no change in PCS-200-013. Future investigations will involve examining the effects of ACPD and ICA-1 on cell motility. Our preliminary results confirms the cell population of melanoma cells have an inversely proportional relationship with the drug concentrations. In conclusion, ACPD and ICA-1 are capable of decreasing the cell proliferation of melanoma cancer cells. 

### Receptors

#4570

Decreased interleukin-17RA expression inhibits the tumorigenesis and independently predicts good prognosis of colorectal cancer patients.

CHIH-YUNG YANG,1 Jeng-Kai Jiang2. 1 _Taipei City Hospital, Taipei, Taiwan;_ 2 _Taipei Veterans General Hospital, Taipei, Taiwan_.

Background:

Interleukin-17 receptor type A (IL-17RA) plays critical role in promoting early colorectal tumor development and affects immune response in tumorigenesis. Our previous study have reported interleukin-17A modulated tumorigenesis and affected metastasis in colorectal cancers, but the roles of its receptor (IL-17R) in colorectal cancers remain unknown. This study explores potential role and function of IL-17RA in colorectal cancers.

Materials and Methods

The expression of IL-17RA was determined in colorectal cancer tissues and adjacent normal tissues using quantitative real-time PCR and immunohistochemistry. The IL-17RA expression level and clinical parameters were then analyzed. To investigate the functional significance of IL-17RA, IL-17RA knockdown cells were analyzed using transwell assay in vitro and implanted subcutaneously in mice, and monitored for primary tumor growth and angiogenesis.

Results

IL-17RA was overexpression in colorectal cancer tissues compared with adjacent normal tissues (P<0.05).The elevated expression level of IL-17RA was associated with poor survival (P<0.001). In vitro, the IL17RA knockdown cells reduced migration and invasion. In mice, the decreased IL-17RA inhibited tumor growth and angiogenesis.

Conclusions

These results demonstrated that IL-17RA plays a critical role in growth, angiogenesis and prognosis of colorectal cancer.

#4571

Semaphorin/plexin signalling promotes nucleocytoplasmic trafficking of nuclear receptors.

Magali Williamson, John R. Masters. _King's College, London, United Kingdom_.

Androgen receptor (AR) signalling is critical to prostate cancer progression and androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer. Acquired resistance to ADT results from androgen-independent reactivation of the AR pathway primarily. Prostate tumours also bypass androgen blockade by the upregulation of the glucocorticoid receptor (GR). Both AR and GR are nuclear receptors that bind their respective ligands in the cytoplasm and then translocate to the nucleus where they act as transcription factors. Plexins are cell surface receptors for semaphorins. PlexinB1 is overexpressed and mutated in several cancers including prostate cancer and deletion of the plexinB1 gene reduces metastasis in mouse models, indicating an oncogenic role for plexinB1 in prostate cancer.

We have found that activation of plexinB1 by semaphorin4D (Sema4D) results in AR phosphorylation at serine 81, activation of AR-transcriptional activity and increased expression of androgen-responsive genes, effects reversed by plexinB1 knockdown. PlexinB1 activation increases the translocation of both AR-GFP constructs and endogenous AR to the nucleus in prostate cancer cells. Furthermore knockdown of AR in the prostate cancer cell line LNCaP reduces Sema4D-induced anchorage-independent growth of these cells.

To determine if the Sema4D/plexinB1 signalling pathway also affects translocation of other nuclear receptors to the nucleus, we have also studied the effect of Sema4D treatment on nuclear trafficking of the glucocorticoid receptor (GR). Prostate cancer cells were transfected with a GR-GFP construct and the subcellular localisation of GR-GFP was monitored upon Sema4D treatment by immunocytochemistry and confocal microscopy. The nuclear translocation of endogenous GR was also assessed following Sema4D stimulation by immunocytochemistry and subcellular fractionation studies.

We found that activation of plexinB1 by Sema4D results in a significant increase in the percentage of cells with nuclear GR-GFP and in the nuclear localisation of endogenous GR in prostate cancer cells.

These findings show that plexinB1 activation has a role in the trafficking and activation of nuclear receptors and so may have a role in resistance to ADT in late stage prostate cancer.

#4572

Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 leads to decreased Rho-induced prostate cancer metastasis.

Kisha A. Scarlett,1 Christopher J. Coke,1 Imani Fennell,2 Cimona V. Hinton1. 1 _Clark Atlanta University, Atlanta, GA;_ 2 _Spelman College, Atlanta, GA_.

BACKGROUND: The binding of the homeostatic chemokine SDF-1 to the G -protein-coupled receptor C-X-C Chemokine Receptor 4 (CXCR4) induces cell migration via heterotrimeric G-proteins of the Gi family. Additionally, activated CXCR4 can stimulate the small GTPase RhoA through Gα13 which is required for directional cell migration, contributing to the metastatic potential of cancer cells. Recently, GPCR hetero-oligomerization has emerged as a means by which new signaling entities can be created, yet how receptor heteromers affect receptor pharmacology remains largely unknown. Here we report that agonist-bound CXCR4 is able to form a heteromer with agonist-bound cannabinoid receptor 2 (CB2) at the cell membrane of prostate cancer (PCa) cells and that receptor interaction inhibits the activation of the cytoskeleton regulator RhoA. EXPERIMENTAL METHODS: To confirm CXCR4-CB2 interactions on the cell membrane, we utilized an in situ proximity ligation assay to detect heterodimer formation on the surface PC-3 cells treated with SDF-1 alone or, simultaneous treatment with SDF-1 and the CB2 agonist AM1241. In order to establish that RhoA activity decreased upon heterodimerization of CXCR4 and CB2, we performed RhoA-GTP pulldown assays in PC-3 cells. We then tested the effects of CXCR4/CB2 heterodimerization on actin cytoskeletal reorganization by performing immunocytochemistry using an anti-Phalloidin antibody to detect F-actin. The ROCK kinase inhibitor Y-27632 was used as positive control of RhoA inhibition in these studies. Next, we assessed the effects of receptor heterodimerization on cellular adhesion to extracellular matrix components as the ability of cancer cells to adhere to the endothelium is critical of transendothelial migration during tumor cell intravasation and extravasation. Intuitively, alterations in adhesion capabilities should affect the ability of PCa cells to migrate through an endothelial cell monolayer. Thus we performed transendothelial migration assays. RESULTS: The proximity ligation assay revealed that CXCR4 and CB2 physically interacted on the cell surface of prostate cancer cells. Furthermore, receptor heterodimerization diminished SDF-1 induced actin cytoskeleton reorganization as characterized by membrane ruffling in a similar manner to the ROCK kinase inhibitor Y-27632. Heterodimer formation, stimulated by simultaneous treatment of cells with SDF-1 and AM1241 also attenuated PCa adhesion and transendothelial migration. CONCLUSION The attenuation of RhoA has multiple functional consequences leading to a less metastatic phenotype. Thus induction of CXCR4-CB2 heterodimerization is a viable pharmacological approach to targeting the CXCR4-RhoA axis as a means of preventing the metastatic dissemination of PCa cells.

#4573

Complex interactions among different types of androgen receptor splice variants and the full-length androgen receptor in mediating castration resistance.

Yang Zhan,1 Guanyi Zhang,1 Xiaojie Wang,2 Yanfeng Qi,1 Haitao Zhang,1 Yan Dong1. 1 _Tulane University, New Orleans, LA;_ 2 _Jilin University, Changchun, China_.

Androgen receptor splice variants (AR-Vs) have been implicated in the pathogenesis and progression of castration-resistant prostate cancer after androgen-directed therapies. Approximately 20 AR-Vs have been identified to date. When expressed alone in cells, some AR-Vs (e.g., AR-V7 and ARv567es) localize primarily to the nucleus, whereas some others (e.g., AR-V4 and AR-V6) distribute equally between the nucleus and the cytoplasm. Significantly, the latter is often co-expressed with the nucleus-localized AR-Vs and the full-length AR (AR-FL). Thus, an important question that needs to be addressed is whether these different types of AR-Vs interact with each other as well as with AR-FL and the consequence of the interactions. In the present study, we used the bioluminescence resonance energy transfer (BRET) assay to show that both AR-V6 and AR-V4 can heterodimerize with AR-V7 and AR-FL. Consequently, AR-V7 and androgen-bound AR-FL induced nuclear localization of AR-V6 and AR-V4, leading to an increased ability of AR-V6 and AR-V4 to transactivate target genes and to confer castration-resistant cell growth. On the other hand, neither AR-V6 nor AR-V4 affected the subcellular localization of AR-V7 or AR-FL. These findings support the ability of AR-V7 and androgen-bound AR-FL to activate AR-V6 and AR-V4. Since AR-FL and nucleus-localized AR-Vs are the predominant forms of AR expressed in clinical specimens, it is likely that most of the AR-Vs in these cells are in the nucleus and are transcriptionally active. Our data therefore highlight the importance of the complex interactions among different AR-Vs and AR-FL in mediating castration resistance.

#4574

C-X-C chemokine receptor 4 and cannabinoid receptor 2 interact to abrogate CXCL12-mediated cellular response.

Christopher Coke, Kisha Scarlett, Kia Jones, Cimona V. Hinton. _Clark Atlanta University, Atlanta, GA_.

Expression of C-X-C Chemokine Receptor 4 (CXCR4) encourages tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, thus facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis is a good strategy to inhibit the metastatic spread and progression of cancer. It has been suggested that homo- or heterodimerization of GPCRs causes decreased signaling through receptor complexes, representing functional desensitization. In the context of cancer treatment, it is plausible that CXCR4 signaling can be silenced through heterodimeric association with other receptors, thereby inhibiting functions that would lead to metastasis. Various studies suggest that cannabis, signaling via cannabinoid receptors, have anti-proliferative and anti-metastatic properties; though a biochemical mechanism describing how this phenomenon has not been described. We've confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) form a heterodimer that decreases cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231, with CXCL12 and AM1241 (synthetic CB2 agonist), desensitized the intrinsic cellular CXCR4 response of cells to migrate towards CXCL12. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we determined increased interaction between the two receptors upon co-stimulation of respective agonists. To further delineate whether co-stimulation of both receptors led to subsequent heterodimerization, CXCR4 receptor activity was inhibited by an antagonist, AMD3100, or expression of endogenous CXCL12 was downregulated by siRNA. An interaction was observed in simultaneously treated AM1241/CXCL12 cells between CXCR4 and CB2, and surprisingly, in AM1241-treated cells. However, upon pretreatment with AMD3100, and CXCL12-siRNA, receptor association was no longer observed in AM1241-treated cells, supporting the therapeutic notion that treating tumors that endogenously secreted CXCL12 with an exogenous AM1241 can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Further, cells pre-treated with CB2 antagonist (AM630) and combinations of CXCL12 and AM1241 the anticipated decrease in migration was no longer seen. Finally, to determine whether simultaneously-treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Therefore, a physical interaction between CXCR4 and CB2 may inhibit the response to CXCL12, leading to a loss in metastatic potential.

#4575

Altered catalytic properties of a subset of Met cytoplasmic domain variants occurring in renal cell carcinoma.

Dinuka De Silva, Young Lee, Arpita Roy, Donald P. Bottaro. _NIH/NCI, Bethesda, MD_.

TCGA provisional data of papillary renal cell carcinoma (PRCC) kidney cancer, a malignant hereditary cancer syndrome, reveals that 23% of patients (n=161) have MET gene amplification and/or mRNA up-regulation (2-fold or greater; RNA-Seq V2) and 10% of patients possess MET mutations. The Met tyrosine kinase is the cell surface receptor for hepatocyte growth factor (HGF). HGF stimulation induces Met protein autophosphorylation and consequently enhances epithelial cell motility, morphogenesis, and proliferation. Chronic and/or overstimulation of Met contributes to tumor growth and metastasis. We determined Km and Vmax for ATP binding to full-length Met protein isolated from the PRCC derived cell line ACHN, which possesses the MET mutation T992I (numbered per SWISSPROT accession P08581) in the juxtamembrane domain, and the clear cell RCC derived cell line Caki-1, which possesses the MET V1220I mutation in the kinase domain. Catalytic constants were determined using two-site immunoassays for total Met and phospho-Met. Both variants were associated with increased Vmax relative to wild type Met protein isolated from B5/589 breast epithelial cells. The observed increased catalytic activity of receptor variants may confer a higher overall rate of signaling at lower ATP concentrations, potentially contributing to enhanced tumor cell motility and proliferation in both papillary and clear cell RCC.

#4576

Hepatocyte nuclear factor 4 alpha suppresses cell proliferation and induces cellular senescence and apoptosis in prostate cancer cells.

Zhu WANG, Franky Leung Chan. _School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors. Recent advances show that it is a key transcriptional regulator of many genes, involved in xenobiotic and drug metabolism and also cancers of gastrointestinal tract. However, the exact functional roles of HNF4α gene in prostate cancer progression are still not fully understood. In this study we used the overexpression (gain-of-function) and knockdown (loss-of-function) approaches to determine the functional roles of HNF4α in prostate cancer. Our results showed that HNF4α exhibited a reduced expression pattern in many prostate cancer cells compared to immortalized prostatic cell lines. Its overexpression could significantly inhibit the cell proliferation of prostate cancer cells, and trigger the cellular senescence and apoptosis by activation of p21 signal pathway in a p53-independent manner. We showed that stable overexpression of HNF4α could significantly induce G2/M arrest in PC3 cells, while shRNA-mediated knockdown of HNF4α could induce G1 growth arrest, suggesting that HNF4α might play a negative role in the cell cycle regulation of PC3 cells. Moreover, stable HNF4α knockdown could confer resistance to paclitaxel treatment and enhance colony formation capacity in prostate cancer cells. Together, our results suggest that HNF4α may play a tumor suppressor function in prostate cancer progression.

#4577

Fibroblast growth factor receptor-4 (FGFR-4) as a novel therapeutic target for pancreatic cancer.

Toshiyuki Ishiwata,1 Hisashi Yoshimura,2 Yoko Matsuda,3 Shunji Ishiwata4. 1 _Nippon Medical School, Tokyo, Japan;_ 2 _Nippon Veterinary and Life Science University, Tokyo, Japan;_ 3 _Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan;_ 4 _Kinki University, Osaka, Japan_.

The fibroblast growth factor receptor (FGFR) family plays crucial roles in development, tissue repair, and malignant tumors. FGFR-1, -2, and -3 each exist in two isoforms, IIIb and IIIc, due to alternative splicing of the extracellular domain, whereas FGFR-4 does not have these isoforms. FGFR-4 is reportedly over-expressed in various cancers such as breast, prostate, hepatocellular, ovarian, gastric, colorectal, and pancreatic cancers, wherein it contributes to tumor progression. Recent studies have shown that a decrease in FGFR-4 levels suppresses the aggressiveness of gastric, colorectal, and ovarian cancers. We found that pancreatic cancer cell lines expressed similar levels of the IIIb and IIIc isoforms of FGFR-1 to -3, but showed different FGFR-4 levels. These findings suggest that the cancer cells surviving after treatment with recently developed anti-FGFR drugs, which target FGFR-1 to -3, tend to express high levels of FGFR-4. Moreover, a single-nucleotide polymorphism (SNP) in exon 9 of the gene encoding FGFR-4 that results in the substitution of glycine with arginine at codon 388 (388 Gly/Arg) in the transmembrane domain is associated with poor outcomes of high-grade soft tissue sarcoma, prostate, lung, head and neck carcinoma, and advanced and treatment-resistant breast cancer. It has been reported that 40-50% of Caucasians carry at least one copy of the 388 Gly/Arg SNP of FGFR-4. In the present study, we examined the expression and roles of the 388 Gly/Arg SNP in pancreatic cancer and tried to clarify whether FGFR-4 may be a novel therapeutic target for cancer treatment. In human pancreatic tissues, FGFR-4 was weakly localized in the normal exocrine and endocrine pancreas, and was strongly expressed in 67 of 136 pancreatic ductal adenocarcinoma cases (49%). FGFR-4 expression positively correlated with larger primary tumors and more advanced stages of pancreatic cancer. FGFR-4 mRNA was expressed in 5 pancreatic cancer cell lines at various levels, and the mutation in codon 388 was detected in 3 of the cell lines, including PK-1 cells. A short hairpin RNA expression vector targeting FGFR-4 was stably transfected to these mutant-expressing PK-1 cells (388 Gly/Arg), which then showed lower growth rates and cell migration and invasion abilities compared to sham vector-transfected cells. DNA microarray analysis showed that decreased expression of FGFR-4 in PK-1 cells altered the expression levels of molecules related to cellular movement, cellular development, and cell-to-cell signaling and interactions. FGF-19—one of the major ligands for FGFR-4—was expressed in all 5 of the pancreatic cancer cell lines tested. These findings suggest that inhibition of the expression of FGFR-4 harboring the 388 Gly/Arg SNP may be a novel therapeutic strategy for pancreatic cancer, especially after treatment with the newly developed anti-FGFR-1 to -3-targeted drugs.

#4578

**Quantification of protein complexes and post-translational modifications in colorectal cancer utilizing combinted MultiOmyx** TM **and PLA assay from a single FFPE slide.**

Qingyan Au, Flora Sahafi, Kathy Nguyen, Raghav Padmanabhan, Edward J. Moler, Nicholas Hoe. _Clarient GE Health Care, Aliso Viejo, CA_.

A comprehensive signaling pathway analysis is critical to characterize cancer pathogenesis, in which malignant cells evolve from nonmalignant cells, in heterogeneous tissues comprised of both healthy and pathological cells. Although significant advances in technologies have led to improved understanding of cancer biology, comprehensive profiling utilizing multiple biomarkers remains a technical challenge due to limited sample availability. GE Healthcare MultiOmyx multiplexed immunofluorescence (IF) assay overcomes this sample limitation and staining up to 60 protein biomarkers has been demonstrated in a single formalin-fixed, paraffin-embedded (FFPE) slide. In this study, proximity ligation assay (PLA) technology is adapted to expand MultiOmyx assay capabilities by enabling detection of protein-protein interactions (PPIs) and post-translational modification (PTMs) from a single FFPE slide.

The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. The PLA technology utilizes a pair of directly conjugated proximity probes to detect proteins of interest. Proximal binding of these probes lead to ligation and DNA amplification using rolling circle amplification (RCA). Amplified DNA is detected by hybridizing Cyanine dye-labeled oligonucleotides.

Herein we report a comprehensive analysis of key receptor tyrosine kinases (RTKs) (HER1, HER2, HER3, cMET, others) along with their downstream signaling proteins (PI3K, phospho AKT, and phospho ERK1/2) in 10 colorectal cancer (CRC) samples using the standard MultiOmyx assay. A PLA-adapted MultiOmyx assay is utilized to detect dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3) and RTK phosphorylation using separate antibodies against the RTK and the phosphorylation site. Protein IF staining revealed heterogeneous expression and activation across different samples. High EGFR and HER3 expression correlated with positive staining for AKT, through EGFR:HER3 dimer. High expression of EGFR correlated with positive staining for phospho Erk1/2, through EGFR:HER2 dimer. Additionally, intra-tumor heterogeneity was observed, with varied expression and activation of EGFR, HER2, HER3, and cMET.

Current IHC and multiplexed IF assays measures the expression levels of individual proteins but overlook the measurement of PPIs and PTMs, which are crucial to understanding the biology of pathway signaling. The PLA-adapted MultiOmyx assay enables true comprehensive pathway signaling analysis at a single cell level by providing spatial context and quantitative analysis of protein expression, protein-protein interactions, and protein activations (phosphorylation).

#4579

Simultaneous activation of C-X-C chemokine receptor 4 and cannabinoid receptor 2 results in decreased cell signaling and tumor cell intravasation.

Imani N. Fennell,1 Kisha A. Scarlett,2 Christopher J. Coke2. 1 _Spelman College, Atlanta, GA;_ 2 _Clark Atlanta University, Atlanta, GA_.

G-protein-coupled receptor (GPCR) heterodimerization has emerged as a means by which new signaling entities can be created with respect to agonist binding. Preliminary data from our lab indicates that agonist-bound GPCR C-X-C Chemokine Receptor 4 (CXCR4) and agonist-bound cannabinoid receptor 2 (CB2R) interact at the cell membrane and that receptor interaction mitigates the effects of CXCL12-mediated CXCR4 signaling. RhoA, Rac1 and Cdc42 are small GTP-binding proteins of the Rho family that are activated downstream of CXCR4 signaling. The effects of RhoA, Rac1 and Cdc42 on the actin cytoskeleton have led to established roles for Rho proteins in cancer cell motility, metastasis and intravasation. Here we report the novel finding that CXCR4/CB2R heterodimerization decreases Rho signaling, resulting in decreased intravasation. To test the effect of receptor heterodimerization on RhoA, Rac1, and Cdc42 activation, PC3 cells were treated with the CXCR4 agonist SDF1α, the CB2R agonist AM1241 or with a combination of SDF1α and AM1241 for 1 minute. Cell lysates were collected and immunoprecipitation experiments were performed to detect RhoA, Rac1 and Cdc42. To test the effects of CXCR4/CB2R heterodimerization on intravsation, we performed a zymography assay for the detection of metalloproteinase-2 (MMP2) and metalloproteinase-9 (MMP9), which are commonly secreted by CXCR4 signaling. Immunopreciptation results indicated an increase in RhoA and Rac1 protein levels after 1 minute in PC3 cells compared to the untreated control. However, when treated with SDF1α and AM1241 for 1 minute, RhoA and Rac1 levels decreased compared to SDF1α independent treatment; there was no observable change in Cdc42 protein levels. The results from the zymography experiments demonstrated that co-treatment with SDF1α and AM1241, which induces CXCR4/CB2R heterodimerization, led to decreased MMP-2 and MMP-9 activity, as compared to the SDF1α-independent treatment. Preliminary results provide novel evidence that CXCR4 and CB2R heterodimerize at the cell membrane, which may result in decreased signaling through RhoA and Rac1, but not through Cdc42. Furthermore, CXCR4/CB2R heterodimerization results in decreased MMP activation, thus abrogating intravsation.

#4580

A blueprint of androgen receptor splice variant transactivation.

Subing Cao,1 Duo Xu,2 Yanfeng Qi,1 Yang Zhan,1 Oliver Sartor,1 Yan Dong1. 1 _Tulane University, New Orleans, LA;_ 2 _Jilin University, Changchun, China_.

Constitutively active androgen receptor splice variants (AR-Vs) have been implicated as one driver of castration-resistant progression of prostate cancer. However, the critical steps leading to AR-V transactivation remain largely unknown. AR-V7 and ARv567es are two major AR-Vs expressed in human prostate cancer specimens. We found that disruption of AR-V7 and ARv567es homodimerization did not affect their nuclear localization, indicating that they can enter the nucleus as monomers. On the other hand, disrupting the heterodimerization between AR-V7 or ARv567es and AR-FL abolished AR-V7 and ARv567es-induced nuclear translocation of AR-FL, indicating that the heterodimerization is required for AR-FL to be piggy bagged into the nucleus. AR-V7 chromatin immunoprecipitation assay further showed that mutating the dimerization interface of AR-V7 did not decrease the ability of AR-V7 to bind to the promoter of its target genes, UBE2C and CCNA2, indicating that dimerization is not required for AR-Vs to bind to DNA. Moreover, mutating the DNA-binding interface of AR-V7 only partially inhibited AR-V7 homodimerization and had no effect on AR-FL/AR-V7 dimerization, suggesting that DNA binding may not be necessary for AR-V/AR-V or AR-FL/AR-V dimerization. Nonetheless, both the dimerization and the DNA-binding mutants of AR-V7 lost trans-activating activity. Taken together, our data suggests that AR-V DNA binding and dimerization are two independent but indispensable steps for AR-V transactivation. AR-V can enter the nucleus as a monomer, and then either forms a dimer before binding to DNA or binds to DNA as a monomer and then forms a dimer to trans-activate gene transcription. Our study may provide insights into developing more precisely targeted strategies to overcome AR-V-driven castration resistance.

#4581

**Combination treatment of orexin-A and NAB-paclitaxel in pancreas cancer:** in vitro **and** in vivo **studies.**

Thierry Voisin, Dina Plaut, Stephanie Dayot, Maxime Bergere, Valerie Gratio, Pascal Nicole, Anne Couvelard, Alain Couvineau. _INSERM, Paris, France_.

Orexin-A (OxA) and orexin-B (OxB) are hypothalamic peptides involved in the sleep/wake control which interact with two class A GPCR, OX1R and OX2R. We have demonstrated that OX1R was highly expressed in digestive cancers including cancer of colon1, pancreas2 and liver. In these cancers, orexin-A induces a mitochondrial apoptosis and a strong inhibition of tumor growth in nude mice xenografted with digestive cancer cell lines1,2. In the present work, we have compared and combined the effect of OxA and NAB-paclitaxel which represents the "gold standard" reference in the chemotherapeutic treatment of pancreas cancer, on their anti-tumoral properties. The incubation of AsPC-1 pancreatic cancer cell line which expressed OX1R, with 0.1µM OxA or 0.1 µM NAB-paclitaxel reveals a cell growth inhibition of 36% and 51%, respectively. The addition of 0.1 µM OxA and 0.1µM NAB-paclitaxel on AsPC-1 cells reveals a significantly cell growth inhibition of 70% suggesting that the double treatment was more efficient than individual treatment. Moreover, the addition of 0.1µM OxA and 0.1 µM NAB-paclitaxel induces 25% of cell apoptosis determined by annexin-V labeling, as compared to single treatment with 0.1µM OxA (18%) or 0.1 µM NAB-paclitaxel (12%). Additionally, we explore the sequential treatment by OxA and NAB-paclitaxel on cell growth of AsPC-1 cells. Our results evidenced than 48h treatment by OxA followed by 48h treatment of NAB-paclitaxel induced an inhibition of 35% of cell growth. In contrast, the reverse treatment (48H NAB-paclitaxel followed by 48h OxA) induces an inhibition of cell growth of 60%. OxA intraperitoneal injection (2 injections/week of 1.12 µmoles/kg OxA and/or NAB-paclitaxel) in nude mice xenografted with AsPC-1 cells, shows that OxA and NAB-paclitaxel induces an inhibition of tumoral volume of 60% and 62%, respectively. Moreover, injection of OxA plus NAB-paclitaxel induces an inhibition of tumoral volume of 70%. Sequential treatments of xenografted tumors in mice with OxA and NAB-paclitaxel was investigated and revealed 72% tumor growth inhibition when mice were treated 30 days with OxA followed by 30 days with NAB-paclitaxel and 83 % tumor growth inhibition when they were treated 30 days with NAB-paclitaxel followed by 30 days with OxA. These results indicate that: 1) the addition of OxA and NAB-paclitaxel improves the effect of individual treatment; 2) the sequential treatment consisting of first OxA treatment followed by NAB-paclitaxel treatment was more efficient than reverse treatment. In conclusion, OxA was close to NAB-paclitaxel treatment in term of response and suggest that combined treatment OxA/ NAB-paclitaxel represents a new promising pancreas cancer therapy

1Voisin et al., Cancer research 2011, 71:3341-51

2Speisky et al., AACR annual meeting, 2014, San Diego, USA

#4582

The role of cytohesins in TGF-alpha dependent activation of EGFR in triple negative breast cancer cells.

Tameka A. Bailey. _University of Arkansas, Fayetteville, AR_.

Epidermal growth factor receptor (EGFR) positive, triple negative breast cancer (TNBC) is the most lethal form of breast cancer and occurs more frequently in African American women, accounting in part for disparate outcomes. Thus, EGFR is an attractive therapeutic target to treat African American patients with TNBC. Previous studies have shown that the EGFR ligand transforming growth factor alpha (TGF-alpha) is more highly expressed in triple negative breast tumors than those of other molecular subtypes. The co-expression of EGFR with TGF-alpha has been shown to establish sustained downstream signaling, which leads to heightened cell proliferation, invasion and metastasis. Cell biologically, the sustained signaling reflects constitutive endocytic recycling of TGF-alpha-activated EGFR. Cytohesins have been identified as modulators of ligand-dependent EGFR activation in lung cancer. However it is unknown whether these regulators of endocytic recycling play a role in TGF-alpha dependent EGFR activation in TNBC. Here, we investigated whether SecinH3, a cytohesin antagonist, impacts the TGF-alpha dependent activation of EGFR in TNBC cells. Western blotting and immunoprecipitation were used to examine the status of tyrosine phosphorylation of EGFR in TNBC cells treated with TGF-alpha, SecinH3 or a combination of TGF-alpha and SecinH3. Our results show that co-treatment with SecinH3 reduced the total and phosphorylated EGFR, suggesting that cytohesins may play an important role in sustaining TGF-alpha-EGFR sustained signaling in TNBC. Additional studies are underway to further link these findings to EGFR recycling in TNBC, elucidate the role of cytohesins in TNBC and to assess the therapeutic potential of SecinH3 in EGFR positive TNBC.

#4583

Potential role of neuropilin-2 in the regulation of c-MET-induced PD-L1 expression in renal cancer cells.

Murugabaskar Balan, Soumitro Pal. _Boston Children's Hospital, Boston, MA_.

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins that act as co-receptors for class III semaphorins and several members of the vascular endothelial growth factor (VEGF) family. In conjunction with VEGF receptors and plexins, NRPs and semaphorins modulate various biological responses. Recent studies suggest that NRPs are up-regulated in many types of cancer cells, and may play a major role in tumor growth. In addition to semaphorins and VEGF, NRP1 and NRP2 can also bind to hepatocyte growth factor (HGF), which is the ligand for the tyrosine kinase receptor c-MET. c-MET is highly over-expressed in renal cancer cells; however, the impact of NRPs on c-MET-mediated tumorigenic pathways have not been studied. Recently, we have reported that HGF-/c-MET-induced signaling increases the expression of immunosuppressive and negative-co-stimulatory molecule programmed death-1 ligand (PD-L1) on renal cell carcinoma (RCC) cells, and it protects tumor cells from immune-mediated killing. In this study, we investigated whether NRPs and its ligands can modulate the expression of HGF-/c-MET-induced PD-L1. We have observed that NRP2, and to a lesser extent NRP1, is over-expressed in human RCC cell lines (786-O, ACHN and Caki-1) compared with normal renal epithelial cells at both mRNA and protein level. siRNA-mediated silencing of NRP1 and NRP2 promoted apoptosis of renal cancer cells, suggesting possible role(s) of NRPs in tumor growth. The knock-down of NRP2 down-regulated the cell surface expression of PD-L1 possibly through the reduced interaction of growth-promoting ligands (like, HGF) with NRP2. Interestingly, SEMA3F, a class III semaphorin ligand for both NRP1 and NRP2, has been reported to have anti-tumorigenic effects. We observed that SEMA3F was significantly down-regulated in RCC cells compared with normal renal epithelial cells; and when we added SEMA3F to the cultures of renal cancer cells, it down-regulated both basal and HGF-/c-MET-induced PD-L1 expression. Together, our findings suggest that NRPs (primarily NRP2) can play a critical role in regulating HGF-/c-MET-induced PD-L1 expression in renal cancer cells to mediate immune escape of tumor cells. Therefore targeting NRP2 and/or its activation through SEMA3F/synthetic ligands may restrict c-MET-PD-L1-induced tumor promoting pathways in renal cancer cells.

#4584

Apoptosis-mediated AXL activation: Contribution to metastatic cancers.

Annelien Zweemer, Aaron Meyer, Douglas Lauffenburger. _MIT, Cambridge, MA_.

AXL is a receptor tyrosine kinase that is widely expressed in a variety of cancers and is predictive of poor patient outcome. This receptor is an essential epithelial-to-mesenchymal transition-induced regulator of cancer metastasis and is related to the occurrence of treatment resistance. AXL activation requires both the lipid moiety phosphatidylserine (PtdSer) and the bridging protein ligand Gas6. PtdSer is only available to mediate AXL activation when it is externalized on cell membranes, an event that occurs specifically during apoptosis. For that reason, we aimed to identify if apoptotic cell debris is capable of activating AXL on cancer cells and how this would affect the metastatic capacity of these cells.

In a non-AXL expressing tumor cell line (HCC827) apoptosis was induced by means of UV-irradiation. We detected AXL activation of AXL-expressing MDA-MB-231 cells that were treated with HCC827 cell debris, suggesting that apoptotic debris of dying tumor cells is capable of activating AXL in surviving surrounding tumor cells. Next we determined the contribution of PtdSer-mediated AXL activation to the migration of MDA-MB-231 cells in a wound scratch assay. The addition of warfarin, which prevents autocrine Gas6 binding to PtdSer, decreased migration to a similar extent as the AXL inhibitor R428.

This study revealed that PtdSer-mediated AXL activation contributes to the migration properties of MDA-MB-231 cells. As we detected AXL activation upon addition of PtdSer-containing apoptotic cells debris, this suggests that the induction of apoptosis by cancer therapeutics might enhance the metastatic capacities of neighboring surviving tumor cells. This finding may broadly impact the oncology field given AXL's widespread expression among different types of cancers, and provides a rationale for the design of novel combination therapies for decreasing tumor metastasis.

#4585

ROR1 sustains caveolae and RTK-mediated survival signaling as a scaffold of cavin-1 and CAV1 in lung cancer.

Tomoya Yamaguchi,1 Can Lu,1 Lisa Ida,1 Kiyoshi Yanagisawa,1 Jiro Usukura,2 Jinglei Cheng,3 Naoe Hotta,1 Yukako Shimada,1 Hisanori Isomura,1 Motoshi Suzuki,1 Toyoshi Fujimoto,3 Takashi Takahashi1. 1 _Division of Molecular Carcinogenesis, Nagoya University Graduate School of Medicine, Nagoya, Japan;_ 2 _Division of Integrated Project, EcoTopia Science Institute, Nagoya University, Nagoya, Japan;_ 3 _Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan_.

TTF-1/NKX2-1, a homeobox-containing transcription factor indispensable for lung morphogenesis, was previously identified by four independent groups including us, as a "lineage-survival" oncogene involved in the pathogenesis of lung adenocarcinoma. We have further shown that TTF-1/NKX2-1 induces expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn plays a "sustainer" role for EGFR-mediated signaling. Interestingly, ROR1 knockdown effectively overcame EGFR-TKI resistance-conferring, HGF-mediated bypass signaling through MET, suggesting that ROR1 sustains not only EGFR but also additional RTK signaling. However, the underlying mechanism remained rather elusive.

We report here an unanticipated function of this receptor tyrosine kinase (RTK) as a scaffold of cavin-1 and caveolin-1 (CAV1), two essential structural components of caveolae. Caveolae are 50-100 nm invaginations of the plasma membrane that play various physiological roles, including function as a platform for insulin-induced signaling in adipose tissue. We found that ROR1 facilitates the interactions of cavin-1 and CAV1 at the plasma membrane in a kinase-independent manner, thereby preventing the lysosomal degradation of CAV1, as well as consequential caveolae formation. Caveolae structures and prosurvival signaling towards AKT through multiple RTKs such as MET and IGF-IR are consequently sustained. The present findings thus provide mechanistic insight into how ROR1 inhibition can overcome EGFR-TKI resistance due to bypass signaling via diverse RTKs, which is currently a major clinical obstacle.

Inhibition of prosurvival signaling and proliferation of lung cancer cells could be recapitulated by knocking down cavin-1 or CAV1, but they are unlikely to be suitable molecular targets for NSCLC treatment due to their crucial physiological functions in various organs even at low expression levels. In this regard, it should be noted that ROR1 is considered to be an onco-embryonic antigen with tumor-specific expression in adults. Taken together, development of novel therapeutic means to inhibit the scaffold function of ROR1 and thereby attack the cancer's "Achilles heel" is thus anticipated to be an attractive approach for improved treatment of this devastating cancer.

#4586

FGFR2 amplification in serous ovarian cancer.

Alexandra Tyulyandina,1 Ilya Tsimafeyeu,2 Irina Demidova,3 Marina Gikalo,3 Sergei Tjulandin1. 1 _N.N. Blokhin Russian Cancer Research Center, Moscow, Russian Federation;_ 2 _Russian Society of Clinical Oncology, Moscow, Russian Federation;_ 3 _Moscow City Clinical Cancer Hospital #62, Moscow, Russian Federation_.

Fibroblast growth factor receptor (FGFR) signaling pathway plays an important role in biology of cancer. Approximately 7% of ovarian cancer (OC) exhibits FGFR1 amplification. Recently the overexpression of FGFR2 was found in 95% of clear cell OC. Up to date there is no data about FGFR2 status in OC. 54 paraffin-embedded blocks from 18 patients with serous OC were analyzed by FISH to identify FGFR2 amplification. Scoring for amplification and polysomy level was adopted from previous studies for gastric cancer (Su X, et al. BJC 2014). Material from each patient had 3 samples: from primary tumor, from primary metastatic lesions, and from relapse. Intratumoral FGFR2 amplification heterogeneity was assessed in sections from all cases with FGFR2 amplification, and was defined as the presence of areas with different FISH scores within the same tumor in full sections.

Amplification was observed in 3 patients (15.7%). Interestingly, in one case amplification was observed in primary ovarian tumor but not in the metastatic nor in relapse samples. In two other cases the FGFR2 amplification was detected in relapse samples and in primary metastatic samples but not in ovary. High-level polysomy (HLP) was observed in 10 patients (55.5%). In all of those patients HLP was revealed in the samples of relapse. In 3 patients HLP was found in all 3 samples, in 3 cases HLP was observed only after relapse, in 4 cases HLP in metastasis sample was the same as in relapse sample, but it wasn't observed in ovary samples. Eight of 13 FGFR2 amplified ovarian cancers or tumors with HLP (61.5%) displayed intratumoral heterogeneity within full sections. In 5 (27.8%) cases neither amplification nor high level polysomy was observed. In conclusion, this is the first study of FGFR2 FISH in serous ovarian adenocarcinoma, demonstrating a FGFR2 amplification and HLP in primary tumor, primary metastases and relapse. Furthermore, we found evidence for intratumoral heterogeneity of FGFR2 amplification and HLP in about 61.5% of ovarian cancers. This study was supported by grant from the "Oncoprogress Foundation".

FGFR2 amplification and high-level polysomy in patients with serous cancer (13 patients)

---

|

N (%), patients | Primary tumor (N samples) | Primary metastasis (N samples) | Relapse (N samples)

Amplification | 3 (15.7%) | 1 | 2 | 2

High level polysomy | 10 (55.5%) | 3 | 4 | 10

#4587

Dynactin dysregulation-induced increase in EGFR on basal-like breast cancer cells.

Gautam Chaudhuri, Smita Misra. _Meharry Medical College, Nashville, TN_.

Aggressive triple-negative basal-like breast cancer (BLBC) cells are often characterized with overabundance of the epidermal growth factor receptor (EGFR). Major objective of our research is to explore the possibility that dysregulation of the function of the dynactin complex as a mechanism of over-abundance of EGFR on BLBC cell surface. Our notion is that molecular defects in the dynactin-mediated retrograde EGFR recycling pathway in the BLBC cells causes the accumulation of this receptor on their cell surface making them hyper-responsive to EGF signaling. Our comparative transcriptome analysis between BLBC cells with higher levels of EGFR and the non-BLBC cells with lower levels of EGFR revealed that the expression of dynactin complex subunits 1, 2, 3, 4 and 6 (DCTN1, 2, 3, 4, 6), are significantly reduced in the EGFR-high aggressive BLBC cells. In addition, EGFR-high BLBC cells also have lower levels of two other dynactin-associated proteins, CLIP1 and LRRK1. Interestingly, elevation of surface EGFR levels in BLBC cells are associated with higher levels of the long non-coding RNA PVT1. Six miRNAs are generated from the precursor of PVT1 RNA during cis-splicing. They are miR-1204, miR-1205, miR-1206, miR-1207-3p, miR-1207-5p and miR-1208. We found that relative levels of these miRNAs are significantly higher in EGFR-high BLBC cells and tissues in comparison to the non-aggressive EGFR-low BLBC cells. Bioinformatics analysis followed by experimental validation revealed that miR-1207-5p inhibits DCTN1, DCTN3 and ACTR1A; DCTN2 is inhibited by miR-1205 and miR-1208. MiR-1208 also inhibits DCTN6 and CLIP1 whereas LRRK1 and CAPZA1 are inhibited by miR-1207-3p and miR-1206, respectively. Moreover beta-spectrin (SPTB), a protein associated with dynactin complex, is also inhibited by miR-1205 and miR-1207-3p. Treatment of the cells with the antagomirs of the miRNAs increased the levels of their respective target proteins with concomitant decrease in the surface level of EGFR. Our data suggest that BLBC cells maintain a robust level of EGFR on the cell surface by repressing the retrograde transport of EGFR through the repression of the proteins in the dynactin complex and thus sustain their higher growth rates. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC.

#4588

Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation.

Eva McGhee,1 Lucy Tran,1 Mengtao Li,2 Yong Wu,1 Seyung Chung,1 Cheryl Araniego,1 Karen Tate,1 Melanie Baker,1 Kamilah Evans,1 Mechelle Rouse,1 Jay Vadgama1. 1 _Charles R. Drew University of Medicine and Science, Los Angeles, CA;_ 2 _UCLA School of Dentistry, Los Angeles, CA_.

The association of human papillomavirus (HPV) with cervical cancer, head and neck cancer is well established, but the involvement of HPV E6/E7 oncoproteins in breast cancer is more contentious. Previous studies suggest that high risk HPV E6/E7 oncoproteins inactivate two tumor suppressor proteins p53 and pRb signaling and increase cell proliferation. There is cumulative evidence that high risk HPV 16 E6/E7 oncoproteins may have causal roles in some human breast cancers. Moreover, some studies have identified HPV DNA in breast tissue and breast cancer specimens. Nevertheless, the route of transmission of HPV in breast cells has not been determined, and the mechanisms by which HPV involvement in breast cancer are not known. We used a non-tumorigenic (MCF12A) epithelial breast cell line transfected with HPV 16 to elucidate the role of oncoproteins E6/E7 in pro-proliferative and anti-apoptotic signaling pathways. We also used Tamoxifen treatment, which is an antagonist of the estrogen receptor in breast tissues through its active metabolite, 4hydroxytamoxifen. We demonstrate that HPV E6/E7 transfection activates the extracellular signal-regulated kinase (ERK) MAPK pathway and Akt/PKB kinase signaling pathways through activating estrogen receptor alpha in MCF12A cells. E6/E7 oncoproteins transfection alone dramatically increases estrogen receptor alpha protein expression. Treatment with tamoxifen, an antagonist of the ER, significantly abolishes E6/E7-increase in ERK and PKB phosphorylation without altering their protein levels, suggesting that the estrogen receptor is a key mediator of HPV E6/E7 mediated ERK and Akt/PKB activation. Our current work show an insightful understanding of the molecular mechanisms of HPV-elicited Akt/PKB activation and its roles in cell proliferation and survival in normal breast epithelial cells. Therefore, these findings indicate that HPV E6/E7 oncoproteins may promote the transforming activities of high-risk HPV E6/E7 proteins and cell proliferation in breast cells. Therefore, suppression of estrogen receptor signaling networks may be used as a therapeutic strategy for HPV associated lesions and cancers such as breast metastasis.

#4589

Perturbation of epidermal growth factor (EGF) receptor internalization by novel aspirin analogues in SW480 cells.

Asma'U I.J. Bashir,1 Sarah Jones,1 Christopher J. Perry,1 Stephen T. Safrany,2 Iain D. Nicholl1. 1 _University of Wolverhampton, Wolverhampton, United Kingdom;_ 2 _RCSI-MUB, Adliya, Bahrain_.

BACKGROUND

Colorectal cancer (CRC) is a global phenomenon with over 135 million new cases and over 600,000 deaths worldwide annually. The EGFR regulates normal growth and differentiation of cells, however its dysregulation is the cause of a wide range of cancers, including CRC. Activation of the EGFR requires phosphorylation at specific sites, which then leads to the recruitment of proteins and ultimately its internalization.

The use of low-dose aspirin as a preventative agent is a controversial topic, particularly as the mechanism of action is hotly disputed. We have previously determined that novel aspirin analogues dramatically reduce EGFR phosphorylation in SW480 CRC cells; these cells express neither COX-1 nor COX-2, the perceived target for aspirin-like compounds. We have seen these aspirin analogues to also perturb the internalization of this receptor.

METHODS

SW 480 CRC cells were cultured in Leibovitz's L-15 medium on glass slides and starved for 48 hours. The cells were then treated with 0.5 mM aspirin analogues (ortho-aspirin, meta-aspirin, para-aspirin, ortho-thioaspirin, meta-thioaspirin and para-thioaspirin) for 30 minutes and then treated with EGF-tagged with AlexaFluor 555 for an hour. The reaction was the quenched with cold PBS, fixed and mounted on microscope slides with VectaShield + DAPI. Confocal microscopy was used to study the effect of these aspirin analogues on EGFR internalization.

RESULTS

EGF increased phosphorylation at all sites tested. We have peviously shown that these effects were prevented by preincubation with thioaspirins more potently than with aspirin and its positional isomers. These novel thioaspirins are also more potent in inhibiting proliferation of SW480 CRC cells. We now show these analogues prevent the internalization of EGFR. Instead they permit the formation of clusters. Aspirin and its analogues can perturb EGFR internalization and signalling in SW 480 cells.

CONCLUSION

The EGFR signalling pathway is a target for aspirin and aspirin analogues. The inhibition of receptor phosphorylation is accompanied by the loss of receptor internalization.

#4590

Mechanism of PI3K activation by the HER3/ErbB3 receptor.

Nicole Michael, Michael Hopkins, Natalia Jura. _University of California San Francisco, San Francisco, CA_.

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) is a receptor tyrosine kinase that lacks catalytic activity but is essential for cellular homeostasis due to its ability to allosterically activate its heterodimeric partners, EGFR and HER2 (also known as HER1/ErbB1 and ErbB2, respectively). HER3 is an important therapeutic cancer target because the signaling emanating from HER3 heterodimers is critical for the progression and maintenance of several human epithelial malignancies. This is primarily due to the potent ability of HER3 to recruit and activate the phosphoinositide 3-kinase (PI3K). This ability has been attributed to the presence of multiple direct binding sites for PI3K located in the intracellular tail of HER3. The advantage of having multiple recruitment sites is unclear and our recent data show that they provide little advantage to the ability of HER3 to activate PI3K. In addition, we have identified an alternative mode by which HER3 can activate PI3K, demonstrating a complex mechanism by which HER3 phosphorylation is coupled to activation of PI3K/Akt signaling pathway. Our results have important implications for therapeutic targeting of a number of human cancers in which HER3/PI3K signaling axis is abnormally activated and contributes to disease progression.

#4591

Estrogen receptor-positive breast cancer: from genomic landscape to treatment approach.

Pradip De, Casey Williams, Amy Krie, Kirstin Williams, Jessica Klein, Jennifer H. Carlson, Nandini Dey, Brian Leyland-Jones. _Avera Research Institute, Sioux Falls, SD_.

Background: Estrogen receptor (ER)-positive or luminal breast tumors represent around two-thirds of all breast cancers. Luminal breast cancer is a highly heterogeneous disease comprising different histologies, gene-expression profiles and mutational patterns, with very diverse clinical courses and responses to systemic treatment. Despite adjuvant endocrine therapy and chemotherapy treatment for patients at high risk of relapse, both early and late relapses still occur, a fact that highlights the unmet medical needs of these patients. A catalogue of molecular aberrations in ER+ breast cancer (BC) is critical for developing and deploying therapies that will improve patients' lives. Methods: Comprehensive genomic profiling from 128 BC patients (February 2014 through October, 2015) were analyzed. Patients were biopsied after consultation and samples were characterized (ER, PR, and HER2 by IHC; FFPE samples for genomic [Foundation One] and proteomic analyses [Theranostics]). We also evaluated mutation distribution in cell free DNA via digital NGS using Guardant 360 panel. Results: Total 97 genes were altered in 83 ER+BC patients and most frequent genetic alterations are PIK3CA (40%), p53 (27.7%) and CCND1 (24%). The PI3K-AKT pathway specific genes (AK1, AKT2, PIK3CA, PIK3CB, PIK3R1, PTEN, MTOR, RICTOR, RAPTOR, TSC2, STK11, MDM2 and MDM4) were altered in 80% of patients. Analyzing the composite alterations in individual ER+BC patients, we observed that 20% of patients had alterations in two or more nodes of the PI3K pathway while alterations of cell cycle pathway genes (CCND1, CCNE1, CDK4, CDKN1B, CDKN2A/B and RB1) were observed in 36% of patients. Interestingly, concurrent alterations of both PI3K and cell cycle pathways were 24%. Hence we tested the anti-tumor efficacy of the combination of isoform-specific PI3K inhibitor (alpha or beta) and LEE011 using ER+ BC cell lines (PIK3CA mutated MCF7, T47D, & PTEN-null MDA-MB415 cells) based model. Data show a synergistic effect of PI3K alpha or beta inhibitor and CDK4/6 inhibitor as determined from the changes in proliferative and apoptotic signals as well as cell cycle arrest. Conclusion: Since a persistent expression of the CDK4-Cyclin D1 pathway activation enables tumor cells survival even in the presence of PI3K inhibitor (Cancer Cell 2014), results of our study demonstrated that the anti-tumor effect of isoform-specific PI3Ki and CDK4/6i be beneficial in RB wt ER+BC patients with a concurrent genetic alterations of these pathways.

#4592

MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression.

Sheema Khan,1 Mara C. Ebeling,2 Mohammad Sikander,1 Murali M. Yallapu,1 Tomoko Ise,2 Satoshi Nagata,2 Stephen W. Behrman,1 Nadeem Zafar,1 Jim Y. Wan,1 Hemendra M. Ghimire,3 Peeyush Sahay,3 Prabhakar Pradhan,3 Meena Jaggi,1 Subhash C. Chauhan1. 1 _University of Tennessee Health Science Center, Memphis, TN;_ 2 _Sanford Research, Sioux Falls, SD;_ 3 _University of Memphis, Memphis, TN_.

Background: Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States and has a very poor survival rate due to late diagnosis. MUC13 is a recently identified high molecular weight glycoprotein that is upregulated in PDAC and its progression is allowed via alterations of multiple signaling pathways. MUC13 is aberrantly expressed in PDAC and generally correlates with increased expression of HER2, however, the underlying mechanism remains poorly understood. MUC13 consists of three EGF-like domains that may serve as a ligand for EGF receptors, such as HER2, and modulate EGFR signaling pathways. We sought to better characterize the interaction of MUC13 with HER2 in PDAC.

Methods: MUC13 and HER2 interaction was studied using reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, Western blotting, co-capping assays in human PDAC cell lines and immunohistofluorescence techniques in human tissues. Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens of PDAC were assessed for expression of MUC13 and HER2 using our own laboratory generated anti-MUC13 mouse monoclonal antibody (MAb) through confocal immunofluorescence. The association of MUC13 and HER2 co-localization with nuclear chromatin organization was analyzed to study the stage or degree of aggressiveness of the pancreatic cancer using Dapi stained confocal images of tissues.

Results: MUC13 co-localizes and interacts with HER2 in PDAC cell lines. The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at Tyrp1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling, motility and invasion of PDAC cells - all collectively contributing to PDAC progression. The interaction between MUC13-HER2 binding resulting in their tumorigenic characteristics likely occurs at the 1st and 2nd but not the 3rd domains of MUC13 as the EGF 1 and 2 deletion mutant constructs of MUC13 failed to promote proliferation and invasion of cells. These phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13. MUC13-HER2 co-localization also held true in PDAC human tissues with a strong functional correlation that contributed to an increased degree of disorder and cancer aggressiveness.

Conclusions: 1) MUC13 can be detected in formalin-fixed paraffin-embedded tissues using our anti-MUC13 MAb. 2) MUC13 co-localizes and activates HER2 and its downstream signaling cascade promoting PDAC progression in both cell lines and human tissue. 3) This process is reversed by depletion of MUC13. 4) This MUC13-HER2 interaction may potentially be manipulated for targeted therapeutics in patients harboring PDAC.

#4593

Analysis of transforming growth factor β receptor trafficking on different signaling transduction pathways.

Evelyn Ng, John Di Guglielmo. _Western University, London, Ontario, Canada_.

Transforming growth factor beta (TGFβ) is a cytokine that regulates cellular adhesion, proliferation and apoptosis. Its canonical downstream effectors include receptor-regulated Smad2/3 proteins, which are phosphorylated and then translocated to the nucleus to alter transcription. Additionally, other atypical pathways are simultaneously initiated by TGFβ, including that of p38 (a mitogen activated protein kinase).

TGFβ acts as a tumour suppressor in its normal epithelial environment, but in tumor cells, it promotes epithelial-mesenchymal transition and cell migration. The manner in which tumor cells overcome the growth suppressive effects of TGFβ is not well-understood, especially since downstream TGFβ signaling is still apparent in these cells. This may point to selective pathway activation as a reason for its dual roles. Thus, it is important to consider membrane trafficking—a central process that directs signaling between various cascades.

Previous studies from our lab demonstrate that perturbation of aPKC signaling alters TGFβ receptor trafficking and Smad signal transduction. Furthermore, we have also observed increased access to the p38 MAPK pathway in cells that have been silenced for aPKC expression. We are currently assessing the trafficking of the TGFβ receptor complex in order to investigate the mechanism(s) responsible differential access to specific signaling pathways. This will be addressed using techniques such as co-immunoprecipitation, immunofluorescence microscopy, subcellular fractionation and western blotting.

Determining these mechanisms will further our understanding on cancerous TGFβ pathway regulation, and may lead to the discovery of promising therapeutic targets.

#4594

Involvement of receptors tyrosine kinase in the modulation of store-operated calcium entry.

Noémie Emeriau. _Universite Catholique de Louvain, Brussels, Belgium_.

A multitude of studies show that alterations in Ca2+ signalling initiate or support the development of hallmarks of cancer. In particular, the role of store-operated Ca2+ entry (SOCE) in tumorigenesis and tumour progression has been subject to intense investigation. SOCE is the main mechanism by which external Ca2+ enters into the cell. It is initiated by depletion of ER Ca2+ stores and mediated by several proteins such as STIM1, an ER transmembrane protein sensing Ca2+ within the ER and ORAI1, a plasma membrane Ca2+ channel. We recently showed that SOC inhibition interfered with EGFR-dependent signalling in non-small cell lung carcinoma cells.

In this study, we observed that STIM1 depletion reduced neuregulin-dependent proliferation of breast cancer cells. Since neuregulin binds to ErbB3 and/or ErbB4 and therefore activates ErbB2, this result prompted us to investigate whether ErbB proteins might modulate SOCE. We observed that lapatinib, a dual inhibitor of EGFR and ErbB2, dramatically inhibited SOCE. As expected, lapatinib also inhibited phosphorylation of EGFR, ErbB2 and downstream pathways of both receptors. Specific inhibition of EGFR by erlotinib or gefitinib had no effect on SOCE. In contrast, specific inhibition of ErbB2 by CP724714 mimicked the effects of lapatinib. We also investigated the role of downstream pathways of ErbB2 in the modulation of SOCE. Inhibition of the MAPK and the JAK-STAT pathways does not modify the amplitude of SOCE. Contrariwise, LY294002 and MK2206, two inhibitors of the PI3K pathway, dramatically reduced SOCE. Interestingly, in silico analysis showed that STIM1 might be phosphorylated by Akt.

All these results suggest that SOCE is important in the proper function of ErbB2-dependent signalling. This raises the possibility to target molecular mediators of SOCE in order to interfere with the proliferation of cancer cells.

#4595

Development and validation of a novel EGF receptor-neutralizing monoclonal antibody.

Krystyna Zuberek Hincman, Christopher A. Manning, Michael R. Nelson, Michael Lewis, Roy Scialdone, Jaime Darce, Stephen R. Lutz, Evans Burford, Herbert Haack, Matthew R. Silver. _Cell Signaling Technology, Inc., Danvers, MA_.

The epidermal growth factor receptor (EGFR) is a cell-surface receptor for members of the epidermal growth factor family (EGF-family). EGFR dimerizes upon ligand binding, which leads to autophosphorylation, downstream signaling and ultimately internalization. In cancer, EGFR mutations and amplification have been linked to cell proliferation and survival. Two EGFR neutralizing monoclonal antibodies are currently FDA approved, which includes Cetuximab (Erbitux, BMS; mouse/human chimeric) and Panitumumab (ABX-EGF, Amgen; fully humanized). However, these antibodies are often unavailable to researchers studying this important pathway. Here we describe the development and validation of a new rabbit monoclonal EGFR neutralizing antibody (clone D1D4J). D1D4J was validated using cell-based models and immunofluorescence imaging. D1D4J binds EGFR and neutralizes EGF-induced activation and internalization. Compared to other Research Use Only (RUO) neutralizing antibodies on the market, D1D4J shows superior performance and is a useful research tool in the EGFR signaling space.

#4596

Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer.

Christiana S. Kappler, Ericka L. Smith, Stephen Ethier. _Medical University of South Carolina, Charleston, SC_.

Although the basal-like 1 (BL1) and BL2 subtypes of triple negative breast cancer (TNBC) share similar genetic and biological characteristics, patients with BL2 type tumors have markedly poorer prognosis. In addition, despite the overexpression of epidermal growth factor receptor (EGFR) characteristically observed in basal-like TNBC, EGFR inhibitors have performed poorly in clinical trials. In order to understand the mechanism of resistance to EGFR inhibition in these tumors, our laboratory utilized cell line models of BL2 (SUM-149 and SUM-229) and BL1 (MDA-MB-468; MM468) TNBC, all of which overexpress EGFR, are PTEN null and exhibit elevated AKT phosphorylation (p-AKT). Compared to MCF10A and MM-468 cells, AKT phosphorylation in the SUM-149 and SUM-229 cell lines was resistant to treatment with the EGFR inhibitor gefitinib, as well as to the PI3K inhibitor BKM120 (BKM) and the mTOR inhibitor KU-0063794 (KU). Even treatment with the allosteric AKT inhibitor MK2206 (MK) only partially reduced p-AKT. RT-PCR and immunoblot analysis of AKT isoform expression showed that in SUM-149 and SUM-229 cells, AKT3 is expressed at higher levels than AKT1 or AKT2 at both the protein and message level. Because AKT3 has a higher IC50 for MK than AKT1 or AKT2, increasing doses of MK were used in an effort to abrogate AKT phosphorylation. The results showed that MK treatment at higher concentrations (5 μM) decreased p-AKT. In addition, shRNA-mediated knock-down of AKT3 sensitized the cells to inhibition of AKT by BKM, KU and MK. Interestingly, MK treatment alone had little effect on colony forming efficiency, even at higher doses, but when added in combination with gefitinib, MK dramatically reduced colony formation. We then investigated the effect of PTEN re-expression on signaling in these cell lines. PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, but resulted in a dramatic reduction in p-AKT in MM-468 cells. Although PTEN expression alone had little effect on p-AKT in SUM-149 and 229 cells, these cells exhibited decreased p-AKT following treatment with gefitinib and were sensitive to inhibition of AKT by MK. In addition, treatment of SUM-149/PTEN cells with gefitinib markedly reduced colony-forming efficiency. Our laboratory has previously demonstrated that in SUM-149 cells, a set of genes that are essential for survival, including PLK1, are uncoupled from regulation by EGFR. Thus, we examined the effect of AKT inhibition on expression of PLK1 and found that inhibition of AKT or re-expression of PTEN was able to recouple EGFR-mediated regulation of PLK1 expression. Our results demonstrate that PI-3 kinase and mTORC2 drive phosphorylation of AKT1 but not AKT3 in SUM-149 cells, which explains their relative resistance to these targeted drugs.

#4597

The G protein-coupled P2Y6 pyrimidoceptor protects colorectal cancerous epithelial cells from apoptosis: Is this receptor a novel target against colorectal cancer.

Morgane Placet, Caroline M. Molle, Guillaume Arguin, Sameh Geha, Fernand-Pierre Gendron. _Université de Sherbrooke, Sherbrooke, Quebec, Canada_.

BACKGROUND. Colorectal cancer (CRC) originates from the accumulation of genetic mutations and epigenetic deregulation of tumor suppressor genes (TSGs) and oncogenes, most prominently APC, K-RAS, and TP53. As a result, transformed intestinal epithelial cells (IECs) acquire an evolutionary growth advantage leading to tumor formation. Like other solid tumors, various types of immune cells, and immunomodulatory molecules, including extracellular nucleotides, such as adenosine 5'-triphosphate (ATP) and uridine 5'-diphosphate (UDP), are found in the vicinity of colorectal tumors and promote tumor formation. In this context, activation of the G protein-coupled P2Y6 receptor (P2Y6R) by UDP could contribute to the formation of tumor-promoting microenvironment by modulating the immune response, as we previously reported, but also by coordinating resistance to apoptosis, one of the hallmarks of human CRC.

METHODS: Colorectal cancer was induced in P2ry6 gene knockout mice (P2Y6-/-) using azoxymethane along with dextran sulfate sodium challenges. Mice were euthanatized and the number and size of tumor measured. Tissues were fixed and stained prior to histological characterization by a pathologist. In vitro, receptor expression level was determined by qPCR analysis in normal and cancerous IECs as well as from CRC tumor biopsies. Cell growth curves and clonogenic soft agar assays were realized to determine the effect of P2Y6R stimulation on cell growth and tumorigenic potential. Finally, the impact of P2Y6R activation on apoptosis was determined by analyzing apoptosis markers by western blotting.

RESULTS: Invalidation of the P2ry6 gene in mice lead to a reduction in the number and size of colorectal tumors, which correlated with the ability of the activated P2Y6R to induce cancerous cell growth in both classical cell growth assays and clonogenic soft agar experiments. Furthermore, P2Y6R stimulation with UDP is not only stimulating cell growth but it also protects colorectal cancerous epithelial cells from TNF-induced apoptosis by a molecular mechanism involving the increase expression of the X-linked inhibitor of apoptosis protein (XIAP).

CONCLUSIONS: In this study, we have shown that sustained activation of P2Y6R could contribute to intestinal tumorigenesis by blocking the apoptotic process and by stimulating cell growth. These results suggest that P2Y6R could be a prime target to reduce colorectal carcinogenesis.

#4598

Androgen receptor acetylation at K618 by ARD1 is essential for dissociation from HSP90 in prostate tumorigenesis.

John S. DePaolo,1 Zehua Wang,1 Jianhui Guo,1 Guanyi Zhang,2 Haitao Zhang,2 Jovanny Zabaleta,1 Wanguo Liu1. 1 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 2 _Department of Pathology and Laboratory Medicine, Tulane University, New Orleans, LA_.

Prostate cancer is an androgen receptor (AR)-driven disease and post-translational modification of AR is critical for its activation in both hormone-sensitive and castration-resistant disease. We previously reported that ARD1 is an oncoprotein in prostate cancer. It acetylates and activates AR to promote prostate tumorigenesis. However, the target residue within AR and the roles and mechanisms of the acetylation event in prostate tumorigenesis remained unknown. Here we show that ARD1 interacts with the AR DNA-binding domain and acetylates AR at lysine 618 (K618) in vivo and in vitro. Inhibition of K618 acetylation by replacing lysine with arginine significantly reduces AR transcriptional activity, prostate cancer cell growth, and xenograft tumor formation due to attenuation of AR nuclear translocation. Constitutive acetylation at K618 by replacing lysine with glutamine reverses these effects. Mechanistically, ARD1 forms a complex with both AR and HSP90. Expression of ARD1 increases levels of AR acetylation and AR-HSP90 dissociation in a dose dependent manner. Moreover, the AR acetylation-defective K618R mutant is unable to dissociate from HSP90 following ligand exposure, and HSP90-dissociated AR is acetylated. This work identifies a new mechanism for ligand-induced AR-HSP90 dissociation and AR activation. Targeting ARD1-mediated AR acetylation may be a potent intervention for AR-dependent prostate cancer therapy. Given that K618 is also present in AR variants, the significance of ARD1-dependent acetylation of AR variants for castration-resistant PCa will be discussed.

#4599

Bile acids modulate estrogen receptor β signaling through nongenomic actions in colorectal cancer.

Jesse Trujillo. _University of Arizona Cancer Center, Tucson, AZ_.

Bile acids deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) have contrasting effects on tumor development. DCA is measurable at increased levels in the serum of colorectal adenocarcinoma patients and is associated with disease progression. DCA is also a well-established tumor promoter. On the other hand, UDCA is a chemopreventative agent revealed to suppress the recurrence of highly dysplastic polyps in a phase III clinical trial. Remarkably, retrospective investigation of the data had shown that UDCA's efficacy as a chemopreventive mediator was gender specific. UDCA significantly lowered the recurrence of advanced lesions in males, but caused a significant increase in the recurrence of advanced lesions in females. Our lab is in pursuit of understanding the gender specific chemopreventive role of UDCA. Estrogen receptor β (ERβ) has been shown to play a significant role in controlling the development of colorectal cancer. Therefore, we hypothesized that the altered regulation of the ERβ pathway by UDCA might explain the gender specificity of this molecule. In order to assess this, we analyzed the effects of UDCA on ERβ to those of the known tumor promoter DCA in both male and female human adenocarcinoma cell lines. Female HT-29 colorectal adenocarcinoma cells induced the phosphorylation of ERβ, as well as up-regulated gene expression of estrogen response elements (EREs), showing stimulated activity of this hormonal receptor. Moreover, we found that pre-treating the cells with PD98059, a MEK 1/2 inhibitor, UDCA and DCA-induced phosphorylation of ERβ, along with increased ERE gene expression, was significantly reduced. Collectively, this data suggests that UDCA and DCA can stimulate ERβ signaling through the non-genomic MAPK signaling cascade; suggesting that UDCA's gender-specific effects may be due, in part, to signaling through hormone receptors.

### Wnt, AKT, and Cell Survival Pathways

#4600

Regulation of Wnt/β-catenin signaling by Wnt co-receptor LRP6 in colorectal cancer cells.

Yonghe Li, Wenyan Lu, Taj D. King. _Southern Research, Birmingham, AL_.

Mutations of APC and CTNNB1 are two major factors for Wnt/β-catenin signaling over-activation in colorectal cancer, however recent studies indicate that the levels of Wnt/β-catenin signaling in colorectal cancer cells could be modulated on the cell surface. The low-density lipoprotein-related protein 6 (LRP6) is an essential Wnt co-receptor for the Wnt/β-catenin signaling pathway. Herein, we reported that LRP6 expression is up-regulated in colorectal cancer. Moreover, LRP6 silencing significantly reduced Wnt/β-catenin signaling in colorectal cancer HCT116 cells (harboring a CTNNB1 mutation) and DLD-1 cells (harboring an APC mutation) and inhibited cancer cell viability. In addition, treatment of HCT116 cells with Wnt3A resulted in LRP6 phosphorylation and activation of Wnt/β-catenin signaling, which was blocked by LRP6 antagonist Mesd. Finally, we found that salinomycin, a potent small molecule inhibitor of LRP6, suppressed LRP6 expression and phosphorylation, inhibited Wnt/β-catenin signaling in colorectal cancer HCT116 and DLD-1 cells, and repressed cancer cell viability and colony formation within the same concentration range. Together, these results indicate that LRP6 plays an important role in over-activation of Wnt/β-catenin signaling in colorectal cancer cells, and that LRP6 is a potential therapeutic target for colorectal cancer.

#4601

Regulation of Hes1 expression by the Wnt transcription factor T-cell factor-4.

Hironori Koga,1 Yasuko Imamura,2 Yu Ikezono,1 Fumitaka Wada,1 Toru Nakamura,1 Hideki Iwamoto,1 Atsutaka Masuda,1 Takahiko Sakaue,1 Hirohisa Yano,1 Takuji Torimura1. 1 _Kurume Univ. School of Medicine, Kurume, Japan;_ 2 _Kurume Univ. Research Center for Innovative Cancer Therapy, Kurume, Japan_.

Background: The Notch effector Hes1 plays a critical role in stemness of cancer stem cells (CSCs). T-cell factor (TCF)-4 is a key transcription factor in Wnt signaling, which is suggested to be linked with Notch signaling. Among our TCF-4 isoforms cloned previously, the TCF-4J and K pair have been characterized based on the presence (K) or absence (J) of SxxSS motif (Exp Cell Res 2010). TCF-4J was highly expressed in poorly differentiated human hepatocellular carcinoma (HCC) tissues, and the TCF-4J-overexpressing HCC cells (J cells) exhibited high tumor-initiating potential, which is one of the features of CSCs (PLoS One 2013). Thus, the AIM of this study was to investigate whether the SxxSS motif of TCF-4 was involved in the regulation of Hes1 expression in HCC cells. Methods: TCF-4K mutants were prepared by site-directed mutagenesis. Sphere formation assay was used to condense CSC-like cells. Results: Hes1 was strikingly expressed in spheres of J cells and K-mutant cells in both protein and mRNA levels, while its expression level was 70% inhibited in K cells. Consistently, protein expression levels of Jagged1 and Notch1 were highest in J cells under both attached and floating conditions. The Notch inhibitor DAPT, a γ-secretase inhibitor, clearly decreased the expression levels of Hes1, suggesting that the Notch-Hes1 axis was functional. Conclusion: Lack of SxxSS motif in TCF-4 robustly upregulated Hes1 expression in HCC cell spheres. The finding strongly suggests that TCF-4 directly regulates Hes1 in HCC spheres, where CSCs abundantly exist.

#4602

A83-01 inhibits TGF-β/Smad3 and Wnt/β-catenin-mediated EMT and tumor metastasis in HER2-overexpressing breast cancer cells.

Yanyuan Wu,1 Tran Trinh,1 Marianna Sarkissyan,1 Sami Dwabe,1 Juri Kim,1 Robin Farias-Eisner,2 Jay Vadgama1. 1 _Charles Drew University of Medicine & Science, Los Angeles, CA; _2 _David Geffen School of Medicine at UCLA, Los Angeles, CA_.

Interactions between Wnt and TGF-β (Transforming growth factor-β) signaling pathways play a critical role in promoting EMT (Epithelial to Mesenchymal Transition) and tumor metastasis. We previously demonstrated that activation of Wnt3/β-catenin signaling pathway promoting EMT is an important mechanism leading to HER2-cells becoming resistant to trastuzumab. In this study, we used HER2-overexpressing breast cancer cells to investigate the interactions between TGF-β and Wnt/β-catenin pathways in regulation of EMT and promoting breast cancer cells invasion. Our data showed that phosphorylation of Smad3 at ser423/425 by TGF-β led to nuclear localization of Smad3 and upregulation of Twist protein expression in HER2-overexpressing breast cancer cells. Cells treated with TGF-β showed poor response to trastuzumab and exhibited EMT-like phenotype; decreased E-cadherin and increased N-cadherin expression. A small molecule inhibitor, A83-01 which inhibits phosphorylation of Smad3 repressed TGF-β-induced nuclear accumulation of Smad3 and Twist expression. The A83-01 also inhibited TGF-β-induced cell migration and invasion and enhanced anti-tumor activity of trastuzumab. The TGF-β/Smad3 induced EMT in HER2-cells was found to be concordant with activation of Wnt3/β-catenin pathway. Following TGF-β treatment, Chromatin immunoprecipitation identified occupancy of Twist at promoter region of Wnt3. This data suggests that Twist in response to TGF-β activated Wnt3 gene transcription in HER2-overexpressing breast cancer cells. Knock-down of Twist by shRNA down-regulated Wnt3 expression and inhibited TGF-β-induced nuclear accumulation of β-catenin. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, VEGF (Vascular endothelial growth factors) and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment.

In summary, our data demonstrated for the first time that the TGF-β/Smad3 mediated Twist results in transcriptional upregulation of Wnt3/β-catenin pathway and promotes EMT and cell invasion. The data suggests that therapeutic targeting of Twist by A83-01 and similar small molecules could inhibit the interaction between TGF-β and Wnt pathways and prevent breast cancer progressing to EMT and metastases.

#4603

TCF7L1 constrains Wnt/β-catenin signaling and tumor growth in pancreatic cancer.

Kathleen M. Kershaw, Michael D. Arensman, Bridgette Ho, Anna R. Lay, Nicole A. Dawson, David W. Dawson. _UCLA, Los Angeles, CA_.

Wnt/β-catenin signaling plays important roles in pancreatic adenocarcinoma (PDA) tumor initiation and progression through its promotion of various pro-tumorigenic phenotypes. While RNF43 mutations increase Wnt signaling activity and confer Wnt growth-dependency in a subset of PDA, other key driver mutations in the Wnt pathway are only rarely encountered in PDA. To gain additional insights into alternative mechanisms contributing to increased Wnt pathway activity in PDA, we examined the expression and activity of LEF/TCF family members, the key transcriptional binding partners for β-catenin in canonical Wnt signaling. Luciferase-based β-catenin-activated reporter (BAR) assays demonstrated variable Wnt activity across a large panel of PDA cell lines, which was used to dichotomize cell lines into groups with high or lower β-catenin/TCF-dependent transcription. Supervised analysis of PDA cell line microarray data and parallel western blot analyses identified distinct patterns of TCF/LEF expression, including increased TCF7 and TCF7L2 (aka TCF4) expression in high BAR lines and increased TCF7L1 (aka TCF3) expression in low BAR lines. RNAi-mediated knockdown of individual or combinations of TCF/LEFs in PDA lines revealed TCF7L1 and TCF7L2 both function to repress β-catenin-activated reporter activity. TCF7L1 protein was also found to be targeted for rapid downregulation following Wnt pathway activation by treatment with either Wnt3A ligand or GSK3β inhibitor. RNAi knockdown of TCF7L1 in PDA lines increased anchorage-independent growth and tumor cell migration in vitro and increased in vivo PDA growth in an orthotopic xenograft tumor model. In summary, differential patterns of TCF/LEF expression are correlated with and functionally responsible for differences in Wnt pathway activation and function in PDA. Furthermore, these data implicate abrogation of TCF7L1 growth suppressive phenotypes via β-catenin-mediated downregulation of TCF7L1 as a potentially important mechanism through which Wnt signaling promotes pancreatic tumorigenesis.

#4604

**Activities of** in vitro **prepared recombinant wnt inhibitory factor-1 (Wif-1).**

Duaa A. Babaer, Xiaofei Wang. _Tennessee State University, Nashville, TN_.

Wnt Inhibitory Factor-1 (Wif-1) is a secreted modular protein that inhibits the activities of Wnt signaling pathway. The latter is involved in many aspects of cancer and embryonic development. Many studies have shown that reduced WIF-1 expression is associated with the development of cancer. Since WIF-1 acts in extracellular space, it is possible to use in vitro expressed WIF-1 as a therapeutic agent for cancer treatment. We tested whether in vitro expressed recombinant WIF-1 could elicit inhibitory effects on Wnt pathway in 3T3 cells by examining the expression of insulin-like growth factor II (Igf2) , Wnt1 inducible signaling pathway protein 2 (Wisp2), (C/EBPδ), and peroxisome proliferator-activated receptor γ (PPARγ) which has been implicated as a suppressor in numerous cancer types including human breast, lung, and colon cancer. 3T3 cells were treated with 0, 1, 5 and 20 ng/ml of recombinant mouse Wif-1 protein. Total RNA was isolated after 24-48 hours of treatment. The levels of Igf2, Wisp2, C/EBPδ, and PPARγ mRNA were analyzed with reverse transcription quantitative polymerase chain reaction. Results are being analyzed to determine whether the in vitro expressed recombinant Wif-1 protein can modulate the expression of these genes and regulate cancer to have a significant inclusion in the field of cancer biology.

#4605

Metastasis-associated tumor cell phenotypes in TNBC is regulated by Wnt-beta-catenin pathway signals.

Nandini Dey, Jennifer H. Carlson, Pradip De, Brian R. Leyland-Jones. _Avera Research Institute, Sioux Falls, SD_.

Introduction: Acquisitions of metastasis-associated phenotypes (MA) by Triple Negative Breast Cancer (TNBC) cells are imparted by genetic alterations which cause deregulation of different signaling pathways associated to those phenotypes. We have recently reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the salient genetic features of TNBC and demonstrated that the WP signaling in TNBC is associated with poor prognosis and metastasis (Dey et al., 2013). We have also identified that the functional upregulation of secreted-MMP7, a transcriptional target of WP in TNBC is associated to the functional loss/absence of PTEN gene (Dey et al., 2013), the most common first event associated with basal-like subtype (Martins et al., 2012). Aim: In order to understand the relevance of WP pathway in the biology of metastasizing TNBC tumor cells, here we have undertaken a study in which the involvement of WP is tested in the context of MA phenotypes including (1) integrin-directed migration, (2) matrigel-invasion and (3) clonogenic growth and proliferation in 15 TNBC cell lines. Methods: CHIR9902, Wnt-C59, XAV939, sulindac sulfide, beta-catenin SiRNA as well as physiological stimulation of the WP (LWnt3A CM) were used for the study. Podia-parameters were studied using confocal microscopy for architecture of filamentous actin while matrigel invasion was studied by MMP7 specific caesin-zymography. Results: The collective % of alterations in CTNNB1, APC and DVL1 genes among total breast invasive carcinomas (TCGA 2012) were 17% in contrast to 46% breast invasive carcinomas, PAM50 Basal. A similar trend was observed among subtypes of BC from brca/tcga/pub2015 (cBioPortal). Wnt-C59, XAV939, sulindac sulfide and beta-catenin SiRNA (1) inhibited fibronectin-directed migration (2) decreased maximum relative distance from the origin and average velocity of the movement by real-time video microscopy, (3) altered cytoskeletal organization of the filamentous-actin and decreased the podia parameters, (4) decreased matrigel-invasion, and (6) inhibited cell proliferation and 3D colony formation. Mechanistically the treatment with sulindac sulfide and beta-catenin SiRNA decreased cellular levels of beta-catenin, active beta-catenin and MMP7 in the same TNBC cell lines wherein fibronectin-directed migration, invasion, and colony formation were found inhibited. LWnt3ACM stimulated cell proliferation, clonogenicity, migration and invasion was perturbed by WP modulators, sulindac sulfide and PI3K pathway inhibitor in brain-metastasis specific TNBC cells. Conclusion: Here we present the first conclusive evidence to demonstrate that the WP activation is functionally responsible for MA phenotypes in TNBC. Our data explains the why different components of the WP is upregulated in TNBC and how WP activation is associated with poor prognosis and metastasis in this subtype.

#4606

Novel mechanism for suppression of human colorectal cancer cell proliferation through induction of Wnt9A and suppression of β-catenin by resveratrol.

Irshad Ali, Bani Medegan, Donald Braun. _CTCA, Zion, IL_.

Dysregulation of the Wnt pathway in histologically distinct cancers has been studied extensively. The canonical Wnt pathway (CWP) utilizing β-catenin is one of the major drivers for proliferation. In contrast, the capacity of the non-canonical Wnt pathway (NCWP) to regulate proliferation has not been studied extensively in neoplastic tissues. Our earlier studies demonstrated that RV-mediated suppression of colorectal cancer (CRC) cell proliferation correlated with upregulated expression of several NCWP components. These results are consistent with the hypothesis that these upregulated NCWP components suppress CWP activity leading to proliferation inhibition. Of the NCWP components measured, the ligand Wnt9A was most prominently upregulated. Since the CWP utilizes β-catenin for its activity, the purpose of the present study was to determine whether induction of Wnt9A and suppression of CRC proliferation by RV can be correlated directly to modulation of β-catenin message and protein levels. This study was conducted on human CRC cells derived from surgical specimens (n=5). Cell proliferation and apoptosis were determined by MTS and Caspase 3 assays, respectively. β-catenin protein levels were determined by ELISA and gene expression using quantitative RT-PCR Wnt pathway gene expression arrays. RV increased Wnt9A expression in CRC cells in a concentration-dependent manner. Wnt9A expression was increased by a mean of 8, 36 and 260 fold (p<0.004, ANOVA) relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively. The increase in Wnt9A expression was associated with decreased total β-catenin protein by 21, 51 and 71 % (p<0.001, ANOVA), and active β-catenin protein by 17, 22 and 50 % (p<0.001, ANOVA). These effects were associated with significant inhibition of proliferation by 35, 54 and 66 % relative to media controls with RV concentrations of 5, 10 and 20 μg/ml, respectively (p<0.006, ANOVA) and a significant increase in apoptosis (p<0.01, ANOVA). Significantly, mRNA levels for β-catenin were not affected by any concentration of RV. Finally, the suppression of CRC cell proliferation by RV was reversed by Wnt9A antibody and IWP-2, a Wnt ligand secretion inhibitor (p<0.02, ANOVA). The induction of Wnt9A with subsequent suppression of β-catenin protein levels by resveratrol represents a novel mechanism for the suppression of canonical Wnt pathway-mediated CRC cell proliferation. These results suggest that interventions that stimulate the induction of non-canonical Wnt pathway components, and specifically Wnt9A, may be therapeutically beneficial for controlling proliferation of colorectal cancers.

#4607

Mediator kinase module as a transducer of oncogenic Wnt/beta-catenin signaling.

Alison Clark,1 Marieke Oldenbroek,1 Yao Wang,1 Jason Spaeth,2 Fangjian Gao,1 Victor X. Jin,1 Thomas Boyer1. 1 _UTHSCSA, San Antonio, TX;_ 2 _Vanderbilt University, Nashville, TN_.

Dysregulation of Wnt/β-catenin signaling promotes colorectal cancer (CRC) and other types of malignancies through unprogrammed changes in gene transcription that drive tumorigenesis. CDK8 is a CRC oncoprotein whose amplification-dependent overexpression identifies a significant subset of CRC patients with poor prognosis. CDK8 kinase activity, along with Cyclin C (CycC), drives tumorigenesis by stimulating β-catenin transcriptional activity. Thus, inhibition of CDK8 kinase function offers a promising therapeutic approach for CDK8-overexpressing CRCs. CycC-CDK8, along with MED12 and MED13, compose a four-subunit "kinase" module within Mediator, a conserved multiprotein interface between gene-specific transcription factors and RNA Polymerase II. We have previously identified a network of physical and functional interactions within the Mediator kinase module critical for oncogenic Wnt/β-catenin signaling. Mechanistically, β-catenin binds directly to MED12 in Mediator, thus activating CycC-dependent CDK8 kinase activity and β-catenin transcriptional activity. More specifically, MED12-dependent CDK8 activation occurs through a direct interaction involving the MED12 N-terminus and a phylogenetically conserved surface groove on CycC. Here, we demonstrate that mutagenic disruption of the MED12/CycC interface in CRC cell lines leads to uncoupling of CycC-CDK8 to MED12 and core Mediator, and concomitant loss of Mediator-associated CDK8 activity. Transcriptome analysis of cells lacking CDK8 kinase function revealed downregulation of Wnt/β-catenin signaling and other oncogenic pathways, consequently impairing CRC cell proliferation and clonogenicity. Our studies therefore identify the MED12/CycC interface as a critical transducer of oncogenic Wnt/β-catenin signaling. By validating MED12/CycC as a potential therapeutic target, our findings have significant implications in current methods of treating CRC as they pave a way for development of novel, targeted inhibitors of Wnt/β-catenin signaling to treat colorectal and other CDK8-driven driven malignancies.

Supported by MH085320-05 (TGB), RP140435 (TGB) and T32DE14318 (ADC).

#4608

Survivin downregulation by α-santalol is not mediated through PI3K-Akt pathway in human breast cancer cells.

AJAY BOMMAREDDY,1 KARRYN CRISAMORE,1 SARAH FILLMAN,1 SARAH BROZENA,1 JAMES STEIGERWALT,1 TERRA LANDIS,1 ADAM L. VANWERT,1 CHANDRADHAR DWIVEDI2. 1 _Wilkes University, Wilkes Barre, PA;_ 2 _SOUTH DAKOTA STATE UNIVERSITY, BROOKINGS, SD_.

α-Santalol, a terpenoid found in sandalwood oil, has been shown to inhibit cancer cell growth in vitro by inducing apoptosis. This study was performed to investigate the anticancer properties of α-santalol associated with the induction of apoptosis in cultured MCF-7 (estrogen receptor (ER) positive, and wild type p53) and MDA-MB-231 (ER-negative and mutant p53) breast cancer cells. Expression of major proteins examined in the study were determined using standard Western blot protocol and analyzed by LICOR-Odyssey infra-red scanner. Total protein levels of survivin were confirmed by survivin ELISA kit. Cell viability was assessed by trypan blue dye exclusion assay, and caspase-3 activity was determined by caspase-3 (active) ELISA kit. Treatment of breast cancer cells for 6 and 9 hour time intervals with α-santalol (20, 40 μM) resulted in statistically significant concentration-dependent downregulation of survivin. pAkt levels were found to be slightly upregulated despite the downregulation of survivin. Pharmacological inhibition of the PI3K-Akt pathway did not result in a synergistic/additive increase in cell death or caspase-3 activity caused by α-santalol. The study reveals that survivin downregulation by α-santalol in breast cancer cells is not mediated through the PI3K-Akt pathway.

#4611

Interrogation of PI3K signaling via multiplex detection of differential phosphorylation of specific Akt isoforms.

Anthony J. Saporita, Melissa Schluter, Debra MacIvor, Jehangir Mistry, Joseph Hwang. _MilliporeSigma, St. Charles, MO_.

The serine/threonine protein kinase Akt is a key node in the PI3K pathway, one of the primary signaling cascades hyperactivated in human cancer. Emerging evidence demonstrates that the three Akt isoforms (Akt1, Akt2, and Akt3) may have unique, isoform-specific roles in key cellular processes such as differentiation and proliferation. We have developed 2-plex assays for each of the Akt isoforms in order to measure the relative levels of phospho- and total Akt1, Akt2, and Akt3. Differential regulation of Akt isoforms was characterized in multiple human cancer cell lines, including SH-SY5Y neuroblastoma cells and MCF-7 breast cancer cells. SH-SY5Y cells were used to study Akt phosphorylation in the context of differentiation. Briefly, SH-SY5Y cells were treated with retinoic acid (RA) for 3 days to induce neuronal differentiation before cells were collected, lysed, and evaluated by Luminex assay. RA-induced differentiation of SH-SY5Y cells stimulated phosphorylation of all three Akt isoforms, with Akt2 displaying the greatest induction. Multi-pathway analysis demonstrated co-induction of phospho-JNK in the RA-treated SH-SY5Y cells, consistent with the role of JNK in RA-mediated differentiation. To study differential regulation of the Akt isoforms in response to growth-stimulatory and growth-inhibitory signals, we used MCF-7 cells. In contrast to SH-SY5Y cells which express all three isoforms, MCF-7 cells only expressed Akt1 and Akt2. Serum-starved MCF-7 cells were cultured in the presence or absence of the PI3K inhibitor LY294002 prior to stimulation with insulin growth factor (IGF). Akt1 showed a greater induction of phosphorylation in response to IGF relative to Akt2. Similarly, whereas LY294002 pre-treatment reduced phospho-Akt1 to baseline levels, it only partially inhibited the IGF-dependent induction of phospho-Akt2. This suggests that Akt1 may be more sensitive to PI3K inhibition than Akt2 in certain human breast cancer cells. Collectively, our results demonstrate that Akt1, Akt2, and Akt3 are differentially regulated in human cancer cells at both the level of phosphorylation and of total protein expression.

#4612

In breast cancer cells IGF-I induces upregulation of DDR1 by suppressing miR-199a-5p via the PI3K/Akt pathway.

Chiara Palladino,1 Roberta Matà,1 Maria Luisa Nicolosi,1 Anna Rita Lo Presti,1 Roberta Malaguarnera,1 Marco Ragusa,2 Daniela Sciortino,1 Andrea Morrione,3 Marcello Maggiolini,4 Veronica Vella,5 Antonino Belfiore1. 1 _University Magna Graecia of Catanzaro, Catanzaro, Italy;_ 2 _University of Catania, Catania, Italy;_ 3 _Jefferson University, Philadelphia, PA;_ 4 _University of Calabria, Rende, Italy;_ 5 _University Kore, Enna, Italy_.

Discoidin Domain Receptor 1 (DDR1) is a collagen receptor tyrosine-kinase that is implicated in tumor progression by contributing to epithelial mesenchymal transition (EMT) and by enhancing tumor cell migration and invasion.

We previously demonstrated that, in breast cancer cells, DDR1 functionally cross-talks with IGF-I receptor (IGF-IR), by affecting IGF-IR expression and trafficking. In these cells, DDR1 was able to enhance the stimulatory effects of IGF-I on mitogenesis and invasion suggesting that the DDR1-IGF-IR cross-talk may play a role in cancer progression.

Herein, we evaluated whether IGF-I may in turn affect DDR1 expression. Indeed, we demonstrated that in breast cancer cells (MCF-7 and MDA-MB-231), IGF-I was able to upregulate DDR1 protein expression in a time and dose dependent manner. In contrast, DDR1 mRNA levels showed non-significant changes after stimulation with IGF-I.

Moreover, we found that DDR1 protein upregulation requires the activation of the PI3K/Akt pathway, while the Erk1/2, the p70/mTOR and the PKC pathways are not involved.

Indeed, IGF-I induced DDR1 protein upregulation through a post-trascriptional mechanism involving miR-199a-5p suppression via Akt. We also found that autocrine IGF-II, induced by stable IGF-II cDNA transfection, is also able to elicit significant DDR1 upregulation in breast cancer cells. Similar effects were induced by IGF-II secreted by human cancer associated fibroblasts.

Finally, we showed that miR-199a-5p transfection in breast cancer cells, decreased DDR1 expression and affected the response to IGF-I in terms of intracellular signaling through the PI3K/Akt and Erk1/2 pathways, and biological effects such as cell proliferation and migration.

These results demonstrate that the protumoral effects of IGF-I include downregulation of miR-199a-5p and consequent upregulation of DDR1 protein. Both these molecules may represent important targets for breast cancers with overactivation of the IGF-IR axis.

#4613

SMYD3-mediated lysine methylation is critical for the activation of AKT1 in human cancer.

Yuichiro Yoshioka, Yusuke Nakamura, Ryuji Hamamoto. _The University of Chicago, Chicago, IL_.

v-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT1), a serine/threonine-protein kinase also known as protein kinase B Alpha (PKB Alpha), is a key mediator of a signaling pathway that governs various cellular processes regulating cell growth, survival, glucose metabolism, genome stability and neovascularization. It is known that dysregulation of AKT1 activity is one of the critical factors for the development and/or progression of a variety of human cancers. Posttranslational modifications of AKT1, including phosphorylation, ubiquitination and glycosylation, are well-analyzed, and it is known that the phosphorylation status of AKT1 is strongly correlated with its oncogenic activity. However, biological functions of methylation on AKT1 have never been analyzed so far. In the present study, we demonstrated that the protein lysine methyltrasnferase SMYD3 methylates lysine 14 in the PH domain of AKT1, which is known to play a critical role in the regulation of AKT1 activity through binding to phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) or phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2), in vitro and in vivo. Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308, which is the major indicator of AKT1 activity, than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 plays a central role in the oncogenic activity of AKT1. Although Phosphatidylinositide 3-kinase (PI3K)-dependent AKT1 phosphorylation by Pyruvate Dehydrogenase Kinase, Isozyme 1 (PDK1) and mTOR Complex 2 (mTORC2) has long been considered to be the primary mechanism accounting for AKT1 activation, these members are not sufficient enough to explain how AKT1 hyperactivation can occur in tumors with normal levels of PI3K/Phosphatase And Tensin Homolog (PTEN) activity. Our findings unveiled the novel mechanism of constitutive hyperphosphorylation on AKT1 in cancer cells, mediated by SMYD3-mediated methylation. The inhibition of this methylation pathway appears to be a promising strategy to develop anti-cancer drugs.

#4615

eIF4B is a key effector of oncogenic Pim and PI3K/Akt/mTOR signaling pathways during Abl-mediated cellular transformation.

Ke Chen, Jianning Li, Jilong Chen. _Chinese Academy of Sciences, Beijing, China_.

Abl oncoproteins, including v-Abl, Bcr-Abl and Tel-Abl, are activated non-receptor tyrosine kinases that are associated with various malignant transformations of hematopoietic cells. Expression of Abl kinase constitutively activates multiple signaling pathways that are critically involved in the regulation of cell proliferation and survival. Despite extensive studies, the precise mechanism by which these Abl oncoproteins induce hematopoietic malignances is not fully elucidated. eIF4B plays a critical role in Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways regulated the phosphorylation of eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Surprisingly, persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers.

#4616

A novel non-canonical Notch-AKT- NF-κB pathways in triple negative breast cancer cells.

Fokhrul Hossain,1 Yin Peng,2 Antonio Pannuti,1 Kandis Backus,3 Todd Eliot Golde,4 Barbara Osborne,5 Lucio Miele1. 1 _LSUHSC, New Orleans, LA;_ 2 _Loyola University Chicago, IL;_ 3 _University of Mississippi, MS;_ 4 _University of Florida, FL;_ 5 _UMass Amherst, MA_.

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases that account for approximately 15-20% of invasive breast cancers. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal-like 1 (BL1) and especially Mesenchymal-Stem-Like (MSL) TNBCs. Expression of Notch1 protein correlates with pAKT and nuclear NF-κB in TNBC specimens. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. NF-κB also plays key roles in promoting cancer cell survival in TNBC. Notch-NF-κB cross-talk through several mechanisms has been demonstrated in T-cell acute leukemia, cervical cancer and Luminal A breast cancer cells, but not TNBC. Here, we demonstrate that Notch1 promotes cell survival in MDA-MB-231 cells, representative of MSL TNBC, in part by activating NF-κB. Notch activation by Jagged1-expressing stromal cells enhances transcription of the anti-apoptotic gene cIAP-2 (BIRC3), a known NF-κB target. This event is dependent on recruitment to the cIAP-2 promoter of NF-κB subunits, IKKα and Notch1. However, Notch1 is recruited to the chromatin later than NF-κB subunits and IKKα, suggesting that early, non-nuclear events contribute to this process. NF-κB activation correlates with AKT activation as revealed by S473 phosphorylation. Pharmacological inhibition of AKT or expression of dominant-negative AKT decreases NF-κB activity. Inhibition of Notch activity through GSIs (γ-secretase inhibitors) leads to decreased AKT phosphorylation. Our results indicate that AKT activation is independent of canonical nuclear Notch1 partner CBF-1. Short term exposure of MDA-MB-231 cells (MSL, PTEN wild-type), but not MDA-MB-468 cells (BL1, PTEN-null) to recombinant Jagged1 leads to AKT phosphorylation within 1 hour. This is suppressed by dual mTORC1/2 inhibitors, PI3K inhibitors and IKKα inhibitors but not Everolimus (mTORC1-selective inhibitor). Pharmacological inhibition of AKT, or mTORC1/2 or IKKα decreases AKT phosphorylation and cell proliferation. These observations support a model where canonical and non-canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF-κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF-κB activation. Notch1 has been shown to activate mTORC2 in T-cells, while IKKα has been reported to activate mTORC2 in other models. Our data indicate that this pathway may be active in some TNBC subtypes as well. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in PTEN-wild type but not PTEN-mutant TNBCs.

#4617

Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion.

Okkyung Rho, Jaya Srivastava, Jiyoon Cho, John DiGiovanni. _The University of Texas at Austin, Austin, TX_.

Considerable evidence indicates a crucial role for Akt and mTORC1 signaling in cancer. Previously, we discovered that the Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. The proline-rich Akt substrate of 40 kDa (PRAS40), a subunit of mTORC1 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246 and this phosphorylation is critical for mTORC1 activity. Moreover, PRAS40 has been identified to negatively regulate mTORC1 signaling.

In this study, transgenic mice were generated that overexpress a mutant form of PRAS40 [PRAS40(T246A)] using the bovine keratin 5 (BK5) promoter to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40(T246A) transgenic mice did not have a discernable gross skin phenotype. However, these mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia. Overexpression of PRAS40(T246A) in the epidermis led to significant suppression of skin tumor development and significant reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis of epidermal protein lysates from BK5.PRAS40(T246A) mice showed that PRAS40(T246A) remained bound to raptor even after TPA treatment. BK5.PRAS40(T246A) mice also displayed reduced mTORC1 signaling, reduced levels of cell cycle proteins and increased autophagy signaling as well as the skin inflammatory response in the epidermis following treatment with TPA. Notably, TPA-induced proliferation and migration of bulge-region label retaining cells were also significantly inhibited in BK5.PRAS40(T246A) mice. In addition, expression of PRAS40(T246A) in basal keratinocytes significantly inhibited keratinocyte migration both in vivo and in vitro and led to delayed wound healing. Decreased migration and impaired wound healing may be attributed to altered expression of EMT markers as identified in PRAS40(T246A) epidermal cells.

The current data using this novel mouse model provides further evidence that mTORC1 specific signaling in keratinocytes contributes significantly to the process of skin tumor promotion by regulating proliferation and migration of keratinocyte stem cells. In addition, the data demonstrate an important role for mTORC1 specific signaling in keratinocytes in regulating skin inflammation. Collectively, using this novel mouse model where mTORC1 signaling is selectively suppressed in keratinocytes has provided further evidence that this signaling pathway regulates several important mechanistic events involved in the process of skin tumor promotion.

Research supported by NIH Grants CA129409, CA037111, and American Cancer Research Center and Foundation

#4618

mTOR signaling complex can be targeted using Torin2 in colorectal cancer to induce efficient apoptosis.

Saeeda O. Ahmed, Maqbool Ahmed, Abdul K. Siraj, Shaham Beg, Saravanan Thangavel, Nasser Al-Sanea, Fouad Al-Dayel, Azhar R. Hussain, Khawla S. Al-Kuraya. _King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia_.

Colorectal cancer (CRC) is the third most common cancer in the world and the 4th most common cause of cancer related death. Despite the considerable advances in awareness, diagnosis and modern therapeutic strategies, the incidence of CRC is increasing in the Middle Eastern population. In order to explore the molecular cause of increasing incidence of CRC in this region, we sought to evaluate the role of mammalian target of rapamycin (mTOR) survival pathway in CRC of this region. mTOR; a component of the phosphatidylinositol 3-kinase (PI3K) cell survival pathway, is known to play an important role in the regulation of many cellular activities leading to cell growth and proliferation through the formation of its two complexes mTORC1 and mTORC2. We therefore examined the expression of mTOR in 700 Saudi CRC cases by immunohistochemistry in a tissue microarray format and found that mTORC1 and mTORC2 were activated in 43.4% (294/677) and 46.2 % (313/677) of cases respectively. mTORC1 activation was found to be significantly associated with Ki67 and mTORC2. In addition, mTORC2 activation was also found to be directly associated with their downstream target; p-AKT. In vitro, we found that treatment with a second generation mTOR inhibitor, Torin2 led to inhibition of cell viability and induction of apoptosis via inactivation of mTORC1 and mTORC2 in majority of CRC cell lines. Inactivation of mTORC1 and mTORC2 following Torin2 treatment also led to dephosphorylation of mTOR downstream targets; P70S6, 4E-BP1, AKT and BAD. Interestingly, one CRC cell line; HT29 that had over-activation of mTORC2 was found to be resistant to Torin2 treatment suggesting that increased activation of mTORC2 confers resistance to Torin2 treatment. As our clinical data demonstrated a significant association between mTORC2 over-expression and activation of AKT in clinical CRC samples, we synergistically targeted HT29 cells with combination of sub-optimal doses of PI3-kinase/AKT inhibitor; LY294002 in combination with Torin2. Our data showed that combination of Torin2 and LY294002 synergistically induced apoptosis in HT29 cells via inactivation of mTORC2 and AKT. These data highlight the utility of targeting mTOR signaling complex using second generation mTOR inhibitors such as Torin2 alone or in combination with other inhibitors such as LY294002 or chemotherapeutic agents to effectively treat these aggressive subgroups of CRC with over-activation of mTOR and AKT simultaneously.

#4619

Sirt2 protein is required for activation of mTOR signaling in colorectal cancer cells.

Qingding Wang,1 Yuning Zhou,2 Heidi L. Weiss,2 B. Mark Evers1. 1 _University of Kentucky Markey Cancer Center and Department of Surgery, Lexington, KY;_ 2 _University of Kentucky Markey Cancer Center, Lexington, KY_.

The human sirtuin proteins (Sirt1-7) possess protein deacetylase activities and are involved in the regulation of metabolism, differentiation and cell survival. Sirt2 is upregulated in some cancers; however, some studies have suggested its role as that of a tumor suppressor. mTOR regulates many processes that control growth, including protein synthesis, oxidative metabolism, glucose homeostasis, cell growth, and lipogenesis. Deptor protein binds mTOR through the PDZ domain and inhibits mTOR activity. Our previous studies have shown that mTOR is a central regulator of colorectal cancer (CRC) cell proliferation and survival. The purpose of this study was to investigate: i) the regulation of Deptor expression by Sirt2, and ii) the role of Sirt2 protein in mTOR activation in CRC and normal intestinal cells. METHODS. Human CRC cell lines, HT29 and Caco-2, the rat normal intestinal cell line, IEC-6, and the human embryonic kidney cell line, HEK293, were transfected with siRNA targeting Sirt2 or control nontargeting siRNA. Cells were also infected with lentiviral vectors containing the control shRNA or shRNA to human Sirt2; stably expressing cells were selected with puromycin. Sirt2 overexpression was achieved by infecting cells with an adenovirus encoding Sirt2. Cell proliferation was assessed by counting the number of viable cells. The expression of Sirt2, Deptor, p-Akt (S473), Akt, p-S6 (S235/236), S6, LC3B, Cyclin D1, c-Myc, and survivin was analyzed by western blot. Deptor mRNA levels were determined by real time RT-PCR. RESULTS. Knockdown of Sirt2 inhibited mTOR signaling, decreased expression of cyclin D1, c-Myc, and survivin, and inhibited CRC cell growth. In agreement with the inhibition of mTOR activation, knockdown of Sirt2 significantly increased Deptor mRNA and protein expression in CRC cells. Conversely, overexpression of Sirt2 decreased Deptor expression and activated mTOR signaling. Knockdown of Sirt2 increased, while knockdown of Deptor decreased, the protein expression of LC3B, an autophagy marker, suggesting Sirt2 inhibition increases autophagy through induction of Deptor. Knockdown of Sirt2 inhibited HT29 cell proliferation; this inhibition was enhanced by treatment with 3MA, an autophagy inhibitor. In contrast, knockdown or overexpression of Sirt2 did not affect either the expression of Deptor or the activation of mTOR in HEK293 and IEC6 cells, suggesting a differential regulation of Deptor/mTOR signaling by Sirt2 in CRC and normal intestinal cells. CONCLUSIONS. We provide evidence that the Sirt2 protein increases mTOR activity and contributes to CRC cell proliferation. Importantly, our results indicate a novel role of Sirt2 in the activation of mTOR signaling in CRC cells but not in normal intestinal cells and suggests that targeted inhibition of Sirt2 could represent a novel and safe strategy for the treatment of CRC.

#4621

Rapamycin inhibits the phosphorylation of mSin1 by targeting a new mTOR complex.

Shile Huang, Yan Luo, Lei Liu. _Louisiana State University Health Sciences Center, Shreveport, LA_.

Current knowledge indicates that mTOR functions as two complexes, mTORC1 and mTORC2, in mammalian cells. mTORC1 comprises mTOR, mLST8 and raptor, and is sensitive to acute rapamycin treatment, whereas mTORC2 consists of mTOR, mLST8, rictor, mSin1 and Protor, and is sensitive to chronic rapamycin treatment in some cases. It is well known that mTORC1 phosphorylates 4E-BP1 and S6K1, while mTORC2 phosphorylates Akt and SGK1. Recently, we and others have observed that rapamycin inhibits the phosphorylation of mSin1. However, little is known about the molecular mechanism by which rapamycin inhibits mSin1 phosphorylation. Here we show that rapamycin inhibits mSin1 phosphorylation in mTOR kinase dependent manner. This is strongly supported by the findings that expression of rapamycin-resistant mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mTOR (mTOR-TE) prevented rapamycin from inhibiting mSin1 phosphorylation; Knockdown of mTOR inhibited mSin1 phosphorylation. However, surprisingly, disruption of either mTORC1 or mTORC2 (by silencing raptor and rictor, respectively) failed to inhibit the phosphorylation of mSin1 as rapamycin did. Similarly, downregulation of S6K1 or Akt did not inhibit mSin1 phosphorylation as rapamycin did either. Of interest, knockdown of mLST8, a component shared by mTORC1/2, inhibited mSin1 phosphorylation. Furthermore, rapamycin treatment reduced the physical interaction of mSin1 with mLST8 and mTOR, but not with rictor. The results suggest that rapamycin inhibits mSin1 phosphorylation, which is not by inhibiting mTORC1 or mTORC2, but via inhibiting a new mTOR complex, which contains at least mTOR and mLST8. (Supported by the FWCC, LSU Health Sciences Center)

#4622

Identification of p62/SQSTM1 as a component of non-canonical WNT VANGL2-JNK signaling in breast cancer.

Jean-Paul Borg,1 Puvirajesinghe Tania,1 François Bertucci,1 Daniel Birnbaum,1 Ashish Jain,2 Terje Johansen,2 Pierluigi Scerbo,3 Laurent Kodjabachian3. 1 _CRCM Inserm-Institut Paoli-Calmettes, Marseille, France;_ 2 _University of Tromsø, Tromsø, Norway;_ 3 _Institut de Biologie du Développement de Marseille, Aix-Marseille Université, CNRS UMR 7288, Marseille, France_.

Planar cell polarity (PCP), an evolutionarily conserved developmental process originally described in Drosophila melanogaster, is essential to the control of tissue morphogenesis and development of polarized structures in metazoans. Its importance in human diseases is best demonstrated in polycystic kidney disease, hearing loss, Bardet Biedl syndrome, neural tube closure defects and cancer. PCP involves a set of evolutionarily conserved PCP genes encoding WNT ligands, transmembrane (vangl2, frizzled, fat, dachsous) and cytoplasmic (scribble, prickle, dishevelled, diego) molecules triggering a genetically well-defined non-canonical WNT-JNK pathway. Recent work has linked defects of this pathway to breast cancer aggressiveness and proposed non-canonical WNT/PCP signaling as a therapeutic target. Here we show that the archetypal non-canonical WNT/PCP protein VANGL2 is overexpressed in basal breast cancers, associated with poor prognosis and implicated in tumor growth. We identify the scaffold p62/SQSTM1 protein as a novel VANGL2-binding partner and show its key role in an evolutionarily conserved VANGL2-p62/SQSTM1-JNK pathway in breast cancer cells and in Xenopus. This proliferative signaling cascade is upregulated in breast cancer patients with shorter survival, and can be inactivated in patient-derived xenograft cells by inhibition of the JNK pathway or by disruption of the VANGL2-p62/SQSTM1 interaction. VANGL2-JNK signaling is thus a potential target for breast cancer therapy.

Puvirajesinghe T.M., et al. Identification of p62/SQSTM1 as a component of non-canonical Wnt VANGL2-JNK signaling in breast cancer. Nature Communications, in press. Sebbagh M. and Borg J.-P. Insight into Planar Cell Polarity. (2014) Invited review in Experimental Cell Research, 328:284-295. Belotti E., et al. The human PDZome: a gateway to PDZ mediated functions. (2013) Mol Cell Proteomics, 12: 2587-603.

#4623

Reevaluation of the role of Notch signaling in melanoma tumor development, melanoma cell survival and drug resistance.

Dareen Mikheil,1 Carlos Rodriguez,2 Ashika Jayanthi,2 Prithvi Yarlagadda,2 Vijaysaradhi Setaluri1. 1 _University of Wisconsin-Madison & William S. Middleton Memorial Veterans Hospital, Madiosn, WI; _2 _University of Wisconsin-Madison, Madiosn, WI_.

Notch is one of the simplest signaling pathways that is required for almost every cell type in the body. It plays several roles such as cell fate decision, differentiation and stem cell maintenance. Notch signaling plays a role in the maintenance of melanoblasts and melanocyte stem cells of the epidermis by preventing their apoptosis. Notch signaling has also been implicated in different aspects of melanoma biology including tumor initiation, progression and metastasis. Accordingly, Notch has been targeted for melanoma therapy both in preclinical xenograft models as well as clinical trials. Notch inhibitors, mainly ɣ-secretase inhibitors, have been widely used for this purpose. However, clinical trials with these agents have not been proven to be highly effective for melanoma treatment. In previously published studies we showed that Notch signaling is involved in regulation of melanoma trans-differentiation, a prognostic feature in primary melanoma. In this study, we reevaluated the role of Notch in melanoma development, cell survival and sensitivity to BRAF and MEK inhibitors. First, our preliminary data using the BRAFV600E/Pten mouse model showed that topical application of ɣ-secretase inhibitor DAPT delays tumor formation and growth. In several early passage metastatic human melanoma cell lines, we found that Notch receptors (Notch 1-3) are highly upregulated compared to normal epidermal melanocytes. Interestingly, mRNA levels for Notch1-3 receptors in normal melanocytes were either higher or comparable to their levels in the cell lines. This suggested that Notch receptor levels are tightly regulated in normal melanocytes by post-transcriptional mechanisms. Presence of cleaved Notch intracellular domain (NICD), both cytoplasmic and nuclear, in melanoma cells, but not in melanocytes, indicated constitutive activation of Notch signaling in melanoma cells. However, inhibition of Notch activation by treatment with ɣ-secretase inhibitor had no significant effect on survival of melanoma cells in vitro. Thus, Notch signaling appears to be dispensable for melanoma cell survival. Additionally, inhibition of Notch activation did not alter the sensitivity of melanoma cells to BRAF(V600E) and MEK inhibitors. Our results highlight the need for re-examination of Notch-regulated melanoma trans-differentiation as a therapeutic outcome.

#4624

Notch signaling in SCLC and other lung NET cell lines.

Ciera Singleton, Judy Crabtree. _Louisiana State University Health Sciences Center New Orleans, New Orleans, LA_.

Neuroendocrine tumors (NETs) of the lung fall into two major categories: slow-growing, well differentiated carcinoids that respond well to treatment and rapidly-growing, poorly differentiated small cell lung cancer (SCLC). SCLC represents 15% of diagnosed lung cancer cases and is most often associated with heavy smoking, expression of neuroendocrine markers, high growth rate, and early metastatic spread. Because patients often present initially with advanced disease characterized by metastases, surgical removal of this tumor type as a treatment option is highly unusual. There are no curative methods for SCLC and first line treatment options vary slightly depending on the disease stage but often include combination chemotherapy along with radiation. Treatment can prolong life 6 -12 months, but within this time frame most patients relapse and the cancer becomes refractive to therapy. The 5 year overall survival is less than 5%. Towards understanding disease progression in lung NETs, the purpose of our research is to investigate the role of Notch receptors and their ligands in SCLC as potential targets for therapy. We have identified Notch receptors and ligands that are elevated in lung NET cell lines, and this increased expression may be playing a role in the rapid proliferation and metastasis observed in SCLC, as well as in the development of chemoresistance. Further, lung NET cell lines treated with gamma secretase inhibitors (GSIs), which inhibit the final proteolytic cleavage event to activate Notch receptors, show altered expression and proliferation profiles, suggesting a critical role for Notch signaling in SCLC tumorigenesis. Knockdown of specific Notch receptors or treatment with GSIs impact downstream expression of target genes and may reveal novel mechanisms or therapeutic approaches for the treatment of SCLC.

#4625

ASIC1 regulates neuronal differentiation of neuroblastoma through Notch signaling pathway.

Mingli Liu, Koichi Inoue, Zhi-gang Xiong. _Morehouse School of Medicine, Atlanta, GA_.

Background: Neuroblastoma arises when neuroblasts of the sympathetic nervous system fails to undergo proper differentiation. The Notch signaling pathway is critical for cell differentiation, growth and proliferation. In neurons, it has been shown that up regulation of Notch activity either inhibited neurite extension or caused retraction of neurites. Conversely, inhibition of Notch1 signaling facilitated neurite extension. Acid-sensing ion channels (ASICs) are proton-gated cation channels. In addition to the role in synaptic plasticity, learning and memory, a good correlation has been found between ASIC1a expression and spine density suggesting that ASIC1a plays an important role in the processes of spine morphogenesis, maintenance and remodeling which are involved in the process of neuronal differentiation. The underlying mechanism however remains elusive. We hypothesize that ASIC1 regulates neurite growth through Notch signaling in neuroblastoma. Methods: 1) We employed proteomic approach and pathway analysis to examine changes in Notch signaling by ASIC gene deletion. By comparison adult ASIC1a-/- and WT mice, quantitative mass spectrometric techniques of spectral counting followed by analysis with SEQUEST were used to identify reliable proteins. 2) We determined the effects of ASIC1a on neurite growth in a mouse neuroblastoma cell line, NS20Y cells by down regulating ASIC1a expression with shRNA or inhibiting ASIC1a function with PcTX, a specific pharmacological inhibitor. 3) We determined the effects of DAPT, a Notch signal inhibitor on neuronal differentiation in NS20Y cells. Results: 1) The enrichment analyses by the bioinformatics system Metacore showed that development Notch signaling pathway (p=1.526E-05), development Notch-induced epithelial-mesenchymal transition (EMT, p=3.902E-05) and development Notch-mediated pathway for NF-κB modulation (p=2.323E-04) were changed in WT mice compared to ASIC1a-/-. 2) Down regulation of ASIC1a in NS20Y cells with shRNA inhibits CPT-cAMP induced neurite growth, while over expression of ASIC1a promotes its growth. 3) Down regulation of ASIC1 by shRNA increased Notch1 and its target gene Survivin in NS20Y cells. 4) DAPT, an inhibitor of Notch significantly protects inhibition of CPT-cAMP induced neurite extension by down-regulation of ASIC1 in NS20Y cells. Conclusion: These data indicate that Notch1 signaling may be required for ASIC1 mediated neurite growth and neuronal differentiation of neuroblastoma. Promoting neural differentiation of neuroblastoma in vitro provides insight into potential clinical applications in the treatment of patients with neuroblastoma (This work is supported by R01NS066027, NIMHD S21MD000101, and U54NS08932)

#4626

In-vitro growth inhibitory effects of diosmetin on human prostate cancer cells.

Rebecca Pakradooni, Riddhi Patel, Janmejai K. Srivastava, Sanjeev Shukla. _Case Western Reserve University, Cleveland, OH_.

Characterization of effective chemo-preventive agents for treatment of prostate cancer is an utmost priority. Diosmetin (5, 7-Trihydroxy-4′-methoxyflavone), a natural flavonoid present in citrus plant, has anti-mutagenic and anti-allergic properties. However, the anticancer properties of diosmetin have not been explored in prostate cancer. Present study demonstrates, cell growth inhibition and induction of apoptosis in human prostate carcinoma LNCaP (androgen-responsive) and PC-3 (androgen-refractory) cells by diosmetin, however, no significant growth inhibition observed in normal prostate epithelial cells (RWPE1). Cell growth is a fundamental biological process, where mTOR (mammalian target of rapamycin) pathway has its role, regulates cell growth by coordinating energy and nutrient signals with growth factor. mTOR protein kinase have two different complexes; complex-I contains major component raptor; a target of rapamycin and complex II; insensitive to rapamycin, has major component rictor. Rictor is important as it phosphorylates Ser-473 of Akt/PKB, which is essential for full Akt/PKB activation. We observed diosmetin treated prostate cancer cells was able to inhibit growth factor (IGF-1) and cytokine (IL-6) induced rictor expression. Furthermore, diosmetin (20μM) treatment to prostate cancer cells prevented rictor nuclear localization and subsequently inhibited phospho-Akt (Ser-473), which subsequently resulted in increased FOXO3a nuclear presence. Downstream pathway p70S6 kinase, which has a critical role in cell cycle, growth and survival, was also inhibited after diosmetin treatment. These effects were associated with a marked decrease in the protein expression of cyclin D1 and their activating partner, cyclin-dependent kinase (cdk) 2 and 4 with concomitant upregulation of KIP1/p27 and INK4a/p16. Additionally diosmetin treatment to these cells modulated cell survival machinery by down regulating key molecules viz., c-Myc, Survivin and XIAP (X-Linked Inhibitor of Apoptosis). Diosmetin treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with an increase in cleaved caspase-3. Taken together, our studies demonstrate that diosmetin-mediated growth inhibition and induction of apoptosis in both LNCaP and PC-3 cells were due to deregulated levels of rictor and Akt signaling cascade and modulation of cell-cycle machinery. We are documenting these evidences for the first time that diosmetin acts against potential molecular targets to alter cellular events to elicit anticancer effects in prostate cancer cells.

#4627

The role of human endogenous retroviral proteins in the development of Merlin-deficient tumors and as potential drug targets.

Emmanuel Maze,1 Shona Reeves,2 David Hilton,3 Lucy Provenzano,2 Robert Belshaw,4 Sylwia Ammoun2. 1 _Plymouth University Peninsula School of Medicine and Dentistry, Plymouth, United Kingdom;_ 2 _Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, United Kingdom;_ 3 _Plymouth University, Derriford Hospital, Plymouth, United Kingdom;_ 4 _School of Biomedical & Healthcare Sciences, Plymouth, United Kingdom_.

Neurofibromatosis Type 2 (NF2) is a genetic disorder caused by loss of the tumour suppressor Merlin leading to the development of multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas. Merlin-deficient tumours may also occur sporadically including all schwannomas, 50-60% meningiomas and 29-38% ependymomas. Loss of functional Merlin have been also observed in other tumours e.g. mesothelioma, melanoma, and a portion of glioma, prostate and breast cancers. Current therapies for Merlin-deficient tumours, comprising surgery and radiosurgery are invasive and not fully effective therefore the emphasis has to be made on the development of new effective drug-based treatments.

Schwannoma is the most common Merlin-deficient tumour, a hallmark for NF2 and a model for all other Merlin-deficient tumours. We have developed the human in vitro model for schwannoma to study tumour pathobiology and for drug testing. Using this model we have shown strong overexpression and activation of ErbB2/ErbB32, platelet-derived growth factor receptor (PDGFR), Insulin like growth factor receptor-I (IGF-IR) and activation of Raf/MEK/ERK, PI3K/AKT and Wnt/β-catenin signalling pathways. Following, we have successfully tested new drugs such as AZD6244, Sorafenib, BEZ235, Lapatinib, Imatinib and Nilotinib. These studies revealed that schwannomas' pathological proliferation, survival and adhesion is regulated by highly complex networks interacting and compensating each other resulting in increased tumour pathology. We suggest that the inhibition of a single target will not be sufficient to stop tumour growth and that multi-targeted therapy would be needed.

Endogenous retroviruses are viruses that have integrated themselves into germ-line chromosomes and thereby become part of the human genome sequence (8%). One group, HERVK, has been found to be overexpressed in several cancers and is currently being investigated as potential immunotherapy targets. Our preliminary data demonstrate that HERVK envelope, capsid, Rec and Np9 proteins are overexpressed in human primary schwannoma cells and tissues. Additionally, HERVK envelope and capsid proteins are released from schwannoma cells. HERVK overexpression is regulated at both the transcriptional and translational level and is partially Merlin dependent. Anti-HERVK antibodies reduced schwannoma proliferation, pERK/pAKT/ pFAK and cyclin D1 levels and increased expression of p53. Also, pre-incubation of schwannoma cells with anti-HERVK antibodies prior to treatment with AZD6244 potentiated drug's efficiency. An HIV/HERVK protease inhibitor, Ritonavir, also decreased proliferation of schwannoma cells and increased the efficiency of AZD6244, Sorafenib and BEZ235 when combined. We suggest that HERVK plays a relevant role in schwannoma and possibly other Merlin-deficient tumours' development and is a promising therapeutic target.

#4628

The RSK inhibitor SL0101 impairs the proliferation of glioblastoma cell lines.

Martín Roffe, Fernanda C. Lupinacci, Luana C. Soares, Glaucia N. Hajj, Vilma R. Martins. _A.C. Camargo Cancer Center, São Paulo, Brazil_.

Glioblastoma (GBM) is the most common and lethal type of brain tumor. The most frequently altered signaling pathway in this tumor is the RTKs-PI3K-Akt-mTORC1 (88% of cases). However, clinical trials for GBM using inhibitors of this pathway exhibited poor clinical responses. One of the main responsibles for this resistance is the compensatory activation of the Ras-ERK1/2 pathway. In fact, GBMs can also present alterations in the Ras-ERK1/2 pathway, such as mutation of NF1 in 18% of the cases. RSK is a direct target of ERK1/2 and has been shown to regulate important functions in different types of tumors, such as prostate and breast. RSK is a family of kinases comprised by four highly-homologous isoforms in humans (RSK1-4) and nothing is known about their functions in GBMs. Here we show that RSK1 levels are highly variable between cells, the levels of RSK2 are less variable, and RSK3 and RSK4 were not detected. To gain insight into the function of RSK, we studied the effects of the RSK inhibitor, SL0101, on the proliferation of four different GBM cell lines (LN18, LN229, U87MG and A172). A suboptimal concentration of SL0101 (50 μM) was able to inhibit basal and EGF-stimulated proliferation, and it was more potent than 100 nM rapamycin in some of the cells. In 3 of the cell lines, SL0101 inhibited serum-stimulated proliferation, and in 2 of them, it was more potent than rapamycin. We have previously shown that SL0101 can inhibit mTORC1 signaling in an ERK1/2-RSK independent manner. However, SL0101 showed a synergistic interaction with rapamycin in all of the cell lines, which suggests that other effects besides RSK-mTORC1 inhibition might be involved in the effect of SL0101. This was further suggested by the modest synergistic interaction between rapamycin and the RSK inhibitor, fmk. Altogether, these results indicate that SL0101 is a potent inhibitor of proliferation in a manner not related with the RSK1 status of GBM cells. Further studies to define the molecular mechanism of the effects of SL0101 are necessary to understand which pathways are indeed modulated.

#4629

The influence of adipose tissue-derived mesenchymal stem cell environment and WNT antagonism on breast tumour cells.

Malini Visweswaran,1 Frank Arfuso,1 Rodney Dilley,2 Philip Newsholme,1 Arunasalam Dharmarajan1. 1 _Curtin University, Perth, Australia;_ 2 _Ear Sciences Centre, University of Western Australia, Perth, Australia_.

Breast cancer is the most commonly diagnosed cancer in Australian women, with an average of 1 in 8 women being affected. Emerging evidence suggests that a close relationship exists between a tumor and the adjacent non-tumor tissue, which considerably affects the overall progression of the disease. Amongst the non-tumor cells, mesenchymal stem cells (MSCs), known to be essential for tissue and organ health, are potentially important in this regard. In our study, we focussed on the interaction between adipose tissue-derived MSCs (ADSCs) and breast tumor cells. Considering the clinical relevance of these interactions, it is imperative to understand the exact mechanisms underlying such cellular crosstalk. Here, we investigated the influence of ADSCs on breast tumor cell lines MDA MB 231 and MCF7 by using both conditioned media and extracellular matrix derived from ADSCs. We observed that the ADSC-conditioned media (ADSC-CM), harvested at specific time-points, inhibited the proliferation of tumor cells as well as their associated cancer stem cells. Further, by using molecular fractionation techniques on ADSC-CM, we identified that the <30KDa fraction of ADSC-CM retained the potential for inhibiting proliferation. We also observed decreased migration of tumor cells when treated with ADSC-CM. Further, we observed an inhibition in proliferation rates of tumor cells when treated with ADSC-deposited extracellular matrix (ADSC-ECM). Since Wnt signaling is an important pathway driving both breast tumor growth and influencing ADSC behaviour, we examined the effect of the Wnt antagonist- secreted frizzled-related protein 4 (sFRP4) and its associated peptides on tumor cells. When tumor cells were treated with sFRP4 or its peptides along with ADSC-CM/ADSC-ECM, we observed an additive inhibitory effect on tumor cell proliferation. We further observed an increase in caspase-dependent apoptosis in tumor cells following treatment with ADSC-CM and in combination with the Wnt antagonists. The effect contributed by the ADSC environment and Wnt antagonists differed in mechanism, based on the tumor cell characteristics. We can conclude that the interaction of ADSCs with tumor cells had a significant impact on tumor cell growth in terms of decreasing proliferation and migration, while increasing the rate of apoptosis, which was further elevated in the presence of Wnt antagonists. This study sheds light on novel therapeutic approaches that could be considered while devising novel anti-cancer strategies. 

## EXPERIMENTAL AND MOLECULAR THERAPEUTICS:

### Cellular Responses to Anticancer Drugs

#4630

The pan-PIM inhibitor PIM447 enhances the antitumor activity of lenalidomide in multiple myeloma cells via synergistic inhibition of c-MYC.

Huaixiang Hao, Dannie Wang, Gary Vanasse, Giordano Caponigro. _Novartis Insts. for Biomedical Research, Cambridge, MA_.

Introduction:

Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide and pomalidomide have significantly contributed to improved overall survivals in multiple myeloma (MM) patients. However, cure remains an elusive goal for the vast majority of MM patients and novel combination regimens are needed. Recently, the mechanism of action of IMiDs was identified to be inducing degradation of two MM transcription factors, IZKF1 and IZKF3. IKZF1/IKZF3 regulates the expression of oncogene c-MYC via another transcription factor-interferon regulatory factor 4 (IRF4). This new finding provides an opportunity to study synergistic combinations of IMiDs with targeted therapies. PIM447 (formerly LGH447, Novartis Pharmaceuticals) is a pan-PIM kinase inhibitor that is in clinical development for the treatment of patients with MM and other hematological malignancies. Based on the observation that several MM lines are sensitive to both IMiD and PIM447, we evaluated the combination effects of PIM447 and lenalidomide in 9 MM cell lines.

Methods:

An 8X8 serial dilution dose matrix of PIM447 and lenalidomide were tested in a panel of 9 MM cell lines. RT-PCR and immunoblotting were performed in selected lines to define possible mechanisms of combination activity.

Results:

Combination activity (synergy score>2) was observed in 4 out of the 9 MM cell lines tested, with MM1-S and NCI-H929 showing the highest synergy scores. Combination activity was only noted in the presence of PIM447 single agent activity and appeared to correlate with PIM2 protein expression. In both MM1-S and NCI-H929 cells, the combination led to increased apoptosis as measured by PARP cleavage. PIM447 did not affect the levels of various targets of lenalidomide, including IKZF1, IKZF3 and IRF4, and lenalidomide did not affect PIM downstream effectors such as phospho-S6RP. Alterations involving c-MYC are amongst the most frequent genetic features found in MM, and the combination of PIM447 and lenalidomide resulted in greater reduction of c-MYC expression. In both MM1-S and NCI-H929 cells, c-MYC knockdown has previously been shown to inhibit cell proliferation, suggesting the combination activity might derive from the observed reduction of c-MYC expression. The mechanisms by which c-MYC expression were synergistically decreased by the combination may involve both enhanced downregulation of c-MYC transcripts and accelerated degradation of c-MYC protein.

Conclusions:

The pan-PIM kinase inhibitor PIM447 enhanced the anti-tumor activity of IMiDs in MM via synergistic inhibition of c-MYC. Our data support in vivo testing of this combination regimen and the design of future clinical trials in MM patients.

#4631

SCFFbxo7 ubiquitin ligase targets cereblon to limit the anti-myeloma activity of IMiDs.

Jiye Liu,1 Wenrong Zhou,2 Yan Feng,1 Tianyu Song,1 Shaobing Gao,1 Junfeng Wu,2 Zhenggang Peng,2 Yong Cang2. 1 _Life Sciences Institute and Innovation Center for Cell Signalling Network, Zhejiang University, Hangzhou, China;_ 2 _Oncology BU, WuXi AppTec, Shanghai, China_.

Multiple myeloma (MM) patients have witnessed significantly improved survival after the approved treatment with the proteasome inhibitor bortezomib and immunomodulatory drugs (IMiDs) including thalidomide and its derivatives lenalidomide and pomalidomide. IMiDs directly bind cereblon (CRBN), an adaptor of the Cul4-DDB1 (CRL4) ubiquitin ligase, and activate CRL4CRBN to target B cell-specific transcription factors IKZF1 and IKZF3 for ubiquitination. IKZF1 and IKZF3 are essential for MM cell survival and their proteasomal degradation induced by IMiDs provides insights into how these drugs work. Paradoxically, IMiDs achieve better clinical response in MM patients when combined with bortezomib, which blocks IKZF1 and IKZF3 turnover and would in principle counteract IMiDs therapy. We performed a CRISPR-based genome-wide knockout screening for regulators of pomalidomide sensitivity in MM cells, and found that CRBN is inherently unstable and its level is controlled by the COP9 signalosome (CSN) to sustain IMiDs cytotoxicity. When unbound to CRL4, CRBN is recruited by the F-box protein Fbxo7 to the Cul1-Skp1 (SCF) ubiquitin ligase for ubiquitination and degradation, a process suppressed by CSN-mediated de-neddylation of Cul1. Deletion of SCFFbxo7 components, or treatment with bortezomib or neddylation inhibitor MLN4924 stabilizes CRBN, and IMiDs further promote the loading of CRBN to CRL4 by inhibiting SCFFbxo7-targeted CRBN degradation, leading to enhanced turnover of IKZF1 and IKZF3 in MM cells and synergistic inhibition of MM cell proliferation. Our findings unravel a dual role of IMiDs in activating CRL4CRBN E3 ligase in MM cells and provide a framework to select patients most susceptible to bortezomib and IMiDs combination therapy.

#4632

Effects of Hsp90 inhibitors on triple-negative breast cancer: BRCA1 as a therapeutic target for TNBC.

Prabhu Ramamoorthy, Parthasarathy Rangarajan, Ossama Tawfik, Shrikant Anant, Roy A. Jensen. _University of Kansas Medical Center, Kansas City, KS_.

Background: Breast cancer is the second leading cause of death for woman. Within breast cancer, those classified as Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. The aim of this study was to elucidate whether the two natural inhibitors of Hsp90, celastrol and triptolide inhibit triple negative breast cancer growth. Both these compounds are terpenoids and were obtained from the Chinese herb "Thunder God of Vine" (Tripterygium wilfordii).

Methods: BT20, BT549, MDA-MB-157 and MDA-MB-231 cells (all TNBC cells), and immortalized human mammary epithelial cells (HMLE) were grown in DMEM containing 10% FBS as per ATCC recommendations. Cell proliferation was assessed by hexoseaminidase activity, and IC50 values calculated using GraphPad Prism5. For clonogenicity, 500 cells were incubated with IC50 concentrations of each compound for 24 h, after which they were allowed to grow and form colonies. For in vivo, BT20 cells were injected into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively.

Results: Celastrol and triptolide treatment suppressed proliferation and colony forming ability of all four TNBC cell lines, but not that of the immortalized HMLE cells. The compounds increased apoptotic cell death, based on increased Annexin V staining. Moreover, there was increased expression of the pro-apoptotic protein Bax but decreased expression of the anti-apoptotic protein, Bcl2 and BclXL. Immunoprecipitation-coupled western blots also showed that the compounds inhibit HSP90/CDC37 complex formation. Interestingly, the coupled immunoprecipitation-western blot analyses showed increased HSP90-BRCA1 interaction after treatment with the compounds. Coupled to this, western blot and immunostaining assays showed increased cytosolic levels and reduced nuclear levels of BRCA1 protein. Similar results were obtained in vivo with BT20 xenografts. In addition to decreased tumor size in response to treatment with celastrol or triptolide, the xenograft tissues showed an increase in cytoplasmic BRCA1 levels following treatment. In addition, there was increased in HSP90-BRCA1 complex formation in the treated xenograft tissues. Finally, knockdown of BRCA1 using specific silencer RNA resulted in partial inhibition in cell growth reduction after celastrol and triptolide treatment in both BT20 and MDA-MB-231 cells.

Conclusion: Taken together, these data suggest that both celastrol and triptolide suppress TNBC cell growth, in part through increasing cytosolic HSP90/BRCA1 complex formation.

#4633

The DNA strand breaking nucleoside analogue sapacitabine sensitizes Brca2-deficient ovarian cancer cells and synergizes with PARP inhibitors.

Xiaojun Liu, Yingjun Jiang, Billie Nowak, Bethany Qiang, Nancy Cheng, William Plunkett. _UT MD Anderson Cancer Center, Houston, TX_.

Sapacitabine, an orally bioavailable prodrug of the deoxycytidine analog, CNDAC, is currently in a Phase III registration trial for elderly AML patients (NCT01303796). CNDAC (as DFP-10917) is in a Phase I/II trial for relapsed or refractory acute leukemia (NCT01702155) with very limited toxicity. CNDAC-induced DNA damage, double-strand breaks converted from initial single-strand breaks, is repaired mainly by the homologous recombination (HR) pathway. Deficiency in HR components (e.g. ATM, Rad51, Xrcc3, Brca1 and Brca2) confers sensitivity to CNDAC. Brca1/2 function is frequently compromised in ovarian cancer (OC). To determine the role of Brca2 in DNA damage repair after CNDAC, we used in this study a Brca2-mutant ovarian adenocarcinoma cell line, PEO1 and its revertant line, PEO1 C4-2, in which Brca2 function is restored due to a secondary mutation. First, the clonogenic sensitivities of the two lines to therapeutic agents were compared. The mutant was 6-10 fold more sensitive to CNDAC than the revertant. In contrast, Brca2 restoration did not confer resistance to cytarabine or gemcitabine, confirming the unique action mechanism of CNDAC among nucleoside analogs. Second, CNDAC-induced chromosome damage was compared in both lines. We found Brca2- mutant cells bearing more chromosomal structural abnormalities (67% metaphases with scorable breaks or fusions, N=51) than Brca2-restored cells (14%, N=50), apparently due to genetic instability when lacking Brca2. The mutant cells exposed to 25 nM CNDAC for 3d manifested massive chromosomal aberrations (100% metaphases unscorable, N=51). In contrast, the revertant cells showed significantly fewer chromosome aberrations (30% scorable, N=50). These results provided cytogenetic evidence for Brca2 involvement in DNA damage repair after CNDAC. Third, the combination effect of CNDAC with two classes of front-line anti-OC drugs, platinum compounds and taxanes, was explored using clonogenic assays and median effect analyses. Both cisplatin and oxaliplatin each had additive cell killing effects with CNDAC (combination index, CI ~1) independent of Brca2 status. Similarly, CNDAC was additive with either paclitaxel or docetaxel in both lines (CI ~1). Finally, two PARP inhibitors (PARPis), rucaparib and talazoparib, greatly sensitized Brca2-mutant cells. Despite the distinctive sensitivities of the two lines, both rucaparib-CNDAC and talazoparib-CNDAC combinations showed synergistic killing effect (CI <1). Since PARPis have promising efficacy in Brca2-deficient OC patients, the synergistic combination of PARPi and sapacitabine could be a novel therapeutic strategy to be tested in clinic. This study provides mechanistic insight into the function of Brca2 in repairing sapacitabine/CNDAC-induced DNA damage, and rationale for sapacitabine-PARPi combination to target Brca2-deficient OC.

#4634

Novel dual HDAC & PI3K inhibitor, CUDC-907, for MYC-driven malignancies.

Kaiming Sun, Ruzanna Atoyan, Mylissa A. Borek, Steven Dellarocca, Maria E. Samson, Anna W. Ma, Guangxin Xu, Troy Patterson, David P. Tuck, Jaye L. Viner, Ali Fattaey, Jing Wang. _Curis, Inc., Lexington, MA_.

MYC family genes are among the most frequently deregulated oncogenic drivers in human cancer. Pharmacologic inhibition of HDAC activity and blockade of the PI3K pathway have both been shown to suppress MYC-induced transcription.

HDAC activity is critical for MYC gene regulation, as MYC represses transcription of target genes through recruitment of HDACs. HDAC inhibitors have been shown to restore expression of genes suppressed by MYC family members and to induce rapid downregulation of expression of MYC itself.

The PI3K pathway plays a central role in regulating MYC at the post-transcriptional level. Activation of PI3K signaling leads to activation of AKT, which phosphorylates and inhibits GSK3β. As GSK3β normally phosphorylates MYC which facilitates the degradation of MYC, activation of PI3K signaling leads to increased stability of MYC, whereas PI3K inhibitors decrease MYC stability. A recent study has demonstrated addiction to MYC signaling and hypersensitivity to PI3K inhibition in PTEN-deficient diffuse large B-cell (DLBCL) cell lines, suggesting that MYC-driven cancers may be particularly sensitive to PI3K inhibition. As HDACs and PI3K regulate MYC protein levels and functions through nonoverlapping mechanisms, simultaneous HDAC and PI3K inhibition may further enhance MYC suppression.

CUDC-907 is an orally bioavailable, small-molecule dual HDAC and PI3K inhibitor that primarily inhibits class I and II HDACs and the PI3Kα, β, and δ isoforms. CUDC-907 shows greater anti-tumor activity in vitro than single-target HDAC or PI3K inhibitors, especially in MYC-dependent cell types, such as DLBCL and NUT midline carcinoma (NMC). In preclinical testing, CUDC-907 treatment leads to a dose-dependent decrease in MYC protein levels, and is also more potent in decreasing MYC than the HDAC inhibitor panobinostat and the pan-PI3K inhibitor pictilisib alone or in combination. Significant antitumor effects have been consistently observed in MYC-driven DLBCL xenograft and genetically engineered mouse models exposed to CUDC907. In particular, certain MYC translocation (Daudi), double-hit (concurrent MYC and BLC2 translocation, WSUDLCL2 and DOHH2), double-expresser (expression of MYC and BCL2 proteins, U2932) xenograft models, and the Eµ-Myc transgenic mouse model achieve tumor growth inhibition of 100%, 69%, 56%, 97% and 72%, respectively.

These findings raise the possibility that hematologic and solid tumors driven by aberrant overexpression of MYC family genes (e.g., MYC-altered DLBCL and NMC) might be more responsive to simultaneous HDAC and PI3K inhibition with CUDC-907 than they are to single-target therapy. Clinical Phase 1 studies are currently testing CUDC-907 in patients with relapsed/refractory (R/R) DLBCL and advanced MYC-aberrant solid tumors. Preliminary data are encouraging and support the planned Phase 2 study in R/R MYC-altered DLBCL, as well as further testing in other MYC-driven malignancies.

#4635

Comparative cancer cell line profiling differentiates the mechanism of action of different PI3K/mTOR, Aurora kinase and EZH2 inhibitors.

Joost C.M. Uitdehaag, Jeroen A.D.M. de Roos, Martine B.W. Prinsen, Judith R.F. de Vetter, Jelle Dylus, Antoon M. van Doornmalen, Suzanne J.C. van Gerwen, Jos de Man, Rogier C. Buijsman, Guido J.R. Zaman. _Netherlands Translational Research Center B.V., Oss, Netherlands_.

[purpose] Profiling of drug candidates in cell line panels is an important tool to compare the selectivity and targeting of new anti-cancer agents. In addition, comparative profiling may be used to repurpose established therapeutics by identifying new mechanisms of action or cross-selectivities.

[experimental procedures] A collection of more than 120 anti-cancer agents, targeting all important oncogenic signaling pathways, including classic cytotoxic agents as well as many targeted kinase inhibitors and epigenetic modulators, was profiled on a panel of 44 or 66 parallel cell line proliferation assays (Oncolines™) [1,2]. The Oncolines™ profiles of the compounds were compared by Pearson correlations of their inhibitor responses. Inhibitor sets were clustered using hierarchical trees. Response profiles were correlated to the genetic background of the cell lines by Analysis of Variance (Anova).

[results] Reproducibility of the NTRC Oncolines™ cell panel was validated by monitoring cell growth rate and the variation in IC50s of replicate profiles over a period of three years. The Pearson correlation between replicates ranged between 0.60 and 0.99 for 16 different inhibitors, depending on dose-response curve shape. Correlation analyses of the > 120 profiled anti-cancer agents revealed separate clusters of, a.o., taxanes, platins, topo-isomerase inhibitors, and EGFR, ABL, MEK and BRAF inhibitors. This demonstrates that the Oncolines™ profiles are an unbiased representation of the compound's mechanisms. The profile of the BTK inhibitor ibrutinib correlated with EGFR inhibitors. In biochemical experiments we showed that this due to its cross-reactivity with EGFR. The six Aurora kinase inhibitors profiled fall into two separate clusters, which are related to their biochemical selectivity. Thus, Aurora A-selective inhibitors are relatively more active in cell lines with mutations in cell cycle checkpoint-related genes such as TP53 and RB1; whereas pan-Aurora inhibitors are more active in cell lines with mutations in growth factor signaling pathways, such as NRAS. Profiling of eleven PI3 kinase and mTOR inhibitors revealed four distinct clusters. PI3Kalpha and PI3Kdelta isoform selective inhibitors each target genetically distinct subgroups of cell lines. Rapamycin-analogs, such as everolimus, specifically target PTEN-mutant cell lines. Finally, profiling of EZH2 inhibitors indicates that there are essentially two classes and that these have a cellular profile that is distinct from HDAC, DOT1L or BET inhibitors.

[conclusions] Comparative cancer cell line profiling is a powerful tool to rapidly explore the pharmacogenomics of drug action in cancer cells and to identify new or previously unnoted activities of compounds.

[references]: [1] Uitdehaag et al. (2014) PLOS ONE 9(3) e92146; [2] Uitdehaag et al. (2015) PLOS ONE 10(6) e0132230.

#4636

Dual targeting of PI3K/mTOR leads to MEK/ERK over-activation in leiomyosarcoma through suppression of mTORC2 inhibition.

Benjamin Fourneaux,1 Vanessa Chaire,1 Marie Karanian,2 Audrey Laroche,1 Antoine Italiano2. 1 _Inserm U916, Institut Bergonié & Université de Bordeaux, Bordeaux, France; _2 _Department of Medical Oncology, Institut Bergonié, Bordeaux, France_.

Leiomyosarcoma (LMS) are an uncommon group of malignant tumors composed of cells showing smooth muscle differentiation that represent 10-15% of all soft-tissue sarcomas and is therefore one of the most frequent sarcoma subtype. From a genetic point of view, LMS are characterized by numerous genomic alterations whose a large region of deletion on chromosome 10 including the tumor suppressor gene PTEN. It is one of the most frequent aberration (40-50% of cases) and is associated with increased activation of the mTOR pathway. Moreover, in vitro and vivo studies have demonstrated that the mTOR pathway plays a crucial role in LMS tumorigenesis. There are no data about the role of PI3K/mTOR targeting in LMS.

The LMS cell lines have been derived from human surgical STS specimens in our laboratory. Cells were treated for 72hrs with a range of increasing concentrations of Everolimus (mTORC1 inhibitor), BKM120 (PI3KCA inhibitor) and BEZ235 (dual PI3K-mTOR inhibitor) to assess anti-proliferative activity by MTT-assay and apoptosis by AnnexinV/PI staining. Effects on mTOR and MAPK pathway signaling were studied by western blot analysis. The LMS cell lines were exposed to increasing doses of BEZ235 in combination with GDC-0994 (ERK inhibitor) and synergy was assessed by Chou-Talalay combination index (CI). Effects of all inhibitors were observed in mice with subcutaneous LMS cell line-derived xenograft tumors for 3 weeks.

Dose-dependent growth suppression was more clearly induced by BEZ235 (range, 0.001 to 0.1 µM) than by Everolimus and BKM120 (range 0.01 to 1.6 µM). Apoptosis was also much more effectively induced by BEZ235. We examined in vivo antitumor activity of the three drugs in mice inoculated with LMS cells. In vivo, the effects of BEZ235 (70% tumor volume [TV] inhibition versus untreated tumors) was also higher than that of BKM120 and Everolimus (30% and 55% TV inhibition). We assessed the effects of BEZ235 on signaling pathways in all cell lines. Strikingly, mTOR pathway was consistently repressed and BEZ235 markedly induced ERK overactivation. This effect was not observed with BKM120 or Everolimus suggesting a role of mTORC2. Knockdown of RICTOR via transfection of siRNA significantly reduced the enhancing effect of BEZ235 on ERK phosphorylation. Combined treatment with BEZ235 and GDC-0994, a potent ERK inhibitor, resulted in synergistic growth inhibition, apoptosis induction and decrease levels of both phosphorylated proteins of mTOR and ERK pathways in vitro and in vivo.

Targeting PI3K and mTOR simultaneously using BEZ235 effectively inhibits LMS cell growth and prolongs survival of mice in comparison of those treated with BKM120 or Everolimus. However, BEZ235 suppress a negative feedback loop mediated by mTORC2, thereby leading to enhanced MEK/ERK pathway activity in LMSs cells. Combining BEZ235 with MEK/ERK inhibitors may be a relevant approach to increase anti-tumor activity and avoid drug resistance.

#4637

Chk1 inhibition: a promising targeted treatment for head and neck squamous cell carcinoma.

Anne M. van Harten, Marijke Stigter-van Walsum, Marijke Buijze, Boudewijn Jm Braakhuis, Ruud H. Brakenhoff. _VU University Medical Center Amsterdam, Amsterdam, Netherlands_.

Over 90% of all malignant tumors in the head and neck region are squamous cell carcinomas (HNSCC), ranking within the top 10 of most common cancers worldwide. HNSCC arise in the mucosal linings of the upper aerodigestive tract, and known risk factors are tobacco use, excessive alcohol consumption and infection with high risk types of the human papillomavirus (HPV). Despite the application of intensive treatment protocols, the 5-years survival rate of HNSCC patients has hardly increased last decades and remained at approximately 50%. Therefore, new therapeutic possibilities are urgently awaited.

Previously, we identified CHEK1 as a candidate therapeutic target for HNSCC in a whole genome siRNA screen (Martens-de Kemp et al, Clin Cancer Res 2013). Additionally, a functional screen using a microRNA expressing library indicated that knockdown of ATM expression may cause a lethal effect in HNSCC cell lines as well (Lindenbergh-van der Plas et al, Clin Cancer Res 2013). The aim of the present study was to investigate whether interference with the ATM/ATR/CHEK1/CHEK2 DNA damage response pathway is indeed a promising therapeutic approach in HNSCC.

RNA interference using siRNAs targeting ATM, ATR, CHEK1 and CHEK2 confirmed that particularly CHEK1 is essential for the survival of a subset of HNSCC cell lines, whereas the cell viability of primary oral keratinocytes and fibroblasts was not compromised. This was confirmed in vitro by treatment with a small molecule inhibitor targeting Chk1, and a therapeutic window between cancer and normal oral mucosal cells was observed. Functional assays indicated an S-phase delay in Chk1 inhibited HNSCC cells, followed by DNA damage. The latter was indicated by elevated phosphorylation of ATM Ser1981 and γH2Ax Ser139, as well as the appearance of chromosomal breaks. The DNA damage response initiated caspase-mediated apoptosis, resulting in programmed cell death.

In summary, our data show that HNSCC cells appear to depend strongly on a functional Chk1 protein during DNA replication. Therefore, Chk1 inhibition seems an interesting therapeutic strategy for HNSCC, at least in a subset of tumors.

#4638

Targeted therapy of precancerous fields in the mucosal linings of the head and neck.

D Vicky de Boer, Sanne R. Martens-de Kemp, Marijke Buijze, Marijke Stigter-van Walsum, Elisabeth Bloemena, C René Leemans, Boudewijn J.M. Braakhuis, Ruud H. Brakenhoff. _VU University medical center Amsterdam, Amsterdam, Netherlands_.

Primary head and neck squamous cell carcinomas (HNSCC) and local relapses develop in preneoplastic fields in the mucosal linings of the upper aerodigestive tract. These fields are characterized by tumor-associated genetic changes, show frequently dysplastic features and are occasionally macroscopically visible as lesions. Currently, there are no adequate treatment options for these fields to prevent tumor development. To develop a targeted therapeutic approach we established four unique preneoplastic cell lines, derived from fields adjacent to HNSCCs. Next, we performed a screen with a selective panel of small interfering RNAs (siRNAs) that are lethal for HNSCC cells, to identify genes essential for survival of preneoplastic cells and not for normal cells. One of these selective essential genes was Polo-like kinase 1 (PLK1). Inhibition of PLK1, both with siRNAs as well as small molecule inhibitors, caused cell death of preneoplastic and HNSCC tumor cells, while primary fibroblasts and keratinocytes were hardly affected. Targeting PLK1 caused a strong G2/M cell cycle arrest in preneoplastic cells accompanied by formation of monopolar spindles during mitosis. Hence, PLK1 is a promising therapeutic target for chemopreventive eradication of preneoplastic fields in patients at high risk of developing HNSCC and local relapses.

#4639

Reproductive hormone levels modulate doxorubicin induced cardiomyopathy in female tumor-bearing spontaneously hypertensive rats.

Kaytee L. Pokrzywinski,1 Elliot T. Rosen,1 Julia Bonanno,1 Thomas Biel,1 Delaram Moshkelani,2 Baikuntha Aryal,1 Steven Mog,3 V. Ashutosh Rao1. 1 _Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD;_ 2 _Division of Process Assessment III, Office of Process and Facilities, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD;_ 3 _Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD_.

The anthracycline doxorubicin (dox) is known to cause cardiomyopathy, at least in part, through the generation of excess reactive oxygen species (ROS) in the mitochondria. Cardiomyocytes have higher concentrations of mitochondria and lower concentrations of antioxidants, making them more sensitive to excessive ROS. The incidence of dox induced cardiac stress is greater in pre-pubescent and post-menopausal females compared to age-matched males but lower in women of reproductive age. Previous studies have associated gender and age differences in cardiosensitivity with estrogen production. This is the first part of a two part study investigating the role that reproductive hormones (estrogen and progesterone) play in dox induced cardiotoxicity in female, tumor-bearing spontaneously hypertensive rats (SHRs). In this portion of the study our aim was to evaluate the effect of hormone therapy on cardioprotection from dox. Reproductively mature ovariectomized animals were implanted with time-release pellets containing a "carrier-matrix" (vehicle), 17-β-estradiol (E2), progesterone (P4), Tamoxifen (Tam) or combinations thereof. The animals were implanted with cells from an SHR-derived syngeneic breast cancer cell line (SST-2). Tumor-bearing animals were then treated with a single dose of saline or dox and monitored for 12 days. Daily vaginal cytologies were obtained to measure estrous cycle status. Serum was collected throughout the study to correlate estrous phase to E2 and P4 levels and evaluate serum protein cTnI as a biomarker for cardiac injury. In all hormone groups, dox exhibited significant anti-tumor activity and resulted in a net decrease in growth rate compared to saline controls. After 12 days exposure, animals treated with dox and implanted with Tam, Tam+E2 or vehicle pellets showed a significant increase in cTnI, indicative of cardiac damage. Dox treated animals with pellets containing E2, P4 or the combination showed a significant decrease in cTnI, demonstrating potential cardioprotection from reproductive hormone therapy. Furthermore, histopathology of cardiac sections from dox-treated groups displayed lower cardiomyopathy scores in animals that received E2, P4 or the combination of the two. We are currently evaluating additional endpoints for cardiotoxicity and cellular stress to assess the mechanism behind E2 and P4 cardioprotection from chemotherapy. Overall, our findings suggest that the hormones estrogen and progesterone reduce dox-induced cardiotoxicity.

#4640

Doxorubicin induced cardiomyopathy associated with natural reproductive hormone cycling in female tumor-bearing spontaneously hypertensive rats.

Kaytee L. Pokrzywinski,1 Elliot T. Rosen,1 Julia Bonnano,1 Thomas Biel,1 Delaram Moshkelani,2 Baikuntha Aryal,1 Steven Mog,3 V. Ashutosh Rao1. 1 _Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Research, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD;_ 2 _Division of Process Assessment III, Office of Process and Facilities, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD;_ 3 _Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Silver Spring, MD_.

Doxorubicin (dox) is a widely used oncology agent known to induce dose-limiting cardiomyopathy, driven partially by the excess production of reactive oxygen species (ROS) in mitochondria. Cardiomyocytes are particularly sensitive to ROS due to higher densities of mitochondria and lower concentrations of antioxidants. The incidence of dox induced cardiomyopathy is lower in reproductively mature women compared to age matched men, while pre-pubescent and post-menopausal females are more sensitive. Previous studies have associated reproductive hormone levels with differences in cardiosensitivity based on gender and age. This is the second part of a two part study investigating the role that reproductive hormones play in dox induced cardiotoxicity in female, tumor-bearing spontaneously hypertensive rats (SHRs). In the current study, we investigated the impact of natural hormone cycling on dox induced cardiomyopathy. Before beginning the experiment, regular hormone cycling was established by monitoring daily changes in the estrous cycle by way of vaginal cytology. The animals were organized by phase (proestrus, estrus, metestrus, or diestrus) and implanted with SST-2 cells, a syngeneic breast cancer cell line derived from a SHR. Tumor-bearing animals were administered a single dose of saline, dox , the cardioprotectant dexrazoxane, or a combination thereof and monitored for 14 days. Daily vaginal cytologies were obtained throughout the experiment to monitor estrous cycling as a means of tracking hormone fluctuations. Serum was collected at various time points to correlate estrous cycling with circulating hormone levels (E2 and P4) and measure the cardiac health indicator cardiac troponin I (cTnI). In all hormone groups, dox treatment resulted in a net loss in bodyweight and significant anti-tumor activity, as measured by tumor volume, compared to respective saline controls. Interestingly, animals treated with saline in metestrus displayed increased tumor volumes compared to animals in different estrous phases. We are currently evaluating cardiac status by echocardiogram, histopathology, and cTnI. We are also measuring serum E2 and P4 hormone levels in an effort to understand the relationship between cardiomyopathy and circulating hormones.

#4641

Bone marrow stromal cells frustrate temozolomide treatment of glioblastoma.

Mark Katakowski, Elizabeth Pikula, Steven Kalkanis, Michael Chopp. _Henry Ford Hospital, Detroit, MI_.

Temozolomide (TMZ) is the most widely used for the treatment of glioblastoma. However, despite the effectiveness of TMZ, progression and recurrence are observed in nearly all patients. Here, we report a heretofore unknown mechanism of TMZ chemo-resistance mediated by circulating exosomes. Exosomes are small microvesicles released by cells that efficiently transfer their molecular cargo to other cells, including tumor. We recently found that in response to TMZ treatment, human bone marrow stromal cells (MSCs) release exosomes that confer TMZ chemo-resistance upon glioblastoma cells.

MSCs produce exosomes which can be found in circulating blood. TMZ is distributed systemically, thus we tested the effect of TMZ upon MSC-produced exosomes, and the profile of miRNAs within the exosomes. In addition, we tested if MSC exosomes altered the response of glioblastoma cells to TMZ. To test our hypotheses, we treated MSCs with TMZ, and performed miRNA PCR arrays upon exosomes produced by the MSCs (tMSC-exo). Furthermore, we exposed glioblastoma cells to tMSC-exo, and measured the effect upon tumor cell viability, and growth in the presence of TMZ. Finally, we collected serum exosomes from patients with glioblastoma upon resection, and after TMZ treatment. We tested if exosomes from these patients before and after TMZ treatment exhibit different chemo-resistive effects, and if TMZ treatment altered the miRNA profile in circulating exosomes.

We report that tMSC-exo elicit a protective effect upon glioblastoma cells treated with TMZ. The miRNA profile of MSC exosomes was also significantly altered by TMZ. Like MSC exosomes, TMZ treatment altered the miRNA profile of circulating exosomes in glioblastoma patients, and resulted in increased chemo-resistance conferred by the exosomes upon tumor cells.

Investigations of TMZ chemo-resistance have largely focused upon characteristics of the tumor cells the agent is intended to kill. Here, we elucidate a novel mechanism of resistance to TMZ which does not arise from the tumor itself. Our findings suggest that circulating exosomes may diminish the efficacy of TMZ treatment of glioblastoma.

#4644

Inhibition of the androgen receptor at two drug-targetable sites on the DNA-binding domain protein surface.

Kush Dalal, Aishwariya Sharma, Mani Roshan-Moniri, Hendrik Borgmann, Nada Lallous, Shannon Awrey, Huifang Li, Fuqiang Ban, Eric LeBlanc, Artem Cherkasov, Paul S. Rennie. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

The androgen receptor (AR) is a hormone inducible transcription factor that continues to be an important drug-target to prevent or slow the progression of prostate cancer. Current small molecule inhibitors, such as Enzalutamide (anti-androgens), compete with naturally occurring steroids that bind to the hormone binding pocket of the AR ligand binding domain (LBD). In advanced or castration resistant prostate cancer (CRPC), mutations in the LBD confer drug-resistance by converting anti-androgens into agonists, prompting research to develop small molecule inhibitors that target different sites on the AR protein surface. Recently, we characterized a set of small molecules that could interact with the DNA binding domain (DBD) of the AR and block its transcriptional activity. Here, we extend the pioneering observations by clarifying the mechanism of two classes of compounds that either block AR-chromatin interactions or reduce AR-dimerization in the cell nucleus. Compound efficacy is demonstrated across multiple prostate cancer cells lines, including enzalutamide resistant forms, with respect to AR transactivation, cell viability and expression of downstream genes. We also characterize the pharmacological properties of the lead compound and its effects on tumour xenografts in mice. Collectively, these results lay the foundation for the development of alternative prostate cancer drugs that interfere with the biochemical function of the activated, nuclear localized AR.

#4645

Cell cycle specific effects and associated DNA damage of selective inhibitors of nuclear export (SINE).

Russell T. Burke,1 Joshua Marcus,1 John DeSisto,1 Yosef Landesman,2 James D. Orth1. 1 _University of Colorado - Boulder, Boulder, CO;_ 2 _Karyopharm Therapeutics, Inc., Newton, MA_.

Nuclear export of proteins is fundamental for cell growth and function. Selinexor is a SINE compound that is in clinical development for the treatment of different cancers. Selinexor forms a slowly reversible covalent bond to Exportin-1 (XPO1), preventing its association with protein cargos, thereby resulting in their nuclear retention. XPO1 cargos include the majority of tumor suppressor proteins (TSP) and cell cycle regulators such as p53, p21 and p27 that have key roles in cancer progression and drug response. It is unclear how selinexor affects cell cycle progression in individual cells and the subsequent stress and fate of those cells. To elucidate selinexor action, we developed cell lines that stably express fluorescent ubiquitin cell cycle reporters (FUCCI), and followed individual cells longitudinally using continuous time-lapse microscopy for 72 hours. We report that in fibrosarcoma-derived HT-1080 cells that express wildtype p53 and p21, 18% of the initial cell population became arrested with >90% in G1- or S-phase and 40% died with 64% from G1- or early S-phase after a cell cycle delay or arrest. We also found that 42% of cells divided, but the progeny died or arrested in G1- or S-phase of the next cell cycle - often after cell cycle arrest or slowed cell cycle progression. Using FUCCI, we tracked the response of cells treated acutely in specific cell cycle stages. Cells treated in G1-phase most often arrested or died in G1- or S-phase, whereas cells treated in G2-phase usually progressed to cell division. As FUCCI revealed S-phase progression defects and associated cell death, we further characterized this phenotype. Using nucleotide incorporation with fluorescent detection, we observed that as early as 2 hours after selinexor treatment only a subset of cells is undergoing DNA replication. Cells that were able to replicate their DNA are doing that inefficiently as both the rate and maximal levels of nucleotide incorporation are significantly reduced. S-phase arrest and progression defects may manifest as DNA double-strand breaks. We find a strong association between S-phase status and DNA damage. In some cells, the damage occurs within hours of selinexor treatment and appears as a striking cluster of foci. At 8 hours, nearly 35-45% of cells contain DNA damage clusters. Importantly, the damage clusters sometimes repair as determined by fixed cell time-course analysis and live-cell microscopy. We are exploring the nature of these DNA damage structures and the mechanisms of their formation and repair. In summary, selinexor is fast acting, shows cell cycle selectivity, results in DNA damage and is highly effective at arresting cell growth and inducing apoptosis in tumor cells. These data suggest that selinexor may exert anti-cancer effects even on slow growing tumors where the bulk of the cell mass presents a G1-like state and that it likely combines well with other cell cycle targeted therapeutics.

#4646

Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer.

Sivan Elloul, Hua Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. _Karyopharm Therapeutics Inc, Newton, MA_.

Introduction: Selinexor (sel) is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports include major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. We have previously shown that HER2+ breast cancer (BC) cells are highly sensitive to sel (SKBR3 and MCF7 IC50 0.21 and 0.073 μM, respectively). Here we tested the hypothesis that inhibition of HER2 signaling with trastuzumab (tras) synergizes with sel as both mediators (PI3K-AKT and Ras-Raf-MEK pathways) and effectors (FOXO3A, MDM2) of the HER2 pathway are among direct cargos of XPO1.

Methods: The effects of sel and tras alone or in combination on cell viability were tested on MCF7 and BT474 HER2+ BC cells using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, BT474 cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of sel and tras alone or in combination and with the therapeutic dose of sel. Tumor Growth (TG) was monitored for 60 days. Xenografts were harvested and analyzed by immunohistochemestry (IHC).

Results: Sel-tras combination was highly effective in-vitro and in-vivo. In MTT assays, both sel and tras demonstrated low IC50 values (nanomolar) and when combined, they synergistically inhibit proliferation. In-vivo, the combination resulted in significant survival benefit and enhanced TG inhibition of 88% compared to the monotherapy groups 57% (sel) and 25% (tras). Interestingly, an increase in pro-survival cytoplasmic FOXO3A protein was observed in the tras-treated group however, in the combination group, FOXO3A, which is an XPO1cargo, was restricted to the nucleus where it can trigger apoptosis. In addition, a synergistic increase in P27 protein, the G1 phase arrest regulator, was observed in the sel-tras-treated group. While in the monotherapy groups p-ERK was not modulated, in the combination group an increase in cytoplasmic p-ERK was observed, suggesting an affect on its downstream regulators of apoptosis. Indeed, apoptosis was evident in the combination samples by increased IHC staining to Cleaved caspase3 and Apoptag. In addition, an increase in PARP protein was observed in the tras-treated group however, in the combination group, PARP was dramatically reduced. We have previously shown that selinxor modulates DNA Damage Response (DDR) proteins suggesting that one mechanism by which the combination enhances cancer cell death is by accumulation of DNA damage that cannot be resolved.

Conclusion: Sel-tras combination is a novel candidate therapy to HER2+ BC modulating DDR and apoptosis. It synergizes to induce survival benefit and TG inhibition. These data provide rational support for study of sel-tras combination in clinical trials.

#4647

BET protein inhibition blocks growth of triple-negative breast cancer by inducing mitotic and cytokinetic dysfunction.

Jennifer M. Brancato,1 Sylvia Gayle,1 Kristen Weber-Bonk,1 Matthew Summers,2 Gurkan Bebek,1 Ruth Keri1. 1 _Case Western Reserve University, Cleveland, OH;_ 2 _Cleveland Clinic, Cleveland, OH_.

Bromodomain and Extra Terminal (BET) proteins are epigenetic "readers" that recognize acetylated histones and mark areas of the genome for transcription. BRD4, a BET family member protein, has been implicated in a number of types of cancer. It has been recently found to associate with super-enhancers, and elevated levels of BRD4 have been linked to increased expression of MYC as well as other oncogenes. BET protein inhibitors are currently being tested for their potential use in the treatment of HIV, heart failure, and cancer, all of which are diseases of aberrant transcription. However, little is known regarding their efficacy in triple-negative breast cancer (TNBC). We found JQ1, a prototypical BET inhibitor, impedes growth of seven TNBC cell lines in a dose-dependent manner within 72 hours of treatment. JQ1 also suppresses growth of three different TNBC xenografts, including a patient-derived model of basal-like breast cancer. Growth arrest, in vitro, is followed by either apoptosis or senescence. However, prior to the induction of these two terminal responses, prolonged treatment results in polyploidy in many of the cell lines, suggesting BETi disrupts mitosis/cytokinesis. Live-cell imaging revealed JQ1 significantly increased the duration of mitosis, and microarray analyses showed a significant JQ1-mediated downregulation of genes critical for cell cycle progression, mitosis, and cytokinesis. In all TNBC cell lines tested, Aurora kinases, proteins critical for proper progression through mitosis and cytokinesis, are suppressed in response to JQ1. Treatment with AZD1152, an Aurora kinase B inhibitor, elicits the same cellular responses as BET inhibition, indicating that the suppression of Aurora kinases plays a key role in the response of TNBC cells to BET inhibitors. These findings reveal that BET inhibitors block the growth of highly aggressive TNBC cells by inducing mitotic dysfunction and that these drugs are promising potential therapeutics for the treatment of TNBC.

#4648

Uncover novel drug combinations for triple negative breast cancer.

Carlotta Costa, Anahita Dastur, Rachel Peterson, Damon Leah, Regina Egan, Ryan March, Cyril Benes, Jeffrey Engelman. _Massachusetts General Hospital, Charlestown, MA_.

Therapeutic options for triple negative breast cancers (TNBC) are limited to multiple lines of chemotherapy with no FDA-approved targeted therapies. In the absence of a clear driver oncogene, the most common molecular alterations, including EGF receptor and genes involved in cell cycle, DNA repair and the PI3K pathway, may represent new therapeutic opportunities in TNBC. Indeed clinical benefits from PARP, EGFR and PI3K inhibitors have been reported in some TNBC patient. However, none of these studies have demonstrated durable responses, indicating that inhibition of additional pathways might be necessary to successfully improve the clinical efficacy of targeted therapies in TNBC. To develop novel, combinatorial therapeutic approaches for TNBC we performed a pharmacological screen testing 672 unique drug combinations in multiple TNBC models. The screen identified previously known and efficacious synergies, such as between EGFR and PI3K inhibitors, supporting the validity of our data and analysis. Importantly, we also identified novel promising drug combinations that we are currently exploring.

#4649

ODM-207, a novel BET-bromodomain inhibitor as a therapeutic approach for the treatment of prostate and breast cancer.

Mari Björkman,1 Elina Mattila,1 Reetta Riikonen,1 Chandra Sekhar,2 Mahaboobi Jaleel,2 Sivapriya Marappan,2 Tarja Ikonen,3 Daniel Nicorici Nicorici,3 Juha Rantala,4 Susanta Samajdar,2 Murali Ramachandra,2 Pekka Kallio,1 Anu-Maarit Moilanen1. 1 _Orion Corporation Orion Pharma, Turku, Finland;_ 2 _Aurigene Discovery Technologies Limited, Bangalore, India;_ 3 _Orion Corporation Orion Pharma, Espoo, Finland;_ 4 _Misvik Biology, Turku, Finland_.

Introduction: BET (bromodomain and extraterminal) family proteins (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that bind to acetylated-lysine residues in histones and recruit protein complexes to promote transcription elongation. In many cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic drivers that are important for cell proliferation and survival. Pharmacologic inhibition of the BET-histone interaction has been shown to result in transcriptional downregulation of a number of oncogenes and inhibition of tumor growth providing a novel strategy for treatment of cancer. Therefore, in this study, we evaluated the in vitro and in vivo antitumor activity of ODM-207, a novel, potent and highly selective BET bromodomain inhibitor using cell lines derived from prostate and breast cancer as well as patient-derived tumor cell cultures of breast cancer.

Methods and Results: ODM-207 has antiproliferative effects on several hematological and solid tumor cell lines. In a panel of prostate cancer cell lines, ODM-207 attenuates cell growth of androgen receptor (AR)-positive cell lines such as VCaP and 22Rv1. RNA-sequencing and Western blot studies revealed that the exposure of sensitive prostate cancer cells to ODM-207 is associated with rapid down-regulation c-Myc expression levels while wtAR was not affected. In 22Rv1 prostate cancer xenograft, which expresses both the full-length androgen receptor and androgen receptor splice variant V7, oral administration of ODM-207 was very efficacious in suppressing tumor growth at well tolerated doses whereas enzalutamide had only a modest effect. Contrary to our findings in prostate cancer cells, BET-inhibitor treatment of estrogen-dependent MCF-7 breast cancer cell line inhibits tumor growth in cell proliferation assays but is associated with down-regulation of ERα while the c-Myc levels are very low, highlighting the context-dependent functional effects of BET inhibition. Interestingly, ODM-207 produces potent antiproliferative effects associated with cell-cycle arrest and cellular senescence in patient-derived breast cancer cells.

Conclusions: In summary, ODM-207 is a new generation BET inhibitor found to possess excellent pharmacological properties and antitumor activity both in vitro and in vivo. Our data suggest the potential utilization of ODM-207 for the treatment of prostate and breast cancer.

#4650

Effect of prescription CYP3A inducers and inhibitors on response to anti-androgen therapy in prostate cancer.

Ranjana Mitra, Oscar B. Goodman Jr. _Roseman University of Health Sciences, Las Vegas, NV_.

Purpose: Prostate cancer affects elderly men who typically have multiple comorbidities requiring concomitant prescription medications, many of which are CYP inducers or inhibitors. Androgen deprivation therapy (ADT) is the mainstay therapy for treating advanced prostate cancer. Recently we demonstrated that CYP3A5 facilitates the nuclear translocation of androgen receptor (AR), promoting prostate cancer growth. As the mechanisms by which intratumoral CYP3A5 regulates AR in prostate cancer are not well understood, the clinical impact of concomitant drugs on the efficacy of ADT is presently unknown. This work describes a platform for assessing the effects of commonly prescribed drugs known to be CYP3A5 inducers or inhibitors on AR signaling and prostate cancer growth.

Methods: We have created a lentiviral LNCaP based reporter system for monitoring changes in AR signaling utilizing a construct comprising a promotor containing AR inducible transcriptional response elements (TREs), fused with firefly luciferase, whereas the negative control (NC) lacks the TREs. We used both a mixed transfected pool and individually selected clones to monitor changes in AR activity.

Results: We tested our assay system by inhibiting CYP3A5 gene expression in LNCaP cells using siRNA and then treating it with DHT. Our data indicate that CYP3A5 siRNA reduced DHT induced AR activity significantly as compared to non-target (NT) siRNA. We further tested our reporter system with rifampicin, phenytoin (both CYP3A inducers) and ritonavir, fluoxetine, amiodarone (CYP3A inhibitors). Rifampicin (antibiotic) and phenytoin (anticonvulsant) showed significant (P≤0.05) induction of AR activity as shown by increased luminescence units. Out of the three inhibitors which were tested amiodarone (antiarrhythmic) and ritonavir (antiretroviral) reduced the AR activity by 50% and 80% respectively whereas fluoxetine (antidepressant) did not result in any significant changes. Both amiodarone and ritonavir also inhibited R1881 (artificial androgen) induced AR activity as compared to control.

Conclusion: Concomitant medications can alter the AR activity thus modifying the response to ADT therapy in prostate cancer patients. These finding support the notion that an CYP3A inhibitor-rich concomitant medical regimen may provide additional clinical benefit for men undergoing ADT and further support the concept of targeting nuclear translocation as a therapeutic adjunct in ADT.

#4651

Docosahexaenoic acid-induced degradation of epidermal growth factor receptor through lysosome and ubiquitin/proteasomal system in human non-small cell lung cancer cells.

Soyeon Jeong,1 Kaipeng Jing,1 Soyeon Shin,1 Soyeon Kim,1 Young-Joo Jeon,2 Jun-Young Heo,2 Gi-Ryang Kweon,1 Seung-Kiel Park,2 Jong-Il Park,2 Kyu Lim3. 1 _Dept. of Biochemistry, School of Medicine, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea;_ 2 _Dept. of Biochemistry, School of Medicine, Chungnam National University, Daejeon, Republic of Korea;_ 3 _Dept. of Biochemistry, School of Medicine, Cancer Research Institute, Infection Signaling Network Research Center, Chungnam National University, Daejeon, Republic of Korea_.

Mutations in EGFR gene frequently occurr in lung cancer and their expressions are related to poor prognosis and drug resistance. Although docosahexaenoic acid (DHA) has anti-cancer activity, the underlying mechanism is not well known in human non-small cell lung cancer (NSCLC) yet. In this study, we show a molecular mechanism of DHA-induced degradation of EGFR in human NSCLC cells. To determine whether DHA modulates EGFR degradation, we used three NSCLC cell lines, which is A549 (EGFR WT), and two mutant cells (PC9 and H1975). We confirmed that the viability of three lung cancer cells was decreased by DHA in a dose- and time-dependent manner. DHA augmented EGFR degradation and its ubiquitin in all three cell lines. Indeed, DHA increased the levels of E3-ligase, c-cbl and endosome-related molecules (Rab5, EEA1, Rab7, and LAMP1). Chloroquin (CQ) effectively blocked the DHA-induced EGFR degradation. Moreover, knockdown of lysosome associated membrane protein (LAMP) by its siRNAs, which regulates lysosome fusion, partially prevented EGFR degradation induced by DHA, and co-localization of EGFR with lysosome in immunocytochemistry, suggesting that DHA-induced EGFR degradation is associated with lysosomal degradation. On the other hand, MG132 (proteasome inhibitor) also protected DHA-triggered EGFR degradation, demonstrating that proteasomes also contribute to EGFR degradation in response to DHA treatment. To confirm the effects of endogenous ω3-PUFAs on EGFR degradation, fat-1-stablyexpressing A549 (f-A549) and PC9 cells (f-PC9) were established (The cells express a C. elegansω3-desaturase converting ω6- to ω3-PUFAs endogenously). The level of EGFR was reduced in f-A549 and f-PC9 cells, indicating ω3-PUFAs diminish EGFR level. The cell growth was retarded and the tumorigenicity was inhibited in nude mice. In addition, EGFR levels were significantly decreased in tumor tissues from f-A549 and f-PC9 cells-bearing mice. Taken together, DHA may induce the degradation of EGFR through lysosome and ubiquitin/proteosomal system in EGFR WT and EGFR mutant NSCLC cells. Thus, these findings provide important preclinical evidence and molecular insight for utilization of ω3-PUFAs in the chemoprevention and treatment of human NSCLC.[This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932)]

#4652

Effects of anti-DLL4 treatment on non-small cell lung cancer (NSCLC) human xenograft tumors.

Alayne Brunner, Fiore Cattaruzza, Wan-Ching Yen, Pete Yeung, Marcus Fischer, Belinda Cancilla, Gilbert O'Young, Raymond Tam, Yu-Wang Liu, Austin Gurney, John Lewicki, Tim Hoey, Min Wang, Ann M. Kapoun. _OncoMed Pharmaceuticals, Inc., Redwood City, CA_.

Background: Non-small cell lung cancer (NSCLC) accounts for the vast majority of lung cancers, the leading cause of cancer-related deaths. Notch signaling has been shown to play an important role in lung cancer initiation and progression. Delta-like ligand 4 (DLL4) activates the Notch pathway and is important for cancer stem cell (CSC) survival. Demcizumab (OMP-21M18) is a humanized IgG2 anti-DLL4 antibody currently being tested in a Phase 2 trial in combination with pemetrexed and carboplatin for first-line treatment of patients with NSCLC. Previously, OMP-21M18 in combination with its mouse anti-DLL4 surrogate has been shown to inhibit tumor growth, decrease cancer stem cell frequency, and cause dysfunctional sprouting of new vessels resulting in an anti-angiogenic effect in patient-derived tumor xenograft (PDX) models in breast, colon, ovarian, and pancreatic cancers. Here we show results from NSCLC PDX models.

Methods and Results: Anti-DLL4 treatment was tested in a series of NSCLC PDX models. Because DLL4 inhibition has been shown to have effects on the tumor as well as the vasculature, the combination of OMP-21M18 (targeting human DLL4) and 21R30 (antibody targeting mouse DLL4) treatment in the PDX models was used to model demcizumab treatment in humans. Treatment with anti-DLL4 in combination with chemotherapy inhibited tumor growth in a series of NSCLC PDX models. Additionally, a tumorigenicity assay showed a decrease in the frequency of tumor-initiating cells following treatment with anti-DLL4 and chemotherapy. Gene expression analysis of tumor samples provided insights into the mechanism of action.

Conclusions: Anti-DLL4 treatment in a panel of NSCLC PDX tumor models in vivo showed inhibition of tumor growth and a decrease in the frequency of tumor-initiating cells. Mechanism of action and gene expression analysis of these models treated with anti-DLL4 will be presented. These findings provide additional evidence supporting demcizumab as an effective treatment for NSCLC patients.

#4653

Combination Azacitidine and histone deacetylase inhibition induces a multi factorial synergistic anti-tumor response in non-small cell lung cancer (NSCLC).

Michael Topper, Christin Hanigan, Michelle Vaz, Katherine Chiappinelli, Julin Justin, Lauren Murphy, Cynthia Zahnow, Stephen Baylin. _Johns Hopkins School of Medicine, Baltimore, MD_.

Epigenetic therapy holds much promise as an emerging paradigm for the treatment of many disparate types of human cancer. However, the data in solid malignancies, particularly, would suggest further optimization is required to extract the true potential of these therapies. In our current study, we have deployed at low, clinically achievable doses, chronic combinatorial administration of the DNA methyltransferase inhibitor Azacitidine in conjunction with both benzamide and hydroxyamic acid based histone deacetylase inhibitors (HDACi) in NSCLC. We have developed our paradigms by sequentially employing chronic HDACi administration after Azacitidine. This strategy achieves durable alteration of the cell transcriptome with corresponding induction of both cell cycle arrest and apoptosis. These events appear driven by alterations, both transcriptionally and post transcriptionally of Myc and Myc regulatory proteins. Epigenetic treatment induced perturbation of Myc is facilitated by Wnt pathway inhibition through induction of Wnt antagonists, namely DKK proteins. Importantly, as part of this scenario of altering a Myc regulatory axis, there is a synergistic cytotoxic response mediated at least in part through downregulation of a critical effector, the Skp2 oncogene. Additionally, we observe significant induction of an interferon responsive viral defense gene signature as the result of epigenetic treatment. This gene signature has been shown to correlate with response to immune checkpoint blockade in the setting of metastatic melanoma. We propose that this combination of viral defense gene signature augmentation in conjunction with potent direct epithelial cancer cytotoxicity holds promise to be a more complete therapeutic paradigm for the treatment of NSCLC.

#4654

A novel proteasome inhibitor, bis-Benzylidine Piperidone (RA190), control hepatocellular carcinoma effectively through inhibiting STAT3.

Rueyshyang Soong. _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

Hepatocellular carcinoma(HCC) is one of the most common malignancy, especially in the HBV endemic Asia area. However, there is still lack of effective chemotherapy drug to treat HCC, expect Sorafenib, tyrosine kinase inhibitor, the only FDA approved chemotherapy drug to treat HCC.

In this study, we explore the chemotherapeutic properties of a novel proteasome inhibitor, bis-Benzylidine Piperidone (RA190), and its ability to manipulate the tumor microenvironment. Unlike the marketed proteasome inhibitor Bortezomib, a chemotherapeutic drug used to treat relapsed multiple myeloma, which inhibits the 20S catalytic core of the 26S proteasome, RA190 irreversibly binds to the ubiquitin receptor RPN13/ADRM1 on the 19S regulatory cap of the 26S proteasome. RA190 inhibits proteasome function and triggers accumulation of polyubiquinated proteins, and has been shown to induce endoplasmic reticulum stress-related apoptosis in HepG2 and Hepa3B hepatoma cell lines. We also found RA190 We observed a significant apoptosis of cancer cells after treatment of RA190. These changes in cancer cell lines were caused by reduction of STAT3 activity. We also proved the treatment effect of RA190 in orthotropic HCC animal models. The initial study of this novel proteasome inhibitor demonstrates its potential as an effective HCC therapy.

#4655

Iron chelation inhibits mTORC1 signaling in tumor cells.

Chaowei Shang, Hongyu Zhou, Shile Huang. _LSUHSC-Shreveport, shreveport, LA_.

Iron chelators are commonly used for treatment of iron overload diseases. Recent studies have shown that iron chelators possess potent anticancer activities. Iron chelators (ciclopirox and 3-AP) are in clinical trials for cancer treatment. However, the molecular mechanism underlying the anticancer effect of iron chelation is not well understood. Mammalian/mechanistic target of rapamycin (mTOR) is a master kinase, regulating cell growth/proliferation and survival through sensing environmental cues. mTOR functions as at least two complexes (mTORC1/2). Here we show that two iron chelators, ciclopirox olamine (CPX) and Dp44mT, inhibit tumor cell proliferation by suppressing mTORC1 signaling in human rhabdomyosarcoma (Rh30) and lung cancer (A549 and A427) cells. This is evidenced by the findings that treatment with CPX or Dp44mT inhibited the phosphorylation of 4E-BP1 and S6K1, two best known effector molecules of mTORC1, which could be blocked by addition of ferrous sulfate. Addition of ferrous sulfate prevented the growth inhibitory effect of CPX. Ectopic expression of constitutively active mTOR also conferred resistance to CPX-induced growth inhibition. Mechanistically, iron chelation inhibition of mTORC1 was partially linked to the activation of AMPK, a negative regulator of mTORC1. Overexpression of a dominant-negative AMPK (DN-AMPK) in Rh30 cells attenuated the inhibitory effect of CPX or Dp44mT on the phosphorylation of S6K1 and 4E-BP1. In addition, iron chelation also transiently stabilized HIF-1α and further upregulated the expression of its downstream effector Bnip3, another negative regulator of mTORC1. Knockdown of Bnip3 partially attenuated the inhibitory effect of iron chelation on mTORC1. Furthermore, we found that iron chelation increased the translocation of mTOR from the cytoplasm to the nucleus. The co-localization of mTOR with the third negative regulator of mTOR, PML (promyelocytic leukemia), increased in the nucleus upon iron chelation. Moreover, iron chelation did not obviously alter the phosphorylation of IGF-1 receptor, PI3K or PTEN. The effect of iron chelation on mTORC2-mediated phosphorylation of Akt was cell line dependent. Taken together, the results suggest that the anticancer effect of iron chelation is in part related to inhibition of mTORC1 signaling, and iron chelation inhibits mTORC1 signaling via multiple mechanisms. (Supported by the FWCC, LSU Health Sciences Center)

#4656

ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells.

Nikhil M. Vad, Dong Wang, Barry J. Maurer. _Texas Tech University HSC, Lubbock, TX_.

The cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased dihydroceramide and reactive oxygen species (ROS) levels and enhanced by co-treatment with safingol (S), an L-threo-dihydroceramide precursor. The ER stress response displays dichotomic effects wherein mild short-term stressors activate responses to neutralize or adapt to stress, but severe or long-lasting stressors activate cell death cascades. Here, we report that ER stress and autophagy are pro-survival responses in glioblastoma multiforme (GBM) cells to unfolded proteins-related cytotoxicity induced by 4-HPR+S in vitro. In T98G and A172 cells, 4-HPR+S induced mixed cell death associated with time-dependent increase of TUNEL-positivity (+12-36 h) and cleavage of caspase-3 (+12-36 h), but pan- caspase inhibitor, boc-D-fmk, did not rescue cell death. Cytotoxicity (+6-24 h) was associated with minimal mitochondrial depolarization or dual-staining of Annexin V/PI. Assays for known non-apoptotic mechanisms were negative. There was time-dependent (+6-24 h) increase of GRP78 protein levels, a key regulator of ER stress response; ER stress sensors, PERK, IRE1α, and ATF6, were activated (+6-24 h); levels of pro-apoptotic transcription factor, CHOP, increased (+6-24 h). Cytotoxicity was increased by PERK pathway inhibitors, salubrinal (+24-72 h, p≤0.05), GSK 2606414 (+24-72 h, p≤0.05), and GSK 2656157 (+48-72 h, p≤0.05), indicating that PERK activation was pro-survival. Treatment increased levels of CHIP, a key co-chaperone targeting unfolded proteins for degradation (+6-24 h). There was strong activation of the ubiquitin-proteasome system (UPS) and autophagy preceding or concurrent with the accumulation of poly-ubiquitinated proteins (+12-24 h). Autophagy was evidenced by increased conversion of LC3B-I to LC3B-II (+6-24 h). LC3B-II levels were increased by lysosomal degradation inhibitor, bafilomycin A1, indicating increased autophagic flux. Cytosolic levels of HDAC6, a coordinator of protein turnover via UPS and autophagy through trafficking of protein aggregates into pre-lysosomal structures (aggresomes) prior to autophagosome engulfment, increased (+6-24 h). siRNA knockdown of CHIP enhanced cytotoxicity (+24-72 h, p≤0.05). siRNA ablation of autophagy (ATG7, BECN1)(+24-36 h, p≤0.05), or pharmacological disruption by mefloquine, increased cytotoxicity (+24-72 h, p≤0.05), indicating that autophagy was pro-survival. Cytotoxicity was increased by siRNA knockdown of HDAC6 (+24-72 h, p≤0.05) and selective HDAC6 inhibitor, ACY1215, (+24-72 h, p≤0.05). Together, these results support that ER stress/unfolded protein response (UPR) is pro-survival in GBM cells exposed to 4-HPR+S. This suggests that co-agents that reduce ER stress response, UPR, or autophagy processes may further sensitize GBM cells to 4-HPR+S.

#4657

Selective inhibitors epigenetically modify and eradicate tumor-initiating stem-like cells through downregulating microRNA 22-mediated TET induction and apoptosis.

Chia-Lin Chen,1 Suresh Swaminathan,2 Ameya Kirtane,2 Jayanth Panyam,2 Keigo Machida1. 1 _University of Southern California, Los Angeles, CA;_ 2 _University of Minnesota, Minneapolis, MN_.

Tumor-initiating stem-like cells (TICs) are a minor population in bulk tumors that play a critical role in tumor recurrence and therapy-resistance. We previously showed that a key stemness marker Nanog is upregulated that regulates self-renewal and pluripotency of embryonic stem cells and TICs. We demonstrated that alcohol-mediated activation of Toll-like receptor 4 (TLR4) by endotoxin resulted in increased expression of pluripotency stem cell transcription factor NANOG that metabolically reprograms tumor-initiating stem-like cells (TICs: cancer stem cells) via inhibition of oxidative phosphorylation and activation of fatty acid oxidation in our recently accepted Cell Metabolism paper. Discovery of a drug that specifically targets TICs would be a vital goal for cancer therapy. To identify selective TIC inhibitors, we conducted a high throughput screening of an FDA-approved drug library using combination of Nanog promoter-GFP-based screening with viability-based screening and their combination screening of each hit compound. Our high-throughput screening identified the best combination of repurposed FDA-approved drugs: all-trans retinoic acid (ATRA) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) that repressed NANOG and induces apoptosis of TICs. Treatment of ATRA inhibits self-renewal ability in vitro. To specifically target the CD133 (+) population, we encapsulated ATRA into nanoparticles conjugated with CD133 antibody using biodegradable poly(D,L-lactide-co-glycolide) (PLGA) polymer. The combination treatment significantly inhibited tumor growth compared to single drug treatments while the ATRA or SAHA monotherapy groups did not reduce or even promoted tumor growth. The combination of ATRA with SAHA specifically induces apoptosis of TICs. Targeting of TICs by NANOG antagonism increases drug susceptibility in tumor-bearing mice and achieves ~90% tumor growth suppression. RNA-seq analysis identified that ATRA+SAHA treatment reduced expression of MicroRNA-22 (miR-22), leading to induction of PTEN and ten-eleven translocation enzymes (TET2), which demethylates DNA. PTEN induction promoted P-FOXO3A and induced BIM, leading to induction of apoptosis. The combined treatment Induced TET1/2 to demethylate p53-binding sites of NANOG promoter, leading to downregulation of NANOG. Taken together, combination treatment of ATRA with SAHA may serve as a novel avenue for HCC treatment. Novel combination of repurposed drugs is cost-effective therapeutic strategy to target microRNA for eradication of TICs, leading to inhibition of metastasis and recurrence.

#4658

Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines.

Ikuko Nagasawa, Satomi Tsukahara, Kazuhiro Kunimasa, Akihiro Tomida. _Cancer Chemotherapy Ctr., Japanese Foundation For Cancer Research, Tokyo, Japan_.

The eIF2α-ΑTF4 signaling pathway termed "Integrated stress response (ISR)" plays an important role in protecting cells against various cellular stresses such as deprivation of glucose and amino acids, virus infection, and deficiency of heme. Under such stress conditions, eIF2α kinases, including PERK, GCN2, PKR and HRI, phosphorylate eIF2α, resulting in reduction of general protein synthesis while facilitating selective translation of ATF4, a central transcription factor that upregulates expression of stress-induced genes for cellular adaptation. Recently, it was reported that BRAF inhibitor vemurafenib triggers activation of the eIF2α-ATF4 signaling in BRAF-mutant melanoma cell lines; however, activation mechanisms of vemurafenib-induced ISR signaling not fully understood.

In this study, we first compared vemurafenib-induced ISR activation in BRAF-mutant and -wild type melanoma cell lines. Consistent with previous studies, vemurafenib activated ISR signaling preferentially in BRAF-mutant cell lines, as determined by phosphorylation of eIF2α and ATF4 expression. We also found that the ISR activation by vemurafenib was impaired by knockdown of BRAF in BRAF-mutant cell lines. These results indicated that existence of mutated BRAF is important for vemurafenib to trigger activation of ISR signaling pathway. We next examined which eIF2α kinase is involved in the ISR signaling activation by vemurafenib. We found that knockdown of GCN2, an eIF2α kinase sensing depletion of amino acids, impaired phosphorylation of eIF2α and ATF4 induction by vemurafenib. In addition, we confirmed that vemurafenib induced phosphorylation of GCN2 on Thr899 that is known to occur in response to amino acids starvation. In agreement with these results, gene expression profiling with ontology analysis revealed that vemurafenib upregulated genes that are involved in amino acids biosynthesis via ISR signaling. Collectively, our results suggested that vemurafenib activated ISR signaling pathway possibly through mimicking amino acid deprivation in BRAF-mutant melanoma cell lines.

#4659

Folic acid-metabolizing enzymes regulate 5-fluorouracil anticancer activity on colorectal cancer cell lines.

Hiroshi Tsukihara, Kenta Tsunekuni, Teiji Takechi. _TAIHO PHARMACEUTICAL CO., LTD., Tokushima, Japan_.

Background: In colorectal cancer chemotherapy, the current standard of care includes 5-fluorouracil (5-FU) and leucovorin (LV) combination therapy. To understand the role of folic acid-metabolizing enzymes in the LV-mediated enhancement of 5-FU's antitumor activity, we suppressed the expression of genes related to folic acid metabolism and evaluated the resultant effect on the proliferation inhibitory effect of 5-fluoro-2′-deoxyuridine (FdUrd). Method: We downregulated the expression of thymidylate synthase (TS), folate receptor 1 (FOLR1), methylenetetrahydrofolate reductase (MTHFR), methylenetetrahydrofolate dehydrogenase (MTHFD), dihydrofolate reductase (DHFR), and phosphoribosylglycinamide formyltransferase (GART) by lipofection of small interfering RNA (siRNA) in DLD-1 and HCT116 cells, and calculated the relative change in IC50 values on 48 hr exposure to FdUrd in comparison with the corresponding IC50 values in control siRNA-treated cells. In cells with TS downregulation, LV-mediated enhancement of sensitivity to FdUrd was also evaluated. Sensitivity to FdUrd was determined using the crystal violet staining method. Results: Treatment with siRNA downregulated the expression of folic acid-metabolizing enzymes. LV enhanced the sensitivity to FdUrd in control siRNA-treated cells in a dose-dependent manner. However, in cells with TS downregulation, LV did not enhance the sensitivity to FdUrd. Downregulation of folic acid-metabolizing enzymes, except TS, decreased the efficacy of FdUrd, although there was little decrease in FdUrd efficacy owing to MTHFR downregulation. Conclusion: The level of TS expression determined the LV requirement, and downregulation of folic acid-metabolizing enzymes, which impaired the folic acid cycle, decreased the efficacy of 5-FU.

Comparison of the IC50 values in target siRNA-treated cells and control siRNA-treated cells.

---

|

DLD-1 cells | HCT116 cells

|

expression (%)* | IC50 (µM) | fold change | expression (%)* | IC50 (µM) | fold change

Non-silencing

Control siRNA | 100.00 | 0.67 | 1.00 | 100.00 | 0.25 | 1.00

TS | 10.58 | 0.01 | 0.01 | 14.84 | 0.03 | 0.12

FOLR1 | 9.14 | 8.65 | 12.91 | 25.12 | 2.59 | 10.36

MTHFR | 25.44 | 1.43 | 2.13 | 48.75 | 0.88 | 3.52

MTHFD | 9.66 | 2.73 | 4.07 | 15.06 | 1.99 | 7.96

DHFR | 8.52 | 4.14 | 6.18 | 17.23 | 1.70 | 6.80

GART | 18.90 | 5.38 | 8.03 | 7.10 | 2.46 | 9.84

Values are means (n = 3). *: Expression (%) is the expression at the beginning of FdUrd treatment (at 48 hr after siRNA treatment).

### Combination Therapies and Approaches to Sensitizing Cancer Cells to Drugs

#4660

Inhibition of p38 enhances ERK inhibitor efficacy in pancreatic ductal adenocarcinoma.

Meagan B. Ryan,1 Kirsten L. Bryant,1 Tikvah K. Hayes,1 Swapnil Kher,1 Kris C. Wood,2 Ahmed A. Samatar,3 Adrienne D. Cox,1 Channing J. Der1. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Duke University, NC;_ 3 _Discovery Oncology Merck Research Laboratories, MA_.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States, with a poor prognosis and limited treatment options. Oncogenic mutation of KRAS in greater than 90% of PDAC leads to aberrant activation of multiple effector pathways including the extra cellular related kinase (ERK)/mitogen activated protein kinase (MAPK) cascade. Hyperactivation of the ERK MAPK cascade has been correlated with poorer prognosis in PDAC patients. We recently showed that direct pharmacological inhibition of ERK1/2 kinases with the ERK1/2-selective inhibitor SCH772984 inhibits the growth of PDAC cell lines both in vitro and in vivo. However, much like the response to ERK/MAPK pathway inhibitors acting at upstream nodes RAF or MEK, resistance to direct inhibition at the level of ERK will also inevitably arise. We performed a novel gain-of-function "Cancer Toolkit" (CTK) genetic screen to identify mechanisms of resistance to the ERK inhibitor SCH772984 in a panel of KRAS-mutant PDAC cell lines. Our CTK screen revealed that expression of either MAPK14 (p38α) or its upstream activator MKK6 could cause resistance to this and other ERK inhibitors. Because we identified activation of the p38 pathway as a mechanism of ERK inhibitor resistance, we tested whether inhibition of p38 would enhance sensitivity to SCH772984. We observed a >2 fold shift to a lower GI50 of SCH772984 upon cotreatment of a panel of established and patient-derived xenograft (PDX) PDAC cell lines with SCH772984 and the clinical candidate p38 inhibitor LY2228820. Consistent with this, we found that SCH772984 induced p38 signaling, marked by phosphorylation of the downstream substrate HSP27, and that combination treatment of SCH772984 and LY2228820 both reversed p38 pathway activation and enhanced PARP cleavage. Results of in vivo testing of the combination treatment as well as the mechanistic basis for p38-driven resistance will be reported. We propose that p38 is a mechanism of resistance to ERK inhibition in PDAC and that p38 inhibitors such as LY2228820 can overcome that resistance to enhance ERK inhibitor efficacy.

#4661

Loss of LKB1 in NSCLC confers sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway.

Tadaaki Yamada,1 Jacob M. Kaufman,2 Joseph M. Amann,3 Seiji Yano,1 David P. Carbone3. 1 _Kanazawa Univ. Cancer Research Inst., Kanazawa, Japan;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _The Ohio State Univeristy Wexner Medical Center, Columbus, OH_.

The use of MEK inhibitors for non-small cell lung cancer (NSCLC) has shown little efficacy in clinical trials, even in the case of tumors with mutant KRAS where one might predict good outcomes. From these data, it is clear that the success of MEK inhibitors is going to rely on finding a biomarker that predicts sensitivity to this type of therapy. Our area of interest is finding a way to treat LKB1 mutant tumors and, surprisingly, an in silico screen of drug sensitivity data for NSCLC cell lines determined that four MEK inhibitors were among the top drugs that were significantly associated with LKB1 loss. We confirmed the effects MEK inhibition by evaluating 23 lung cancer cell lines with known LKB1 status. The results of the study showed that MEK inhibition with trametinib led to a significant reduction in cell viability in LKB1 mutant cell lines when compared to cell lines with wild type LKB1. In addition, we investigated MEK sensitivity by restoring wild-type (Wt) LKB1 in lung cancer cell lines with LKB1 loss, or by silencing LKB1 in lung cancer cell LKB1-Wt lines. Transduction of LKB1 resulted in significant MEK resistance in three of the five LKB1 add-back lines, while silencing LKB1 induced MEK sensitivity in all five LKB1-Wt lines tested. The mechanism behind these observed results appears to be through phosphorylation of AKT and its downstream target FOXO3, which are important determinants of the apoptotic response to MEK inhibition. With LKB1 transduction into mutant cell lines we see an increase in the activating phosphorylation of AKT, a protein involved in survival mechanisms, and an increase in the deactivating phosphorylation of FOXO3, a transcription factor implicated in increased levels of apoptosis. Our data suggest that the identification of LKB1 activity may be promising biomarker for the sensitivity to MEK inhibition by regulating activation of AKT-FOXO3 pathway in NSCLC.

#4662

Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis.

Monique B. Nilsson,1 Huiying Sun,1 Lixia Diao,1 Pan Tong,1 Youhong Fan,1 Hai Tran,1 Diane Liu,1 Guillermo Armaiz Pena,1 Jing Wang,1 Phil Rowe,2 Alan Webster,2 Jack Lee,1 Daniel Gomez,1 Waun Ki Hong,1 Ignacio Wistuba,1 Anil Sood,1 John Heymach1. 1 _UT MD Anderson Cancer Ctr., Houston, TX;_ 2 _AstraZeneca UK, London, United Kingdom_.

Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers.

#4663

Combination of c-Met inhibitor tepotinib (MSC2156119J) and a third-generation EGFR inhibitor can overcome double resistance mediated by EGFR T790M mutation and c-Met amplification in non-small cell lung cancer models.

Manja Friese-Hamim, Giuseppe Locatelli, Friedhelm Bladt. _Merck Biopharma Research & Development, Darmstadt, Germany_.

In clinical practice, acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) causes EGFR TKI treatment failure. Several resistance mechanisms have been identified; EGFR T790M mutation and aberrant activation of c-Met through c-Met gene amplification and/or HGF upregulation are the two most common mechanisms. We have previously shown that combining the highly selective c-Met inhibitor tepotinib with 1st or 2nd-generation EGFR TKIs overcomes gefitinib resistance caused only by c-MET amplification. Third-generation EGFR TKIs such as rociletinib have been developed specifically to overcome T790M-mediated resistance to EGFR TKIs. We hypothesized that combining a 3rd-generation EGFR TKI with tepotinib could overcome resistance to a 1st or 2nd-generation EGFR TKI (e.g. gefitinib) regardless of the mechanism of resistance.

We assessed the efficacy of tepotinib and a Merck-synthesized version of rociletinib (MSR) as monotherapy or in combination in a NSCLC cell line xenograft model (HCC827-GR-T790M) with an EGFR del 19 mutation, c-Met amplification, and exogenous T790M expression, and in a patient-derived xenograft model (DFCI081) with an EGFR del 19 mutation and c-Met amplification, but not a T790M mutation.

Single-agent tepotinib, erlotinib, and afatinib did not show antitumor activity in HCC827-GR-T790M xenografts and tumors progressed on treatment (median tumor volume [TV] changes >73%). The combinations of tepotinib + erlotinib and tepotinib + afatinib delayed tumor regrowth more than single-agent therapy, but were no more effective than single-agent MSR. In contrast, the combination of tepotinib + MSR had strong antitumor activity in this model with double resistance due to c-Met amplification and exogenous T790M expression, resulting in complete tumor regression (median TV change -97%).

In the patient-derived xenograft model DFCI081, tumors progressed under single-agent therapy with the EGFR TKIs erlotinib, afatinib, and MSR, as expected given that this cell line expresses c-Met. In contrast, tepotinib induced complete tumor regression as monotherapy and when combined with each of the EGFR TKIs (median TV change -100%). Complete regression was maintained until the end of the 60-day observation period after treatment termination.

These findings are compatible with the hypothesis that combining tepotinib with a 3rd-generation EGFR TKI to overcome double resistance to 1st and 2nd-generation TKIs mediated by T790M and c-Met in NSCLC is more effective than using either agent alone. Furthermore, whereas EGFR TKIs were not effective in tumor models showing c-Met amplification and EGFR del 19 mutation but not T790M mutation, ie resistance is c-Met mediated, tepotinib monotherapy induced complete tumor regression.

#4664

Co-targeting HGF-cMET signaling with MEK inhibitors in metastatic uveal melanoma.

Hanyin Cheng, Ken Kageyama, Timothy Purwin, Connie Liao, Mizue Terai, Takami Sato, Andrew Aplin. _Thomas Jefferson University, Philadelphia, PA_.

Metastatic uveal melanoma (UM) patients usually die within one year, emphasizing an urgent need to develop new treatment strategies. Mitogen-activated protein kinase kinase (MEK) inhibitors improve survival in cutaneous melanoma patients but have limited efficacy in UM patients. Our previous work show that HGF-cMET signaling provides resistance to MEK inhibitors in metastatic UM cells. In this study, we further investigated the mechanisms of HGF-driven resistance to MEK inhibitors in metastatic UM cells. Cellular signaling pathway analysis was carried out to determine the role of downstream Bcl2 family members in overcoming trametinib-mediated apoptosis by HGF in metastatic UM cells. A new class of clinical grade cMET antibody and inhibitor were tested for their capacity to revert the resistance to trametinib mediated by HGF and tumor microenvironment. Selective inhibitors were utilized to determine the PI3K isoform dependency of metastatic UM cells in HGF-mediated protection against trametinib in metastatic UM cells. We demonstrate that the expression of two BH3-only family proteins, Bim-EL and Bmf, is upregulated in trametinib treated cells, but returns to basal levels upon HGF treatment in these cells. Targeting HGF-cMET signaling with LY2875358, a neutralizing and internalizing anti-cMET bivalent antibody, and LY2801653, a dual cMET/RON inhibitor overcomes resistance to trametinib provided by exogenous HGF and by conditioned medium from primary hepatic stellate cells. PI3Kβ-sparing inhibitor GDC0032 effectively blocks HGF-induced AKT phosphorylation and HGF-mediated resistance in trametinib treated cells in growth assays. In conclusion, our data suggest that HGF promotes resistance to MEK inhibitors through modulating Bim-EL and Bmf expression in UM cells. Our data also support the notion that selectively blocking PI3K isoform activities or cMET signaling may delay the onset of resistance to MEK inhibitors provided by tumor microenvironment in metastatic uveal melanoma.

#4665

**The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance** in vitro **and** in vivo **in non-small cell lung cancer.**

Ratnakar Singh,1 Yuehong Wang,2 Liguang Wang,3 Monique Nilsson,1 Ruchitha Goonatilake,1 Pan Tong,4 Lerong Li,4 Uma Giri,1 Jing Wang,4 John V. Heymach,1 Faye M. Johnson1. 1 _Thoracic H &N Med Onc-Rsch, University of Texas, MD Anderson Cancer Center, Houston, TX; _2 _College of Medicine, Zhejiang University, Hangzhou, China;_ 3 _Shandong Provincial Hospital, Shandong University, Jinan, China;_ 4 _Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX_.

Introduction: Activation of epidermal growth factor receptor (EGFR) leads to the development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (EGFR-TKI) are effective therapy for NSCLC with activating EGFR mutations. However, acquired resistance to EGFR-TKIs is unavoidable and occurs through various molecular mechanisms including the development of T790M secondary EGFR mutations, MET amplification, epithelial-mesenchymal transition (EMT) and other mechanisms. One of potential strategy to overcome EGFR-TKIs is through inhibition of polo-like kinase 1 (PLK1). PLK1 is one of the key regulators of mitotic progression and a DNA damage recovery checkpoint. PLK1 inhibitors are at various stages of clinical development. Our previous studies showed that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibitor than epithelial ones. This study examines the efficacy of PLK1 inhibition in NSCLC cell lines with acquired resistance to the EGFR-TKI erlotinib.

Methods: Erlotinib resistant cell lines were developed in EGFR mutant, erlotinib sensitive cell lines PC9, HCC4006 and HCC827 by chronic exposure to stepwise increased concentrations of erlotinib. The effects of the PLK1 inhibitor volasertib alone or combined with erlotinib on proliferation, apoptosis, cell cycle, EGFR-dependent signaling, DNA damage and DNA damage response signaling were evaluated using CellTiter-Glo assay, TUNEL assay, BrDU incorporation assay, comet assay, western blot and immunofluorescence. Sensitivity of volasertib alone or in combination with erlotinib was also detected in the human tumor xenograft model. The combination index (CI) was calculated by Calcusyn software.

Results: Acquired erlotinib resistant NSCLC cell lines with endogenous EGFR mutations (PC9, HCC4006 and HCC827) showed diverse resistance mechanisms: T790M EGFR mutation, MET amplification and EMT. Four of the 6 ER clones that underwent EMT became more sensitive to volasertib. Volasertib and erlotinib demonstrated synergistic effects only in cells bearing T790M EGFR mutations where we observed more pronounced G2M arrest and increased apoptosis. Volasertib alone or in combination showed enhanced DNA damage, activation of CHK1/ATR and CHK2/ATM pathways and an increase in γH2AX foci but only a modest effect on canonical signaling downstream of EGFR. The combination treatment exhibited a pronounced reduction in the size of tumor compared to single agent treatment in subcutaneous xenograft model generated with T790M mutant EGFR TKI-resistant PC9 cell line.

Conclusions: These data demonstrate that PLK1 inhibition alone induces apoptosis and DNA damage in EGFR-TKI resistant cell lines with EMT. The combination of volasertib and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo.

#4666

Phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitors demonstrate broad efficacy and synergy in head and neck cancer cell lines and patient-derived xenografts.

Anthony C. Nichols,1 Laurie Ailkes,2 Ren Sun,2 Morgan Black,1 Alessandro Datti,3 Frederick Vizeacoumar,4 Nicole Pinto,1 Kara Ruicci,1 John Yoo,1 Kevin Fung,1 Danielle MacNeil,1 Joe Mymryk,1 Paul C. Boutros,2 John W. Barrett1. 1 _Western University, London, Ontario, Canada;_ 2 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 3 _Lunenfeld-Tanenbaum, Toronto, Ontario, Canada;_ 4 _University of Saskatchewan, Regina, Saskatchewan, Canada_.

Background: There is an urgent need for improved therapeutics in head and neck squamous cell cancer (HNSCC) to improve survival and decrease treatment morbidity. The Phosphatidylinositol 3-kinase (PI3K) pathway is frequently altered in HNSCC, particularly in human papillomavirus (HPV) positive disease. PI3K pathway inhibitors are under active investigation in preclinical models and clinical trials, however it is not clear which patient population will benefit most from these agents.

Methods: Thirty unique HNSCC cell lines including five HPV positive lines were characterized with whole exome sequencing and copy number arrays. All lines were treated over a 10-point dose range with a PIK3CA specific inhibitor (BYL719), a pan PI3K inhibitor (GDC-0941), a combined pan-PI3k and mTOR inhibitor (BEZ-235), mTOR inhibitor (everolimus) and a combination of BYL719 and everolimus. Five unique patient derived xenografts (PDX) were treated with BYL719 and everolimus alone and in combination.

Results: The selective PIK3CA inhibitor BYL719 was preferentially active in cell lines with PIK3CA activating mutations and amplifications (>100% relative PIK3CA amplification), however HRAS mutant lines were resistant. These genomic markers of sensitivity and resistance did not apply to the pan-PI3K inhibitor GDC-0941 or combined PI3K-mTOR inhibitor BEZ-235, however these agents were more active in HPV-positive cell lines (P<0.05). BYL719 and everolimus were individually effective at decreasing tumor growth in four PDX models including a model with an activating PIK3CA mutation, two PIK3CA amplified models and a PIK3CA wild-type model. Combined PIK3CA and mTOR inhibition appeared to be synergistic and evolved resistance never developed to this treatment, however the combination caused significant weight loss in the PDX models.

Interpretation: PI3K inhibitors were broadly active in HNSCC cell lines and PDXs and their application should not be solely restricted to tumors with activating PIK3CA mutations or amplifications. Agents with broader isoform activity and combined PI3K and mTOR treatment were more potent, however combined inhibitor treatment appeared toxic in the PDX models. This trade off of toxicity and efficacy needs to balanced in the clinical setting.

#4667

pCR induced by triplet therapy with low-dose afatinib, cetuximab and bevcizumab in lung cancer cells harboring EGFR T790M.

Kadoaki Ohashi, Kenichiro Kudo, Eiki Ichihara, Hiroe Kayatani, Hisao Higo, Yuka Kato, Yasuko Kurata, Daisuke Minami, Takashi Ninomiya, Toshio Kubo, Katsuyuki Hotta, Mitsune Tanimoto, Katsuyuki Kiura. _Okayama University, Okayama, Japan_.

Background. EGFR T790M plays a major role on the resistance for 1st or 2nd-generation EGFR-TKIs (e.g. erlotinib, afatinib) in lung tumors with activating EGFR mutations. 3rd generation EGFR-TKIs (e.g. AZD9291) show an excellent response in lung tumors with EGFR T790M, while the acquired resistance is inevitable (NEJM 2015). The combination of afatinib and cetuximab also induced significant response in the lung cancers in clinical trial (Cancer Discov. 2014). However, there is clearly still room for improvement in the combination therapy. Bevacizumab, a key drug in lung cancer treatment, is thought to have a wide variety of effects, including anti-vascularization and improved drug delivery. Thus, we hypothesized that the dosage of afatinib and cetuximab could be reduced by combining with bevacizumab, and that the triple therapy might produce better tumor inhibition with tolerable toxicity.

Methods. Two lung cancer cell lines, H1975, with EGFR L858R and T790M, and RPC-9, with EGFR exon 19 deletion and T790M, were injected subcutaneously into nude mice as xenograft models. Mice were divided into four groups 10 days after tumor cell inoculation (vehicle, afatinib, afatinib + cetuximab, afatinib + cetuximab + bevcacizumab). Compared with previous reports (afatinib 25 mg/kg, 5 times/week, cetuximab 1 mg/mouse, every 3 days; J. Clin. Invest. 2009), the low dose of afatinib (10 mg/kg, 5 times/week; i.e., 60% less drug) or cetuximab (0.1 mg/mouse, once/week; i.e., 90% less drug) were administered to the mice for 1 month. Bevacizumab was injected at 2 mg/kg twice/week. Following the therapies, the mice were observed for 1 month without treatment.

Results. Surprisingly, the triple therapy induced a pathological complete response (pCR) in H1975 and RPC-9 cell xenograft tumors; in contrast, the tumors treated with single or double therapy were inhibited only partially. AZD9291 monotherapy did not reproduce pCR. Furthermore pCR was maintained during the observation period in the triple therapy group. There was no body weight loss in any group. Frozen tumors were obtained from RPC-9 xenograft models after 1 week treatment with each regimen. Expression levels of pEGFR, pAKT, and pERK were decreased in the triple therapy group. CD31-positive blood vessels and Ki-67-positive cells in tumors with triple therapy were reduced significantly versus the tumors in other groups. Cleaved caspase-3 expression in the tumors with triple therapy was positive in a higher number of tumor cells than in the other groups. There were no difference of afatinib concentration among the tumors.

Conclusions. We demonstrated that a low-dose of afatinib and cetuximab combined with bevacizumab induced pCR in EGFR T790M mutated cell xenograft tumors with no obvious adverse events. We suggest that the suppression of neovascularization and induction of apoptosis may play important roles in the triple therapy.

This project is supported by KAKEN(No26893155)

#4668

Silencing PTGER3 enhances chemotherapeutic responses.

Emine Bayraktar,1 Cristian Rodriguez-Aguayo,1 Junhua Mai,2 Cristina Ivan,1 Cristina Ivan,1 Arturo Chavez-Reyes,3 Anil K. Sood,1 Mauro Ferrari,2 Haifa Shen,2 Gabriel Lopez-Berestein1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Houston Methodist Research Institute, Houston, TX;_ 3 _National Polytechnic Institute (CINVESTAV del IPN), Monterrey, Mexico_.

Inflammation is involved in the pathogenesis of cancer and inflammatory mediators such as prostaglandins (PGs) are abundantly present in the tumor microenvironment. Prostaglandin E(2) (PGE(2))-prostaglandin E2 receptor (EP)3 (PTGER3) signaling appears critical for tumor-associated angiogenesis, tumor growth and chemoresistance. However, the precise mechanism of this phenomenon in ovarian cancer remains unknown. In this study we hypothesized that down modulation of PTGER3 regulates drug resistance through the regulation of Ras-MAPK/Erk-Est-ELK1 axis in OC cells, resulting in decreased migration, proliferation and increased apoptosis. We analyzed a PTGER3 data set of OC patients from the Cancer Genome Atlas. Furthermore, the analysis of a panel of ovarian cancer cell lines showed that PTGER3 at mRNA and protein levels were higher in cisplatin-resistant cells when compared with their cisplatin sensitive counterparts. In vitro cell viability, growth, cell-cycle progression, migration, invasion and apoptosis, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following siRNA-mediated PTGER3 silencing in cisplatin-resistant ovarian cancer cells. The experimental validation identifies PTGER3 associated with OC survival. Significant inhibition of cell growth and viability, cell-cycle arrest, and activation of apoptosis were observed upon siRNA-mediated PTGER3 suppression. Furthermore, combination treatment with MSV/DOPC-siRNA-PTGER3 and cisplatin inhibited growth of A2780-CP20 tumors, which were otherwise resistant to cisplatin treatment. These findings identify PTGER3 as a potential therapeutic target in ovarian chemo-resistant cancers expressing high levels of this oncogenic protein.

#4669

Targeting hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells.

Fernanda Faiao-Flores,1 Debora Kristina Magalhaes Alves-Fernandes,1 Paula Comune Pennacchi,1 Silvana Sandri,1 Anna Luiza Silva Almeida Vicente,2 Cristovam Scapulatempo-Neto,2 Vinicius de Lima Vazquez,2 Rui Manuel Reis,2 Keiran S. Smalley,3 Silvya Stuchi Maria-Engler1. 1 _School of Pharmaceutical Sciences, University of São Paulo, Sao Paulo, Brazil;_ 2 _Barretos Cancer Hospital, Barretos, Brazil;_ 3 _The Moffitt Cancer Center & Research Institute, Tampa, FL_.

Melanoma represents the deadliest of all skin cancers being responsible for more than 75% of skin cancer-related deaths. Although the results with BRAF inhibitors have been very promising, practically all of the patients treated thus far have developed resistance to these drugs. The Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies, including melanoma. However, there are no reports on the activation of this pathway in the setting of vemurafenib-resistance. Herein, we first identified that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, showed increased levels of Hh pathway component genes glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared to naïve cells. We also observed these findings in melanoma samples from patients. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with non-canonical Hh signaling, involving TGFβ/SMAD signaling. Knockdown of GLI1 and GLI2 restored the vemurafenib-resistant cells to sensitivity, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a 3D skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphtalmia transcription factor (MITF) upregulation. Our findings also demonstrated that GLI1/GLI2 modulation could be a useful strategy to prevent drug resistance. Alternating pre-treatment with vemurafenib and Gant61 significantly reduced IC50 of subsequent vemurafenib treatment in naïve melanoma cells, demonstrating a promising approach for the control of vemurafenib-resistance in patients with unresectable melanoma. The modulation of vemurafenib chemosensitivity was triggered by GLI1/GLI2 repression during the acquisition of resistance, which did not occur in the treatment protocols with only vemurafenib continuously or in alternate days. The alternating treatment regimen with vemurafenib and Gant61 was able to suppress GLI expression, delaying or decreasing vemurafenib resistance. Taken together, our data demonstrated an unprecedented mechanism of vemurafenib resistance by GLI1/GLI2 upregulation, shedding light on the development of Hh pathway inhibitors as a promising strategy for melanoma treatment.

#4670

Combinations of kinase siRNAs potentiates paclitaxel sensitivity in ovarian cancer.

Hailing Yang, Xiaoyan Wang, Ahmed Ahmed, Lakesla Iles, Geoffrey Bartholomeusz, Zhen Lu, Robert C. Bast Jr.. _UT MD Anderson Cancer Center, Houston, TX_.

Drug resistance imposes a major obstacle in ovarian cancer treatment. All patients with newly diagnosed ovarian cancer are treated with a combination of paclitaxel and carboplatin. While 70% of ovarian cancers respond to carboplatin, less than 50% respond to paclitaxel. Inhibition of kinases that modulate primary paclitaxel resistance, could enhance response to therapy. A Kinase siRNA library was screened in ovarian cancer cell lines to determine which kinases regulate paclitaxel sensitivity. Among 45 hits from high throughput screening, 14 target proteins (AATK, ACRBP, BMP2K, CHUK, EDN2, IKBKB, ILK, RAPGEF3, RAPGEF4, RFP, SIK2, STK24, STK39 and TBK1) regulated sensitivity to paclitaxel and were differentially expressed or overexpressed in a fraction of ovarian cancers. siRNAs against each of these kinases were tested for the ability to enhance sensitivity to paclitaxel in each of 12 ovarian cancer cell lines that reflected the heterogeneity observed in ovarian cancers including mutations of TP53, BRCA1/2, KRAS, BRAF, PI3K and PTEN. Knockdown of 10 individual genes enhanced paclitaxel sensitivity by at least two folds in different cell lines. A subscreen using combinations of these 14 kinase siRNAs in the six most responsive cell lines were carried out to select pairs of kinase siRNAs which could further potentiate paclitaxel sensitivity. IKBKB and STK39 kinase siRNAs had the greatest activity. We discovered that knockdown of IKBKB and STK39 stabilized microtubules judged by a microtubule fractionation assay and levels of acetylated and detyrosinated tubulin. IKBKB and STK39 kinase siRNAs also induced apoptosis. Double knockdown of microtubule stabilizing kinases IKBKB and STK39 resulted in greater enhancement in microtubule stability and apoptosis, leading to additive sensitization of paclitaxel. Small molecule inhibitors are available for IKBKB. As inhibition drugs become available for STK39, these observations can be translated to the clinic.

#4671

Inhibition of phosphatidylinositol 3-kinase suppresses propagation of drug-tolerant cancer cell subpopulations enriched by 5-fluorouracil.

Satoshi S. Nishizuka,1 Kaoru Ishida,1 Yukimi Ohmori,1 Kohei Kume,1 Chie Ito,1 Akari Konta,1 Yuka Koizumi,1 Mamoru Nukatsuka,2 Takashi Kobunai,2 Teiji Takechi,2 Takeshi Iwaya1. 1 _Iwate Medical Univ. School of Medicine, Iwate, Japan;_ 2 _Taiho Pharmaceuticals Co. Ltd., Tokyo, Japan_.

The majority of advanced gastric cancer has been treated with 5-fluorouracil (5-FU)-based chemotherapy after complete resection, but a considerable number of patients experience relapse within 5 years after the resection. It has been considered that extremely small drug-tolerant cancer cell subpopulations are responsible for the relapse after chemotherapy. Previously, we established a 5-FU-tolerant gastric cancer cell line, MKN45T, which requires a higher drug concentration to suppress its growth relative to its parental, MKN45. Using the pair of cell lines, we developed an orthotopic xenograft (OX) model that mimics advanced human gastric cancer by transplantation of the cells into the gastric submucosal layer of nude mice. The OX of the 5-FU-tolerant cell line, MKN45T, showed an aggressive tumorigenicity, metastasis, and peritoneal dissemination, in contrast to the parental cell line, MKN45. Proteomic screening using a colony lysate array, a special format of reverse-phase protein array that allows us to quantify the protein levels in individual drug-tolerant subpopulations (i.e., colonies), revealed the level of phosphatidylinositol 3-kinase (PI3K) phosphorylation was increased according to the 5-FU dose-escalation. The prevalence of cells with active PI3K also increased with 5-FU treatment in OX. In contrast, phosphorylation of Akt and mTOR proteins were slightly decreased by the 5-FU treatment. Interestingly, PTEN protein expression was almost completely suppressed in OX after the 5-FU treatment. To see whether PI3K inhibitors have potency in the reduction of tumors with persistent growth after 5-FU treatment, PI3K inhibitors, including LY294002, Queracetin, Wortmannin, GNE493, and GDC0941, were tested by means of colony formation assay. Among them, simultaneous administration of 5-FU and GDC0941 significantly suppressed colony formation, which led us to examine the inhibitory effect of GDC0941 for tumor growth in the OX model. Sequential administration of 5-FU and GDC0941 prohibited the propagation of 83% (5/6) of OX tumors in the stomach as well as other organs. Western blot was then performed to clarify the growth suppression mechanisms by GDC0941 for PI3K-activated tumors triggered by 5-FU. The Akt/PI3K/mTOR and PTEN pathway analysis in response to single/combinational drug administration revealed that the majority (75%, 6/8) of gastric cancer cell lines tested exhibited an increased level of PI3K phosphorylation with 5-FU administration. The increased level of PI3K phosphorylation with 5-FU, which subsequently induces Akt and mTOR activation, was effectively suppressed by GDC0941 administration, particularly with a considerably decreased level of p70 S6 kinase phosphorylation. These results suggest that administration of 5-FU followed by GDC0941 may suppress relapse after complete resection with 5-FU-based chemotherapy for gastric cancer.

#4672

**Targeting MEK/MAPK1/2** in vivo **to eradicate antiestrogen resistance.**

Jason Conger, Matthew Manning, Kingsley Anosike, Ahmed Elsherbini, Brandon Ware, Emily Bass, Mathusamy Thangaraju, Darren Browning, Patricia V. Schoenlein. _Georgia Regents University, Augusta, GA_.

Tamoxifen (TAM), an antiestrogen and selective estrogen receptor modifier (SERM) has become a widely utilized therapeutic drug for treating ER+ breast cancer, however many cases develop TAM resistance. In previous in vitro studies, TAM combined with the selective Mitogen/Extracellular signal-regulated Kinase pathway (MEK) inhibitor U0126 (U) led to increased levels of the proapoptotic protein BimEL. This combined treatment was more effective at killing estrogen receptor expressing (ER+) breast cancer cells than either agent alone via a Bim-dependent mechanism [Periyasamy-Thandavan el al., Breast Cancer Res. 14(2), 2012]. In this study, we aimed to recapitulate the superior efficacy of this combined treatment strategy with the clinically relevant MEK inhibitor Selumetinib (SEL) using the TAM sensitive, ER+ MCF-7 breast cancer cell line. In addition, we utilized MCF7-TAM resistant cells (TR5), created in vitro through exposures to incremental increases in TAM concentration(100ng to 5.0μM). MCF-7 cells that were treated with TAM, SEL, U, or combination therapies in vitro demonstrated a similar efficacy between U and SEL with the lowest cell viability resulting from TAM plus U or SEL compared to TAM, U, or SEL as single agents. Western blot analysis of U- or SEL-treated MCF7 cells showed a similar robust upregulation of BimEL in the presence or absence of TAM. Comparison of the TR5 TAM resistant cells to parent MCF-7 cells showed elevated levels of active MAPK1/2 protein (p-MAPK) that mediated the phosphorylation and subsequent proteasomal degradation of BimEL. Importantly, inhibition of MEK/MAPK1/2 by SEL or U in TR5 cells in vitro significantly increased BimEL levels and TAM induced apoptosis as evidenced by increased levels of cleaved PARP and/or lamin A. In a pilot in vivo study, we further demonstrated that TR5 cells were highly tumorigenic with the xenografts being estrogen independent and insensitive to TAM inhibition. In contrast, the TR5 xenografts were sensitive to SEL when delivered in the presence or absence of TAM. Further, tissue analyzed from SEL-treated TR5 xenografts showed significantly higher levels of BimEL than tissue analyzed from TAM- or E2-treated xenografts. These initial studies provide strong data that MEK1/MAPK1/2 inhibition will be required in vivo to raise BimEL levels to negatively affect breast cancer growth. These studies also support the concept of combining SEL with antiestrogen treatment (as a combined adjuvant therapy) to reduce the emergence of antiestrogen resistant cells and to include SEL as part of a regimen to treat antiestrogen resistant breast cancer.

#4673

A novel way to sensitize pancreatic cancer cells for gemcitabine treatment.

Jingwu Xie, Dongsheng Gu, Xiaoli Zhang, Yanfei Jia, Gabi Chiorean. _Indiana University School of Medicine, Indianapolis, IN_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, with a dismal median survival of 6 months. While combining albumin-bound paclitaxel with gemcitabine has shown clear therapeutic advantage, numerous attempts using novel targeted therapies did not yield clinical benefits. Gemcitabine is still the first-line treatment for patients with PDAC. We hypothesize that strategies to increase gemcitabine sensitivity may be effective in PDAC treatment. In this study, we report that cancer stem cell regulator Sox2 is highly induced in acquired gemcitabine resistant pancreatic cell lines, and down-regulation of Sox2 sensitizes cancer cells to gemcitabine treatment both in the cultured cells and in mouse models. Down-regulation of Gli1/2 was associated with reduced Sox2 expression, decreased expression of cancer stem cell markers. We found Gli1/2 proteins in association with a Sox2 promoter sequence with a putative Gli binding site, suggesting a direct regulation of Sox2 by Gli1/2. We further showed that down-regulation of Gli1 or Gli2 was as effective as Sox2 knockdown in sensitizing cancer cells to gemcitabine treatment. The relevance of Gli1/2-Sox2 signaling axis to PDAC was suggested by association of high Sox2 expression with poor survival in stage II PDAC patients. Taken together, our data indicate an important role of non-canonical hedgehog signaling in regulation of gemcitabine sensitivity, and down-regulation of Gli1/2 transcription factors, together with gemcitabine, may be effective in pancreatic cancer treatment.

#4674

Vitamin D enhances erlotinib response in EGFR-mutant non-small cell lung cancer.

Tatiana Shaurova, Suzanne F. Shoemaker, Pamela A. Hershberger. _Roswell Park Cancer Institute, Buffalo, NY_.

Background. Despite remarkable initial therapeutic responses, most patients develop resistance to the first generation of EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, within one year of treatment initiation. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms of resistance to EGFR TKIs. We recently demonstrated that vitamin D promotes epithelial phenotype in NSCLC cells. We hypothesized that we could prevent/overcome EMT-driven resistance by combining vitamin D with EGFR TKIs.

Methods. EGFR-mutant, erlotinib sensitive HCC827 cells were treated with: vehicle control, 100nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1ng/ml TGF-β, or 1,25(OH)2D3+TGF-β for 14 days. Expression of EMT markers was determined by q-RT PCR and immunoblot. Sensitivity to erlotinib was analyzed by MTT assay. For the in-vivo study, HCC 827 tumor xenografts were established in vitamin-D deficient mice. Mice were stratified based on tumor volume to receive diets containing 25 IU vitamin D3/kg (deficient) or 10,000 IU vitamin D3/kg (supplemented). Erlotinib treatment (12.5 mg/kg) was initiated two weeks after the diet switch and administered 5 days per week for the duration of the study. Tumors were collected two weeks after the diet switch (baseline), 48 hours after the erlotinib initiation (acute response) and upon treatment failure (outgrowth).

Results. Vitamin D attenuated expression of mesenchymal markers in the presence of TGF-β in-vitro and opposed TGF-β driven resistance to erlotinib. Erlotinib EC50 values were 9.85 nM, 122 nM and 3.83 nM for vehicle, TGF-β, and 1,25(OH)2D3+TGF-β treated cells, respectively. In-vivo, vitamin D supplementation enhanced acute response to erlotinib. We documented a greater decrease in tumor volume after 48h of erlotinib initiation (1.1% of baseline ±19.3% in vitamin D deficient diet and 25.9% ±29.5% in supplemented diet). Greater efficacy was accompanied by increased suppression of STAT3 phosphorylation and decreased vimentin expression in the vitamin D supplemented group. Although we observed a trend towards a longer progression-free survival in animals maintained on vitamin D supplemented diet (8 vs 5.5 weeks), statistical significance was not achieved. The outgrowth tumors in both groups maintained their epithelial phenotype but exhibited a significant increase in the expression of AXL tyrosine kinase. However, outgrowth tumors from the vitamin D supplemented group had lower AXL expression than those from the vitamin D deficient group.

Conclusion. Vitamin D supports epithelial phenotype in-vitro and enhances acute response to erlotinib in-vivo, possibly, by suppressing STAT3 signaling. Vitamin D supplementation also attenuates overexpression of AXL tyrosine kinase in erlotinib resistant tumors. The clinical implication of our work is that patients diagnosed with EGFR mutant NSCLC who receive erlotinib may benefit from vitamin D supplementation.

#4675

Synergistic effect of pacritinib with erlotinib on JAK2-mediated resistance in epidermal growth factor receptor mutation-positive non-small cell lung cancer.

Nobuaki Ochi,1 Hideko Isozaki,2 Masami Takeyama,1 Jack W. Singer,3 Hiromichi Yamane,1 Yoshihiro Honda,1 Katsuyuki Kiura,2 Nagio Takigawa1. 1 _Kawasaki Medical School, Okayama, Japan;_ 2 _Okayama University, Okayama, Japan;_ 3 _CTI Biopharma, Seattle, WA_.

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR) benefit from treatment with EGFR tyrosine kinase inhibitors (TKIs). However, almost all of the patients eventually develop resistance to EGFR-TKIs within approximately one year. The resistance mechanisms of EGFR-TKIs have not been fully elucidated. We previously established an erlotinib-resistant NSCLC cell line named PC-9/ER3 after continuously exposing PC-9 cells, which harbor an in-frame deletion in EGFR exon 19, to erlotinib. PC-9/ER3 cells were 136-fold more resistant to erlotinib than the parental cells. Although the PC-9/ER3 cells did not carry the T790M mutation or MET amplification and had similar levels of pSTAT3, pJAK2 increased in the resistant cells (Cancer Sci 103, 1795-802; 2012). Meanwhile, pacritinib is a novel macrocyclic pyrimidine-based JAK2/FLT3 inhibitor with clinical activity in patients with myelofibrosis and lymphoma, which will be used as a standard therapy for myelofibrosis. In this study, we evaluated the synergistic effect of pacritinib combined with erlotinib on JAK2-medatited EGFR-TKI resistant NSCLC.

Drug sensitivity was determined using an MTT assay and the combination index was calculated according to the methods based on the median-effect analysis. The combination of pacritinib with erlotinib showed synergistic effects on JAK2-medated EGFR TKI-resistant PC-9/ER3 cells in some cases. Western blotting showed that the combination treatment markedly suppressed pAKT and pERK although pSTAT3 was equivalent regardless of treatment with the pacritinib, erlotinib, pacritinib plus erlotinib, or control in PC-9/ER3 cells. Receptor tyrosine kinase array profiling demonstrated that pacritinib suppressed MET in the PC-9/ER3 cells. The combined treatment of pacritinib and erlotinib in PC-9/ER3 xenografts showed more tumor shrinkage compared with each drug as monotherapy. Western blotting revealed that pMET in the tumor samples was inhibited. These results suggest MET suppression by pacritinib may play a role in overcoming the EGFR-TKI resistance mediated by JAK2 in the PC-9/ER3 cells.

In conclusion, pacritinib combined with EGFR-TKI might be a potent strategy against JAK2-mediated EGFR-TKI resistance.

#4676

Overcoming EGFR-induced resistance to enzalutamide in castration-resistant prostate cancer.

Thomas M. Steele,1 Maitreyee K. Jathal,1 Salma Siddiqui,2 Paramita M. Ghosh1. 1 _UC Davis Medical Center, Sacramento, CA;_ 2 _VA Northern California Health Care System, Mather, CA_.

Background: The androgen receptor (AR) remains a major therapeutic target in patients with castration-resistant prostate cancer (CRPC). Enzalutamide, an AR inhibitor that is FDA-approved for patients with CRPC, prevents ligand induced AR transcriptional activity, but some initial responders eventually become resistant to the drug. The expression and activity of the EGFR/ErbB family of receptor tyrosine kinases also increases in CRPC patients. The present work was undertaken to determine whether the activation of EGFR family (EGFR/ErbB2/ErbB3/ErbB4) may be responsible for enzalutamide resistance and whether treatment with receptor tyrosine kinase inhibitors would overcome this effect.

Methods: Human-patient-derived androgen-dependent and CRPC prostate tumor cells were grown in 10% Fetal Bovine Serum (FBS). Protein expression and phosphorylation status of the EGFR family were determined by immunoblotting techniques. MTT assays were used to determine the viability of cells treated with enzalutamide, lapatinib (HER2/EGFR inhibitor), erlotinib (EGFR inhibitor), or dacomitinib (pan-ErbB inhibitor). A Luciferase Assay kit (Roche) was used to determine AR transcriptional activity. Lapatinib was obtained from LC Laboratories while erlotinib and dacomitinib were obtained from Selleck Chemicals. Enzalutamide was kindly provided by Medivation Inc.

Results: In viability assays, erlotinib and dacomitinib were more effective than lapatinib in sensitizing CRPC cells to enzalutamide. Enzalutamide suppressed AR transcriptional activity, either alone or in combination with lapatinib, erlotinib, or dacomitinib. Erlotinib, lapatinib, and dacomitinib equally inhibited EGFR and ErbB3 phosphorylation. However, in EGF stimulated cells, erlotinib and dacomitinib—but not lapatinib—suppressed ERK 1/2 phosphorylation at Tyr202/Thr204, indicating a stark difference in downstream inhibition and potentially proliferation.

Conclusions: The above results indicate that ERK 1/2 may play an important role in reducing the efficacy of enzalutamide by itself and in combination with lapatinib. Furthermore, lapatinib's inability to prevent ERK phosphorylation upon EGF stimulation may play a role in its ineffectiveness in prostate cancer. Our preliminary preclinical data indicate that co-administration of enzalutamide with an EGFR targeted inhibitor may be a suitable therapeutic approach towards overcoming enzalutamide resistance in vitro. We are testing whether ERK 1/2 or MEK may be effective in reducing this ERK specific enzalutamide resistance. These strategies may potentially prolong enzalutamide sensitivity in CRPC patients.

#4677

Tumor suppressor p53 reactivation by oncolytic adenovirus reverses chemoresistance in human osteosarcomas.

Kazuhisa Sugiu,1 Hiroshi Tazawa,1 Joe Hasei,1 Shuhei Osaki,1 Yasuaki Yamakawa,1 Toshinori Omori,1 Tadashi Komatsubara,1 Kouji Uotani,1 Tomohiro Fujiwara,1 Toshiyuki Kunisada,1 Yasuo Urata,2 Toshifumi Ozaki,1 Toshiyoshi Fujiwara1. 1 _Okayama university, Okayama, Japan;_ 2 _Oncolys BioPharma, Inc., Tokyo, Japan_.

Background: Osteosarcoma is a primary malignant bone tumor. Despite recent advances in multi-agent chemotherapy and aggressive surgical resection, the poor response to chemotherapy often contributes to poor prognosis in osteosarcoma patients. Therefore, the development of novel strategies for reversing the chemoresistance is a pivotal approach to improve the clinical outcome for osteosarcoma patients. We recently developed a tumor suppressor p53-expressing oncolytic adenovirus, OBP-702, which drives the adenoviral E1 gene under the control of the human telomerase reverse transcriptase promoter for tumor-specific virus replication and induces profound p53 expression for tumor-specific cell death. We recently found that OBP-702 effectively kills human osteosarcoma cells. In this study, we investigated the therapeutic potential of OBP-702 as a chemosensitizing reagent in human osteosarcoma cells with different p53 status.

Methods: We used 4 human osteosarcoma cell lines with different p53 status, including U2OS (p53 wild-type), MNNG/HOS (p53 mutant), 143B (p53 mutant), SaOS2 (p53 null). We also used the doxorubicin (DOX)-resistant U2OS cells, which were established by sequential exposure to DOX over 3 months. We performed the XTT assay to examine the antitumor effects of DOX and OBP-702. Combination efficacy between DOX and OBP-702 was assessed by calculating the combination index using CalcuSyn software (BioSoft, Inc.). We further investigated the DOX- and OBP-702-mediated apoptosis in parental and DOX-resistant U2OS cells using Western blot analysis.

Results: OBP-702 improved the sensitivity to DOX in a dose-dependent manner in all 4 osteosarcoma cell lines. The calculation of combination index revealed the synergistic effect in all 4 osteosarcoma cell lines. Combination with DOX and OBP-702 induced more profound apoptosis than monotherapy in all 4 osteosarcoma cell lines. Moreover, in DOX-resistant U2OS cells, OBP-702 induced the cytopathic effect as well as parental U2OS cells. Synergistic effect was also observed in DOX-resistant U2OS cells when we treated with DOX and OBP-702. Although DOX-resistant U2OS cells was more resistant to the DOX-mediated apoptosis than parental cells, OBP-702 enhanced the DOX-mediated apoptosis in DOX-resistant U2OS cells as well as parental cells.

Conclusions: These results suggest that OBP-702-mediated p53 reactivation reverses the chemoresistance in human osteosarcomas.

#4678

Inhibition of CDK4 sensitizes multidrug resistant ovarian cancer cells to paclitaxel by increasing apoptosis.

YAN GAO, Jacson Shen, Rosemary Foster, Francis J. Hornicek, Zhenfeng Duan. _Massachusetts General Hospital, Boston, MA_.

Purpose: Overexpression of cyclin-dependent kinase (CDK) 4 has been shown in a variety of cancers and contributes to tumor cell growth and proliferation. However, the effect of inhibition of CDK4 in ovarian cancer is unknown. Here we investigated the therapeutic effect of CDK4 inhibitor, palbociclib, in combination with paclitaxel in ovarian cancer cells. Experimental Design: Cell viability was determined by MTT assay after incubating with different dosages of palbociclib and/or paclitaxel. Western blot, immunofluorescence, and Calcein AM assays were conducted to determine the potential effects and mechanisms of palbociclib in combination with paclitaxel. CDK4 siRNA was used to validate the outcome of targeting CDK4 in ovarian cancer cells. Immunohistochemistry was performed to evaluate the clinical relevance of CDK4 expression in an ovarian cancer tissue microarray. Results: The combination of palbociclib and paclitaxel significantly enhanced drug sensitivity in both Rb positive (Rb+) and Rb negative (Rb-) ovarian cancer cells. Palbociclib suppressed the phosphorylation of pRb (Ser 807-811, Ser 795, and Ser 780) in multidrug resistant SKOV3TR (Rb+) cells. When combined with paclitaxel, palbociclib induced significant apoptosis in both SKOV3TR (Rb+) and OVCAR8TR (Rb-) cells. Palbociclib also inhibited the activity of P-glycoprotein (Pgp). Additionally, transfection of CDK4 siRNA showed effective knockdown of CDK4 expression and sensitized multidrug resistant SKOV3TR and OVCAR8TR cells to paclitaxel. Furthermore, primary ovarian cancer patients with high expression of CDK4 tended to have unfavorable clinical outcomes. Conclusions: Inhibition of CDK4 either by palbociclib or by specific siRNA can increase the in vitro paclitaxel sensitivity in either Rb+ or Rb- multidrug resistant ovarian cancer cells through increased apoptosis. Therefore, CDK4 may be a promising target in the treatment of ovarian cancer.

#4679

Acquisition of cytarabine resistance leads to increased glucocorticoid sensitivity in AML.

Disha Malani,1 Astrid Murumägi,1 Bhagwan Yadav,1 Mika Kontro,2 Samuli Eldfors,1 Ashwini Kumar,1 Krister Wennerberg,1 Caroline Heckman,1 Kimmo Porkka,2 Maija Wolf,1 Tero Aittokallio,1 Olli Kallioniemi1. 1 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 2 _Hematology Research Unit Helsinki, University of Helsinki and Helsinki University Hospital Comprehensive Cancer Center, Helsinki, Finland_.

Acquired resistance to standard chemotherapeutic agents, such as cytarabine, is a major challenge in the treatment of acute myeloid leukemia (AML). Here, we hypothesized that development of resistance to one chemotherapeutic agent may lead to increased sensitivity to other drugs. Hence, we sought to identify novel drug vulnerabilities that arise during the development of cytarabine resistance using both cytarabine resistant AML cell lines and samples from AML patients who had relapsed during cytarabine containing chemotherapy.

We developed resistant variants of AML cell lines MOLM-13 and SHI-1 by long-term drug treatment with increasing doses of cytarabine. Profiling data from the in vitro generated cytarabine resistant cell line variants were systematically compared with corresponding data from 31 chemorefractory AML patient samples. All samples were subjected to genomic and transcriptomic profiling and high-throughput drug sensitivity and resistance testing with a panel of 250 chemical compounds (each in five doses).

Cytarabine resistant AML cell line variants and patient samples showed co-resistance to other nucleoside analogues, such as cladribine, clofarabine and gemcitabine. Genomic profiling showed deletion of the deoxycytidine kinase gene DCK, a well-known genetic lesion related to cytarabine resistance, in both MOLM-13 and SHI-1 cytarabine resistant cell lines and in one chemorefractory AML patient. Importantly, comprehensive drug testing revealed that cytarabine resistant SHI-1 cell variants developed increased sensitivity to glucocorticoids, such as dexamethasone, methylprednisolone and prednisolone when compared to parental cells. This was accompanied by up-regulation of the glucocorticoid receptor NR3C1. We also observed acquisition of glucocorticoid sensitivity in paired samples from two AML patient cases who had relapsed after cytarabine containing chemotherapy. Systematic ex vivo drug testing of 31 relapsed and chemorefractory AML patient samples showed high sensitivity to dexamethasone in five (20%) and to prednisolone and methylprednisolone in four (13%) patient samples.

In conclusion, our results from both cytarabine resistant AML cell lines and chemorefractory patient samples indicate that a subset of AML samples develop sensitivity to glucocorticoids. This novel finding indicates the need of detailed investigation of glucocorticoid efficacy in the clinic.

#4680

Restoration of EGFR-TKI resistance by endocytosis inhibitor PAO in lung cancer with wild-type EGFR.

Boyeon Kim,1 Jae Sook Sung,1 Yeul Hong Kim2. 1 _Cancer Research Institute, Korea University, Seoul, Republic of Korea;_ 2 _Department of Oncology/Hematology, Korea University Anam Hospital, Seoul, Republic of Korea_.

Therapeutic efficacy of epithelial growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is currently limited in selected lung cancer patients who have TKI sensitive EGFR mutation. However, the potential molecular mechanism that expands the therapeutic benefit of EGFT-TKI in patients who have wild-type EGFR non-small cell lung cancer (wtEGFR NSCLC) remains unclear. Recently, several studies revealed EGFR endocytosis mechanism could mediate signal transductions that influence in cancer cell proliferation. Moreover, our previous study demonstrated that EGFR endocytosis is related to gefitinib sensitivity in wtEGFR NSCLC and EGFR endocytosis could be a novel therapeutic target in lung cancer with wtEGFR (Oncotarget. 2014 15;5(5):1265-78). In this study, we investigated whether EGFR-TKI resistance could be restored in wtEGFR NSCLC cell lines by endocytosis inhibitor phenylarsine oxide (PAO). To investigate effects of PAO in vitro, we analyzed EGF-induced EGFR internalization and performed Annexin V and propodium iodide (PI) staining by flow cytometry. As a result, EGF-induced EGFR endocytosis is decreased and apoptotic cell death is induced accompanied by G0/G1 arrest after PAO treatment. In addition, we observed that cell viability is reduced significantly when PAO and EGFR-TKIs (gefitinib, erlotinib) treated together. Furthermore, we verified signaling transduction that associated with proliferation and apoptosis by western blot. To confirm combination effects of PAO and EGFR-TKI in vivo, we established xenograft mouse model using gefitinib-insensitive SNU1327 cell lines. After Tumor sizes reached 100-200mm3, mice were treated with PAO or gefitinib alone, or combined treatment for 3 weeks. We observed that tumor sizes of combination group were more decreased than other groups.In these result, we validate endocytosis inhibitor PAO is potential drug for overcoming therapeutic limitation of EGFR-TKI in gefitinib-insensitive wtEGFR NSCLC cell lines, though further experiment is needed to explain more accurate mechanism. This study was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2057538).

#4681

Oseltamivir phosphate completely ablates tumor refractoriness to conventional chemotherapeutics abraxane and gemcitabine in xenografts of tumors in mouse model of human pancreatic cancer.

Myron R. Szewczuk. _Queen's University, Kingston, Ontario, Canada_.

Pancreatic cancer, the fourth leading cause of cancer-related death worldwide, is highly aggressive and associated with a poor prognosis. Resistance to drug therapy, along with high rates of metastasis, contributes to the low survival rates in patients diagnosed with pancreatic cancer. Gemcitabine, a chemotherapeutic agent, is the current standard of care for patients with the disease. Although gemcitabine has higher success rates than any other chemotherapeutic in use, patients receiving gemcitabine still only have progression-free survival in the range of 0.9-4.2 months. Given the poor response rate to gemcitabine, pancreatic cancer cells develop rapid resistance to this drug. Here, we investigated the proof of concept for a reported entirely new anticancer drug, oseltamivir phosphate (OP), to overcome tumor refractoriness to gemcitabine and abraxane in heterotopic xenografts of pancreatic MiaPaCa-2 tumors growing in RAGxCγ double mutant mice. The data clearly indicate that mice treated with 50mg/kg abraxane alone (1x weekly, i.p.) at Day 33 post implantation slightly restrained tumor growth until Day70 at which time the tumor developed resistance to abraxane and rapidly increased in tumor volume and spread to the lungs. In contrast, abraxane in combination with OP 20mg/kg (3x weekly, i.p.) abrogated the tumor resistance to abraxane and impeded tumor growth and metastatic spread to the lung. Very interestingly, with comparable results to abraxane/OP, mice treated with 30mg/kg gemcitabine (1x weekly, i.p.) at Day31 post implantation developed tumor resistance at Day50 to gemcitabine and rapidly increased in tumor volume and spread to the lung. Gemcitabine in combination with 20mg/kg OP (3x weekly, i.p.) completely impeded the tumor growth, chemoresistance and spread to the lungs with long term survival at Day 240 with no relapse and metastasis. The significance of these findings is the first to show that OP in combination with standard, clinical chemotherapeutics can impede (a) human pancreatic tumor growth, (b) tumor neovascularization, (c) liver and lung metastases, and (d) tumor refractoriness to conventional chemotherapeutics, as well (e) significantly extending survival rates with no relapse and metastasis.

#4682

Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase.

Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth D. Beaman. _Rosalind Franklin University of Med. Science, North Chicago, IL_.

The development of resistance to cisplatin drugs significantly hinders successful ovarian cancer treatment. An emerging centralized mechanism of chemo-resistance is a dysregulation in tumor cell pH (alkaline intracellular, acidic extracellular). Vacuolar-ATPase (V-ATPase) is a key proton pump that alters the pH and mediates metastasis/chemo-resistance in tumor cells. The major pH sensing unit of the V-ATPase complex is the 'a2' isoform(V0a2) and has been previously shown to promote metastasis in ovarian cancer cells. Here, we show that V0a2 is over-expressed in acquired cisplatin resistant ovarian tumor cells (cis-A2780 and cis-TOV112D) at both mRNA and protein levels. The shRNA mediated inhibition of V0a2 activity in cisplatin resistant cells (sh-V0a2-cis) sensitized the cells to cisplatin with a 3 fold reduction in inhibitory concentration 50 % [IC50] (sh-V0a2-cis, IC50= 11.47 ± 3.02 µM, p<0.05) compared to control resistant cells [IC50= 37.75 ± 6.62 µM] as determined by Alamar blue cell cytotoxicity assay. Suppression of V0a2 activity strongly reduced the cytosolic pH in the resistant tumor cells (cis-A2780- pH= 7.19 ± 0.03; sh-V0a2-cis- pH=6.87 ± 0.04; cisplatin sensitive cells- pH= 6.96± 0.01). This cytosolic acidification significantly enhanced DNA damage by cisplatin-DNA adduct formation as revealed by FACS and immuno-fluorescence analysis (p<0.05). To further define the cisplatin mediated multi-factorial responses in resistant cells upon V0a2 inhibition, we performed a PCR array for cell death pathway related genes. Several pro-apoptotic genes including caspase 3, caspase 9, Apaf-1, FASL, CD40,CD40L, TNF, TNFR1 were up-regulated (p<0.05). Notably, caspase-1α was significantly up-regulated upon cisplatin treatment in sh-V0a2-cis compared to control resistant cells (p<0.05). Caspase-1α is known to induce apoptosis of neurons and macrophages and is recently indicated to be pro-apoptotic in ovarian cancer cells. Additionally, several autophagy related genes such as LC3A, IGFR, ESR1, IRGM were also found up-regulated upon V0a2 inhibition. Impaired autophagy in sh-V0a2-cis was further confirmed at protein level by FACS analysis using autophagy detection kit. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 inhibits autophagy and activates caspase-1α upon cisplatin treatment in resistant ovarian cancer cells. V0a2 inhibition can therefore serve as a therapeutic strategy to overcome cisplatin resistance and in improving the treatment efficacy in chemo-resistant ovarian carcinoma.

#4683

Verapamil and tamoxifen modulate ABCB1 expression in multidrug-resistant cells and resensitize them to conventional chemotherapy.

Georgia Limniatis, Elias Georges. _McGill Univ., Ste-Anne-de-Bellevue, Quebec, Canada_.

Multidrug-resistant (MDR) tumors are an increasingly important obstacle in cancer treatment, wherein many cases are associated with the overexpression of P-glycoprotein 1 (P-gp1, ABCB1). Previous efforts to sensitize ABCB1-expressing tumor cells to anti-cancer drugs were associated with severe side effects, so interest in clinical trials using these drugs has dwindled.

Studies have shown P-glycoprotein-overexpressing cells to be hyper sensitive (or collaterally sensitive) to a diverse group of non-toxic compounds. Given earlier results demonstrating a significant increase in ABCB1 expression post-chemotherapeutic treatment of cancer patients (e.g., breast, ovarian, myeloma, and acute myeloid leukemia), and the role of ABCB1 in drug resistance, it was determined that targeting ABCB1-expressing tumor cells with effective collateral sensitivity drugs should increase the effectiveness of current chemotherapeutic treatments.

In this report, it was of interest to determine the effect of the collateral sensitivity drugs verapamil and tamoxifen on P-glycoprotein 1 expression at the level of cells and individual cell clones. Clones from P-gp1-overexpressing Chinese hamster ovarian cell lines (CHRC5) or triple negative breast cancer cells (MDA-MB-231/400 nM doxo) were treated with varying levels of collateral sensitivity drugs and ABCB1 expression was determined by Western blots and ELISAs. The sensitivity of each cell clone to anti-cancer or collateral sensitivity drugs was determined using cytotoxicity assays.

Our results demonstrate a drop in ABCB1 expression in each clone, together with decreased collateral sensitivity and overall increased sensitivity to anti-cancer drugs in cell lines selected with verapamil and tamoxifen. Work is in progress to study the various mechanisms responsible for this drop in ABCB1 expression and its impact on treatment outcome.

#4684

Modulating the function of multidrug resistance ABCG2 transporter by tyrosine kinases receptor inhibitor cabozantinib.

Guannan Zhang, Yun-Kai Zhang, Yi-Jun Wang, Zhe-Sheng Chen. _St. Johns University, New York, NY_.

Cabozantinib (XL184) is an oral tyrosine kinases receptor inhibitor which inhibits MET and vascular endothelial growth factor receptor 2 (VEGFR2). Cabozantinib has been approved by Food and Drug Administration for treating advanced medullary thyroid cancer and it is also tested in clinical trials on other solid tumors, including prostate, bladder, ovarian and breast cancer. In the present study, we evaluated the ability of cabozantinib in modulating the function of ABCG2 transporter in drug-selected H460/MX20 cell line and ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2 and ABCG2-482-T7. Cabozantinib at non-toxic level can sensitize the ABCG2-overexpressing cells to antineoplastic drugs mitoxantrone, SN-38 and topotecan. Our results indicated that cabozantinib reversed ABCG2 mediated multi-drug resistance by antagonizing the drug efflux function of ABCG2. However, this reversal effect was not attributed to reduced expression of ABCG2 protein, because ABCG2 protein level was unchanged after treatment of 4μM cabozantinib for 72 hours. Immunofluorescence result showed that cabozantinib did not alter the cellular localization of ABCG2 transporter. Docking analysis indicated that cabozantinib binds to the drug-binding site of ABCG2 transporter. Finally, cabozantinib at 4μM did not significantly change the resistance of ABCB1 overexpressing cell line SW620/AD300 to antineoplastic drug paclitaxel. Overall, our finding demonstrated that cabozantinib potentiates the cytotoxic effects of various antineoplastic drugs that are substrates of ABCG2 and that this is due to modulating the function of ABCG2 transporter.

Keyword: Cabozantinib; ABCG2; Multidrug resistance;

#4685

**Metformin enhances trastuzumab efficacy for HER2** + **gastric cancer cells.**

Jin-Soo Kim, Geum Ock Kim, Mi Young Kim. _Boramae Medical Center, Seoul, Republic of Korea_.

Background: Trastuzumab is an effective monoclonal antibody against advanced gastric cancers (GCs) with HER2 amplification. Recently, HER2 directed therapies, such as pertuzumab or lapatinib are actively investigated in clinical trials, but showed disappointing results in patients with HER2+ GC. Unfortunately, the efficacy of trastuzumab is limited due to primary and acquired resistance to this agent. Metformin is a widely used antidiabetic drug and shows promising anti-tumor effect in several cancers.

Material and Methods: The following GC cells were evaluated: HER2 amplification -positive (HER2+) and trastuzumab-sensitive NCI-N87 and OE19, HER2+ trastuzumab-resistant OE33 and HER2 negative AGS, H1750 and SNU601. We assessed response of these GC cells to trastuzumab alone or in combination with metformin by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. MGI (mean growth inhibition) was calculated by dividing the colony number of the treated group by that of the untreated control group. Western blots were performed to assess biochemical changes, especially those related with HER2 signaling components.

Results: We observed that treatment with trastuzumab in combination with metformin induced a statistically significant decrease in the number of colonies formed in soft agar by N87 and OE19 cells, as compared to the numbers formed by control cells or cells in the single-treatment groups (Kruskal-Wallis test, p = 0.025, respectively). No growth inhibitory effect was detected in OE33 cells with Compared to trastuzumab alone, the combination of metformin resulted in decreased phosphorylation of HER2 and downstream targets, such as AKT or ERK, in trastuzumab-sensitive HER2+ cells.

Conclusion: Collectively, these data support that trastuzumab and metformin are synergistic in trastuzumab-sensitive HER2+ GC cells.

Combination effect on soft agar colony forming assays

---

Cell Lines | Trastuzumab | |

Metformin | |

Combination | |  | Index

|

MGI | P | MGI | P | Expected | Observed | P

|

NCI-N87 | 0.800 | 0.021 | 0.816 | 0.031 | 0.653 | 0.607 | <0.001 | 1.077

OE19 | 0.600 | 0.005 | 0.565 | 0.003 | 0.339 | 0.152 | <0.001 | 2.234

OE33 | 0.925 | 0.677 | 0.823 | 0.103 | 0.761 | 0.900 | 0.470 | 0.846

#4686

**Intermittent exposure to EGFR tyrosine kinase inhibitors selects less** EGFR **T790M mutant clones than continuous exposure in lung cancer cell lines.**

Youngjoo Lee, Kyoung-Yeon Kim. _National Cancer Ctr. Korea, Goyang-si, Republic of Korea_.

Background: Drug-resistant cell lines are essential tools for investigating the mechanisms of resistance to molecular-targeted anti-cancer drugs. However, little is known about how to establish clinically relevant drug-resistant cell lines. Our study examined the impact of a drug-free period on the establishment of a cell line with clinically relevant resistance to molecular-targeted drugs.

Methods: We used PC-9 cells, a lung cancer cell line carrying EGFR mutation, because this is a validated target for EGFR tyrosine kinase inhibitors (TKI). PC-9 cells were intermittently or continuously exposed to increasing concentrations of gefitinib (0.01 µM to 1.0 µM) and the emergence of the most common acquired resistance mutation in EGFR, T790M, was determined.

Results: T790M was detected at a 25-fold lower drug concentration in cells continuously exposed to gefitinib (PC-9/GRc) than in cells intermittently exposed to gefitinib (PC-9/GRi) (0.04 µM vs 1.0 µM, respectively). The mutation frequencies at those drug concentrations were 19.8% and 8.0% in PC-9/GRc and PC-9/GRi cells, respectively. After drug-free culture for 8 weeks, resistance to gefitinib decreased in the PC-9/GRi cells but not in the PC-9/GRc cells. In the PC-9/GRc cells, the frequency of the T790M mutation was consistently about 20% from 0.04 µM to 1.0 µM of gefitinib. In the PC-9/GRc cells, the T790M mutation was detected in all single-cell clones, at frequencies ranging from 7.0% to 37.0%, with a median of 19.5% (95% confidence interval, 17.3%-20.9%).

Conclusion: Intermittent exposure to EGFR-TKIs reduces the emergence of the EGFR T790M mutation in a lung cancer cell line.

#4687

Rigosertib synergistically enhances the anticancer activity of Cisplatin in various preclinical models of upper gastrointestinal cancers.

Priya Pancholi,1 Tanmay Dichwalkar,1 Samhita Bapat,1 V. J. Rajadhyaksha,2 Benjamin S. Hoffman,2 Manoj Maniar,2 Vikas Sehdev1. 1 _Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY;_ 2 _Onconova Therapeutics Inc., Newtown, PA_.

Background: Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to overactive intrinsic mechanisms that mediate drug resistance. ] Rigosertib is an investigational anticancer agent, that inhibits cellular signaling by acting as a Ras mimetic to effect the activation of multiple pathways associated with oncogenic transformation and drug resistance. Previous studies have demonstrated the inhibitory effect of rigosertib with conventional platinum based anticancer agents in various pre-clinical cancer models. We hypothesized that the novel mechanism of action of rigosertib would complement the DNA-damaging activity of Cisplatin (CDDP) , the standard of care in UGC. To test this hypothesis, we investigated the potential therapeutic benefit of rigosertib alone and in combination with CDDP in P53 wild type and mutant models of UGCs. Methods: For this study, we evaluated the effect of rigosertib treatment alone and/or in combination with CDDP on AGS (P53 wild type) and FLO-1 (P53 mutant) UGC cell viability, survival, and expression of apoptotic markers. The MTT cell viability assay and Compusyn mediated median effect plot analysis (MEPA)(Chou and Talaly) were used to determine synergistic drug combinations of rigosertib and CDDP in AGS and FLO-1 UGC cells, respectively. Results: The cell viability data and MEPA indicated that rigosertib and CDDP show optimal synergistic anticancer activity in both AGS and FLO-1 UGC cells at a ratio of 1:10, respectively. The clonogenic cell survival assay data showed that in comparison to treatment with Rigosertib (AGS: 100nM; FLO-1: 50nM) or CDDP (AGS: 1000nM; FLO-1: 500nM) alone, the combination treatment at a ratio of 1:10 significantly increased (p<0.05) inhibition of cellular survival in AGS and FLO-1 UGC cell lines. The Combination Index of 0.5 suggests a strong synergism between the two drugs. Similarly, the western blot data after treatment with Rigosertib (100nM) and/or CDDP (1000nM) for 24h induced higher levels of P53/P73, P21, and cleaved PARP with Rigosertib (100nM) and CDDP (1000nM) combination treatment in UGC cells. Conclusions: Our in vitro data indicate that rigosertib, as a single agent, is an effective therapeutic strategy for inducing apoptosis in UGC cells Additionally, rigosertib in combination with CDDP synergistically enhances the anti-tumor activity of CDDP against UGC cells. The observed synergism can translate into lower drug toxicity as significant anticancer activity can be achieved with much lower doses of rigosertib and CDDP when used in combination.

#4688

HPV+ cervical cancer cells are selectively killed by the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the cyclin-dependent kinase-9 inhibitor (CDK9i) dinaciclib.

David S. Kotlyar, Yoshimi E. Greer, Donna Voeller, Lidia Hernandez, Christina M. Annunziata, Stanley Lipkowitz. _NCI/NIH, Bethesda, MD_.

Background: Cervical cancer affects more than 520,000 women worldwide yearly, kills over 250,000 and disproportionately affects younger women. With optimal systemic treatment, the median overall survival of patients with metastatic disease is approximately 1.5 years. Previously, a cyclin dependent kinase-9 inhibitor and TRAIL have shown synergistic efficacy in a variety of cancer cell lines in vitro and in vivo. We investigated whether TRAIL combined with dinaciclib (also a CDK9 inhibitor) was selective for HPV+ tumor cells and also explored the mechanisms by which this combination killed HPV+ killed these cells.

Methods: We used a colorimetric MTS assay to assess viability after 24 h of treatment with CDK9i and TRAIL on 3 HPV+ cervical cancer lines (HeLa, SiHa, CaSki) and one HPV- cervical cancer line. Synergy was assessed by the CompuSyn program. The pan-caspase inhibitor ZVAD-FMK was used to evaluate caspase-dependence. Morphological changes were evaluated by light microscopy.

Results: The combination of TRAIL and dinaciclib resulted in greater growth inhibition than either alone in the HPV+ cell lines HeLa, Caski, and Siha. By contrast, TRAIL + dinaciclib did not inhibit the growth of the HPV - cervical cancer cell line C33a more than dinaciclib alone. The growth inhibition by TRAIL + dinaciclib was synergistic in the HPV+ lines but not the HPV- line as calculated by the Combination Index (CompuSyn). Treatment with the pan-caspase inhibitor ZVAD-FMK completely blocked TRAIL-mediated loss of viability and blocked the increased toxicity when TRAIL was added to dinaciclib, but only partially rescued dinaciclib induced loss of viability. When TRAIL and dinaciclib were combined, microscopic analysis was consistent with the synergistic killing of the HPV+ cell lines, but not the HPV- cell line. Morphologically, most cells showed signs of apoptotic cell death due to TRAIL + dincaciclib. However, in 2 HPV+ cell lines (CaSki and Hela) in addition to evidence of shrinkage of some cells with dense nuclei, seen in apoptosis, there was evidence of other cells with cell swelling with heterogeneous nuclei, which can be seen in necroptosis.

Conclusions: Our results demonstrate synergistic killing in HPV+ cells by the combination of TRAIL and dinaciclib. As one HPV- line did not show any effect of TRAIL nor synergistic killing, this is also suggestive of potential HPV selectivity of killing. Dinaciclib induces both caspase-dependent apoptosis, and a non-caspase dependent mechanism of cell death. Direct examination of the cells by light microscopy suggests dinaciclib being associated with apoptosis and necroptosis. TRAIL alone appears to solely cause apoptosis. Future experiments will investigate the mechanisms of cell death and will test these drugs in mouse xenografts.

#4689

Successfully targeting triple negative breast cancer using combination therapy of Didox and Doxorubicin with reduced cardiotoxicity.

Elizabeth A. Wilson,1 Howard L. Elford,2 Jesika S. Faridi1. 1 _University of the Pacific, Stockton, CA;_ 2 _Molecules for Health, Inc., Richmond, VA_.

Triple-negative breast cancer (TNBC) is an aggressive histological subtype that has limited treatment options with chemotherapy being the only systemic therapy for these patients. The most common chemotherapy regimen used includes the anthracycline Doxorubicin (DOXO). Although DOXO is a highly effective agent, its efficacy is limited due to acquired drug resistance and cardiotoxicity. We have previously identified that ribonucleotide reductase M2 (RRM2) is upregulated in TNBC cells and is a key contributor to acquired drug resistance. The ribonucleotide reductase inhibitor Didox has been shown to be a powerful free radical scavenger and iron chelator that provides the capabilities to alleviate the cytotoxic profile of DOXO especially the cardiotoxicity as well as enhance the DOXO efficacy. In this current study, MDA-MB-468 and MDA-MB-231 TNBC cells were treated with DOXO alone or DOXO plus Didox. Synergy assays were performed and IC50 values were determined. Here we report that Didox worked synergistically with DOXO and reduced the DOXO IC50 values by more than one-half in each cell line. In order to determine the in vivo benefit of adding Didox to DOXO therapy, ncr-nude mice implanted with MDA-MB-468 xenografts were treated with (1) vehicle, (2) daily Didox alone (425 mg/kg), (3) DOXO alone (10 mg/kg/week for 2 doses and 2 mg/kg/week for 3 additional doses), or (4) the combination of DOXO and Didox. Tumor volumes over time were determined for each of the treatment groups. At endpoint, we performed necropsy, collected serum, and dried and weighed the hearts. Treatment with DOXO alone prevented tumor growth and provided about a 70% lower tumor volume as compared to vehicle treatment. Treatment with Didox alone yielded about 50% lower tumor volume as compared to vehicle treatment. However, adding Didox in combination with DOXO significantly inhibited the tumor size and resulted in a 90% lower final tumor volume as compared to vehicle treatment. One indicator of DOXO cardiotoxicity is increased heart weight. We found that the heart weight of DOXO alone treated animals was significantly increased and the inclusion of Didox in the treatment prevented this increase. Collectively, these results identify that adding the ribonucleotide reductase inhibitor Didox in combination with DOXO may be used to treat TNBC by enhancing the efficacy on tumor inhibition and by alleviating the cardiotoxic effects of DOXO. 

### Epigenetic Agents

#4690

Novel Ring1B-Bmi1 small molecule inhibitors target leukemia stem cells.

Shirish Shukla, Felicia Gray, Weijang Ying, Hyoje Cho, Qingjie Zhao, Hongzhi Miao, Trupta Purohit, Jolanta Grembecka, Tomasz Cierpicki. _University of Michigan, Ann Arbor, MI_.

Leukemia is a clonal neoplastic disorder which possesses a rare population of cells, termed leukemia stem cells (LSCs), capable of limitless self-renewal and are necessary to initiate, sustain or regenerate the disease. Targeting LSCs self-renewal not only would offer substantial therapeutic benefit but also likely to be essential and sufficient for selective eradication of LSCs. BMI1, polycomb group protein, has been identified and shown to be required for self-renewal of LSCs. Bmi1 alongwith Ring1B constitute enzymatic active unit of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (H2AK119ub). Considering Bmi1 as a key regulator of self-renewal, targeting Bmi1 activity could impair LSCs self-renewal ability and may represent a new therapeutic approach for leukemia. Thus, in this study we aimed to develop and characterize novel small molecule inhibitors for Ring1B - Bmi1. Using NMR fragment-based screening and extensive medicinal chemistry optimization we developed WY332 that binds to Ring1B-Bmi1 with low micromolar affinity. Further characterization of the WY332 demonstrated selective reduction in Ring1B-Bmi1 mediated H2AK119ub levels in in vitro and in leukemic cells. Contrary, structurally similar and less potent compound displayed similar effect at much higher concentrations establishes that higher binding affinity of potent inhibitor is associated with blocking Ring1B-Bmi1 E3 ligase activity. Further evaluation of lead inhibitor, WY332, in colony formation assay using leukemic cells demonstrated significant decrease in the number and size of the colonies indicated that interference with Ring1B- Bmi1 activity alter the frequency of LSCs colony forming potential. Moreover, expression analysis of CD11b and CD34, cell surface markers associated with myeloid differentiation and AML LSCs phenotype, in WY332 treated TEX cells revealed marked increase in CD11b expression and significantly decreased CD34 expression establishes the specific effect of our Ring1B-Bmi1 small molecule inhibitors on induction of differentiation in leukemic cells. Interestingly, gene expression analysis of TEX cells treated with WY332 revealed significant increase in expression of genes suppressed in LSCs. Overall, these compounds represent the novel small molecule inhibitors of Ring1B-Bmi1 which inhibit E3 ubiquitin ligase activity and impair leukemic stem cell self-renewal capacity.

#4691

Identification and characterization of BRPF1 bromodomain inhibitors.

Bernard Haendler,1 Léa Bouché,1 Stephan Siegel,1 Amaury E. Fernandez-Montalvan,1 Tatsuo Sugawara,1 Julia Meier,2 Stefan Knapp,3 Vicki Gamble2. 1 _Bayer Pharma AG, Berlin, Germany;_ 2 _University of Oxford, Oxford, United Kingdom;_ 3 _University of Frankfurt, Frankfurt, Germany_.

BRPF (bromodomain and PHD finger-containing) proteins represent a subgroup of the bromodomain protein family with three closely related members, namely BRPF1, -2, and -3. They are found in different histone acetyltransferase complexes and regulate development and cell differentiation. Despite their high similarity, they have unique, non-redundant functions. Knock-out of BRPF1 in mice leads to embryonic lethality at E9.5 due to vascular defects whereas BRPF2 deficiency leads to embryonic lethality at E15.5 due to anemia. BRPF proteins contain multiple functional regions including a PZP domain which binds to unmodified H3K4, a PWWP domain which binds to H3K36me3 and a single bromodomain which recognizes acetyl-lysines in histones. BRPF1 is overexpressed in a variety of tumors, suggesting a role in cell proliferation. As first steps towards understanding the role of BRPF1, we performed knock-down studies in different tumor cell lines and initiated the search for potent and selective inhibitors of the BRPF1 bromodomain.

BRPF1-specific siRNAs were designed and used for gene silencing experiments in bladder and ovarian cancer cell lines. Following BRPF1 knock-down, marked inhibition of proliferation was observed in the CAL-29, 5637 and JMSU-1 bladder cancer cell lines, and in the ES-2 and OVCAR-8 ovarian cancer cell lines. In order to further delineate the function of BRPF1, we sought to identify selective BRPF1 bromodomain inhibitors. A collection of 5000 compounds was selected by virtual screening based on the BRPF1 bromodomain crystal structure (4LC2) and tested in a biochemical TR-FRET assay for inhibition of the interaction between BRPF1 bromodomain and an acetylated histone H4-derived peptide. Two potent hit clusters (IC50 < 150 nM) were identified and subsequent optimization led to BAY-140 and BAY-496 which were selected for further analyses. The IC50 values of BAY-140 and BAY-496 for inhibition of BRPF1 bromodomain binding were 30 and 20 nM, and for BRPF2 bromodomain binding 380 and 160 nM, respectively. Importantly, no interaction was seen for either BRD4 bromodomain (IC50 >20 µM). This binding and selectivity profile was confirmed in cellular NanoBRET assays where IC50 values for BAY-140 and BAY-496 were 1300 and 810 nM for BRPF1 bromodomain, 3600 and 2310 nM for BRPF2 bromodomain, and >10 µM for full-length BRD4. In vitro proliferation assays performed with these compounds showed single digit micromolar inhibition for the ovarian cancer ES-2, small cell lung cancer DMS-53 and acute myeloid leukemia THP-1 cell lines. However, no anti-proliferative activity was observed in a number of other tumor cell lines derived from a variety of hematological and solid tumors.

Altogether, these data show that inhibiting BRPF1 bromodomain has only a limited anti-proliferative effect on tumor cell lines, suggesting that other regions than the bromodomain of the protein account for the anti-proliferative effects seen in knock-down experiments.

#4692

Targeting the BET bromodomain proteins and anti-apoptotic protein BCL2 in small cell lung cancer.

Lloyd T. Lam, Xiaoyu Lin, Emily Faivre, Ziping Yang, Xiaoli Huang, Ricky Bellin, Xin Lu, Yu Shen, Tamar Uziel. _AbbVie, North Chicago, IL_.

10% to 15% of all lung cancers are small cell lung cancer (SCLC), named for the size of the cancer cells when seen under a microscope. SCLC often starts in the bronchi near the center of the chest. It usually grows and spreads quickly to distant parts of the body before it is diagnosed. Unfortunately, no new treatment has been identified in the past 30 years for patients with SCLC. The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic transcription factors that drive disease pathology. Pharmacologic inhibition of BET binding to acetylated proteins has been shown to inhibit tumor growth in MYC-dependent cancers, such as acute myeloid leukemia, multiple myeloma and neuroblastoma. Here, we demonstrate that ~40% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor ABBV-075. Whereas most SCLC cell lines undergo cell cycle arrest with ABBV-075 treatment, a few cell lines undergo apoptosis. Sensitivity does not correlate with MYC status (amplification/expression). However, downregulation of anti-apoptotic protein BCLxL and BCL2 by ABBV-075 was observed in sensitive SCLC cells. Synergy was observed when co-treating BET inhibitor and BCL2 inhibitors, venetoclax (ABT-199) or navitoclax (ABT-263), and it positively correlated with BCL2 expression. Thus, high BCL2 protein expression could potentially be a biomarker predictive of these therapeutic combinations. The potential higher synergy with venetoclax may be explained by downregulation of BCLxL with BET inhibition and potent activity of venetoclax against BCL2. Our studies have provided evidence for treating SCLC by BET inhibition and/or combination with BCL2 inhibitors and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need.

Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#4693

Mechanism-based combination strategies for BET inhibitors in NUT midline carcinoma.

Anastasia Wyce,1 Peter Soden,2 Daniel J. Felitsky,1 Jeanne J. Matteo,1 Susan Korenchuk,1 Gary Thripp,2 Kathryn Keenan,1 Charles F. McHugh,1 Rab Prinjha,2 Christopher Carpenter,1 Nicholas Smithers,2 Olena Barbash1. 1 _GlaxoSmithKline, Collegeville, PA;_ 2 _GlaxoSmithKline, Stevenage, United Kingdom_.

NUT midline carcinoma (NMC) is a highly aggressive squamous cell cancer that responds poorly to standard chemotherapuetic approaches. NMC is characterized by translocations involving the NUT (nuclear protein in testes) protein, which in a majority of cases is fused to the BET (bromodomain and extra-terminal) protein family members BRD3 or BRD4. BET proteins (BRD2, BRD3, BRD4, BRDT) are epigenetic readers that modulate expression of genes involved in cell growth and oncogenesis. Selective small molecule inhibitors of BET proteins, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated chromatin and inhibit the expression of BET target genes. Here we describe the activity in I-BET762 and other BET inhibitors in pre-clinical models of NMC. Consistent with previous reports, we observe profound growth inhibition and cytotoxicity in NMC cell lines in vitro, as well as significant tumor growth inhibition or tumor regression in cell line xenografts of NMC. I-BET762 treatment in NMC cell lines results in transcriptional changes affecting MYC and other pathways critical for cancer cell growth. We explore the contribution of these changes to the anti-proliferative effects observed in NMC models, and identify rational combinations to improve upon the efficacy of I-BET762 as a monotherapy. Taken together, our data highlight novel mechanisms through which BET inhibitors impact NMC cell growth and survival, and suggest potential treatment strategies to improve response in this highly aggressive disease. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

#4694

ABBV-075, a novel BET family inhibitor, disrupts critical transcription programs that drive prostate cancer growth to induce potent anti-tumor activity in vitro and in vivo.

Emily J. Faivre, Denise M. Wilcox, Paul Hessler, Tamar Uziel, Paul Tapang, Terry Magoc, Daniel H. Albert, Guowei Fang, Saul Rosenberg, Keith McDaniel, Keith McDaniel, Warren Kati, Yu Shen. _Abbvie, Inc., North Chicago, IL_.

The novel bromodomain inhibitor, ABBV-075, is being tested in a Phase I study for the treatment of solid tumors. Here, we show that potent inhibition of the BET (bromodomain and extra-terminal) family with ABBV-075 is a highly efficacious therapy in pre-clinical models of prostate cancer. The single-digit to low nanomolar anti-proliferative IC50s and potent in vivo tumor growth inhibition of ABBV-075 is mediated in part via inhibition of androgen receptor (AR)-dependent transcription. Prostate tumor incidence and CRPC clinical progression are driven by aberrant activation of the AR transcription program. Gene expression profiling and qPCR results indicate that ABBV-075 inhibited DHT-stimulated transcription of AR target genes without significant effect on AR protein expression. Further, ABBV-075 disrupted DHT-stimulated recruitment of the BET family member BRD4 to gene regulatory regions co-occupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition led to the disassembly of AR occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and additionally downregulated enhancer RNA (eRNA) transcription. ABBV-075 displayed potent anti-proliferative activity in multiple models of resistance to the second generation anti-androgen Enzalutamide, including the F876L, L702H AR ligand binding domain mutations and the AR-V7 splicing variant. In addition to blocking the transcription activation downstream of AR, ABBV-075 is also a potent inhibitor of MYC and the TMPRSS2-ETS fusion proteins. Thus, we propose that ABBV-075 may provide a promising therapeutic option for CRPC patients who have developed resistance to second-generation anti-androgens.

#4695

Functional group elaboration of a low molecular weight fragment to yield the novel BET family bromodomain inhibitor ABBV-075.

Keith McDaniel, Le Wang, George Sheppard, Steve Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Robert Mantei, Chang Park, Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Scott Warder, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise Wilcox, Rongqi Wang, Terry Magoc, Ganesh Rajaraman, Andrew Petros, Sanjay Panchal, Chaohong Sun, Guowei Fang, Steven W. Elmore, Saul Rosenberg, Yu Shen, Warren Kati. _AbbVie, North Chicago, IL_.

Phenotypic cell-based screening assays combined with affinity chromatography and mass-spectrometry identified the BET family of bromodomains as potential targets for blocking proliferation in a variety of cancer cell lines. Lead-finding investigations included screening a library of compounds with an average molecular weight of 225 for binding to the 13C-labeled 2nd bromodomain of BRD4 using 2-dimensional NMR. A pyridazinone fragment emerged from this effort that possessed weak binding affinity (Kd = 130 uM). The binding affinity was improved by roughly 100,000-fold through an X-ray structure enabled medicinal chemistry program that included moving to a pyrrolopyridone core along with judicious placement of additional functional groups. Antiproliferative potencies strongly correlated with potencies in a cell-based target engagement assay, suggesting that the antiproliferative effects resulted from the inhibition of BRD4/BET protein function. In vitro metabolite ID studies in rat liver microsomes helped identify sites of oxidative metabolism. The addition of fluorine atoms at these locations improved rat in vitro microsomal stability that translated to low in vivo clearance and high oral exposure in rat. The final molecule, ABBV-075, exhibited long half-lives and low unbound clearances in rat, mouse, dog and monkey. ABBV-075 demonstrated significant tumor growth inhibition in mouse flank xenograft studies representing diverse hematological and solid tumor malignancies and recently entered Phase I clinical studies.

Disclosures:

All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

#4696

The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia.

Min Ye,1 Mike Liu,1 Jin Lu,1 Yvonne Lo,1 Margaret Favata,1 Gengjie Yang,1 Melody Diamond,1 Valerie Dostalik,1 Paul Waeltz,1 Melissa Ann Fischer,2 Chunhong He,1 Liangxing Wu,1 Xiaochuan Shan,3 Hong Chang,1 Maryanne Covington,1 Yanlong Li,1 Tim Burn,1 Richard Wynn,1 Wenqing Yao,1 Gregory Hollis,1 Reid Huber,1 Peggy Scherle,1 Michael Savona,2 Martin Carroll,3 Bruce Ruggeri,1 Sang Hyun Lee1. 1 _Incyte Corporation, Wilmington, DE;_ 2 _Vanderbilt University, Nashville, TN;_ 3 _University of Pennsylvania, Philadelphia, PA_.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients.

#4697

The covalent CDK7 inhibitor THZ1 can counteract apoptotic resistance and suppress the transcription of genes attributed to chronic lymphocytic leukemia malignancy.

Austin Young Shull,1 Jeong-Hyeon Choi,1 Jordan Bauer,1 Lirong Pei,1 Farrukh T. Awan,2 Huidong Shi1. 1 _Georgia Regents University Cancer Center, Augusta, GA;_ 2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by the progressive accumulation of CD19+ B-cells capable of overcoming their regulated life cycle. Because this disease is highly heterogeneous and utilizes several mechanisms to resist apoptotic death, CLL currently remains incurable. Based on the ongoing efforts to interpret the transcriptional dynamics of CLL pathogenicity as well as prior reports demonstrating the efficacy of cyclin-dependent kinase (CDK) transcriptional inhibitors, we sought to characterize the molecular response of CLL cells treated with the covalent CDK7 inhibitor THZ1 and determine the therapeutic potential of CDK7 inhibition in CLL. We first observed that THZ1 was able to inhibit growth in malignant B-cell lines MEC1 and MEC2 in both a dose dependent (IC50: .045uM & .030uM, respectively) and time dependent manner. The inhibition in cell growth corresponded with both G1-mediated cell cycle arrest as well as apoptosis in the respective cell lines. We also observed dose-dependent reduction in cell viability through apoptosis in patient-derived CLL B-cells after 24 hours. Because of the observed outcomes of THZ1-treated CLL cells, we then wanted to determine the specific transcriptional targets significantly suppressed by THZ1 treatment. To identify the THZ1-sensitive transcripts, we performed a time course RNAseq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of covered reads over a 12-hour period, we determined that the top 50 transcripts greatly diminished in both MEC1 and MEC2 contain significant enrichment in genes attributed to glycolytic metabolism (ex: ALDOC, SLC2A1, TPI1, GAPDH, ENO2). Along with the overlapping comparison between the two cell lines, we next compared the downregulated transcripts from the treated cell lines against the RNAseq expression profile of 47 CLL patients and 5 healthy donors. We observed that THZ1 was able to suppress transcripts upregulated in CLL patient samples including ENO2, FGR, WNT10A, and CBX7 in MEC1 and ENO2, MALAT1, CCR7, and PLCG1 in MEC2. Finally, we performed H3K27Ac ChIPseq in MEC1 to identify super enhancers that may overlap with THZ1-sensitive transcripts. From this comparison, we determined that super enhancers exist within reported oncogenic drivers CBX7, WNT10A, and FGR. Overall, we observe that THZ1 can effectively overcome the anti-apoptotic phenotype of CLL B-cells as well as directly suppress transcription of genes that can drive CLL malignancy. With the addition of CDK7 ChIPseq, our ultimate goal is to provide clarity regarding CDK7-mediated transcriptional regulation in CLL and demonstrate the molecular consequences of therapeutic CDK7 inhibition in CLL.

#4698

BET protein inhibitors prevent TKI-induced drug resistance in solid tumors.

Yuan Zhao, Yiyuan Wu, Hexiang Wang, Bo Ren, Ye Liu, Min Wei, Changyou Zhou, Lusong Luo. _BeiGene(Beijing) Co., Ltd., Beijing, China_.

Acquired drug resistance is one of the major challenges for clinical application of tyrosine kinase inhibitor (TKI) in cancer treatment. TKI-induced drug resistance could develop as a result of adaptive cell reprogramming that leads to pathway feedback activation in the same signaling pathway or parallel pathways or gatekeeper mutations abolishing drug binding. The findings of reversible drug response and lack of genetic mutations in TKI-resistant tumor cells suggest epigenetic basis during resistance development process. Targeting epigenetic modifiers and readers, in particular, the bromodomain and extra-terminal (BET) protein family, has shown promising efficacies against both primary and drug-resistant hematological malignancies via modulating transcription profile and suppressing the MYC oncogene. BGB-3619 is a novel, potent inhibitor of BET family proteins (BRD2, BRD3, BRD4 and BRDT) with IC50 ranging from 20 to 49 nM. It demonstrates superior anti-proliferation activity than iBET762 in multiple leukemia and lymphoma cell lines. In this study, we investigated the effect of BGB-3619 and iBET762 on solid tumor cells during and after TKI-resistance development. From cell killing assays, BET inhibitors were found to be much more potent to TKI-resistant cells (vemurafenib-resistant A375, HT29, SK-MEL-5, cells and erlotinib-resistant HCC827 cells) than their parental cells. Notably, long-term colony formation assays showed that combination of low dose BET inhibitors and TKI effectively suppressed the expansion of TKI-tolerated cell populations. The enhanced sensitivity of TKI-resistant cells to BET inhibitor was due to strong G1 phase arrest rather than induction of apoptosis. Furthermore, BET inhibitors blocked cell proliferation independent of c-Myc inhibition, suggesting they might affect a broad transcription network and provide favorable epigenetic state to induce cell cycle arrest. Taken together, these findings suggested potential therapeutic activity of BET inhibitor in TKI-resistant solid tumors and a strategy of combining BET inhibitors with TKIs to overcome resistance to targeted cancer therapies.

#4699

Superior lethal activity of single agent BET protein PROTAC compared to bromodomain inhibitor or in combination with JAK inhibitor against post-myelofibrosis secondary AML cells.

Dyana T. Saenz,1 Warren C. Fiskus,1 Taghi Manshouri,1 Jing Lu,2 Yimin Qian,2 Kanak Raina,2 Baohua Sun,1 Stephanie S. Krieger,1 Simrit Parmar,1 Srdan Verstovsek,1 Kapil N. Bhalla1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Arvinas, Inc, New Haven, CT_.

Myeloproliferative neoplasm, myelofibrosis (MPN-MF), exhibits increased JAK-STAT signaling and often progresses (~15-20%) to AML (sAML). JAK inhibitor (JAK-I) ruxolitinib (Rux) or standard induction chemotherapy is only modestly active against sAML, culminating in treatment-refractory relapse. Genetic alterations commonly documented in sAML include the co-occurrence of JAK2 V617F and mutant TP53. Here, we demonstrate that treatment with ARV-825 (Arvinas, Inc.), a BET (bromodomain and extraterminal) protein PROTAC (proteasome activating chimera) which degrades (through the proteasome) BET proteins by binding and recruiting E3 Ubiquitin ligase cereblon activity to them, caused efficient, and prolonged depletion of the levels of the BET protein BRD4. In contrast, treatment with the bromodomain inhibitors (BET-Is) JQ1 or OTX015 induced the levels of BRD4. ARV-825 treatment also mediated greater and more sustained attenuation than OTX-015 of the mRNA and protein expressions of BCL-xL, CDK4/6, PIM1, p-STAT5 and p-STAT3 levels, while concomitantly inducing p21 and p27 in the cultured sAML (HEL92.1.7 and SET2) cells. This correlated with high level of apoptosis in the cultured and patient-derived post-MPN-MF sAML cells, with relative sparing of the normal CD34+ progenitor cells. Compared to treatment with each agent alone, co-treatment with ARV-825 and the JAK-Is Rux (100 to 1000 nM) or pacritinib (250 to 1000 nM), was synergistically more lethal against the cultured sAML cells (CI of < 1.0 on the isobologram analyses). Co-treatment with ARV-825 and Rux caused a marked inhibition of p-STAT5, p-STAT3, MYC, CDK4/6, BCL-xL and PIM1 in the sAML cells. HEL92.1.7 and SET2 cells harbor JAK2 V617F as well as express mutant TP53 (M133K in HEL92.1.7 and R248W in SET2 cells). Co-treatment with ARV-825 or JQ1 and the heat shock protein (HSP) 90 inhibitor AUY922, which is known to down-regulate the levels of mutant-TP53, JAK2, c-RAF, p-STAT5, p-STAT3 and p-AKT, was synergistically lethal against HEL92.1.7 and SET2 cells. We have isolated JAK-I-resistant HEL92.1.7 cells (> 10-fold resistant to ruxolitinib; HEL/JIR cells) under the in vitro selection pressure of a continuous exposure to JAK-I. Notably, compared to the parental HEL92.1.7, HEL/JIR cells were highly and collaterally sensitive to both ARV-825 and AUY922. Furthermore, co-treatment with ARV-825 and AUY922 was also synergistically lethal against HEL/JIR cells (CI < 1.0). Taken together, these findings demonstrate that ARV-825 is a highly active agent alone or in combination with JAK-Is against post-MF sAML cells expressing mutant p53. Combined therapy with ARV-825 and an HSP90 inhibitor is synergistically lethal against sAML cells resistant to JAK-Is. Based on these finding, in vivo testing of the BRD4 PROTAC-based combinations against post-MF sAML cells is warranted.

#4700

BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer.

Lina Gao, Daniel J. Coleman, Carly J. King, Jacob Schwartzman, Nicholas Wang, Amanda Esch, Joshua Urrutia, Archana Sehrawat, Laura M. Heiser, Joshi J. Alumkal. _Oregon Health & Science University, Portland, OR_.

Background: While treatment options for patients with castration-resistant prostate cancer (CRPC) are expanding, one American man is still predicted to die every 19 minutes from this disease this year. Moreover, more widespread use of novel and more potent AR-targeting agents has led to increased clinical frequency of virulent androgen and AR-independent CRPC subsets(Small, Huang et al. 2015). Currently, there are limited treatment options for men with CRPC who are resistant to these AR-targeting agents, clearly demonstrating an urgent need to develop more effective therapies for CRPC patients.

Recent published reports demonstrate an important role for BET bromodomain chromatin reader proteins in prostate cancer models that are androgen or AR-dependent (Gao, Schwartzman et al. 2013; Wyce, Degenhardt et al. 2013; Asangani, Dommeti et al. 2014; Chan, Selth et al. 2015). However, there was limited information about the anti-tumor activity of this class of drugs in androgen-independent, enzalutamide-resistant, or AR-independent CRPC models. The studies reported herein were designed to address that deficit.

Methods: We treated a panel of CRPC cell lines with dose escalation of the BET bromodomain inhibitor JQ1 and measured cell viability. To clarify molecular mechanisms that contribute to the anti-tumor effect, we treated CRPC cell lines with JQ1 and measured gene expression changes with RNA-sequencing. Finally, to determine the anti-tumor activity of JQ1 in vivo, we implanted enzalutamide resistant or AR-null CRPC xenografts in immunocompromised mice and treated them with JQ1.

Results: All cell lines were sensitive to JQ1 with similar GI50 values (all < 1µM). Our analysis of RNA-seq data after JQ1 treatment identified a BET bromodomain response signature that was conserved across the population of cell lines. Further, we applied Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to identify novel Master Regulator transcription factors that mediate response to JQ1(Aytes, Mitrofanova et al. 2014). Finally, JQ1 treatment of enzalutamide resistant or AR-null CRPC xenografts implanted in vivo suppressed tumor growth without appreciable toxicity.

Conclusions: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal CRPC. Our work using a broad panel of CRPC models sheds further light on pharmacodynamic markers of response and target proteins whose function is impacted by BET bromodomain inhibition. These findings may have implications for the design of BET bromodomain inhibitor clinical trials in men with CRPC and interpretation of on-target effects in those trials.

#4701

A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds.

Hanghang Zhang,1 Noël J.-M Raynal,1 Takahiro Sato,1 Yasuyuki Okamoto,1 Judit Garriga,1 George Morton,2 Wayne Childers,2 Marlene A. Jacobson,2 Stephen B. Baylin,3 Xavier Graña,1 Magid Abou-Gharbia,2 Jean-Pierre J. Issa1. 1 _Fels Institute for Cancer Research, Philadelphia, PA;_ 2 _Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, Philadelphia, PA;_ 3 _The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD_.

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. In an effort to identify novel targets that can reactivate hypermethylated silenced genes, our lab developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. By screening an NDL-3040 library and grouping the molecules based on chemical structures, we were able to identify three main drug classes. The Moulder Center then synthesized 60 new analogs based on class #1's lead's structure and 15 are positive in the YB5 system. The most potent analog can induce 10% GFP+ cells upon 500nM treatment. All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes can be reactivated upon drug treatment. Also, GFP positive cells show higher endogenous gene reactivation than unsorted and GFP negative cells. By performing RNA-seq analysis upon class #1 top lead treatment followed by connectivity mapping, we identified X as the class #1 drug target. The on-target effect can be validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Proliferation assays showed differential sensitivities of a panel of colon cancer cell lines compared to normal cells. These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Besides class #1 drugs, a novel class of LSD1 inhibitors was identified and the most active drug can induce 15% GFP at 5uM. Consistent with LSD1 inhibition, many known LSD1 target genes can also be upregulated. Like known LSD1 inhibitors, these compounds significantly inhibited proliferation of AML cells. We also identified some known natural compounds that have epigenetic activities, including arsenic trioxide, cardiac glycosides, and toyocamycin. Thus, many novel epigenetic drug classes derived from natural compounds were identified and can be developed by targeting silenced gene expression.

#4702

**Combination of BET inhibitor INCB054329 and LSD1 inhibitor INCB059872 is synergistic for the treatment of AML** in vitro **and** in vivo **.**

Xuesong Liu, Matthew Stubbs, Min Ye, Roberts Collins, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Yvonne Lo, Chunhong He, Liangxing Wu, Andrew Combs, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Phillip Liu, Sang Hyun Lee. _Incyte Corporation, Wilmington, DE_.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML an altered

epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Indeed, a screen for

essential genes in the murine MLL-AF9 retroviral model of leukemia identified the epigenetic regulators BRD4 and LSD1 as potential therapeutic targets. Furthermore, independent studies have demonstrated that inhibition of BRD4 or LSD1 by small molecules induced myeloid differentiation and suppressed leukemic stem cell phenotype in AML models. Recently it has been demonstrated that combinations of inhibitors that target distinct epigenetic regulators can exhibit synergistic effects on target gene transcription and cancer cell growth. Therefore we investigated the potential combinatorial effects of a novel FAD-directed LSD1 inhibitor, INCB059872, together with the BET inhibitor, INCB054329. The effects of single agent LSD1 and BET inhibitors or their combination were assessed using human AML models in vitro and in vivo. INCB054329 as monotherapy inhibited cell proliferation and induced apoptosis in human AML cell lines, while INCB059872 induced cellular differentiation in these cell lines as determined by the induction of myeloid differentiation markers, CD86 and CD11b. The combination of INCB054329 and INCB059872 enhanced myeloid differentiation and apoptosis in human AML cell lines compared with the single agents. Interestingly, c-myc expression was down-regulated to greater extent with the combination of both compounds compared to either agent alone. Enhanced anti-tumor efficacy with favorable tolerability was observed in human AML xenograft models when both agents were administered simultaneously or sequentially, with INCB059872 dosing regimens preceding INCB054329 administration demonstrating the greatest efficacy. These ongoing studies demonstrate that concurrent inhibition of two distinct families of epigenetic regulators, BET and LSD1, is active in preclinical AML models, and provide a rationale for the clinical evaluation of this novel, epigenetic doublet therapy in AML.

#4703

The BET inhibitor BAY 1238097 shows efficacy in BRAF wild-type and mutant melanoma models.

Bernard Haendler, Kathy A. Gelato, Laura Schöckel, Tatsuo Sugawara, Pascale Lejeune, Heidrun Ellinger-Ziegelbauer, Amaury E. Fernandez-Montalvan, Simon Holton, Stephan Siegel, Melanie Heroult, Annette Walter, Stuart Ince, Matthias Ocker. _Bayer Pharma AG, Berlin, Germany_.

BET proteins recognize histone acetylation marks and play an essential role in transcription elongation. BRD4, the best studied family member, binds to the regulatory regions of oncogenes such as MYC, thereby controlling its expression and that of the downstream transcriptome. Following the identification of the first selective inhibitors, BET proteins have been shown to play essential roles in hematological and, more recently, in solid tumors. Here we studied the efficacy of the novel BET inhibitor BAY 1238097 in melanoma models.

BAY 1238097 was a potent BET inhibitor with IC50 values of 47 and 295 nM for BRD4 BD1 and BD2, respectively. In NanoBRET assays, the interaction between BRD4, BRD3 or BRD2 with histone H4 was blocked in intact cells with IC50 values of 65 nM, 294 nM and 642 nM, respectively. In addition to its strong anti-proliferative activity in several hematological tumor models, BAY 1238097 was also effective in several melanoma cell lines with GI50 values below 500 nM in BRAF wild-type (CHL-1, COLO-792, B16F10, IPC-298, MeWo) as well as BRAF mutant (A375, G-361, SK-MEL-30, LOX-IMVI, SK-MEL-5, MEL-HO) models. Resistant cell lines with GI50 values above 10 µM were identified in both the BRAF wild-type and mutant groups. ChIP analysis performed in BRAF wild-type CHL-1 cells showed that BAY 1238097 displaced BRD4 from regulatory regions of oncogenes such as MYC, leading to loss of expression. The oxygen consumption rates of the melanoma cell lines were measured and we found that cell lines sensitive to BAY 1238097 depended on oxidative phosphorylation rather than glycolysis for energy production.

In vivo efficacy was determined for BAY 1238097 in three patient-derived melanoma models harboring the wild-type BRAF gene. Daily, oral treatment with 7.5 mg/kg BAY 1238097 led to significantly reduced tumor growth (39% T/C) in one model. Interestingly, this model was resistant to dacarbazine given i.p. at 100 mg/kg daily. No biologically significant anti-tumor activity was observed for the two other models treated with the same conditions (62% and 70% T/C, respectively).

Altogether the results show BAY 1238097 to be a potent inhibitor of BRD4 binding to histones. The compound has strong anti-proliferative activity in different melanoma models, regardless of the BRAF mutation status but related to the metabolic activity. One out of three patient-derived melanoma models with wild-type BRAF responded to BAY 1238097 treatment in vivo. Future studies will help to better characterize the impact of BET inhibition on melanoma.

#4704

The evaluation of INCB059872, an FAD-directed inhibitor of LSD1, in preclinical models of human small cell lung cancer.

Sang Hyun Lee, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Margaret Favata, Chunhong He, Liangxing Wu, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri. _Incyte Corporation, Wilmington, DE_.

Methylated histone marks on H3K4 and H3K9 are generally coupled with transcriptional activation and repression, respectively. Altered levels of these histone methylation marks lead to abnormal gene expression and are associated with oncogenesis. Lysine specific demethylase 1 (LSD1) catalyzes demethylation of mono and di-methylated lysine 4 and 9 of histone H3 via an FAD-dependent redox-process. Deregulated LSD1 activity perturbs normal gene expression and can lead to cellular transformation. In particular, the function of LSD1 has been reported to maintain stem cell-like gene expression patterns in various cancers, most notably small cell lung cancer (SCLC), a cancer type that is characterized by a highly undifferentiated state and with high unmet medical need. INCB059872 is a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. The effect of INCB059872 in SCLC cell lines was assessed in vitro and in vivo. In these studies, the proliferation of a panel of SCLC cell lines was inhibited by INCB059872, with EC50 values ranging from 47 to 377 nM. Non-tumorigenic cells, such as IL-2 stimulated T cells from normal donors, by contrast, were significantly less sensitive with IC50 values > 10 μM. Oral administration of INCB059872 on once daily (QD) and alternative day (QoD) dosing regimens inhibited tumor growth in the NCI-H526 and NCI-H1417 human SCLC xenograft models. Consistent with previous reports, INCB059872 treatment in these models led to the induction of the FEZ1 and UMODL1 genes relative to vehicle-treated animals, as part of a gene signature in SCLC cell lines that is predictive of LSD1 responsiveness. Moreover, serum levels of the neuroendocrine marker pro-GRP were markedly reduced at all efficacious dosing regimens in the NCI-H1417 human SCLC xenograft model, suggesting that serum pro-GRP levels could be used as a surrogate pharmacodynamic marker of LSD1 inhibition. Currently, the efficacy of INCB059872 in combination with standard of care therapies for SCLC is under evaluation in these preclinical models. These data support the clinical evaluation of INCB059872 in patients with SCLC.

#4705

Therapeutic activity of bivalent BRD4 inhibitor AZD5153 in hematological cancers.

Huawei (Ray) Chen,1 Maureen Hattersley,1 Garrett Rhyasen,1 Austin Dulak,1 Wendy Wang,1 Phil Petteruti,1 Ian Dale,2 Tony Cheung,1 Shenghua Wen,1 Lilian Castriotta,1 Deborah Lawson,1 Mike Collins,1 Miika Ahdesmaki,2 Graeme Walker,3 Al Rabow,3 Jonathan Dry,1 Corinne Reimer,1 Paul Lyne,1 Steve Fawell,1 MIke Waring,3 Mike Zinda,1 Ed Clark,1 Ed Clark1. 1 _AstraZeneca R &D Boston, Waltham, MA; _2 _AstraZeneca R &D, Cambridge, United Kingdom; _3 _AstraZeneca R &D, Alderley Park, United Kingdom_.

The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Across a number of tumor models pharmacological targeting of BRD4 bromodomains by small-molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor. AZD5153 is a candidate drug that possesses an unprecedented bivalent binding mode among reported BET inhibitors, which allows AZD5153 to ligate the tandem bromodomains in BRD4. The avidity resulted from the bivalent binding interaction translates into markedly enhanced cellular potency. Although AZD5153 demonstrates broad activity across a cancer cell line panel comprising solid and hematologic subtypes, there is enriched antitumor activity against hematologic cell lines, including acute myeloid leukemia (AML), multiple myeloma (MM), and diffuse large B-cell lymphoma (DLBCL). The activity of AZD5153 in hematologic tumors was further confirmed in five selected xenograft models of AML, MM, and DLBCL where AZD5153 treatment led to tumor stasis or regression, accompanied by concomitant modulation of BRD4 pharmacodynamic markers, such as MYC and HEXIM1. In order to characterize the transcriptional consequences elicited by AZD5153, we carried out transcriptional profiling of 11 hematologic tumor lines and identified the robust modulation of MYC, and E2F transcriptional programs. Moreover, our transcriptional data was used to identify candidate clinical PD biomarkers. The suitability and dynamic range of the top two candidate biomarkers for AZD5153 was confirmed using human whole blood from normal healthy volunteers. To identify protein biomarkers associated with sensitivity to AZD5153 treatment, we deployed reverse-phase protein array (RPPA) technology to quantitatively examine the level of 182 proteins following AZD5153 treatment. Our findings indicate that cell lines sensitive to AZD5153 uniquely exhibit a marked decrease in the level of mTOR-pathway associated proteins following AZD5153 treatment. Conversely, MYC modulation was observed in both sensitive and resistant groups. Thus, these data suggest that in hematologic malignancies, mTOR pathway downregulation may serve as an appropriate biomarker of sensitivity to BRD4 inhibitors such as AZD5153.

Our study establishes AZD5153 as a novel and potent BRD4/BET inhibitor possessing a unique bivalent binding property. We have characterized the pharmacological consequences of BRD4/BET inhibition by AZD5153 via unbiased transcriptional and proteome profiling. These efforts are the first to identify mTOR modulation as a putative biomarker of sensitivity to BET bromodomain inhibition in hematologic tumors and may help to inform future clinical evaluation of AZD5153 and other BET bromodomain inhibitors.

#4706

ABBV-075 exhibits robust in vitro and in vivo activities against the ABC and GCB subtypes of DLBCL.

Xiaoyu Lin, Xiaoli Huang, Aparna Sarthy, Terry Magoc, Daniel Albert, Lloyd Lam, Tamar Uziel, Xin Lu, Mai-Ha Bui, Emily Faivre, Denise Wilcox, Steven Elmore, Keith McDaniel, Warren Kati, Yu Shen. _Abbvie, North Chicago, IL_.

Diffuse Large B-Cell Lymphoma (DLBCL) consists of activated B-cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subtypes. Among these, the ABC subtype of DLBCL often harbors mutations that cause abnormal NF-κB activation, which subsequently activates genes important for cell survival and disease progression in ABC DLBCL. The bromodomain and extraterminal domain (BET) protein BRD4 binds acetylated NF-κB and regulates NF-κB dependent transcription. Accordingly, targeting BET family proteins using small molecule inhibitors such as ABBV-075 might provide therapeutic benefit in ABC DLBCL by blocking the NF-κB pathway. In this study, we examined the activity of ABBV-075 in both the ABC and GCB subtypes of DLBCL. As expected, ABBV-075 diminished NF-κB reporter activity, down-regulated NF-κB target genes, inhibited proliferation and survival of ABC DLBCL cells in vitro, and delayed the growth of ABC DLBCL tumors in vivo. Interestingly, ABBV-075 also exhibited robust anti-proliferative activities in GCB DLBCL cells in vitro and inhibited the growth of GCB DLBCL tumors in vivo. Further characterization of the responses of GCB DLBCL cells to ABBV-075 indicated that ABBV-075 induced strong apoptosis in GCB DLBCL cells that are resistant to the Bcl-2 inhibitor venetoclax (ABT-199), but rarely in venetoclax-sensitive GCB DLBCL cell lines. Our results indicate that ABBV-075 could be active against both the ABC and GCB subtypes of DLBCL. The complementary activity of ABBV-075 and ABT-199 further suggests that ABBV-075 maybe an interesting option for ABT-199 resistant/refractory DLBCL patients.

#4707

Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC).

Darmood Wei, Christopher J. Ricketts, Laura S. Schmidt, Youfeng Yang, Cathy D. Vocke, William M. Linehan. _National Cancer Institute, Bethesda, MD_.

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors such as CDKN2A/p16. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects on two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC cell lines both in vitro and in vivo. Our preliminary data suggests EZH2 inhibition results in reduced growth of ccRCC cell lines with PBRM1 mutations.

#4708

Neuroendocrine gene transcript expression is associated with efficacy to lysine-specific demethylase-1 inhibitor RG6016 in small cell lung cancer-derived cell lines.

Francesca Milletti,1 Wei-Yi Cheng,1 Tamara Maes,2 Serena Lunardi,2 Mark DeMario,1 William E. Pierceall,1 Fiona Mack1. 1 _Roche Innovation Center New York, New York, NY;_ 2 _Oryzon Genomics, Barcelona, Spain_.

Small cell lung cancer (SCLC), which comprises 15% of lung neoplasms, is an aggressive malignancy with limited treatment options. Survival in refractory settings is typically less than one year, exemplifying the need for more effective therapeutics. Recent published results suggest that epigenetic modulation mediated by Lysine Specific Demethylase-1 (LSD1) inhibition can impact this disease setting (Mohammed et al., Cell, 2015 28(1):57-69). RG6016 is a potent and selective inhibitor of LSD1 that promotes growth inhibition in vitro and in vivo SCLC xenograft models. Here we describe the identification of a gene expression profile associated with neuroendocrine lineages that predicts responses to RG6016 in SCLC cell lines. RG6016 was screened on a panel of 19 SCLC cell lines; potent sub-nanomolar to nanomolar activity was exhibited on a subset of these cell lines. Growth inhibitory responses were greater in classic compared to variant SCLC cell lines (p-value 0.0055). 9 of the 11 classic SCLC cell lines tested were responsive to growth inhibitory effects of RG6016, while 7 of the 8 variant cell lines were insensitive to RG6016 treatment. Differential gene expression analysis comparing classic to variant cell lines ranked neuroendocrine lineage-associated markers, such as ASCL1, amongst the highest differentially expressed genes (adj. p-value = 2.6 X 10-23). ASCL1 is a transcription factor required for proper development of pulmonary neuroendocrine cells, and was recently identified as essential for the survival of a majority of small cell lung cancers (Augustyn et al., Proc Natl Acad Sci USA 2014 111(41):14788-93). Two other established neuroendocrine markers, DDC and GRP, ranked within the top ten differentially expressed genes. We also found that a subset of insensitive SCLC cell lines harbor c-MYC amplifications, suggesting a potential pathway for resistance to LSD1 inhibition. An RG6016 response gene expression pattern that highly correlates with growth inhibition was defined using baseline expression levels of neuroendocrine lineage transcripts and c-MYC amplification. In vitro activity also correlated with in vivo responses; RG60106 treatment inhibited xenograft growth of response signature positive cell line NCI-H510A, while the response signature negative NCI-H526 xenografts were refractory to RG6016 exposure. Similar gene expression patterns were observed within SCLC cell lines and a panel of SCLC patient samples, suggesting that use of the RG6016 response gene signature may increase the likelihood of identifying patients who will clinically benefit from LSD1- based therapies.

#4709

Broad activity for the combination of BET and MEK inhibitors across solid and hematologic cancers.

Anastasia Wyce, Daniel J. Felitsky, Xi-Ping Zhang, Jeanne J. Matteo, Susan Korenchuk, Lijoy K. Mathew, Melissa Musso, Sakina Khaku, Victoria Ortiz, Kathryn Keenan, Melissa Stern, Yan Degenhardt, Ramona Plant, Charles F. McHugh, Peter J. Tummino, Christopher Carpenter, Olena Barbash. _GlaxoSmithKline, Collegeville, PA_.

BET (bromodomain and extra-terminal) family proteins are epigenetic readers that modulate expression genes involved in cell growth and oncogenesis. Selective small molecule BET inhibitors, such as the GSK I-BETs (I-BET762, I-BET151), abrogate binding of BET proteins to acetylated chromatin and inhibit transcription of BET target

genes. We and others have previously demonstrated single agent activity for BET inhibitors in a number of pre-clinical solid and hematologic tumor models. Transcriptomics

and mechanistic studies from several of these tumor types indicate that BET inhibitors influence numerous signaling pathways at the transcriptional level, including RAS/RAF/MEK signaling. Here we describe the synergistic effects of combining BET and MEK inhibitors in various solid and hematologic cancer models. We observe synergistic growth inhibition and apoptosis in a subset of cell lines representing multiple tumor types, as well as patient-derived xenograft models treated with the combination ex vivo. Additionally, combination of BET and MEK inhibitors results in improved tumor growth inhibition in cell line xenograft models compared to either single agent therapy. Further exploration of the combination in sensitive tumor types highlights multiple mechanisms potentially driving synergy, and suggests possible markers associated with sensitivity to the combination. Taken together, our data highlight the potential of BET/MEK inhibitor combinations to improve upon the efficacy observed for these agents as monotherapies in a wide variety of preclinical cancer models.

All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents.

#4710

BRD4 degradation by PROTACs represents a more effective therapeutic strategy than BRD4 inhibitors in ovarian cancer.

Kanak Raina,1 Jing Lu,1 Yimin Qian,1 Martha Altieri,1 Hanqing Dong,1 Jing Wang,1 Xin Chen,1 Andrew Crew,1 Kevin Coleman,1 Craig Crews,2 James Winkler1. 1 _Arvinas, New Haven, CT;_ 2 _Yale University, New Haven, CT_.

BRD4, a member of the bromodomain and extraterminal domain (BET) family of chromatin readers, localizes to super-enhancer regions of DNA, and turns on the expression of a number of oncogenes. Therefore, it has attracted considerable interest as a potential drug target in a variety of cancers, and several inhibitors that block its binding to acetylated histones have been discovered. The BRD4 genomic region is amplified in approximately 17% of ovarian serous adenocarcinomas, and a recent shRNA screen identified it as a bona fide target in high-grade serous ovarian carcinoma (HGSOC). BRD4 inhibitors were shown in the same study to be efficacious in cell culture and PDX models of HGSOC. However, the inhibitor paradigm of drug discovery has several limitations, for example, the requirement of high target occupancy for activity, and the inability to block potentially critical scaffolding functions of the drug target. To circumvent these limitations, we have designed small molecule BRD4 degraders based on our PROTAC (PROteolysis TArgeting Chimera) platform, and assessed their activity across a panel of ovarian carcinoma cell lines.

BRD4 PROTACs, containing a BRD4 ligand connected to a VHL E3 ubiquitin ligase binding moiety, successfully promote proteasome-mediated degradation of BRD4 at sub- to low-nM concentrations in a number of ovarian cancer cell lines. Furthermore, this BRD4 degradation results in a very potent anti-proliferative effect in the cells. All the cell lines tested showed modest anti-proliferative effects when treated with BRD4 inhibitors (ED50 ~ 0.5 µM-1µM). In contrast, when treated with BRD4 PROTACs the cell lines separated into three profoundly different groups (most sensitive, moderately sensitive, least sensitive), with a difference of several orders of magnitude (ED50 ~ 0.03nM-500nM). Thus BRD4 PROTACs are up to 10,000-fold more potent than BRD4 inhibitors in the most sensitive cell lines. Moreover, the ED50 curves closely track with BRD4 degradation, and result in a much more profound apoptotic response than BRD4 inhibition. In addition, we have also shown that the anti-proliferative effect of BRD4 PROTACs is decoupled from their effect on the levels of cMYC. Finally, we have carried out deep RNA sequencing in five cell lines of variable sensitivity to BRD4 PROTACs in order to thoroughly define the molecular subtype of the malignancies represented and have identified gene signatures that correlate with efficacy of BRD4 PROTACs and BRD4 inhibitors in these cells.

In summary, PROTACs designed to target BRD4 for proteasomal degradation are highly potent in ovarian cancer cells, and elicit a powerful pro-apoptotic response in the same setting. The cells can be grouped by their level of sensitivity to PROTAC-mediated BRD4 degradation, and carry gene expression biomarkers which we hope will enable a patient selection strategy as BRD4 PROTACs are advanced into the clinic.

#4711

Broad anti-tumor activity of a novel BET bromodomain inhibitor.

Yan Ma, Ben Powell, Jiazhong Zhang, Ying Zhang, Heidi Carias, Ullrich Schwertschlag, Gaston Habets, Prabha Ibrahim, Wayne Spevak, Chao Zhang, Gideon Bollag. _Plexxikon, Inc., Berkeley, CA_.

Inhibitors against the bromodomain and extra terminal domain (BET) family of proteins have been pursued as promising oncology agents based on growing understanding of epigenetic control of disease processes. Through scaffold-based and crystallography-guided drug design, we discovered PLX51107, a potent and selective small molecule inhibitor of the BET family bromodomains. PLX51107 is structurally unrelated to the benzodiazepines such as JQ1, I-BET762, and OTX015 and other published BET inhibitors. PLX51107 exhibits low nanomolar potency in blocking interactions mediated by the four BET family proteins BRD2, BRD3, BRD4, and BRDT. Pharmacologic inhibition of BET proteins by PLX51107 suppresses the transcription of genes essential for tumor growth and survival and leads to selective killing of cancer cell lines across a broad range of hematologic malignancies (e.g. leukemia, lymphoma and multiple myeloma). A subset of solid tumors (e.g. melanoma and SCLC) is also sensitive to growth inhibition by the BET inhibitor PLX51107. Novel biomarkers in these diseases have been identified. PLX51107 is well tolerated and has sufficient potency and oral bioavailability to demonstrate in vivo efficacy in animal models of a variety of tumor types, representing both hematologic and solid tumors of diverse genetic backgrounds. In combination studies, PLX51107 showed potential to improve the efficacy (response rates and duration of response) of other anticancer treatments without increased toxicity. These results support further development of PLX51107 as an epigenetic-based therapy for a variety of cancer indications.

#4712

Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML.

Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. _Incyte Corporation, Wilmington, DE_.

Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape,

often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we

describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML.

#4713

BET bromodomain degradation as a therapeutic strategy in drug-resistant multiple myeloma.

Geoffrey M. Matthews, Yiguo Hu, Michal Sheffer, Paul J. Hengeveld, Dennis L. Buckley, Megan A. Bariteau, Haley Poarch, James E. Bradner, Constantine S. Mitsiades. _Dana Farber Cancer Institute, Boston, MA_.

Multiple myeloma (MM) is an incurable malignancy with an unmet need for novel therapeutic modalities. Moreover, acquired or de novo resistance to established or novel therapeutics remains a major challenge in this, and other, neoplasias. BET Bromodomain inhibitors (BBIs), including JQ1, have potent anti-MM activity in vitro and in in vivo, but do not provide curative outcome and do not induce apoptosis in most cell types. We sought to investigate dBET, a class of BBIs that induce degradation of BET Bromodomains (BRDs) through CRBN-mediated ubiquitination and proteasomal degradation, in drug-resistant MM. Additionally, we posited that resistance to dBET treatment would emerge through genetic perturbations and wished to uncover potential mechanisms prior to its clinical utilization. To address this, we compared effects of optimized lead compound, dBET6, with JQ1 on a panel of MM cell lines, including clones resistant to JQ1 or bortezomib and assessed viability using CS-BLI/CTG assay and BRD/c-MYC expression by western blot. Using an open-ended unbiased genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)-associated Cas9 approach, we examined whether we could uncover genes associated with resistance to dBET6. MM1.S cells were transduced with Cas9 and pooled lentiviral particles of the GeCKO library, consisting of 2 pooled sgRNA sub-libraries (~120,000 sgRNAs; targeting ~19,000 genes and ~1800 miRNAs). Using this CRISPR/Cas9-based approach we sought to expedite the isolation of MM cells resistant to dBET6. We treated the pool of cells thrice with dBET (≥IC80), allowing regrowth between treatments and maintaining a coverage of 1000 cells/sgRNA. dBET6-resistant cells were processed to quantify sgRNA enrichment or depletion, using deep sequencing. We observed dBET6 to have significantly greater potency against MM cells than JQ1, or its combination with lenalidomide, and that MM1S.CRBN-/- cells were resistant to dBET6. Resistance to neither JQ1 nor Bortezomib conferred resistance to dBET6. We observed dBET6 to induce rapid (<4hrs) degradation of BRD2, BRD3 and BRD4 and complete absence of c-MYC protein, in contrast to JQ1 which caused dose-dependent down-regulation of c-MYC, and apparent upregulation of BRD4. As predicted, our CRISPR/Cas9 screen identified significant enrichment of sgRNAs targeting CRBN, as well as the Cullin-RING ligase (CRL) complex, known to play a critical role in E3 ubiquitin ligase activity. In summary, our data support the development of dBET for the treatment of drug-resistant MM. Additionally, our results demonstrate that loss of function of CRBN or the CRL complex induces dBET resistance by perturbing dBET-mediated BRD4 degradation. However, it is plausible that additional CRBN/CRL-independent mechanisms of dBET resistance exist that allow cells to survive despite complete degradation of BRDs and this will be a key question to be answered in future studies.

#4714

In vivo **efficacy of BET inhibitor BAY 1238097 in preclinical models of melanoma and lung cancer.**

Olaf Klingbeil, Bernard Haendler, Antje Stresemann, Claudia Merz, Annette Walter, Amaury Ernesto Fernández-Montalván, Matthias Ocker, Stephan Siegel, Pascale Lejeune. _Bayer Pharma AG, Berlin, Germany_.

Bromodomain and extra terminal domain (BET) family proteins which bind to acetylated lysines of histones and regulate gene transcription are promising targets in oncology. We have previously shown that the BET inhibitor BAY 1238097 possesses strong activity in a number of hematological models, both in vitro and in vivo (AACR 2015). Here we show that BAY 1238097 is also able to inhibit the growth of solid tumors such as melanoma and lung tumors.

BAY 1238097 was evaluated in vitro against a panel of melanoma cell lines and was effective in BRAF wild-type as well as BRAF mutant models. When given to C57BL/6 mice at the maximum tolerated dose (MTD) of 15 mg/kg, p.o., qd, BAY 1238097 showed efficacy against the B16/F10 syngeneic model with a T/C of 31% on day 12 post tumor inoculation (T/C≤42%=active according to NCI criteria). In this study, dacarbazine was less active than BAY 1238097 with a T/C of 44%. This was also the case in a patient-derived melanoma model resistant to dacarbazine where BAY 1238097 was found active with 39% T/C. Potent efficacy of BAY 1238097 was also observed against the human LOX-IMVI model in SCID mice either using a daily dose of 15 mg/kg or a q3d schedule at 45 mg/kg (MTD), leading to 10% and 13% T/C, respectively on day 12. Dose-dependent down-regulation of the MYC oncogene was demonstrated ex vivo in B16/F10 tumors as well as in mice peripheral blood mononuclear cells (PBMCs) with maximal effect observed between 3 to 6h post oral treatment with BAY1238097. In addition, strong up-regulation of HEXIM1 mRNA was observed in tumors and PBMCs from the mice.

BAY 1238097 was also evaluated in a number of in vitro and in vivo studies in lung cancer cell lines. It was strongly active in KRAS mutant non-small cell lung cancer (NSCLC) (DV-90, NCI-H1373, LCLC-97TM1) and small cell lung cancer (SCLC) models (NCI-H69, NCI-H146, NCI-H526) with IC50 values below 1 µM. Down-regulation of c-Myc protein expression upon treatment with 1 µM was observed in all three sensitive NSCLC models. In the NCI-H1373 model, an induction of caspase 3/7 activity was observed upon 24h of treatment in vitro. In the same model implanted in vivo, a strong reduction of tumor growth was observed following daily oral application of BAY 1238097 (12 mg/kg). The treatment was well tolerated with a T/C of 16% at day 15 after start of treatment. A similar antitumor activity was obtained by applying the compound intravenously twice a week at 100 mg/kg and ex vivo analysis revealed time-dependent regulation of MYC and HEXIM1 after treatment. In vivo efficacy was not limited to NSCLC as daily oral application of BAY 1238097 (10 mg/kg) to the SCLC xenograft model NCI-H526 resulted in high efficacy with 7% T/C on day 21.

Taken together, these positive results in preclinical models of melanoma and lung tumors warrant further evaluation of BET inhibition in solid tumors in the clinic.

#4715

A novel small molecule with both anti-angiogenic and anti-tubulin mechanisms of action as an oral treatment for glioma.

Frank L. Sorgi,1 Aleem Gangjee2. 1 _FLAG Therapeutics, Inc., Raleigh, NC;_ 2 _Duquesne University, Pittsburgh, PA_.

FLAG-003 is a novel small molecule (molecular weight of 283) that has been designed as a bi-functional compound exhibiting both anti-angiogenic and anti-tubulin effects. Binding studies have been performed to fully elucidate the mechanisms of action. The compound exhibits a selective inhibition of EGFR, VEGFR-2 and PDGFR-ß kinase activity only. The anti-tubulin activity is achieved by selective binding to the colchicine site on tubulin, thereby inhibiting tubulin proliferation.

Preclinical data from the NCI 60 cell line panel showed tumor cell inhibition in the 10-50 nM range across a wide variety of cell lines. Early MTD studies suggested the possibility that this molecule could cross the blood brain barrier (BBB). Although preliminary animal studies demonstrated potent activity in triple negative breast cancer and NSCLC models, DMPK studies suggested an ability to penetrate tight junctions and showed high levels of brain penetration so a glioma model was considered. Animal studies with 3 different glioma strains demonstrated the ability to shrink established glioma tumors located in the flank or in the brain following IP injections.

Brain cancers are typically unresponsive to most chemotherapeutics, with the exception of alkylating agents. This is likely due due to their small molecular weight and ability to penetrate the BBB but the utility of alkylating agents to treat gliomas are predominantly limited to those tumors with a damaged (methylated) MGMT (methyl guanidine DNA methyltransferase) gene. Tumors with inactivated MGMT genes are susceptible to treatment with temozolomide (TMZ) but this only represents approximately 30% of all glioma patients. MGMT repairs damage caused by alkylating agents and patients with an intact MGMT gene who are administered TMZ (an alkylating agent) fair no better clinically that those patients who are untreated. These 70% of glioma patients with active MGMT genes have no current approved therapy beyond surgery and radiation therapy and therefore represents a huge unmet medical need. Several new treatments have centered around ways to inactivate the MGMT gene and make TMZ therapy more effective, rather than seek new agents that are more effective in treating more glioma patients.

This new class of dual-acting molecules exhibits their cytotoxic effect via tubulin inhibition rather than via alkylation and therefore may be applicable in treating all glioma patients without regard to their MGMT status. Preclinical DMPK studies have demonstrated the target selectivity and brain penetration attributes necessary to warrant further development. The molecule demonstrates oral bioavailability in three animal species and the pharmacokinetic properties lend themselves to daily dosing. Therefore, FLAG-003 is a viable candidate to progress into clinical trials and test in patients as a possible therapy for the treatment of glioma.

#4716

Dimethyl fumarate impairs breast cancer growth and inhibits the nuclear factor κB pathway in breast cancer cells by covalent modification of p65.

Irida Kastrati, Marton I. Siklos, Esther L. Calderon-Gierszal, Gregory R. J. Thatcher, Jonna Frasor. _University of Illinois at Chicago, Chicago, IL_.

Despite major advances in breast cancer treatment, successful therapy outcome is limited to early detection of cancer at the primary organ. Therapy options for aggressive, advanced stage, metastatic and recurring cancers are limited and this translates to poor patient outcome contributing to over 40,000 deaths each year in the United States. A key underlying factor of aggressive breast cancers is activation of the inflammatory nuclear factor κB (NFκB) pathway because it promotes numerous phenotypes such as cell survival, migration, invasion, angiogenesis, stem cell-like properties, and resistance to therapy. Therefore, adding an anti-inflammatory/anti-NFκB agent to breast cancer treatment would be beneficial, but no such drug is approved as either a mono- or adjuvant therapy. One option is Tecfidera® (dimethyl fumarate, DMF), the anti-inflammatory drug already in clinical use for multiple schlerosis. DMF is neuroprotective and is proposed to act via inhibition of the NFκB and activation of Nrf2 pathway. Most importantly, DMF has a proven safety in humans. This makes DMF an attractive candidate for NFκB inhibition in breast cancer therapy. Moreover, its therapeutic potential in treating breast cancer has yet to be explored.

We find that DMF inhibits proliferation of breast cancer cells in vitro. In addition, DMF abrogates mammosphere formation, a functional measure of cancer stem cell (CSC) properties. This prompted us to examine its activity in a murine breast cancer xenograft model. We find that DMF (30mg/kg daily) significantly impairs MDA-MB-231 tumor growth in athymic nude mice. As an anti-inflammatory agent, we show that DMF effectively blocks NFκB activity in multiple breast cancer cell lines. Mechanistically, DMF prevents p65 nuclear translocation and attenuates its DNA binding activity, but has no effect on upstream proteins in the NFκB pathway. Dimethyl succinate, the inactive analog of DMF that lacks the electrophilic double bond of fumarate, is unable to inhibit NFκB activity. Also, the cell permeable thiol, N-acetyl L-cysteine, reverses DMF's inhibition of the NFκB pathway, supporting the notion that the electrophile, DMF, acts via covalent modification. To determine whether DMF directly interacts with p65, we synthesized and used a novel chemical probe of DMF by incorporating an alkyne functionality, and found that DMF covalently modifies p65 at the cysteine 38 residue.

Altogether, these results establish DMF as a safe and effective NFκB inhibitor in breast cancer cells and elucidate its mechanism of action. These in addition to DMF's anti-tumor activity support the advancement of DMF as a therapeutic option to treat aggressive breast cancers in the clinic.

#4717

Clinical implications for m-tyrosine, an isomer of p-tyrosine, for the treatment of aggressive prostate tumors.

Geraldine Gueron,1 Nicolás Anselmino,1 Paula Chiarella,2 Emiliano Ortiz,1 Alejandra Paez,1 Jimena Giudice,3 Federico Schuster,1 Daiana Leonardi,1 Felipe Jaworski,1 Estefania Labanca,4 Verónica Manzano,5 Javier Cotignola,1 Roberto Meiss,6 Norma D´Accorso,5 Nora Navone,4 Raul Ruggiero,6 Elba Vazquez1. 1 _School of Sciences- Univ. of Buenos Aires - IQUIBICEN- CONICET, Buenos Aires, Argentina;_ 2 _IMEX-CONICET - National Academy of Medicine, Buenos Aires, Argentina;_ 3 _Baylor College of Medicine, Houston, TX;_ 4 _MDAnderson Cancer Center, Houston, TX;_ 5 _School of Sciences- Univ. of Buenos Aires -CIHIDECAR-CONICET-UBA, Buenos Aires, Argentina;_ 6 _IMEX- CONICET, National Academy of Medicine, Buenos Aires, Argentina_.

Clinical and experimental evidence suggest that primary tumors may exert a controlling action on its metastases. The phenomenon, by which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis, is known as concomitant tumor resistance (CR).

We have previously showed in murine T-lymphoma (LB) tumors, that meta-tyrosine (m-Tyr) an isomer of tyrosine not present in normal proteins, is the main serum anti-tumoral factor responsible for CR. In this work, we assess for the first time the CR phenomenon in human prostate cancer (PCa). Athymic nude mice were inoculated with PC3 cells (primary implant) and after 14 days the animals received a second inoculation (secondary implant). Strikingly, the growth of the secondary implant was significantly reduced after 27 days, in animals carrying the primary xenograft. When phenylalanine (Phe), a protective amino acid highly present in primary tumors, and precursor of p-tyrosine, was periodically inoculated at the site of a secondary tumor implant (otherwise inhibited by CR), this secondary implant grew similarly to controls. On the contrary, when m-Tyr was inoculated at the site of a primary tumor implant or systemically, this implant did not grow. Tumor inhibition was associated with low expression of Ki-67 and STAT3.

In vitro analyses demonstrate the higher inhibitory activity of the serum from tumor-bearing mice on PC3 cell proliferation, compared to serum from control animals. m-Tyr could account for most of the growth-inhibitory activity present in the serum. Furthermore, we observed an increase in the frequency of Gr1+ CD11b+ MDSCs in bone marrow, spleen and lymph nodes from tumor-bearing mice compared to control mice. This expansion correlated with a significantly higher production of reactive oxygen species and enhanced suppressor function upon CD8+ T cell proliferation. Further, in vitro studies also showed that exposure of PC3 cells to m-Tyr inhibited cell growth, induced G0/G1 cell cycle arrest, altered the expression levels of survivin, Ki67 and Hes1; impaired the NFκB/STAT3 pathway and induced autophagy; effects reversed by Phe treatment. Strikingly, m-Tyr periodic intravenous administration provoked a dramatic reduction of experimental lung metastases generated in mice bearing PC3 human tumors. Altogether, we demonstrate for the first time that RC occurs in experimental human solid tumors, that this effect is mediated by m-Tyr, a non-cytotoxic metabolite with high potential clinical implications for metastatic PCa.

#4718

ABBV-075, a novel BET family bromodomain inhibitor, represents a promising therapeutic agent for a broad spectrum of cancer indications.

Aparna Sarthy, Leiming Li, Daniel H. Albert, Xiaoyu Lin, Warder Scott, Emily Faivre, Mai H. Bui, Xiaoli Huang, Denise M. Wilcox, Terry Magoc, Fritz G. Buchanan, Paul Tapang, George S. Sheppard, Le Wang, Steve D. Fidanze, John Pratt, Dachun Liu, Lisa Hasvold, Paul Hessler, Tamar Uziel, Lloyd Lam, Ganesh Rajaraman, Guowei Fang, Steven W. Elmore, Saul H. Rosenberg, Keith McDaniel, Warren Kati, Yu Shen. _Abbvie, North Chicago, IL_.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins have emerged as a promising option for cancer therapy. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 clinical trials. It binds bromodomains of BRD2/4/T with similar affinities (Ki of 1-2.2 nM), but exhibits roughly 10-fold weaker potency towards BRD3 (Ki of 12.2 nM). ABBV-075 is highly selective for 18 bromodomain proteins tested (Kd > 1 μM; more than 600-fold selectivity vs. BRD4) and has moderate activity towards CREBBP (Kd = 87 μM; 54-fold selectivity vs. BRD4). ABBV-075 exhibited robust single agent activity in cell viability assays across cancer cell lines derived from solid tumors, leukemia and lymphomas. Further characterization of cancer cell responses to ABBV-075 indicated that ABBV-075 manifested diverse mechanisms of action in different cancer settings. These include 1): disruption of cell cycle control leading to G1 arrest followed by senescence, 2) inhibition of oncogenesis drivers leading to apoptosis and 3) potentially targeting tumor microenvironment to provide additional therapeutic benefit. Consistent with its broad spectrum of activities in vitro, ABBV-075 has comparable or superior efficacies to standard of care agents in flank xenograft mouse models of non-small-cell and small cell lung cancers, pancreatic, breast, prostate, head & neck cancers, multiple myeloma, diffuse large B cell lymphoma and leukemia. These results support the development of ABBV-075 in diverse hematological malignancies and solid tumor indications.

#4719

Varlitinib demonstrates potent antitumor efficacy in patient-derived gastric cancer xenograft models.

A. G. Lisa Ooi,1 Bertil E. Lindmark,1 Mark McHale,1 Huynh T. Hung,2 Richard Ong2. 1 _ASLAN Pharmaceuticals, Singapore, Singapore;_ 2 _National Cancer Centre Singapore, Singapore, Singapore_.

Varlitinib is a potent, reversible pan-HER inhibitor of the ATP-binding site of EGFR (IC50 = 7 nM), HER2 (IC50 = 2 nM) and HER4 (IC50 = 4 nM). Gastric Cancer is the fifth most common cancer worldwide, with 952,000 cases diagnosed in 2012, constituting 6.8% of total cancer diagnosis. Prognosis for gastric cancer is poor, with the overall 5-year survival rate for gastric cancer in the US at ~29%. Current clinical guidelines recommend use of trastuzumab combined with chemotherapy for HER2+ (amplified) patients with advanced gastric cancer. However the majority of patients (~80%) do not have HER2 amplification . Studies have implicated dysregulation of HER signaling in gastric cancer and there is strong scientific rationale for testing of pan-HER therapies in Gastric Cancer. We tested the anti-tumour activity of Varlitinib as a single agent in patient-derived gastric cancer xenograft in SCID mice (GC22-0808 & GC11-0414) with over-expression of HER proteins. Varlitinib is very active in tumour growth inhibition studies, causing dose-dependent inhibition with tumour stasis observed at dosing of 50 mg/kg BID and tumour regression observed at dosing of 100 mg/kg BID. Tumour protein lysates after two-day treatment with Varlitinib and vehicle were also analysed to elucidate the mechanism of action. Multiple proliferation and anti-apoptosis pathways were inhibited by Varlitinib, including AKT, PI3K and Survivin pathways. In view of the robust anti-tumour activity inhibiting multiple survival and proliferation pathways, as well as the safe tolerabilty profile of Varlitinib, ASLAN has initiated Phase 2 clinical trials of Varlitinib in gastric cancer.

### HDAC, Methyltransferase Inhibitors, and Novel Anticancer Agents

#4720

Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and panobinostat in a mouse model of multiple myeloma.

Sivan Elloul, Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. _Karyopharm Therapeutics Inc, Newton, MA_.

Introduction: Selinexor is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports over 200 cargos, including major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. XPO1 also mediates the export of key signaling molecules in multiple myeloma (MM) including c-MYC mRNA and NF-κB. Selinexor is currently being investigated in phase 2 clinical trials in MM in combination with dexamethasone, pomalidamide, lenalidomide, bortezomib and carfilzomib. In preclinical studies selinexor has been shown to synergize broadly with these anti-MM drugs making it an excellent candidate partner for combination therapies in MM. To identify additional synergistic pairings, we investigated the use of selinexor in combination with panobinostat, a pan-histone deacetylase inhibitor (HDAC) recently approved by the FDA in combination with bortezomib for 3rd line treatment of MM, as a potential treatment for MM both in-vitro and in-vivo.

Methods: The effects of selinexor and panobinostat alone or in combination on cell viability were tested on MM1.S and NCI-H929 cell lines using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, MM.1S cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of selinexor and panobinostat alone or in combination and with the therapeutic dose of selinexor. Tumor growth was monitored for 17 days and % Tumor Growth Inhibition (%TGI) was determined. Xenografts were harvested and analyzed microscopically and by immunohistochemestry (IHC).

Results: Selinexor-panobinostat combination was highly effective both in-vitro and in-vivo. In MTT assays, both selinexor and panobinostat demonstrated low IC50 values (nanomolar) and when combined, they synergistically/ additively inhibit proliferation. Gene expression and western blot analyses showed that the combination treatment leads to a synergistic reduction in c-MYC mRNA and protein levels. In addition, an overall reduction in expression of anti-apoptotic signaling molecules including NF-κB and p21 and an increase of expression of pro-apoptotic molecules including cleaved caspase 3 and BAX were observed. Importantly, in-vivo, while %TGI of sub-therapeutic doses of selinexor and panobinostat measured 58% and 52% respectively, the combination showed a synergistic effect, measuring 93%. Importantly this high %TGI exceeded the effect of the therapeutic dose of selinexor alone 79%.

Conclusion: Selinexor-panobinostat combination synergizes to induce apoptosis in MM cells and amplifies anti-tumor effect in a MM xenograft model. These data provide rational support for study of selinexor/ panobinostat combination in clinical trials.

#4721

Enhancement of doxorubicin cytotoxicity by histone deacetylase inhibition in human sarcoma cells.

Maria Grazia Tupone,1 Daniela Trisciuoglio,1 Marianna Desideri,1 Marta Di Martile,1 Simonetta Buglioni,1 Barbara Antoniani,1 Rossella Loria,1 Rita Falcioni,1 Michele Milella,1 Virginia Ferraresi,1 Gemma Di Pompo,2 Nicola Baldini,2 Roberto Biagini,1 Donatella Del Bufalo1. 1 _Regina Elena National Cancer Institute, Rome, Italy;_ 2 _Istituto Ortopedico Rizzoli (IOR), Bologna, Italy_.

Introduction: Soft tissue and bone sarcomas are rare malignancies of mesenchymal origin that account for about 1% of all adult solid tumors. Surgical resection is the first treatment option but is heavily hampered by delayed diagnosis. Due to few therapeutic treatments available, novel and efficacious therapy is urgently needed. Histone deacetylase inhibitors (HDACi) are emerging as a prominent class of therapeutic agents for several cancers; however, little is known about HDACi activity in sarcomas. By using human sarcoma models, we studied the therapeutic activity of ITF2357, a novel histone deacetylase inhibitor, as an anti-cancer agent alone or in combination with doxorubicin, on both wild-type and mutated p53-bearing sarcoma cell lines.

Materials and methods: A wide panel of sarcoma cell lines, including both established and patient-derived sarcoma from different histotypes, were used. Western Blot analysis was performed to evaluate the effect of ITF2357 on histone and non-histone protein acetylation and expression of potential downstream targets. Cell viability was tested by MTT and CellTiterGlo assays. Apoptosis induction and cell cycle perturbation were assessed by flow cytometry and Western Blot analysis. Autophagy was assessed by analysis of autophagosome formation in EGFP-LC3B expressing cells and Western Blot analysis. Pharmacological interaction between ITF2357 and Doxorubicin was assessed by conservative isobologram analysis. In vivo efficacy of drug combination was evaluated in nude mice bearing sarcoma xenografts.

Results: In sarcoma cell lines, HDAC inhibition induced H3 histone and alpha-tubulin acetylation, cell proliferation inhibition and activation of apoptotic program in a dose-dependent manner. ITF2357 cytotoxicity was independent on p53 status, despite its ability to inhibit the expression of mutated p53 protein. Moreover, ITF2357 induced a canonical-autophagic process, which protects sarcoma cells from apoptotic cell death and enhances cell survival. The combination of doxorubicin and ITF2357 was found to be highly synergistic in terms of cell proliferation inhibition and was related to the engagement of the apoptotic mitochondrial pathway. Noteworthy, treatment with both drugs also showed synergistic inhibition of cell viability in a doxorubicin-resistant osteosarcoma line, as compared with either agent alone. The effect of the drug combination was corroborated in sarcoma stem cells obtained from sarcoma patients. In vivo experiments in mouse xenograft models confirmed that co-treatment increased both inhibition of tumor growth and apoptosis, as compared with either agent alone.

Conclusions: Overall, HDAC inhibition in sarcoma may provide an effective therapeutic modality capable of overcoming the doxorubicin resistance, and thus may lead to more durable responses in the clinic.

#4722

Selective inhibition of DNA methyltransferases and efficacy of novel DNMT inhibitors in leukemia xenografts.

Dat Nguyen, Melinda Hollingshead, Larry Rubinstein, James Doroshow, Sherry X. Yang. _National Cancer Institute, Bethesda, MD_.

Background. DNA methyltransferase (DNMT) 1 plays an important role in tumorigenesis and is responsible for maintenance and propagation of DNA methylation patterns, and DNMT3A and DNMT3B are involved in de novo DNA methylation. 4'-Thio-5-aza-2'-deoxycytidine (5-aza-T-dCyd) and 4'-thio-2'-deoxycytidine (T-dCyd) are novel DNMT inhibitors in development. In this investigation, we test the hypothesis that DNMTs are differentially modulated by DNMT inhibitors that may contribute to treatment efficacy. Methods. Animals (5 per group) bearing HL-60 leukemia cell xenografts were treated with saline or aza-T-dCyd at 2 mg/kg with and without 100 mg/kg tetrahydrouridine (ThU) for 5 days weekly for 2 weeks and cycle repeat after 9-day rest. Two other groups were dosed with T-dCyd at 4 mg/kg and decitabine at 0.75 mg/kg, respectively. Tumor growth was assessed by measuring tumor size. Expression of DNMT1, DNMT3A and DNMT3B was examined by immunohistochemistry in paraffin-embedded tumors resected at day 11. Results. Expression of DNMT1 was high, DNMT3B intermediate, and DNMT3A at low levels in saline control group. Significant growth delay was achieved in the aza-T-dCyd (86%) and aza-T-dCyd plus ThU cohorts (45%), compared with the controls. In the aza-T-dCyd treated xenografts, histological examination confirmed a significant reduction (77%) in tumor area (P=0.002). Expression of DNMT1 and DNMT3B (n=2 in aza-TdCyd group) was reduced in aza-T-dCyd cohort (P=0.01 and P=0.04) as well as aza-T-dCyd in combination with ThU group (P=0.01 and P= 0.03). In contrast, no growth inhibition but decrease in DNMT3A and DNMT1 were observed by dosing with decitabine. There were neither delays for tumor growth nor changes of all three DNMTs by T-dCyd treatment with the dose administered. Conclusions. Aza-T-dCyd and aza-T-dCyd plus ThU treatments result in significant tumor growth inhibition in HL-60 leukemia xenografts. The anti-tumor activity is associated with simultaneous inhibition of the two drug targets — DNMT1 and DNMT3B. The preclinical results provide compelling evidence to advance aza-T-dCyd for clinical development and perhaps in the context of companion biomarker approach.

#4723

Innovative cancer treatment of human lung cancer cells PC-9 with a synthetic retinoid Am80 and EGCG via inhibition of HDAC4 and HDAC5.

Masami Suganuma,1 Yukiko Oya,1 Sonthaya Umsumarng,1 Keisuke Iida,1 Anchalee Rawangkhan,1 Ryo Sakai,1 Hiroyuki Kagechika,2 Koichi Shudo,3 Hirota Fujiki4. 1 _Saitama University, Saitama, Japan;_ 2 _Tokyo Medical and Dental University, Tokyo, Japan;_ 3 _Japan Pharmaceutical Information Center, Tokyo, Japan;_ 4 _Saga University, Saga, Japan_.

A synthetic new retinoid Am80 (tamibarotene) is used for treatment of acute promyelocytic leukemia (APL) patients with resistance of all-trans-retinoic acid (ATRA). To potentiate efficacy of Am80, we challenged combination treatment of Am80 and EGCG on human lung cancer cell line PC-9. It is now well accepted that EGCG acts as synergist with numerous anticancer compounds, and that the average reduction of tumor volume for in vivo genograft mouse models implanted using various human cancer cell lines was 70.3% with combinations of 13 anticancer compounds and EGCG. We previously reported that combinations of NSAIDs (sulindac and celecoxib) or other retinoids (ATRA, 13-cis-RA and 9-cis-RA) and EGCG synergistically induced apoptosis of the cells along with strong expression of growth arrest and DNA-damage inducible gene 153 (GADD153) and death receptor (DR5) gene. Our objective was to investigate the mechanism of the synergistic induction for GADD153-DR5 apoptosis pathway. The proteomic analysis of lysine-acetylated proteins was conducted with a shotgun analysis using nanoLC-ESI-MS/MS in PC-9 cells. We found that the numbers of acetylated proteins were different: 551 acetylated proteins were found in non-treated cells, 331 proteins in cells treated with combination, and 59 proteins were common in both cases. Acetylation was mainly found in non-histone proteins, chaperons, transcription factors, and enzymes. The combination increased acetylated p53 (Lys382) about 3-fold compared with non-treated cells. In addition, combination significantly decreased the protein amounts of histone deacetylase (HDAC) 4 and HDAC 5 to 50% and 20%, respectively, but not HDAC6 among Class II HDACs. Class I HDACs (HDAC1, 2, and 3) were not decreased. All the results suggest that combination of Am80 and EGCG induces GADD153-DR5 apoptotic pathway, and reduces the amounts of HDAC4 and HDAC5 resulting in changing protein acetylation.

#4724

Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 synergize with DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML).

Chengyin Min, Steven N. Quayle, Pengyu Huang, Jeffrey R. Shearstone, Simon S. Jones, Min Yang. _Acetylon Pharmaceuticals, Boston, MA_.

AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain generally poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor VIDAZA™ (azacitidine) is approved by European Medicines Agency for the treatment of elderly patients with AML. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML.

HDAC inhibitors are promising agents for the treatment of AML. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have suggested beneficial clinical activity in AML. However, the toxicity profiles of non-selective HDAC inhibitors in the combination settings limit the clinical benefit. In this work, we describe the preclinical development of highly selective HDAC inhibitors, which are hypothesized to have improved safety profiles than non-selective HDAC inhibitors, for combination therapy in AML. We demonstrate that selective HDAC1/2 inhibitors are highly effective and efficacious both as single agents and in combination with azacitidine in preclinical models of AML, including established AML cell lines, bone marrow samples freshly derived from AML patients and in vivo xenograft models of human AML. At the molecular level, gene expression profiling, DNA methylation mapping and chromatin immunoprecipitation analysis were performed on AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the drug combination. Molecular signatures and genes involved in myeloid cell differentiation, apoptosis and cell cycle arrest were identified as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients.

#4725

Inhibition of DNA methyltransferase by RX-3117 (fluorocyclopentenylcytosine) leads to upregulation of hypomethylated targets.

Godefridus J. Peters,1 Dzjemma Sarkisjan,1 Joris J. Julsing,1 Ahmed Hassan,1 Kees Smid,1 Ietje Kathmann,1 Young Lee,2 Deog J. Kim2. 1 _VU University Medical Center, Amsterdam, Netherlands;_ 2 _Rexahn Pharmaceuticals, Rockville, MD_.

RX-3117 (Fluorocyclopentenylcytosine; FCPEC) is an orally bioavailable novel cytidine analog which is currently being evaluated in a Phase I clinical study. Downregulation of DNA methyltransferase 1 (DNMT1) by RX-3117 was shown in various cell lines with different histological backgrounds. In the present study we determined the effect of RX-3117 on DNMT1 at the DNA, RNA, protein and enzyme activity, and reactivation of suppressed target genes, including p16INK4A, methylguanine methyltransferase (MGMT) and the proton coupled folate transporter (PCFT).

In the moderately sensitive non-small cell lung cancer (NSCLC) cell lines such as A549 and SW1573, 5-50 µM RX-3117 downregulated DNMT1 protein expression by 5-20% after 24 hr exposure and >90% after 48 hr. DNMT1 mRNA was not affected after 24 hr exposure but was affected moderately after 48 hr. In the sensitive ovarian cancer cell line A2780, protein down regulation was already observed after 24 hr at 1 µM RX-3117. DNMT1 activity was inhibited by 1 µM RX-3117 by 32% which was similar to the percent inhibition with 5 µM of the reference compound 5-aza-2'-deoxycytidine (DAC, 31%). In A549 cells 5 µM RX-3117 decreased overall methylation of DNA (detected by an antibody against 5-methylcytosine) by 25% after 48 hr exposure, while 5 µM DAC only inhibited 9%. For several genes known to be affected by methylation, we evaluated their protein expression and activity. In A549 and SW1573 cells a 24 hr exposure to 5 µM RX-3117 increased the expression of the cell cycle protein p16INK4A and of the DNA repair enzyme MGMT.

For PCFT we evaluated its functional activity in CCRF-CEM leukemic cells which have a highly methylated PCFT promoter and in CEM-MTX cells which are deficient for the reduced folate carrier (RFC). PCFT is a specific folate transporter responsible for uptake of folic acid and the folate analogs methotrexate (MTX) and pemetrexed. Incubation of both CEM and CEM-MTX cells with either 29.6 µM RX-3117 or DAC as a positive control markedly increased PCFT mediated transport of MTX. This was more pronounced in CEM-MTX cells, 10-11-fold increase for both RX-3117 and DAC, compared to a 4-fold increase in CEM cells.

In conclusion, the down-regulation of DNMT1 by RX-3117 was accompanied by a decrease in DNMT1 protein expression and enzyme activity, resulting in up-regulation of proteins of MGMT, PCFT, and the tumor suppressor gene p16INK4A. These data underline DNMT1 inhibition as a novel mechanism of RX-3117.

#4726

Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell-mediated lysis of prostate and breast carcinoma cells.

Sofia R. Gameiro,1 Anthony S. Malamas,1 Massimo Fantini,1 Kwong Y. Tsang,1 Soldano Ferrone,2 James W. Hodge1. 1 _NCI-CCR, Bethesda, MD;_ 2 _Department of Surgery, Massachusetts General Hospital, Boston, MA_.

The clinical promise of cancer immunotherapy relies on the premise that the immune system can effectively recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition and thus promote long-lasting tumor control when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. Although epigenetic therapies have shown limited clinical benefit for patients harboring solid tumors, recent clinical reports suggest a strong potential for clinical synergy when combined with immunotherapy. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate and breast carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC-1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

#4727

ST7612AA1: focus on a drug candidate, front-runner of a new generation of HDAC inhibitors.

Giuseppe Giannini, Ferdinando Maria Milazzo, Gianfranco Battistuzzi, Loredana Vesci. _Sigma-Tau, IFR, S.p.A., Pomezia, Italy_.

After more than 20 years of research in the field of HDAC inhibitors, the oncological armamentarium has significantly enriched with new approved drugs. Currently, four drugs, Vorinostat (Zolinza; 2006), Romidepsin (Istodax; 2009) Belinostat (Beleodaq; 2014) and Panobinostat (Farydak; 2015) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and multiple myeloma, respectively. A fifth, Chidamide (Epidaza; 2015), has been recently approved by the China Food and Drug Administration for the treatment of PTCL.

Beyond them, more than twenty are currently under pre-clinical and clinical investigation as single agent and in combination therapies against different cancers and in other several novel therapeutic indications. However, it is to emphasize a feature: among all these, no one, with the exception of the natural product Romidepsin, is a thiol derivative.

After efforts lasted years, now we have selected ST7612AA1 - a new thiol-ω(γ-lactam amide) SAHA analogues, potential front-runner of a new generation of HDAC inhibitors, [1] highly competitive respect to other HDAC inhibitors drugs, orally administered with a negligible toxicity.

ST7612AA1 displays low-nanomolar inhibitor activity on HDACs, being especially powerful on HDAC6 isoform, associated to a wide spectrum of antitumor activity, both in vitro and in vivo [2]; it can be industrially developed with a quick and easy synthetic process [3]. Moreover, it has also been investigated as an enhancer in antiretroviral therapy against HIV, aimed to eradicate the viral reservoirs [4].

All these results encouraged further enrichment experiments with ST7612AA1 as a drug candidate, which is currently in a phase of pre-clinical evaluation.

The overall profile of ST7612AA1, including synthesis and a comprehensive pharmacological characterization, will be presented.

[1] G. Giannini et al. J Med Chem. 2014, 57, 8358-8377

[2] L. Vesci et al. Oncotarget 2015, 6, 5735-48

[3] G. Battistuzzi, G. Giannini. Bioorg. Med. Chem .Lett. 2015, submitted

[4] R. Badia Antiviral Research 2015, 123, 62-69

#4728

Histone deacetylase inhibitors induce apoptosis in multiple tumor types through induction of ATF3.

Anderly C. Chueh,1 Janson Tse,1 Paul Ioannidis,1 Lars Tögel,1 Bee S. Tan,1 Mercedes Davlos-Salas,1 Rebecca Nightingale,1 Matthew R. Thompson,2 Bryan Williams,2 Michael Dickinson,3 Amardeep S. Dhillon,3 John M. Mariadason1. 1 _Olivia Newton-John Cancer Research Institute, Melbourne, Australia;_ 2 _Monash University, Melbourne, Australia;_ 3 _Peter MacCallum Cancer Institute, Melbourne, Australia_.

Histone deacetylase inhibitors (HDACi) are approved for the treatment of cutaneous T-Cell lymphoma (CTCL) and Multiple Myeloma. In colon cancer cells HDACi-induced apoptosis is linked to induction of a specific transcriptional response involving upregulation of the immediate-early (IE) response genes FOS, JUN, EGR1, EGR3 and ATF3. To determine if this transcriptional response underpins HDACi-induced apoptosis across multiple tumour types, including CTCL and MM, 50 cell lines derived from common solid and haematological cancers were screened for sensitivity to HDACi-induced apoptosis, and response correlated with IE gene induction. HDACi treatment robustly induced IE gene mRNA and protein expression in multiple tumour lines. IE gene induction was sustained over 24 hours and dependent on the Sp1/Sp3 transcription factors and was also observed in vivo in CTCL patients treated with the HDACi panobinostat. The magnitude of induction of FOS, JUN and ATF3 expression correlated significantly with HDACi-induced apoptosis. The induction of ATF3 was critical for HDACi induced apoptosis as ATF3 downregulation markedly attenuated HDACi-induced apoptosis in multiple tumour lines, and ATF3 knockout MEFs were refractory to HDACi-induced apoptosis. To identify downstream targets of ATF3, the magnitude of ATF3 induction following HDACi treatment was correlated with altered expression of pro and anti-apoptotic genes involved in the intrinsic apoptotic pathway. The magnitude of ATF3 induction correlated inversely with repression of the anti-apoptotic gene Bcl-XL, and ATF3 knockdown attenuated HDACi-mediated Bcl-XL repression. Notably, combinatorial treatment of refractory cell lines with an HDACi and a Bcl-XL inhibitor significantly enhanced HDACi-induced apoptosis. This study demonstrates that IE gene induction is a universal feature of HDACi-induced apoptosis, and that ATF3 as a key driver of HDACi-induced apoptosis through repression of Bcl-XL. These findings establish a method for rapid assessment of HDACi response, and identify a combination strategy for enhancing the activity of these compounds.

#4729

Class-selective histone deacetylase inhibitors differentially promote translation-dependent HER2 transcript decay in HER2-positive breast cancer cells.

Gary K. Scott, Katya Frazier, Sadaf Malik, Mariah Alejo, Birgit Schilling, Christopher Benz. _Buck Institute for Research on Aging, Novato, CA_.

Dysregulation of translational control is emerging as an essential aspect of tumorigenesis ripe for targeted therapeutic intervention. We have recently shown that polysome assembly can be differentially targeted by class IIB/HDAC6 selective histone deacetylase (HDAC) inhibitors (Wilson-Edell et al., Oncotarget, 2014), while translation-dependent and 3'UTR-targeted decay of oncogenic transcripts like HER2 is induced by pan-HDAC inhibition (Scott et al., Mol Cancer Res, 2008). To learn more about the HDAC dependency of oncogenic transcript stability, HER2-positive SKBr3 breast cancer cells were treated (2-24 h) with a pan-inhibitor of both class I and II HDACs (trichostatin-A/TSA), a class IIB > class I selective inhibitor (ACY-1215/Ricolinostat), class I/HDAC1-3 selective inhibitors (Entinostat, ACY-1035), and class IIB/HDAC6 selective inhibitors (ACY-775, Tubacin) for their respective abilities to induce HER2 mRNA decay (Northern blotting), reduce HER2 protein levels, induce acetylation of histone 2B and/or tubulin (Western blotting), and to reduce cell culture growth (MTT cell viability). In parallel studies, protein candidates potentially mediating HER2 transcript decay were identified by mass spectrometry of complexes specifically binding to the 3' UTR sense strand of HER2 mRNA. Polysome profiling demonstrated that pan-HDAC inhibition promoted protein candidates hnRNP-A2/B1 and the DEAD Box Helicase 5 (DDX5) to co-migrate with the 40S ribosome, and candidate hnRNP-K to co-migrate with higher (>60S) polysome fractions. Additionally, immunoprecipitates of these candidates were found to be acetylated following pan-HDAC inhibitor treatment. With regard to reducing cell viability (at 72 h) and HER2 protein levels (at 24 h), TSA and ACY-1215 proved to be the most potent inhibitors, causing >50% reductions at >0.5 uM doses, while equimolar doses of the HDAC6-selective inhibitors showed minimal effects and the class I inhibitors (Entinostat, ACY-1035) showed only intermediate effects on both cell viability and HER2 protein reduction. Interestingly, only the pan-HDAC inhibitor TSA and the class I/IIB selective inhibitor ACY-1215 appeared capable of activating HER2 mRNA decay within 6 h of treatment; and both of these HDAC inhibitors produced similar enhancement of hnRNP-A2/B1 and DDX5 co-association with the 40S ribosome fraction. In summary, while the translational machinery including ribosome (40S, 60S) and polysome protein complexes can be rapidly dysregulated by either class IIB/HDAC6 selective or pan-HDAC inhibitors, it appears that the rapid induction of HER2 mRNA decay requires inhibition of both class I and class IIB/HDAC6 HDACs. These findings suggest that the investigational HDAC inhibitor ACY-1215/Ricolinostat possesses optimal class-selective properties for use as an adjunct to treat HER2-positive breast cancers.

#4730

The histone deacetylase inhibitor panobinostat sensitizes cyclin E-amplified ovarian cancer cells to poly ADP-ribose polymerase inhibitor olaparib.

Kofi Sarfo-Kantanka,1 Andrew J. Wilson,2 Alexandra Steck,2 Jeanette Saskowski,2 Dineo Khabele2. 1 _Meharry Medical College, Nashville, TN;_ 2 _Vanderbilt University Medical Center, Nashville, TN_.

Objective: Ovarian cancer is the second most common gynecological cancer and the number one cause of cancer death in gynecological cancers. In the United States alone, there are about 21,290 expected new cases in 2015 and about 14,180 expected deaths. Approximately 95% of ovarian malignancies are epithelial ovarian cancer (EOC). Most (50%) of these EOC tumors have BRCAness features - contain molecular defect in homologous recombination (HR) DNA repair pathways. About 20% of the non-BRACness subtypes of EOC exhibit increased expression of cyclin E (CCNE1) which is associated with resistance to DNA damaging drugs and increased mortality. A first-in-class drug, a poly(ADP-ribose) polymerase inhibitor (PARPi) olaparib was approved by the FDA for the treatment of advanced ovarian cancer patients with BRCA mutations who have had three or more lines chemotherapy. PARPi efficacy is lower in non-BRCAness tumors but may be enhanced by other drug combinations. Our group has shown that epigenetic histone deacetylase inhibitor (HDACi) therapy sensitizes ovarian cancer cells to various chemotherapeutic drugs. Our goal is to expand olaparib therapy to patients with chemoresistant tumors by sensitizing CCNE1 amplified EOC with the potent HDACi, panobinostat. Method: Ovarian cancer cell lines used were OVCAR-3 (CCNE1-amplified) and SKOV3 (CCNE1-non-amplified). They were co-treated with 0.01% DMSO vehicle, olaparib, panobinostat or olaparib/panobinostat combination. Sulforhodamine B (SRB) assays assessed cytotoxicity and immunofluorescence (IF) assessed markers of apoptosis (cleaved caspase-3). Images were analyzed using the Adobe Photoshop counting tool. Data analysis was performed via the Microsoft Excel average and percentage functions. Figures and p-value analysis were created using GraphPad Prism. Results: Our previous studies have demonstrated through western blot analysis of cleaved PARP expression that panobinostat enhances the pro-apoptotic effect of olaparib in CCNE1 amplified OVCAR-3 ovarian cancer cells. In this current study, IF was used to assess the established marker of apoptosis, cleaved caspase-3, in OVCAR3 and SKOV3 cells. Compared to vehicle-treated cells, olaparib alone induced no difference in the number of cleaved caspase-3 expression in both SKOV3 and OVCAR3 cells. However, there was significant upregulation in the percentage of cells positive for cleaved caspase-3 expression in combination treatments compared to single drug treatments in both cell lines. Conclusion: The response of ovarian cancer cells to the PARPi olaparib is enhanced by co-treatment with HDACi panobinostat in vitro. This indicates that, responses to olaparib in HR-proficient EOC can be improved by combination therapy with panobinostat. Thus we have identified a novel way to expand the use of olaparib for the treatment of advanced ovarian cancer to patients with non-BRCA tumors.

#4731

Targeted therapies for prostate cancer: Strategies for efficient combinatorial approaches.

Nasir A. Butt,1 Swati Dhar,1 Avinash Kumar,1 Agnes M. Rimando,2 Xu Zhang,1 Anait S. Levenson1. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _United States Department of Agriculture, Oxford, MS_.

Metastasis-associated protein 1(MTA1) is a cancer progression- related epigenetic regulator, which is overexpressed in hormone-refractory and metastatic prostate cancer (PCa). MTA1/HDAC unit is a part of the multi-protein nucleosome remodeling and deacetylation (NuRD) complex. In our previous studies we found that MTA1 expression is significantly increased in prostate-specific Pten-null model, and that potent natural analog of resveratrol, pterostilbene (PTER), exerts its anti-tumorigenic effects by blocking MTA1-associated inactivation (deacetylation) of tumor suppressors. SAHA (suberoylanilide hydroxamine, vorinostat) is a histone deacetylase (HDAC) inhibitor that has been shown to be an effective inhibitor of tumor cell growth. We hypothesize that targeting MTA1/HDAC unit of NuRD complex using PTER in combination with SAHA could act additively or synergistically to block prostate tumor progression in vivo with higher efficacy and lower toxicity. In the current study, we utilized the prostate-specific Pten-null mouse model to evaluate the MTA1/HDAC mediated anti-cancer efficacy of combinatorial approach for dietary PTER and clinically approved HDAC inhibitor, SAHA. After a series of carefully designed breeding strategies and genotyping, we collected 30 prostate-specific luciferase expressing Pten knockout (Pten f/f; Rosa26Luc/+ ; Pb-Cre4) male mice for our experiments. Prostate-specific luciferase expression allowed non-invasive monitoring of prostate tumor growth in these animals. Mice were randomized into four groups: Vehicle control (10% DMSO); PTER (10 mg/kg bw) alone, SAHA (50 mg/kg bw) alone, and PTER + SAHA. Compounds were injected daily, i.p., starting at 8 weeks of age. Mice were sacrificed at week 18. Histopathological (H&E, SMA, CK-8), immunohistochemical (Ki-67, cleaved caspase-3, CD31) and molecular evaluation (MTA1, pAkt, AR) of prostate tissues showed beneficial effects of treatments alone and more so in combination compared to the control group.

#4732

A novel analog of Leonurine inhibits melanoma growth and survival through STAT3 signaling pathways.

Manoj K. Pandey, Jacek Krzeminski, Deepkamal Karelia, Arun K. Sharma, Shantu G. Amin. _Penn State University College of Medicine, Hershey, PA_.

Previous studies have shown that a major alkaloid of Leonurus japonicus Houtt, Leonurine, displays a variety of biological effects including inhibition of HMG-CoA reductase, kidney fibrosis, and osteoporosis. However, the full potential of this alkaloid as a cancer therapeutics has not been explored, primarily due to the lack of potency. A limited reports however, do suggest that Leonurine induces apoptosis in lung and liver cancer cells. We hypothesized that this natural product's structure can be optimized to create a more potent and drug-like molecule. A recent structure-activity relationship (SAR) study in our laboratory based on Leonurine structure has led to the identification of three novel analogs which were >100 times potent than Leonurine in killing solid cancer cells. The aim of the current study is to determine whether the novel analogs of Leonurine exhibit anti-proliferative effects against tumor cells through suppression of the signal transducer and activator of transcription 3 (STAT3) activation pathway. We investigated the effects of Leonurine analogs on constitutive STAT3 activation, modulation of tyrosine kinases and phosphatases in STAT3 activation, STAT3-regulated gene products, and growth modulation of tumor cells. We found that these compounds inhibited constitutive STAT3 activation in metastatic melanoma cells. The suppression was mediated through the inhibition of activation of the upstream kinases Janus-like kinase (JAK) 1, and JAK2. Leonurine derivatives down-regulated the expression of STAT3-regulated gene products such as survivin, Bcl-xL, Bcl-2, cyclin D1, and Mcl-1 leading to the suppression of proliferation and induction of apoptosis. Moreover, we found that these Leonurine analogs significantly inhibited the proliferation of variety of cells derived from solid tumors including breast, pancreatic, prostate, and colon cancer. Overall, these results suggest that these new compounds are novel blockers of STAT3 activation and thus may have potential to suppress melanoma cell proliferation and chemoresistance.

#4733

An optimised lead of the proteasome deubiquitinase inhibitor b-AP15 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells.

Padraig B. D'Arcy,1 Magdalena Mazurkiewicz,2 Ellin-Kristina Hillert,2 Stig T. Linder2. 1 _Linköping University, Linköping, Sweden;_ 2 _Karolinska Institute, Stockholm, Sweden_.

Inhibition of deubiquitinase (DUB) activity is a promising strategy for cancer therapy. VLX1570 is an inhibitor of proteasome DUB activity developed from b-AP15 (D'Arcy et al., Nature Med 17 (2011) 1636), and is currently in clinical trials for relapsed multiple myeloma. We here report that VLX1570 binds to ubiquitin-specific protease-14 (USP14) in vitro using surface plasmon resonance and induces thermostabilization of USP14 in myeloma cell lines. Both binding and inhibitory activity towards UCHL5 (ubiquitin-C-terminal hydrolase-5) was comparatively weaker. Treatment of multiple myeloma cells with VLX1570 induced the accumulation of proteasome-bound high molecular weight polyubiquitin conjugates and a subsequent apoptotic response. Sensitivity to VLX1570 was moderately affected by glutathione depletion and altered drug uptake but was unaffected by overexpression of BCL2-family proteins or inhibition of caspase activity. Finally, VLX1570 showed potent anti-neoplastic activity in xenograft models of multiple myeloma. Our findings demonstrate promising antiproliferative activity of VLX1570 in multiple myeloma, primarily associated with inhibition of USP14 activity.

#4734

Inhibitory effects of metformin on epithelial-mesenchymal transition of CD44+/CD117+ ovarian cancer stem cells.

Gary G. Xiao, Rongrong Zhang, Ping Zhang, Hong Wang, Dongming Hou, Wentao Li, Chenwei Li. _Dalian University of Technology, Dalian, China_.

Background: Although metformin, a first-line drug for treating diabetes, may play an important role in inhibition of epithelial ovarian cancer cell growth and cancer stem cells (CSCs), metformin at low dose showed less effect on proliferation of ovarian cancer cells. In this study, we evaluated the effect of metformin at low dose on ovarian CSCs in order to understand the molecular mechanisms underlying ovarian tumorigenesis.

Methods: The inhibitory effects of metformin at los dose on proliferation and population of ovarian cancer cells including SKOV3 and A2780 were assessed by cell proliferation assay and flow cytometry. Quantitative real-time PCR assays on expression of Bcl-2, Survivin and Bax were performed to determine the effect of the metformin at low dose on epithelial-mesenchymal transition (EMT) of cancer cells and CSCs. Tumor sphere formation assay was also performed to evaluate the effect of metformin on spheres forming ability of CSCs. The therapeutic efficacy and the anti-CSC effects of metformin at low dose were investigated by using both SKOV3 cells and primary tumor xenografts. In addition, the CSC frequency and EMT in tumor xenograft models were also assessed by flow cytometry and quantitative real-time PCR.

Results: Metformin at low dose did not affect proliferation of ovarian cancer cells, however, it inhibited the population of the CD44+/CD117+ selectively, neither CD133+ nor ALDH+ cells. It suppressed expression of snail2, twist and vimentin significantly in cancer cells and CD44+/CD117+ CSCs in vitro. Low dose of metformin reduced survivin expression in CSCs leading to arrest of cell cycle. Metformin at low concentration inhibited the secondary and the tertiary tumor sphere formation, decreased the growth of either SKOV3 or primary ovarian tumor xenograft, enhanced the anticancer effect of the cisplatin, and lowered the proportion of the CD44+/CD117+ CSCs in the xenograft tissue. Metformin was also associated with a reduction of snail2, twist, and vimentin in CD44+/CD117+ ovarian CSCs in vivo.

Conclusions: Our results implicate that metformin at low dose inhibits selectively CD44+/CD117+ ovarian CSCs through inhibition of EMT and potentiates the effect of the cisplatin.

#4735

Designing novel warheads for targeted therapies: SAR and efficient strategies for the synthesis of analogues of tubulysin.

Iontcho Vlahov, Fei You, Paul Kleindl, Marilynn Vetzel, Joseph Reddy, Christopher Leamon. _Endocyte, W. Lafayette, IN_.

Background: Tubulysins are natural products isolated from myxobacterial species. They are potent mitotic poisons as they inhibit the polymerization of tubulin into microtubules. Structurally, tubulysins are linear tetrapeptides comprised of N-Me pipecolic acid (Mep), isoleucine (Ile), tubuvaline (Tuv) and tubutyrosine (Tut). All natural tubulysins possess an acid-, base-, and enzyme-sensitive N-acyloxymethyl substituent, as well as a labile acetate group. For the natural tubulysins, both of these functional groups are essential for their potent cytotoxicity. The focus of this presentation is on design and synthesis of chemically stable analogs of tubulysin with improved biological activity.

Methods: To explore the influence of tubulysin's functional groups on its cytotoxicity, we designed novel analogs, incorporating ether, thioether, amide and carbamate-based side chains. We also suggested basic SAR-rules for the activity of these synthetic analogs.

Results: Structural features, critical for the tubulysin's cytotoxicity, were investigated. e.g., changing the (R)-OAc group of Tuv to (S)-NHAc completely abolished cytotoxicity, while analogs possessing (R)-NHAc on the Tuv moiety still exhibited activity. On the other hand, exchange of Tuv's (R)-OAc group with (R)-OMe had no influence activity. Replacement of the O-acyl part of N-acyloxymethyl substituent (dotted circle) with an alkyl or alkylthiol group resulted in N,O-acetals or N,S-thioacetals possessing similar or higher activity. Tup and Tut are interchangeable in terms of the activities of their respective tubulysin analogs.

Conclusion: We designed and synthesized tubulysin analogs which are less prone to degradation under acidic and basic conditions, as well as enzymic influences. Structural features, essential for the cytotoxicity of the tubulysin analogs, were established. Some of the new compounds, especially those with N,S-thioacetal side chains, have increased cytotoxicity in comparison to natural tubulysins. These novel tubulysin analogs were used as warheads for folate receptor(FR)-targeted therapy.

#4736

PDE10A overexpression in cancer cells and tumors as compared to normal cells and tissues.

Ashley S. Lindsey,1 Kevin Lee,1 Bing Zhu,1 Ritu Arora,1 Dennis Otali,2 Veronica Ramirez-Alacantara,1 William Grizzle,2 Gary Piazza1. 1 _University of South Alabama Mitchell Cancer Institute, Mobile, AL;_ 2 _University of Alabama at Birmingham School of Medicine, Birmingham, AL_.

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is expressed in areas of the brain related to motor function and cognition. An elevation in neural PDE10A expression has been strongly linked to many diseases that affect cognition, including schizophrenia and Huntington's disease. Recently, high levels of PDE10A were shown to be expressed in colon tumor cells in vitro and in vivo compared with cells derived from normal colon and normal colonic mucosa (Li et al., Oncogene 2015). PDE10A inhibition by small molecules or genetic silencing, attenuated tumor cell growth by a mechanism involving cGMP/PKG activation, the suppression of β-catenin and a decrease in TCF transcriptional activity (Li et al., Oncogene 2015). These observations suggest that PDE10A may play an important role in tumorigenesis and PDE10A could serve as a novel therapeutic target for colorectal cancer. However, the expression and role of PDE10A in other cancer cell types has yet to be elucidated. Here we report that PDE10A expression is elevated (compared to controls) in tumor cells and solid tumors from xenograft, as well as, genetic models from the colon, breast, and lung using western blot and immunohistochemistry. The human colon adenocarcinoma cell line, HT29, the human breast adenocarcinoma cell line, MDA-MB-231, and the human lung carcinoma cell line, A549, showed elevated expression of PDE10A as compared to the normal colon mucosal epithelial cell line, NCM460, normal human mammary epithelial cells, and normal human airway epithelial cells, respectively. Solid tumors collected from the colons of heterozygous APCmin mice displayed an elevation in PDE10A levels as compared to uninvolved mucosa from the APCmin mice and wild type colon mucosa. Solid tumors collected from the mammary glands of viral transgenic FVB-MMTV-Her2 mice also possessed high levels of PDE10A as compared to FVB wild type mice. These findings suggest that PDE10A could be a novel therapeutic target for not only colorectal cancer, but breast and lung cancers as well.

#4737

New platinum (Pt)-based anticancer agents, bearing a PtN2SO coordination core, for treating advanced melanoma.

Kenneth K.W. To, Peace Chong, Steve C.F. Au-Yeung. _The Chinese University of Hong Kong, Hong Kong, Hong Kong_.

The aim of this project is to demonstrate the potential utility of a new series of platinum (Pt) compounds having a PtN2SO coordination core in treating advanced melanoma. There is currently an unsatisfied medical need for a more effective treatment of advanced melanoma, which is notorious for its prominent resistance to conventional chemotherapeutic drugs due to elevated DNA repair capacity and dysregulation of cancer apoptotic pathways. Platinum (Pt)-based anticancer drugs, exemplified by cisplatin, are key component in combination chemotherapy. They bind to DNA and form mainly the DNA-Pt bifunctional crosslink, subsequently leading to apoptosis to mediate cell death. A series of new Pt(II) compounds possessing a bidentate ligand modified from oxaliplatin has been synthesized, with one of the oxygen ligating atom substituted for a sulfur atom (resulting in a PtN2SO coordination core structure). This novel design is to minimize competition from protein and other bionucleophiles for the Pt drug but maximize drug-DNA interaction. We hypothesize that the unique features of the new PtN2SO compounds may make them good candidates for treating advanced melanoma by (i) overcoming DNA repair-mediated drug resistance; and (ii) generating excess reactive oxygen species (ROS) to selectively trigger apoptosis in melanoma cells. The new compounds were found to be more cytotoxic than cisplatin in a panel of human and mouse melanoma cell lines. Importantly, the new compounds were also found to be minimally affected by Pt resistance, which is presumably caused by the formation of unique DNA-Pt monoadduct and reduced DNA repair. DNA-platination study in melanoma cell lines revealed that the new PtN2SO compounds are able to form more DNA-Pt adducts and there was less repair than treatment with cisplatin. The new compounds were also found to cause a greater induction of the MAPKs (p38, JNK and ERK) than cisplatin to mediate apoptosis. On the other hand, at equipotent concentration, some of the new Pt compounds were found to generate more ROS than cisplatin as indicated by a general fluorescent dye-based oxidative stress indicator. Interestingly, the cytotoxicity of the new compounds were blocked to a greater extent than cisplatin by ROS scavengers, suggesting a more prominent role played by ROS in mediating the cytotoxicity of the new compounds. Our study thus advocates further investigation of the new PtN2SO compounds in treating advanced melanoma.

#4738

The BET family bromodomain inhibitor ABBV-075 is a promising therapeutic agent for acute myeloid leukemia and myelodysplastic syndrome.

Mai H. Bui, Xiaoyu Lin, Xiaoli Huang, Leiming Li, Aparna Sarthy, Daniel Albert, Terry Magoc, Lloyd Lam, Paul Hessler, Tamar Uziel, Steven Elmore, Keith McDaniel, Warren Kati, Yu Shen. _Abbvie INC., North Chicago, IL_.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) proteins exhibit interesting activities in preclinical models of various cancer indications, and several of these inhibitors are currently under clinical investigation. ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered Phase 1 studies. Upon characterizing cellular responses to BET inhibitors across a large panel of cancer cell lines, we identified AML/MDS as one of the few cancer indications where BET inhibitors triggered robust apoptosis in cancer cell lines and in patient-derived cancer cells. The induction of apoptosis by BET inhibitors in AML/MDS cells may be partly attributed to their abilities to down regulate Bcl-XL and Bcl-2, and combining BET inhibitors with the Bcl-2 inhibitor venetoclax (ABT-199) resulted in robust cytotoxicity in AML/MDS cells. ABBV-075 exhibited significant antitumor efficacy as a monotherapy in flank xenograft models of AML and MDS. Furthermore, combining low doses of ABBV-075 with the standard of care agent azacitidine in the SKM1 model led to significant tumor regression, and the combination regimen was better tolerated than BETi monotherapy at doses that produced a similar degree of therapeutic benefit. Expression profiling of SKM1 tumors from mice treated with the ABBV-075/azacitidine combination or each of these agents as monotherapies revealed that ABBV-075 and azacitidine regulated a common set of biologic pathways. The combination of ABBV-075/azacitidine led to a more robust impact on these common pathways, which may partially contribute to the enhanced efficacy of the ABBV-075/azacitidine combination in the SKM-1 model.

#4739

**The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer** in vitro **.**

Jennifer Bourn, Maria Cekanova. _University of Tennessee, Knoxville, TN_.

Several types of cancer, including transitional cell carcinoma (TCC) overexpress several receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR), c-kit receptor, epidermal growth factor receptor (EGFR), as well as the vascular endothelial growth factor receptor (VEGFR). Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with high expression of RTKs. Cyclooxygenase-2 (COX-2) is highly expressed in bladder cancer as well other types of cancers and is one of the key proteins during tumorigenesis. In this study, we validated the effects of RTKIs, Masitinib® (AB1010) and Axitinib on cell proliferation of human (UMUC-3 and T24) and canine (K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) bladder TCC cells. Using WB analysis, tested human and canine TCC cells, except T24 cells, expressed PDGFR and c-kit receptors. There was no detectable EGFR and VEGFR expressions in any of tested cells by WB analysis. The K9TCC#1Lillie and K9TCC#5Lilly cells had a high expression of COX-2, however K9TCC#2Dakota and human T24 had moderate expression of COX-2, and no COX-2 expression was detected in UMUC3 cells by WB analysis. Both RTKIs, Masitinib® and Axitinib inhibited cell proliferation of the tested human and canine bladder TCC cells in a dose-dependent manner by MTS and apoptosis assays. Interestingly, both tested RTKIs, Masitinib® and Axitinib increased COX-2 protein expressions in tested canine TCC cells. Combined treatment of RTKIs with COX-2 inhibitors decreased cell proliferation of tested TCC cells more effectively as either treatments alone. Our results indicate a possible role of COX-2 signaling pathway in developed RTKIs-resistance in bladder cancer cells in vitro. Co-treatment of RTKIs with COX-2 inhibitors might indicate better clinical outcomes in treatments of patients diagnosed with bladder cancer.

#4740

Targeting Ref-1/APE1 pathway inhibition in pancreatic cancer using APX3330 for clinical trials.

Melissa L. Fishel,1 Derek P. Logsdon,1 Michelle L. Grimard,1 Claudiu T. Supuran,2 Nicholas Zyromski,1 Mircea Ivan,1 Mark R. Kelley,1 Fenil Shah1. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _University of Florence, Florence, Italy_.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ~95% die within five years, with most surviving less than six months. Targeted therapies including Gemcitabine (GemzarTM), FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin), and sustained release, nab-paclitaxel (AbraxaneTM) offer modest improvement in survival, albeit at an increase in side effects and unwanted toxicities. Data is presented on redox factor-1 (Ref-1) and specific Ref-1 inhibitor APX3330.

Ref-1 regulates multiple transcription factors involved in pancreatic cancer survival signaling due to its redox-coactivator activity on HIF-1α, NFkB, NRF2 and STAT3. High expression levels of Ref-1 indicate decreased survival in PDAC as well as other cancers. APX3330 has been shown in multiple in vitro and in vivo pancreatic cancer models to be effective in reducing tumor growth and metastases as a single agent. The mechanism of action has been extensively investigated and characterized for its specific activity on Ref-1, as well as its preclinical PK/PD and ADME. The safety and dose administration of APX3330 have been established by Eisai pharmaceutical company through a previous development program including toxicology, phase I, and phase II clinical evaluation in non-cancer patients in Japan. We have partnered with ApeX Therapeutics to develop APX3330 for cancer treatment (phase I trial anticipated start date early 2016).

While developing APX3330 for single agent use, we studied interactions of Ref-1, APX3330, convergent pathways; i.e. HIF-1 α and STAT3, and downstream targets like CAIX. Initially, we performed in vivo studies demonstrating single and combination effects of APX3330 with Gemcitabine (Gem) showing significantly decreased tumor volume in the APX3330 and Gem combination treatments compared to the single-agents alone. We also tested single and combination studies of APX3330 in an ex vivo 3-D tumor-stroma model system using patient derived tumor cells along with patient derived cancer-associated fibroblasts (CAFs). We used the CAIX inhibitor SLC-0111 and JAK2 inhibitor, Ruxolitinib; both agents in clinical trials. In our ex vivo 3D co-culture system, APX3330 decreases the tumor area and intensity in a dose-dependent manner. The combination of APX3330 with Gem demonstrated an additive enhancement effect in the tumor. Blocking both Ref-1 redox-signaling activity with APX3330 and CAIX activity via SLC-0111 demonstrated enhanced tumor killing in our models. APX3330 along with Ruxolitinib also demonstrated enhanced tumor killing.

These data demonstrate APX3330 single agent efficacy in our 3D patient PDAC model and enhanced tumor killing when pathways regulated by Ref-1, HIF-1 α and STAT3 are blocked. Additional drug combinations focused on pathways that are dependent on Ref-1 signaling will also be presented.

#4741

Targeting tumor growth in a medullary thyroid carcinoma mouse model using a combinatorial treatment with a tyrosine kinase inhibitor and histone deacetylase inhibitor.

Karine Pozo,1 Stefan Zahler,2 Chunfeng Tan,1 Keisuke Ishimatsu,1 Masaya Takahashi,1 James Bibb1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany_.

Neuroendocrine tumors are rare forms of cancers arising from hormone-producing cells that are scattered throughout the body. Medullary thyroid carcinoma (MTC) originates from the thyroid gland parafollicular cells. Treatment options for progressive, metastatic MTC patients are limited to the recently FDA-approved tyrosine kinase inhibitors (TKIs), Vandetanib and Cabozantinib, which target multiple receptor tyrosine kinases, including VEGFR2. However, the efficacy of TKIs is limited and development of TKI resistance is common.

Here we examined, the effect of a combinatorial drug therapy using the TKI, Nintedanib, and the histone deacetylase (HDAC) inhibitor, Romidepsin, on MTC growth in the NSE/p25-gfp bi-transgenic mouse line, an inducible MTC mouse model (see Pozo et al. 2013). The TKI- and HDAC inhibitors were administered intraperitoneally for 3 weeks and tumor growth progression was monitored using a T2 weighted magnetic resonance imaging on a 7 Tesla system. Tumor tissues were analyzed for oncogenic signaling pathways by immunoblotting and immunohistochemistry.

We find that co-administration of Nintedanib and Romidepsin stops tumor growth and increases the number of necrotic foci within the tumor tissue. Interestingly, Nintedanib treatment alone reduced tumor vascularization as shown by decreased density of the vascular marker, CD31, but did not stop parafollicular cell proliferation. Taken together, these results suggest that a combinatorial drug therapy with a TKI- and an HDAC inhibitor may be a more efficient strategy to target MTC progression and overcome recurrence.

#4742

Comparison of enhancement of the antitumor effect of CDDP by PXR antagonists.

Shuichi Kishimoto,1 Megumi Yasuda,2 Megumi Tomita,1 Yuki Yamane,1 Ryosuke Suzuki,1 Shoji Fukushima1. 1 _Kobe Gakuin Univ. Faculty of Pharm. Sciences, Kobe, Japan;_ 2 _Hyogo Univ. of Health Sciences, School of Pharm., Kobe, Japan_.

We have already found that a nuclear receptor PXR can modulate the cytotoxicity of CDDP for cancer cells, and ketoconazole, a PXT antagonist, can be an agent enhancing the antitumor activity of CDDP due to increase of the intracellular platinum content as the mechanism. However, nuclear receptor ligands have a variety of functions, and there is a possibility that PXR antagonist may enhance the cytotoxicity of CDDP through the mechanism other than the inhibition of transporter-mediated efflux of CDDP. The goal of this study was to compare the enhancing ability of three PXR antagonists, ketoconazole (KTZ), phenethyl isothiocyanate (PEITC) and metformin (MFM), for the antitumor effect of CDDP and clarify the contribution of inhibition of the efflux transporter as the mechanism of PXR antagonist. Cell lines used in this study were human hepatoma cell line HepG2. Changes in mRNA expression were assessed by real-time RT-PCR, and the ability of apoptosis induction was assessed as caspase-3 activity. Total intracellular platinum contents were determined by ICP-AES. When HepG2 cells were treated for 24 hr with 10 μM of CDDP, no change of caspase-3 activity was observed when compared to control cells. Also, 30 μM of KTZ, 30 μM of PEITC or 5 mM of MFM had no affect on the caspase-3 activity in treated cells compared with control cells. When KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 24-hr treatment with 10 μM of CDDP, the caspase-3 activity was significantly increased to 461%, 355% and 326% of the control, respectively, in comparison with that in CDDP alone. When HepG2 cells were treated for 8 hr with 25 μM of CDDP, the intracellular platinum content was 110 ng/mg protein. On the other hand when KTZ, PEITC or MFM was exposed to HepG2 cells from 24 hr before treatment and during 8-hr treatment with 25 μM of CDDP, the intracellular platinum content was apparently increased to 227%, 491% and 161% of CDDP alone, respectively. Three PXR antagonists resulted in an increase in the intracellular platinum content that seems to be due to inhibition of the platinum efflux transporter, but there is no correlation between increase in intracellular platinum content and enhancement of antitumor activity by PXR antagonists. These results concluded that PXR antagonist can be an agent enhancing the antitumor activity of CDDP, but the ability of PXR antagonist for enhancing antitumor activity of CDDP can not be determined from only the increase in the intracellular platinum content.

#4743

Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640.

Timothy S. Heuer, Richard Ventura, Julie Lai, Joanna Waszczuk, Claudia Rubio, Glenn Hammonds, Marie O' Farrell, Douglas Buckley, George Kemble. _3-V Biosciences, Menlo Park, CA_.

Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming.

TVB-2640, TVB-3166, and TVB-3664 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Analysis of gene expression data from tumor cell lines and human tumors, both primary and patient-derived xenografts, has allowed for the classification of FASN sensitivity by tumor type, histology, and molecular genetic markers. Discoveries from these analyses are being characterized further using in vitro and in vivo studies.

Combined inhibition of FASN and microtubule function with taxane treatment, e.g. paclitaxel, results in synergistic inhibition of tumor growth. Indeed, in Phase I clinical investigation, TVB-2640 combined with paclitaxel has shown promising early signs of clinical activity. Previous in vitro studies revealed that FASN inhibition causes changes in beta-tubulin expression and disrupts the organization of cellular microtubule structures in varied tumor cell types such as CALU-6 non-small-cell lung and 22Rv1 prostate tumor cell lines. Extending our investigation of the mechanism of FASN/taxane synergy, we now show that FASN inhibition prevents beta-tubulin palmitoylation. This likely plays a significant role in the observed effects on beta-tubulin expression and microtubule architecture. As disruption of protein palmitoylation is believed to contribute significantly to the anti-tumor activity of FASN inhibition in general, we expanded the analysis of protein palmitoylation following inhibition of FASN with TVB-3166 and TVB-3664 to include key oncogenic drivers of cell growth, proliferation, and survival such as K-Ras and EGFR. Additionally, the efficacy of FASN inhibition in combination with additional, non-taxane approved cancer therapies, including immunomodulatory agents and bevacizumab, is being investigated.

#4744

Targeting nitric oxide signaling with nNOS inhibitors as a novel strategy for the therapy and prevention of human melanoma.

SUN YANG,1 Zhen Yang,2 Richard B. Silverman,3 Thomas Poulos,4 Frank L. Meyskens5. 1 _Chapman University, Irvine, CA;_ 2 _Shandong Provincial Hospital, Jinan, China;_ 3 _Northwestern University, Department of Chemistry, IL;_ 4 _University of California Irvine, Irvine, CA;_ 5 _University of California Irvine, School of Medicine, CA_.

The incidence of cutaneous melanoma (CM) has increased markedly over the past four decades although there have been some dramatic advances recently in the treatment of advanced melanoma. The development of novel therapeutic interventions blocking melanoma progression would be of high impact. Recently, our group has identified that neuronal NO synthase (nNOS) activated by UV radiation and growth factor plays an important role in melanoma progression, in parallel with generating constitutive NO stress. Knockdown of nNOS significantly reduced tumor growth and lung metastasis in vivo.

The newly developed nNOS inhibitors HHs (HH044 and HH045) exhibited potent anti-melanoma activity both in vitro and in vivo. The IC50s of HH compounds in human melanoma cells are less than 10 µM, which are comparable or even better than that of chemotherapeutic drug cisplatin (4.2µM and 14.3µM in A375 and Sk-Mel28 cells, respectively). Notably, the inhibition by HHs is more predominant in metastatic melanoma A375 cells compared to primary early stage Wm3211 cells, which supports our hypothesis that nNOS/NO signaling is more critical to melanoma progression than in the initiation phase. In a melanoma xenograft tumor model, we further determined the effects of HH044 and HH045 in tumor growth in vivo. Treatments with HH044 and HH045 (50mg/kg i.p for 21 days) significantly reduced the tumor size to 12% and 19% of control respectively with no apparent systematic toxicities observed. The body weight in treated mice was even moderately higher than the control's. Taken together, these results are consistent with our hypothesis that targeting nNOS is an efficient and practicable approach for human melanoma therapy.

#4745

Repurposing of valproic acid and simvastatin combination as anticancer agents in prostate cancer: synergistic interaction with docetaxel and suppression of docetaxel resistance.

Federica Iannelli, Rita Lombardi, Biagio Pucci, Maria Rita Milone, Chiara Ciardiello, Alessandra Leone, Elena Di Gennaro, Alfredo Budillon, Francesca Bruzzese. _Istituto Nazionale per lo Studio e la Cura dei Tumori "Fondazione G. Pascale" – IRCCS, Naples, Italy_.

Although docetaxel (DTX) remains a standard of care for advanced prostate cancer (PCa), limited long-term responses, side effects and resistant disease suggested the need of novel combination strategies. Increased expression of histone deacetylases (HDAC) and alteration of the mevalonate pathway (MVP) are common aberrations in PCa. In this study, we analyzed the antitumor effect of DTX in combination with valproic acid (VPA), an anticonvulsant with HDAC inhibitory activity, and simvastatin (SIM), a cholesterol-lowering drug inhibiting the rate-limiting enzyme of MVP HMG-CoA reductase, on androgen-dependent 22RV1 and LNCAP and androgen-independent PC3 and DU145 PCa cells, as well as on the highly aggressive SIM-resistant DU145R80 subline developed in our laboratory from DU145 cells (Milone MR et al. Cell Death Dis. 2013; Milone MR et al. Oncotarget 2014). We first showed a potent synergistic anti-proliferative effect of VPA/SIM combination, assessed by calculating combination index (CI) according to the method of Chou and Talalay, on all cell lines, including SIM-resistant cells, whatever schedule of administration (simultaneous vs sequential) we used, confirming our previous data on the combination between the HDAC inhibitor (HDACi) panobinostat and zoledronic acid, the latter also targeting the MVP pathway (Bruzzese F. et al, Cell Death Dis. 2013). Notably, exposure to triple combinations (VPA/SIM/DTX) resulted in a further strong synergistic anti-proliferative effect, with sequential exposure with 24h delay between VPA/SIM and DTX as the best schedule. The synergistic interaction of VPA/SIM and DTX combination involved apoptotic effect, measured by FACS analysis of sub-diploid DNA content and caspase 3/7 cleavage, and DNA damage induction, assessed by γH2AX expression. Synergistic effect of VPA/SIM and DTX combination was confirmed by soft agar clonogenic assay and by 3D culture on self-assembled PCa spheroids. Significantly, VPA/SIM combination was also able to revert DTX-resistance in DTX-resistant PC3 and DU145 sublines developed in our laboratory from the parental cells. All together these findings suggested that the combination of two safe generic drugs such as VPA and SIM can improve DTX efficacy, representing a novel therapeutic approach that warrant clinical investigation in advanced PCa patients. Our study also suggests a new strategy to overcome resistance to standard taxane-based therapy in PCa patients.

#4746

Pharmacological characterization of BAY-876, a novel highly selective inhibitor of glucose transporter (GLUT)-1 in vitro and in vivo.

Charlotte Kopitz,1 Luisella Toschi,1 Carolyn Algire,1 Mélanie Héroult,2 Anna-Lena Frisk,1 Kirstin Meyer,1 Arndt Schmitz,1 Eleni Lagkadinou,1 Heike Petrul,1 Iring Heisler,3 Roland Neuhaus,1 Bernd Buchmann,1 Herbert Himmel,3 Marcus Bauser,1 Andrea Haegebarth,1 Karl Ziegelbauer3. 1 _Bayer AG, Berlin, Germany;_ 2 _Bayer AG, Leverkusen, Germany;_ 3 _Bayer AG, Wuppertal, Germany_.

One hallmark of cancer is the accelerated metabolism, high energy requirements, and increased glucose uptake by the tumor cells, the latter being the first and rate-limiting step for glucose metabolism. Glucose transport into the tumor cell is mediated by facilitative high-affinity glucose transporter (GLUT) proteins. Among the 14 GLUT proteins, expression of GLUT1 in normal organs is nearly exclusively restricted to the blood brain barrier, while other GLUTs are also expressed in a wide variety of vital organs such as liver and heart. Interestingly, GLUT1 expression is highly regulated by hypoxia-inducible factor (HIF)-1α, a key driver of tumor progression. In line with this finding, GLUT1 over-expression was found to be associated with tumor progression and poor overall survival in various tumor indications. Consequently, GLUT1 represents a potential target for cancer treatment. Therefore, we have developed a highly-selective GLUT1 inhibitor, namely BAY-876, with selectivity over GLUT2, 3, and 4 of 4700-, 800-, and 135-fold, respectively. We here show for the first time the pharmacological characterization of BAY-876, comprising inhibition of glucose-uptake, anti-proliferative activity in vitro, and anti-tumor efficacy in vivo in models of different tumor indications in monotherapy as well as first results on the combinability of BAY-876. Furthermore, at the therapeutic dose, BAY-876 treatment did not show any relevant finding on the behavior of treated mice in the Irwin test, assuming no or only minor effects on brain function. In conclusion, BAY-876 is the first GLUT1-selective inhibitor which reduces glucose uptake and growth of tumor cells with sufficient tolerability at the efficacious dose in preclinical models.

#4747

Novel HER2-targeted gold nanoparticles; integration of antibody therapy and nanotechnology.

Tetsushi Kubota, Shinji Kuroda, Toshiaki Morihiro, Hiroshi Tazawa, Shunsuke Kagawa, Toshiyoshi Fujiwara. _Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Targeted therapies utilizing monoclonal antibodies for malignant tumors has been increasingly developed and applied to clinical practice. Trastuzumab (Tmab) is a humanized monoclonal antibody which binds to the human epidermal growth receptor 2 (HER2), which leads to strong antitumor effects by inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and inhibiting HER2 signaling pathway. While Tmab is clinically applied to HER2-positive breast and gastric cancers, there still remain some issues such as low HER2 expression (only 20%) and resistance to Tmab. Recent progress in nanotechnology has brought significant benefits to medical fields. Gold nanoparticles (AuNPs), marked by the in vivo stability and easy surface functionalization, have been developed as therapeutic and contrast agents in the medical field, and some AuNPs reportedly show some cytotoxicity by inducing autophagy and apoptosis, which indicates that AuNPs could be attractive therapeutic agents in combination with tumor-targeting antibodies. In this study, we created Tmab-conjugated AuNPs and evaluated the antitumor effects on HER2-positive but Tmab-resistant gastric cancer cells (MKN7) and HER2-negative gastric cancer cells (MKN74) in addition to HER2-positive and Tmab-sensitive esophagogastric cancer cells (NCI-N87, TE4, OE19) in vitro and in vivo. IC50 dose of Tmab-AuNPs was 6.5 times smaller than free Tmab on NCI-N87 cells. Tmab-AuNPs exhibited stronger antitumor effects on MKN7, NCI-N87, TE4 and OE19, HER2-positive cells, than the other controls on XTT assay, but not such strong effects on MKN74 and NHLF, HER2-negative cells. Notably, Tmab-AuNPs showed potent antitumor effects even on MKN7, HER2-positive but Tmab-resistant cells. Moreover, once HER2 was overexpressed on MKN74 cells by Ad-HER2/ECD, Tmab-AuNPs became effective on artificially-HER2-overexpressed MKN74 cells. More effective intracellular uptake of Tmab-AuNPs was observed by dark field microscopy and transmission electron microscopy on HER2-positive cells, which seemed to cause stronger induction of oxidative stress, autophagy and apoptosis. Finally, stronger antitumor effects of Tmab-AuNPs were also confirmed in mouse subcutaneous xenograft models using HER2-positive cells. In conclusion, Tmab-AuNPs demonstrated potent antitumor effects on Tmab-resistant cells as well as Tmab-sensitive cells, and if combined with HER2/ECD overexpression, Tmab-AuNPs became effective even on HER2-negative cells. These findings suggest that Tmab-AuNPs could be promising HER2-targeted nanodrugs which were able to overcome shortcomings in Tmab-based therapy.

#4748

Targeting colon cancer stem cells: Novel marmelin analog THB suppresses DCLK1 and Notch Signaling.

Dharmalingam Subramaniam,1 Sivapriya Ponnurangam,1 Prasad R. Dandawate,1 Ossama W. Tawfik,1 Roy A. Jensen,1 Scott J. Weir,1 Subhash B. Padhye,2 Shrikant Anant1. 1 _University of Kansas Medical Center, Kansas City, KS;_ 2 _M. C. E. Society's Interdisciplinary Science and Technology Research Academy (ISTRA), Pune, India_.

Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Previously, we have reported that the identification of a novel compound, HDNC or Marmelin from Aegle marmelos with potent anti-colon cancer activity. We have now developed a novel marmelin analogue THB and made it water soluble THB using β-cyclodextrin (THBCD). The current study is designed to determine whether THB affects stem cells and to identify a mechanism.

Method: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we have performed HCT116 cells induced tumor xenografts. Immunohistochemistry was determined for stem cell markers and Notch signaling proteins.

Results: THB treatment induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. To demonstrate THB effects on stem cells, we performed colosphere assays. THB treatment significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where THB reduced the number of DCLK1+ cells. We next determined whether THB affects the Notch signaling pathway, a pathway that is important in maintaining CSC population. Notch receptor and its ligands are up-regulated in human colon cancer tissues. THB treatment significantly downregulated the expression of Notch1, its ligand Jagged1 and downstream target protein Hes1. Notch activation requires cleavage by the γ-secretase complex. THB treatment inhibits the expression of γ-secretase complex proteins Presenilin1, Nicastrin, APH1 and PEN2. Moreover, ectopic expression of the Notch Intracellular domain (NICD) rescued the cells from THB mediated growth suppression. These data demonstrate that THB mediated effects of colon cancer stem cells is in part through downregulating Notch1 activation. To determine the effect of THB on tumor growth in vivo, mice carrying HCT116 tumor xenografts were administered the compound intraperitoneally (5mg/kg bw) every day for 21 days. THB treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition of CSC marker proteins DCLK1, LGR5 and CD44 and also the Notch signaling proteins in the THB-treated xenograft tissues.

Conclusion: Together, these data suggest that THB treatment suppresses colon cancer growth that targets stem cells by inhibiting Notch1 signaling pathway.

#4749

Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma.

Patricia J. Keller,1 Andrew R. Conery,1 Richard C. Centore,1 Archana Bommi-Reddy,1 Karen E. Gascoigne,2 Barbara M. Bryant,1 Jennifer A. Mertz,1 Robert J. Sims1. 1 _Constellation Pharmaceuticals, Inc., Cambridge, MA;_ 2 _Genentech, Inc., South San Francisco, CA_.

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. We demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network. 

### Molecular Characterizations of Tumors and Translational Studies

#4750

Characterization of the cytoskeletal protein MACF1 in lung cancer.

Najlaa Afghani, Quincy A. Quick. _Tennessee State University, Nashville, TN_.

Genetic variation between cancer patients with the same tumor and intratumor genetic heterogeneity have broadened the complexity of factors that contribute to clinical therapeutic resistance and poor response outcomes. This has made it imperative to identity atypical expressed outliers that influence the maintenance and progression of cancers. We have identified and investigated the spectraplakin protein, Microtubule Actin Cross-Linking Factor 1 (MACF1) which functions as an actin-microfilament and microtubule crosslinker for its role in lung cancers. Genomic data generated by the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) indicate that the MACF1 gene is mutated and/or amplified in ~15% of squamous lung cancers and 25% of lung cancers, respectively. Further examination with immunohistochemistry expression analysis revealed that MACF1 is highly expressed in several lung cancer subtypes, particularly adenocarcinomas and squamous cell carcinomas, as compared to normal lung tissue. Additionally, functional studies showed that suppression of MACF1 with RNA interference significantly impaired the reproductive capacity of these solid tumors. Taken together, MACF1 represents a unique target with genetic alterations and diagnostic biomarker potential in lung cancers, the leading cause of cancer deaths in the United States.

#4751

Genetic subtype-specific prognostic significance of genetic alterations in lower-grade gliomas.

Kosuke Aoki,1 Hiromichi Suzuki,1 Hideo Nakamura,2 Keitaro Matsuo,3 Kazuya Motomura,4 Fumiharu Ohka,4 Masahiro Mizoguchi,5 Yasutomo Momii,6 Yasuhiro Muragaki,7 Satoru Miyano,8 Toshihiko Wakabayashi,4 Atsushi Natsume,4 Seishi Ogawa1. 1 _Kyoto University, Kyoto, Japan;_ 2 _Kumamoto University, Kumamoto, Japan;_ 3 _Aichi Cancer Center Research Institute, Aichi, Japan;_ 4 _Nagoya University, Aichi, Japan;_ 5 _Kyushu University, Fukuoka, Japan;_ 6 _Oita University, Oita, Japan;_ 7 _Tokyo Women's Medical University, Tokyo, Japan;_ 8 _The University of Tokyo, Tokyo, Japan_.

BACKGROUND:

Lower-grade gliomas (WHO grade II and III gliomas) are usually slowly progressive but rarely curable brain caners, for which optimal therapy is still controversial. Recently, we have delineated a comprehensive picture of genetic alterations in lower-grade gliomas and revealed that they could be clearly classified into three genetic subtypes characterized by distinct genetic and clinical features: IDH1/2 mutated tumors with 1p/19q co-deletion (Type−I), IDH1/2 mutated tumors without 1p/19q co-deletion (Type−II) and IDH1/2 wild-type tumors (Type−III). Median survival of each genetic subtype is 16.4, 7.77, and 2.12 years in Type−I, −II, and −III tumors, respectively. Given a higher layer of inter-tumor heterogeneity inferred from the presence of additional genetic lesions on each subtype, there still remains a possibility that within each subtype, we could find one or more subgroups showing distinct biological behaviors and clinical outcomes. In the current study, we analyzed a large data set of patients with lower-grade gliomas fully genotyped for major genetic lesions to determine the prognostic significance of additional genetic alterations..

METHODS:

We analyzed genetic alterations detected by exome and/or targeted sequencing as well as SNP-array karyotyping obtained from clinically well-annotated 724 patients aged ≥18 years with supratentorial lower-grade gliomas from the Japan and The Cancer Genome Atlas (TCGA) cohorts. We performed univariate and multivariate Cox hazard proportional analyses for overall survival; multivariate models incorporated age at the time of surgery (≥60 vs. <60 years old), WHO grade (grade II vs. III), and extent of resection (gross total resection vs. partial resection or biopsy) with stratification of cohorts (Japan or TCGA) and were performed with stepwise selection of variables based on the Akaike information criterion.

RESULTS:

In the multivariate Cox proportional-hazards regression analysis, we revealed that several genetic alterations have subtype-specific prognostic significance: NOTCH1 mutation in Type-I [hazard ratio (HR) = 2.57, 95% confidence interval (CI) = 1.40-4.70], high-level focal amplification of CDK4 [HR = 9.40, 95% CI = 3.08-28.7], PIK3R1 mutation [HR = 8.13, 95% CI = 2.70-24.52], and loss of chromosome 9p [HR = 1.98, 95% CI = 1.16-3.38] in Type-II, and loss of chromosome 10 [HR = 2.26, 95% CI = 1.28-3.97] in Type-III tumors. In Type-III tumors, WHO grade [HR = 5.79, 95% CI = 2.79-12.0] showed a much higher prognostic value than other prognostic factors. Most of these genetic alterations are components of three major signaling pathways or copy number variations frequently affected in glioblastomas, suggesting a close link of disease progression to glioblastoma pathophysiology.

CONCLUSIONS:

Subtype-specific genetic lesions can be used to further stratify patients in each genetic subtype for overall survival to improve management of lower-grade gliomas.

#4752

Overexpression of enhancer of zeste homolog 2 (EZH2) in endometrial carcinoma: evidence from an NRG Oncology/Gynecologic Oncology Group trial.

Lauren Krill,1 Shamshad Ali,2 Ramez Eskander,1 Meenakshi Singh,3 David Mutch,4 Susan Zweizig,5 Michael Birrer,6 Heather Lankes,2 Bang Hoang,7 Leslie Randall1. 1 _University of California, Irvine, Orange, CA;_ 2 _NRG Oncology Statistics and Data Management Center, Roswell Park Cancer Institute, Buffalo, NY;_ 3 _University of Kansas Medical Center, Kansas City, KS;_ 4 _Washington University, St Louis, MO;_ 5 _University of Massachusetts, Worcester, MA;_ 6 _Massachusetts General Hospital, Boston, MA;_ 7 _Montefiore Medical Center, The University Hospital for Albert Einstein College of Medicine, Bronx, NY_.

Objectives: Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that mediates epigenetic silencing of tumor suppressor genes. It is commonly over-expressed in several solid tumors and has been shown to be a prognostic biomarker in breast, renal, and gastric cancers. This study aims to investigate patterns of EZH2 expression in endometrial cancer.

Methods: Evaluation of expression of EZH2 was completed on both early and advanced stage endometrioid adenocarcinoma tissues and a subset of matched normal mullerian tissue samples, from participants enrolled in GOG protocol 210, using real time reverse transcription polymerase chain reaction (RT-PCR) and western blot (WB) analysis. Investigators were blinded to clinical data. One-way ANOVA, F test and Fisher's exact test were used to assess differences in log mRNA and protein expression respectively with known clinical and pathologic prognostic factors. Kaplan Meier survival analysis was used to evaluate differences in progression free survival (PFS) based on EZH2 expression.

Results: Eight-seven specimens were analyzed including 60 tumors and 27 normal tissue specimens. EZH2 mRNA and protein expression in tumor specimens was significantly higher than in matched controls (paired t test, p < 0.001). EZH2 expression was associated with lympho-vascular space invasion (p = 0.044) but other clinical and pathologic factors including age, stage, grade, nodal involvement or disease status were not associated with significant differences in expression. However, there does appear to be a trend towards decreased progression free survival among patients with increased EZH2 expression levels.

Conclusions: Our results confirm that there is differential expression of EZH2 in uterine cancers compared to normal tissues and suggests a potential role of the EZH2 as prognostic marker of aggressive disease in patients with endometrial cancer. In view of the substantial portion of early stage low grade tumors expressing EZH2, it will be interesting to investigate if increased EZH2 protein levels may also have therapeutic implications, since targeted inhibition of EZH2 expression has been shown to repress tumor cell growth in other solid malignancies. Further investigation into this potential prognostic biomarker and therapeutic target is warranted.

#4753

Ovarian cancer stem cell marker ALDH1A2 is a candidate biomarker for patient selection for intraperitoneal chemotherapy.

Brandon-Luke L. Seagle,1 Monica Dandapani,1 Chen Hui Luo,2 Claire Hoppenot,2 Robert Samuelson,1 Kevin H. Eng,3 Kunle Odunsi,3 Shohreh Shahabi2. 1 _Western Connecticut Health Network, Danbury, CT;_ 2 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Objective. To evaluate ovarian cancer stem cells as predictors of survival we analyzed associations of cancer stem cell markers ALDH1, CD117 and CD133 with survival outcomes among patients with high grade serous ovarian cancer (HGS OvCa).

Methods. Data from HGS OvCa patients who were surgically staged prior to treatment with IP (n = 90) or IV-only (n = 398) chemotherapy was obtained from The Cancer Genome Atlas. Multivariate Cox proportional-hazards regression tested associations of tumor mRNA expression of 5 cancer stem cell markers with progression free survival (PFS) and overall survival (OS). Expression was analyzed as a continuous variable by restricted mean survival (RMS) analysis. Mean PFS or OS were compared between IP and IV groups by permutation testing stratified by expression. P-values were two-tailed.

Results. Increased tumor ALDH1A2 expression was associated with decreased OS among patients treated with IP chemotherapy, HR 2.36 (1.16-4.80), p = 0.017. IP treated patients with upper 20th percentile ALDH1A2 expression had decreased OS compared to bottom 20th percentile expression (30.4 versus 51.1 months, -20.7 months difference, p = 0.017). RMS curves for PFS and OS of IP versus IV treated patients crossed at high ALDH1A2 expression, indicating that IP treated patients had decreased survival compared to IV-only treated patients. Patients with upper 20th percentile ALDH1A2 expression treated with IP versus IV-only chemotherapy had no difference in mean PFS (21.3 versus 21.6 months, -0.3 months difference, p = 0.67) and decreased OS (30.4 versus 35.3 months, -4.9 months difference, p < 0.0001). Upper 10th percentile ALDH1A2 expression was associated with decreased PFS (19.5 versus 22.0 months, -2.5 months, p = 0.005) and OS (27.1 versus 35.2 months, -8.1 months, p < 0.0001) comparing IP versus IV groups. Increased CD133 expression was associated with increased PFS among IV-only patients, HR 0.85 (0.74-0.97), p = 0.016. CD117 and the ALDH1 genes ALDH1A1/3 were not associated with survival.

Conclusions. High primary tumor ALDH1A2 expression was independently associated with significantly decreased OS among patients treated with IP chemotherapy. High ALDH1A2 was associated with lack of benefit or decreased survival among patients treated with IP compared to IV-only adjuvant chemotherapy.

#4754

Overexpression of TSH and ERBB2 (HER2) receptors is associated with sharply decreased survival in high grade serous ovarian cancer.

Brandon-Luke L. Seagle,1 Monica Dandapani,1 Chen Hui Luo,2 Claire Hoppenot,2 Robert Samuelson,1 Kevin H. Eng,3 Kunle Odunsi,3 Shohreh Shahabi2. 1 _Western Connecticut Health Network, Danbury, CT;_ 2 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Objective. To test if hormone or growth factor receptor expression is associated with survival among patients with high grade serous ovarian cancer (HGS OvCa) we analyzed tumor mRNA expression microarray and clinical data from The Cancer Genome Atlas (TCGA).

Methods. HGS OvCa patients were surgically staged prior to treatment with IP (n = 90) or IV-only (n = 398) chemotherapy. Multivariate Cox proportional-hazards regression tested associations of expression of 9 hormone or growth factor receptors with progression free survival (PFS) and overall survival (OS). Hazard ratios are hazard per each one standard deviation increase in gene expression. Expression was analyzed as a continuous variable by restricted mean survival analysis. Mean PFS or OS were compared between IP and IV groups by permutation testing stratified by expression. P-values were two-tailed.

Results. Expression of ESR1, PGR, FSHR, LHCGR, MET, and EGFR were not associated with PFS or OS. TSHR expression was associated with decreased OS (HR 1.22 (1.06-1.41), p = 0.005) and PFS (HR 1.18 (1.05-1.33), p = 0.006) among IV-only treated patients. ERBB2 expression was associated with decreased OS (HR 1.48 (1.05-2.10), p = 0.027) among patients who received IP chemotherapy. Using multigene (ESR2, PGR, TSHR, and ERBB2) analysis, among the IP group, ERBB2 (HR 1.76 (1.11-2.79), p = 0.015) and ESR2 (HR 0.28 (0.08-0.93), p = 0.037) were associated with OS. Only TSHR expression was associated with decreased OS (HR 1.22 (1.06-1.41), p = 0.005) and decreased PFS (HR 1.18 (1.05-1.33), p = 0.007) on multigene analysis of IV-only treated patients. OS and PFS decreased steeply at high expression of TSHR and ERBB2. Among patients with upper 10th percentile TSHR expression, no significant difference in mean OS or PFS was observed between patients treated with IP versus IV-only chemotherapy. Patients with lower TSHR expression (< 90% percentile) experienced a mean 13.7-month increased OS and 16.4-month increased PFS associated with IP chemotherapy (p < 0.0001).

Conclusions. Among HGS OvCa patients treated with IV-only chemotherapy, increased tumor TSHR expression was associated with decreased OS and PFS. High TSHR expression characterized patients who did not benefit from IP chemotherapy.

#4755

Correlation of expression of EGFR, cMET and mTOR signaling pathway proteins with each other and their impact on prognosis in non-small cell lung cancer patients.

Zachary Crees,1 Caleb Shearrow,1 Leo Lin,1 Jennifer Girard,1 Kavin Arasi,1 Aayush Bhoraskar,1 Andrew Nowak,1 Bonnie Sheu,1 Gagan Chhabra,1 Shylendra Sreenivassappa,2 Connie Vitali,3 Odile David,4 Neelu Puri1. 1 _University of Illinois at Chicago, Rockford, IL;_ 2 _OSF Saint Anthony Center for Cancer Care, Rockford, IL;_ 3 _Rockford Memorial Hospital Dept. of Pathology, Rockford, IL;_ 4 _University of Illinois at Chicago, Chicago, IL_.

NSCLC tumors acquire resistance to EGFR-TKIs, and studies have suggested that co-localization of c-MET and EGFR may be a modality of acquired resistance. Upregulation of alternative signaling pathways such as Wnt or mTOR have been shown to be associated with poor prognosis and are a potential mechanism of resistance. This study aims at examining these signaling pathways and EGFR/c-MET co-expression in 100 patients with stage I-IV NSCLC. We have data on 50 patients, and are working on the remaining samples. Tumor tissue from biopsies or resections have been obtained with IRB approval, processed, sectioned, and mounted on microscope slides. Total and active forms of EGFR, c-MET, mTOR, S6K, beta-catenin, and Axin2 were detected using singleplex or multiplex IHC staining procedures, and stains were graded by an independent pathologist using a validated scoring system. We selected Stage IV NSCLC (n=32) patients to correlate EGFR/c-MET expression with overall survival, and analyzed them for months to mortality based on high or low EGFR expression. Patients with high EGFR expression (n=16) showed lower overall survival compared to those with low EGFR expression (n=16). Expression of c-MET is linked to decreased survival in Stage IV NSCLC patients (n=4). Patients with EGFR/c-MET co-localization (n=19) showed decreased overall survival compared to patients without EGFR/c-MET co-localization (n=9). Elevated mTOR and p-mTOR are associated with worse prognosis in Stage IV NSCLC patients. Patients categorized with either low mTOR expression (n=10) or high mTOR expression (n=19) showed increased mortality with high mTOR expression (5.9 months) compared to patients with low mTOR expression (13.5 months). A similar trend was seen in patients with either low (n=4, 7.5 months) or high (n=24, 15.9 months) p-mTOR expression. Patients with low beta-catenin expression (n=4) showed improved survival in comparison to patients with high beta-catenin expression (n=18), 9.4 months vs 6.3 months, respectively. To determine correlations in expression of these proteins we found that EGFR/c-MET co-expression is inversely correlated with active beta-catenin and directly correlated with a negative regulator of beta-catenin, Axin-2 suggesting EGFR/c-MET co-expression is associated with a downregulation of Wnt activity. In contrast, elevated EGFR/c-MET co-expression and co-activation is statistically significantly correlated with elevated mTOR-S6K expression and activation suggesting EGFR/c-MET co-expression is associated with an upregulation of the mTOR pathway activity. Elevated mTOR pathway activation at the time of diagnosis is statistically significantly associated with poor prognosis in patients with stage IV NSCLC. These preliminary results suggest that mTOR inhibition therapy in addition to EGFR/c-MET inhibition therapies may be beneficial in this population.

#4756

N-cadherin signaling predicts prognosis in medulloblastoma subtypes.

Maria Sverdlov, Michael P. Downing, Shuqi Kang, Anjen Chenn. _University of Illinois Chicago, Chicago, IL_.

Identification of signaling pathways that drive the clinical behavior of tumors will guide specific molecular therapies. Medulloblastoma, the most common malignant pediatric solid tumor, shows considerable clinical and molecular diversity, providing impetus for specific targeted therapies of predicted molecular drivers. Here we show a gene signature derived from in vitro experimental inhibition of N-cadherin in medulloblastoma (MB) cells predicts survival in human MB in two large independent patient cohorts. Patients with high N-cadherin signature scores had earlier death and reduced overall survival. We found novel heterogeneity in proposed MB molecular subgroups, with significant differences in survival predicted by the N-cadherin signature in sonic hedgehog (SHH) and c2 tumors. In cultured MB cells, N-cadherin cell-cell interactions activated beta-catenin signaling and increased expression of the cancer stem cell regulator Sox2, known to be critical in the growth and relapse of SHH MB. Inhibition of N-cadherin reduced beta-catenin signaling and Sox2. Together, these findings indicate N-cadherin activity predicts the survival of patients with MB and suggest that differential activity of cellular pathways can identify novel disease subgroups and reveal molecular mechanisms with therapeutic significance.

#4757

Identification of biomarkers that predict response of head and neck squamous cell carcinoma to mitotic inhibitors.

Ming Zhang, Shaohua Peng, Tuhina Mazumdar, Vaishnavi Sambandam, Li Shen, Pan Tong, Lerong Li, Lauren Byers, Curtis Pickering, Mitchell Frederick, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. _University of Texas, MD Anderson Cancer Center, Houston, TX_.

Objectives: It is urgent to explore novel biomarkers and therapeutic targets for that are relevant to head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cancer worldwide. Based on a prior drug screen, we identified 3 mitotic inhibitors (AZD7762, AZD1775, volasertib) as effective therapies for HNSCC. Our objective with this study is to identify mechanisms of response and potential biomarkers of response and

Methods: Cell viability assays were performed by the CellTiter-Glo Luminescent method in a panel of 68 fingerprinted HNSCC cell lines using the 3 drugs at concentrations of 0.018 to 9.613 µM. Cell cycle, apoptosis and altered pathway protein expression after cells treated by the polo-like kinase 1 (PLK1) inhibitor volasertib were investigated by FACS, TUNEL and western blots respectively. An orthotopic mouse model of HNSCC was used to confirm the antitumor effects of PLK1 inhibition in vivo. To determine the mechanisms of drug sensitivity, we analyzed the correlation between gene expression, protein expression, gene mutation and drug sensitivity using modified two-sample t-tests were performed. The beta-uniform mixture (BUM) model was used to control false discovery rate (FDR). For correlations between drug sensitivity and gene mutations, we performed Fisher's exact test.

Results: Using the IC80 values with the peak plasma concentration of each drug as the cut-off to determine sensitivity, 34, 44 and 20 HNSCC cell lines were sensitive AZD1775 (Wee inhibitor), AZD7762 (CHK1/2 inhibitor) and volasertib (PLK1 inhibitor) respectively. HNSCC harboring AJUBA mutations were more sensitive to these 3 inhibitors and those with RAS mutations more resistant. PLK1 inhibition led to G2/M arrest, but only sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. Decreases of the levels of phosphorylated TCTP were observed following treatment with volasertib confirming PLK1 inhibition. There was a significant decrease of tumor volumes and prolongation of survival in the mice bearing orthotopic HNSCC tumors treated with volasertib in vivo.

Conclusions: PLK1 inhibition was an effective therapy in vitro and in vivo models of HNSCC. We identified the AJUBA and RAS mutations as potential candidate biomarkers of response to these mitotic inhibitors in HNSCC. This study identified the therapeutic potential of PLK1 as a novel therapeutic target for HNSCC.

#4758

Benzaldehyde suppresses multiple signal pathways in cancer cells by regulating 14-3-3ζ-mediated protein-protein interactions.

Jun Saitoh, Hideyuki Saya. _Keio University School of Medicine, Tokyo, Japan_.

Benzaldehyde is the simplest aromatic aldehyde constituent of almonds and many fruits. Anticancer effect of Benzaldehyde was first reported in 1980, and multi-institutional clinical trials were performed in those days in Japan. However trial was over without determination of effectiveness, only its safety was confirmed. The underlying mechanism why Benzaldehyde specifically suppresses growth of some particular cancer cells but not that of normal cells has not been elucidated. Therefore, we attempted to clarify the mechanism of anticancer effect of Benzaldehyde. In pancreatic cancer cell BxPC3 and in non-small cell lung cancer cell A549, we found that Benzaldehyde inhibits PI3K/AKT/mTOR, STAT3, NFκB and ERK pathways, those are major signaling pathways activated in cancer cells. Effects of Benzaldehyde on multiple signaling pathways are found to be derived from regulation of 14-3-3 family proteins which interact with phosphorylation sites of various proteins of multiple signals. In BxPC3 cells, Benzaldehyde treatment reduced the phosphorylation levels of 14-3-3-binding sites. Furthermore, we ectopically expressed seven isoforms of 14-3-3 in HEK293T cells and found that Benzaldehyde treatment significantly suppressed association of 14-3-3ζ with client proteins such as mTOR, Rictor, cRaf, STAT3 and FOXOs. The interaction of other isoforms of 14-3-3 with their client proteins was also partially reduced. But, the expression levels of those seven 14-3-3 isoforms were not significantly changed. Those data indicate that Benzaldehyde suppresses the interaction of 14-3-3ζ with its client proteins. Recent reports have shown that 14-3-3ζ is overexpressed in many cancers and acts as a signaling hub controlling the network of oncogenic pathways, suggesting that 14-3-3ζ associates with carcinogenesis, metastasis and resistance for chemotherapy and radiation. Hence, Benzaldehyde is considered to be a new class of anti-cancer agent inhibiting 14-3-3ζ-mediated tumor promoting and/or maintaining signals.

#4759

Identification of ALK, ROS1, FGFR2 and NRG1 fusions and validation with targeted inhibitors in lung and ovarian PDX models.

Xuzhen Tang,1 Hua Dong,1 Yibo Gao,2 Zhixiang Zhang,1 Xiangnan Qiang,1 Baoyuan Zhang,1 Yuxin Qin,1 Shuqun Yang,1 Yunbiao Yan,1 Qingyang Gu,1 Norman Zhang,1 Jie He,1 Qunsheng Ji1. 1 _Wuxi AppTec Inc., Shanghai, China;_ 2 _Chinese Academy of Medical Sciences, Beijing, China_.

Chromosomal rearrangement mediated gene fusions and activation of oncogenic signaling pathways have become druggable targets for therapeutic intervention, with successful development of Imatinib as an inhibitor of Bcr-Abl in treatment of Philadelphia chromosome positive chronic myelogenous leukemia. A recent study by Stransky et. al. reported systemic analysis of the transcriptome of nearly 7000 human tumor samples from the Cancer Genome Atlas and revealed the landscape of near twenty kinase fusions across twenty solid tumor types. Identification of corresponding PDX models that carry given kinase fusions would be of paramount interest for preclinical validation of candidate inhibitors. We have established about 1000 PDX models across 20 tumor types. Here we report the identification of ALK, ROS1, FGFR2 fusions in lung cancer PDX models and a NRG1 fusion in the ovarian cancer models by RNA using Sequencing data combined with the Chimerascan and Star-Fusion software for fusion calling.. The gene fusions, as validated by PCR and Sanger Sequencing, led to over-expression of the affected kinases, supporting the notion that kinase activation may be the oncogenic driver in the corresponding tumors. We further validated that ALK inhibitors Crizotinib, AP26113 and LDK378 exhibited robust antitumor activities in the ALK fusion model. Current works on validation of ROS1, FGFR2 and NRG1 inhibitors in the pertinent fusion models are under progression. These models should provide means for pre-clinical validation of novel kinase inhibitors and for experimenting novel combinatorial therapeutic strategies.

#4760

Efficacy of EGFR/HER2 duel-kinase inhibitors in PDX models harboring known and novel HER2-mutations.

Michael J. Wick, Monica Farley, Teresa Vaught, Justin Meade, Michaels Glassman, Alyssa Moriarty, Anthony W. Tolcher, Drew Rasco, Amita Patnaik, Kyriakos P. Papadopoulos. _START, San Antonio, TX_.

Background: Neratinib and afatinib are small molecule therapies that irreversibly bind to the kinase domain of the epidermal growth factor receptors 1 (HER1/EGFR) and 2 (HER2/ERBB2). Afatinib is currently approved for non-small-cell lung cancers harboring acquired EGFR mutations; however both agents have recently reported clinical activity in HER2-mutated breast cancer. HER2 mutations have also been reported in melanoma and colorectal cancer and at least two mutations (S310F/V777L) have been shown to affect EGFR/HER2 signaling. Whether other reported or novel mutations are important in EGFR/HER2 signaling in colorectal and other cancer types is unclear. To better understand effects of HER2 mutations we characterized a panel of patient-derived xenograft tumor models representing colorectal, ovary, pancreas and endometrial cancers using RNA- and DNA-based sequencing. EGFR and HER2 expression was also quantitated using immunohistochemistry (IHC). Models identified positive for HER2 mutation by sequencing were evaluated in vivo, testing antitumor activity of neratinib, afatinib and other targeted therapies.

Methods: START-PDX models were established from tissue or fluid samples as previously described. DNA or RNA were extracted and subjected to NGS and HER2 and other cancer-related mutations reported; growth factor receptor densities were interrogated using standard IHC. Drug studies were performed evaluating sensitivity of models to single agent neratinib, afatinib, lapatinib, trastuzumab and T-DM1 administered on standard treatment regimens. Study endpoints included tumor volume and time from treatment initiation with T/C values and tumor regression reported at study completion.

Results: DNA and RNA sequence analysis of 300 models identified eleven HER2 mutations including six previously reported in COSMIC (S310F, A386D, H473R, D582N, V777L, R678Q) and five novel variants (R330Q, G366R, Q398K, I628T, A1216D) in thirteen total models. High EGFR staining was reported in two colorectal models: ST427 (HER2V777L) and ST428 (HER2A1216D) while high EGFR and HER2 staining was reported in a lung model designated ST1243 (HER2S310F). In vivo treatment with neratinib or afatinib resulted in tumor growth inhibition in some tested models including ST022 (HER2G366R/R678Q) ovary and ST204 (HER2A386D) pancreas and tumor regressions were reported with either agent in the ST427 (HER2V777L) colorectal line. Neratinib or afatinib were also found active in one each of four tested endometrial models. Lapatinib, trastuzumab and T-DM1 were inactive in all tested HER2-mutant models.

Conclusion: We have sequenced a panel of PDX models and identified six previously reported and five novel HER-2 mutations in thirteen models which we screened in vivo for sensitivity to EGFR/HER2 duel kinase inhibitors and HER2-targeting therapies with the colorectal model ST427 (HER2V777L) identified as most sensitive to neratinib and afatinib.

#4761

Translational regulation by eIF4E and its contribution to tamoxifen resistance in breast cancer.

Chun Gong,1 Ka Chun Mok,1 Yuen-Nei CHEUNG,1 Pui-Sum Man,1 EWF Lam,2 Ui-Soon Khoo1. 1 _The University of Hong Kong, Hong Kong, Hong Kong;_ 2 _Imperial College London, London, United Kingdom_.

Breast cancer is one of the prevalent causes of cancer in women and its occurrence has been rising in the last few decades. Two thirds of breast cancer patients are ER-positive and can receive tamoxifen (TAM) treatment. Unfortunately, many of the patients eventually develop resistance to tamoxifen. Identifying a molecular marker associated with tamoxifen resistance would help in designing better therapeutic strategies to overcome this. In eukaryotes, translation initiation is the most regulated step. Abnormal translation machinery underlies a variety of human diseases including cancers. Eukaryotic translation initiation factor 4E (eIF4E) is the least abundant initiation factor and is believed to be the rate limiting step for translation initiation. Overexpression of eIF4E in cells selectively promotes translation of mRNAs involved in cell proliferation, apoptosis and tumor progression. Recently, eIF4E overexpression has been implicated in drug resistance in melanoma1. We hypothesized that eIF4E may also be involved in tamoxifen resistance. The aim of this study was to characterize the role of eIF4E in tamoxifen resistance and identify novel molecular targets that may be translationally regulated.

Immunohistochemistry (IHC) staining of eIF4E showed up-regulation in human breast cancer samples compared with non-tumor counterparts. In silico analysis predicted that ERα and FOXM1 have a long and highly structured 5'-UTR, which are sensitive to the expression level of eIF4E. Polysomal fractionation confirmed that ERα and FOXM1 mRNA were more actively translated in MCF7 than in MCF10A. The correlation between eIF4E and ERα/FOXM1 were further confirmed in tissue microarray (TMA). The role of eIF4E in conferring TAM resistance was studied by MTT assays. Overexpression of eIF4E induced TAM resistance while knockdown eIF4E sensitized cells to TAM. Additionally, overexpression of eIF4E upregulated ERα/FOXM1 expression at protein level while knockdown eIF4E downregulated their expression at protein level.

Our results show that eIF4E induced TAM resistance possibly through translational regulation of ERα/FOXM1. eIF4E is a promising biomarker for anti-estrogen drug responsiveness and a future therapeutic target for treating drug resistant breast cancer patients.

Reference:

1. Zhan Y, et al. The role of eIF4E in response and acquired resistance to vemurafenib in melanoma J Invest Dermatol. 2015 May;135(5):1368-76.

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#4762

Gremlin 2 is repressed in invasive endometrial cancer and inhibits cell growth in vitro.

Hiroshi Tsubamoto, Kayo Inoue, Kazuko Sakata, Hiroaki Shibahara, Yasuo Miyoshi. _Hyogo College of Medicine, Nishinomiya, Japan_.

Background: There are limited therapeutic opportunities for the treatment of advanced or metastatic endometrial cancer (EC), and limited diagnostic tools of myometrial invasion for fertility sparing management of early stage EC. Novel approach based on molecular profiling of EC cells are required. Patients and Methods: We used microarray analysis of EC tumour samples in order to identify tumour-specific changes in gene expression. Identified the most frequently and lowest repressed gene was subjected to immunohistochemistry of tissues of EC. MTT assay and migration assay using EC cells were also conducted to evaluate the antitumor activity of the identified gene. Results: The most significantly suppressed gene in EC compared with the control was Gremlin 2 with an 8.5-fold reduction (p<0.001). Immunohistochemistry revealed that Gremlin 2 was expressed in 4 of 4 tissues of normal endometrium, in 4 of 4 tissues of atypical endometrial hyperplasia and 5 of 5 tissues of low grade EC with FIGO stage IA without myometrial invasion. Among 13 patients with low-grade FIGO stage I with myometrial invasion, Gremlin 2 was partially expressed in the myometrial invasion front in 11 (85%) patients. The MTT assay revealed the proliferation of Ishikawa cells incubated with 1.0 μg/ml and 5.0 μg/ml of Gremlin 2 and HEC-1A cells incubated with 5.0 μg/ml of Gremlin 2 were significantly reduced (P = 0.029, P = 0.025, and P = 0.048, respectively). The proliferation of Ishikawa cells was significantly attenuated after incubation with Gremlin 2 for 72 h (P = 0.024). On the other hand, the migration assay did not show influence of Gremlin 2 on Ishikawa or HEC-1A cells. Conclusion: Gremlin 2, formerly known as protein related to Dan and cerberus (PRDC), has been implicated in development of the embryo and the ovarian follicle in animals, however, there is no report of Gremlin 2 in association with neoplasm or cancer. Gremlin 2 downregulation may lead to carcinogenesis and progression of EC. We suggest that re-activation of Gremlin 2-associated pathways could suppress EC progression, and should thus be explored as a potential novel therapeutic approach as well as diagnostic tool of myometrial invasion.

#4763

Targeted therapy by MET inhibitors against small-cell lung cancer with aberrant activation of HGF/MET pathway.

Hirokazu Taniguchi,1 Shinji Takeuchi,1 Koji Fukuda,1 Tadaaki Yamada,1 Shuichi Sakamoto,2 Manabu Kawada,2 Seiji Yano1. 1 _Divisions of Medical Oncology, Cancer Research Institute, Kanazawa, Japan;_ 2 _Institute of Microbial Chemistry, Numazu, Japan_.

Small-cell lung cancer (SCLC) is one of the most aggressive malignancy, characterized by rapid growth and metastatic spread to multiple organs. Approximately 70% of SCLC patients have distant metastases at the diagnosis. Recent studies reported some genomic aberrations in SCLC and several molecular target drugs have been evaluated in clinical trials. However, no molecular target agents have been approved yet for SCLC.

Hepatocyte growth factor (HGF), also known as scatter factor, is only one ligand for MET receptor. HGF/MET pathway plays the crucial role in the growth of various types of cancers. The purpose of this study is to examine the role of HGF/MET pathway activation in SCLC and to evaluate therapeutic potential of MET inhibitors, including crizotinib and golvatinib, against metastasized SCLC.

We used eight human SCLC cell lines. Expression of HGF and MET was evaluated by ELISA and western blot, respectively. Cell viability was examined by MTT assay. Four cell lines produced discernible levels of HGF and four cell lines expressed phosphorylated MET, respectively. MET inhibitors, crizotinib and golvatinib, remarkably inhibited the viability of three cell lines, SBC-5, DMS273 and DMS273B1. Knockdown of either MET or HGF by specific siRNA suppressed the growth of these cell lines, indicating that HGF/MET pathway activation is essential for survival of these SCLC cell lines. These observations suggest that MET-TKIs may be useful for a subset of SCLC with aberrant HGF/MET pathway activation.

We recently established in vivo imaging model of multiple organ metastasis (including liver and bone metastases) with SBC-5 cells. We are now evaluating the anti-metastatic effect of MET-TKIs, crizotinib and golvatinib, in the in vivo imaging model. At the annual meeting, we will present the results of MET-TKI treatment and discuss their potential against multiple-organ metastasis produced by SCLC with aberrant HGF/MET pathway activation.

#4764

Development of a multigenic bioluminescence imaging system to detect prostate cancer cells and assess their response to therapy.

Pallavi Jain, Bertrand Neveu, Yves Fradet, Frédéric Pouliot. _Centre de Recherche du CHU de Québec, Quebec, Quebec, Canada_.

BACKGROUND

Currently, liquid biposies for imaging single cancer cell to provide personalised medicine is gaining importance. In the last years, molecular imaging techniques using transcriptional amplification systems have been developed but a system enabling both PCa cell detection and treatment response assessment is lacking. PCA3 RNA is a unique PCa biomarker that has been widely studied for PCa screening and detection while the PSA gene is another biomarker of high clinical significance as it gives an account of response to androgen deprivation treatments (ADT). In this study, we have developed and studied an imaging system based on the combined transcriptional activities of the PCA3 and PSA gene promoters for single PCa cell detection and ADT response assessment from patients body fluids.

METHODS

Adenoviruses (Ad) were constructed utilizing the ability of site-specific recombination of the Cre-Lox system. The PCA3 and PSA promoters were integrated into a single Ad backbone with one promoter driving the expression of CRE recombinase and the other driving the Two Step Transcriptional Amplification system and the Firefly luciferase gene (fl) to generate a new system that we named the Multigenic Integrative Transcriptional Amplification System (MP-ITSTA). PCa cells specificity and ADT response was tested by transient infection. To detect cells in body fluid, 22Rv1-GFP cells were spiked in urine or blood of healthy control, infected with MP-ITSTA after purification and single cell imaging was done using the LV200 bioluminescence microscope.

RESULTS

We show that the PCA3-TSTA driven fl expression is specific to PCa cells (22Rv1, LAPC4, PC3, DU145) giving 8.5-108.4 fold higher expression when compared to SW780 bladder cancer cells. Contrary to PCA3-TSTA, the PSA-TSTA activity is regulated by androgen treatment but is not prostate cancer-specific as it is active in AR responsive breast cancer cells (CAMA-1 and ZR-75). We show that MP-ITSTA reporter expression is dependent on the combined activation of two promoters (PCA3 and PSA promoter) in a DHT dependent manner. MP-ITSTA could therefore also give an account of responsiveness to bicalutamide or enzalutamide treatments PCa cells. The signal obtained by MP-ITSTA system is 2.3 and 1.6 times higher than PCA3-TSTA in 22Rv1 and LAPC4, respectively proving that MP-ITSTA has the ability to enhance the reporter gene expression from a weak but PCa specific PCA3 promoter. Finally, MP-ITSTA could specifically target spiked 22Rv1-mcherry cells isolated from urine while no signal was found in non-spiked samples.

CONCLUSIONS

MP-ITSTA therefore represents a prostate cancer specific and non-invasive tool to target with high accuracy PCa cells and to detect their response to ADT cell per cell from body fluids.

#4765

Impact of TP53 status and functional classification on molecular profiles in breast cancer subtypes.

Swati Garg, Mahadeo A. Sukhai, Maksym Misyura, Mariam Thomas, Tong Zhang, Lillian L. Siu, Philippe L. Bedard, Tracy L. Stockley, Suzanne Kamel-Reid. _Princess Margaret Cancer Centre, Toronto, Ontario, Canada_.

Breast cancer is a multifaceted disease with several clinical, pathological and molecular attributes contributing to disease prognosis or treatment outcome. Treatment measures in breast cancer are based on hormone/growth factor receptor -estrogen/progesterone receptor (ER/PR) or human epidermal growth factor receptor 2 (Her2) status. TP53 pathway inactivation in breast cancer is well-established. Although TP53's therapeutic relevance is well-recognized, it remains under-utilized in patient-management, since all TP53 mutants are treated equally in the diagnostic context. In reality, enormous heterogeneity exists in nature, type and functional impact of TP53 variants. Therefore, understanding the diversity of TP53 variants in breast cancer subtypes may enhance its diagnostic utility in this cancer.

We utilized clinical NGS data, obtained using commercially available targeted panels, TruSeq Amplicon Cancer Panel (Illumina) and Ion AmpliSeq Cancer Hotspot Panel v2 (Thermofisher) to analyze tumor DNAs from cancer patients at the Advanced Molecular Diagnostic Laboratory (Princess Margaret Cancer Centre, Toronto, Canada). We focused on data from 105 advanced breast cancer patients. We consolidated several schemes proposed in the literature to classify TP53 variants, and evaluated patient molecular profiling and pathology data based on: (1) presence of TP53 variants; (b) coding effect; and (c) transcriptional activity. We further investigated whether TP53 variants were associated with reportable variant load, co-occurrence with other molecular changes and hormone/growth-factor receptor status.

In our study group, 70.4% cases carried one or more variants. TP53 alterations were prevalent (40.9%) in our cohort, followed by PIK3CA variants (36.2%). 15/105 cases (14.3%) carried variants in both genes. Unlike in other cancer types, where missense TP53 variants predominate (e.g., colorectal, 72.6%), missense (49%) and nonsense/frameshift (42%) variants were similarly distributed in breast cancers. Gain-of-Function (GOF) and Loss-of-Function (LOF) TP53 variants were also equally distributed (32% vs. 33%). However, TP53mut PIK3CAmut breast cancer cases were more likely to carry missense and/or LOF variants (10/15 cases). TP53 variants were also associated with hormone/growth-factor receptor status. A greater proportion of ER- vs ER+, PR- vs PR+, and ER-PR-Her2- vs ER+PR+Her2- breast cancer cases carried missense GOF TP53 variants respectively when compared to missense LOF and variants of unknown significance taken together(80-85% vs 50-55%; p<0.0001). Finally, TP53mut cases were more likely to carry multiple variants in contrast to TP53wt cases (37.2% vs. 12.9%).

Taken together, we define a stratification strategy for TP53 that takes into account the diversity of TP53 variants, and demonstrate its application to molecular profiling and clinico-pathological data in breast cancer.

#4766

Establishment of drug sensitivity and resistance testing using focused drug libraries to develop targeted therapies for patients with leukemia.

Apurvi Patel, Justin J. Montoya, Daniel H. Wai, Robert J. Arceci, David O. Azorsa. _Phoenix Children's Hospital, Phoenix, AZ_.

Despite major advancements in pediatric leukemia therapy, toxicities and morbidity from chemotherapy regimens have been detrimental. Advances in genomic research as well as precision medicine have helped identify specific mutations that may be susceptible to targeted therapy in leukemic patients. Overall, the use of targeted drug therapy is rising in the pediatric oncology field. To complement genomic analysis of leukemia patients, our laboratory is working on the development of a drug sensitivity and resistance screening library, that would provide functionally relevant drug response data of the patient's leukemia cells. Development of this assay included the preparation of pre-dosed library plates allowing for rapid initiation of the assay. Cells were treated for 72 hours and cell viability was determined. Preliminary work in our laboratory used a library of 56 anti-cancer and targeted agents, which were tested on 16 AML cell lines and 10 ALL cell lines. Biological replicates were preformed for each cell line and drug response data in the form of IC50s and Area Under the Curve (AUC) were calculated. Hierarchical clustering was performed to determine similar responses between cell lines. Our results identified several cell lines that showed hypersensitivity to specific drugs. Among them, the AML cell line KG-1a showed sensitivity to three FGFR inhibitors including Ponatinib, which was previously reported. Validation of the screening results was done using a 10-concentration drug dose response assay. Further testing will include expansion of our drug library and application to ex vivo patient samples. The results from these studies can lead to the establishment of a drug sensitivity and resistance assay that could provide specifically engineered therapies for each patient. This form of personalized therapy would lead to diminished toxicities from systemic therapies as well as allow increased cure rates.

#4767

Generating and characterizing novel prostate cancer cell lines that employ the alternative lengthening of telomeres (ALT) telomere maintenance mechanism.

Mindy K. Graham, Jacqueline Brosnan-Cashman, Anthony Rizzo, Michael Haffner, Alan Meeker, Christopher Heaphy. _Johns Hopkins School of Medicine, Baltimore, MD_.

A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by telomere shortening. The majority of cancers overcome this critical barrier by up-regulating the enzyme telomerase, a telomere-specific reverse transcriptase. However, for a subset of cancers that lack telomerase, telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is thought to be dependent on homologous recombination. In a variety of tumor types, our laboratory and others have reported a strong correlation between the ALT phenotype and recurrent cancer-associated somatic inactivating mutations ATRX or DAXX, genes encoding chromatin remodeling proteins. In a previous comprehensive survey of the ALT phenotype in cancer, we reported that the ALT phenotype is highly prevalent in some tumor types (e.g. astrocytoma, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive cases in over 1,000 cases of primary prostate cancer. Notably, however, we found a subset of metastatic prostate cancer harbors the ALT phenotype, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer. Here, we have created the first prostate cancer cell lines exhibiting the ALT phenotype, with the purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic approaches for men with lethal metastatic prostate cancer harboring this phenotype. These novel cell lines were generated by inactivating ATRX using the CRISPR cas9 nickase system in a genetically well characterized prostate cancer cell line that normally expresses telomerase, as well as wild-type ATRX and DAXX. In this new model, abolishing ATRX expression is sufficient to induce the ALT phenotype, as assessed by multiple biomarkers of ALT, including bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and c-circles. Interestingly, compared to the parental line, cells with compromised ATRX function have significantly lower telomerase activity and a five-fold decreased expression of telomerase reverse transcriptase (TERT). Ultimately, we will use these isogenic cell lines to further characterize and elucidate the basic biology of cancers harboring ALT, and pharmacologically target the molecular features unique to the ALT phenotype. The identification of ALT-specific drugs will pave the way for the development of new targeted treatments for a subset of men with lethal metastatic prostate cancer that harbor this unique molecular phenotype, and more broadly, other cancers that share the ALT phenotype in common.

#4768

Determination of Rab GTPase-mediated pathways critical for the antimyeloma activity of Rab GGTase inhibitors.

Kaitlyn M. Dykstra, Cheryl L. Allen, Sarah A. Holstein. _Roswell Park Cancer Institute, Buffalo, NY_.

Multiple myeloma (MM) is a plasma cell malignancy characterized by the production of high levels of monoclonal protein (MP). This leads to an increased protein folding burden in the endoplasmic reticulum (ER) in MM cells, resulting in a constitutively upregulated unfolded protein response (UPR) and a lower threshold for ER stress-mediated apoptosis. We have previously shown that inhibitors of Rab GGTase, the enzyme responsible for Rab GTPase geranylgeranylation, disrupt MP trafficking in MM cells, leading to an accumulation of MP in the ER and induction of the UPR and apoptosis. There are over 60 different Rab GTPases in mammalian cells involved in regulating a wide range of membrane trafficking events including exocytic, endocytic, and lysosomal pathways. In this study, we aimed to further define how disruption of RabGTPase function in MM cells leads to apoptosis by knocking down individual Rab proteins. Given the accumulation of MP and induction of UPR, our initial focus was on secretory Rabs. Knockdown of Rab1A/B (>50%) using siRNA in RPMI 8226 cells lead to moderate increases in intracellular lambda light chain in Rab1A/B knockdowns after 72 hours (~120-140% of control levels by ELISA). However, despite the increase in intracellular MP, no increases in UPR or apoptosis markers were seen by western blot analysis or annexin-V/propidium iodide flow cytometry. Extended time course experiments (up to 120 hours) also did not exhibit any increase in UPR or apoptosis. Knockdown of Rab6A (>60%), another RabGTPase associated with the secretory pathway, did not lead to increases in intracellular lambda light chain or induction of apoptosis. These results suggest that although individual knockdown of Rab1 is sufficient to partially disrupt MP trafficking, either more complete knockdown of the individual Rabs or knockdown of multiple Rabs is necessary for induction of the UPR and apoptosis. Additional optimization of the knockdowns will be performed in order to verify these results. These studies support the further development of Rab GGTase inhibitors that globally effect Rab GTPase geranylgeranylation as a novel anti-myeloma strategy.

#4769

Inhibition of ribosomal protein L9 expression suppresses colorectal carcinoma cell growth in vitro and in vivo.

In Hye Baik,1 Keon Uk Park,2 Ilseon Hwang,2 Hun-Mo Ryoo,3 Yun-Han Lee1. 1 _Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Keimyung University School of Medicine, Daegu, Republic of Korea;_ 3 _Daegu Catholic University, Daegu, Republic of Korea_.

Background: Ribosomal protein L9 (RPL9), a component of the 60S subunit, is upregulated in human colorectal cancer (CRC). We thus hypothesized if targeting of RPL9 with small interfering (si) RNA could inhibit CRC progression. We also investigated molecular mechanism to mediate CRC cell death caused by RPL9 silencing. Methods: HCT116 and HT29 human CRC cells were transfected with RPL9 siRNA and tested for growth inhibition and apoptotic induction using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to RPL9 knockdown, global changes in gene expression were examined using RNA sequencing. Results: RPL9 silencing caused inhibition of CRC cell growth through induction of apoptotic cell death. RNA sequencing revealed that RPL9-specific knockdown led to dysregulation of 622 genes in HCT116 and 2882 genes in HT29 cells. Among those, 256 genes showed the same directional regulation (128 up- and 168 down-regulated genes), including up-regulation of tumor suppressors such as KLF6 and ATF3, and were considered as a common RPL9 knockdown signature. Western blotting proved that downregulation of RPL9 was accompanied with the decrease in the levels of PARP-1 and pro-caspase 3 delaying cell cycle progression and accelerating apoptotic signaling. Of importance, targeting RPL9 significantly inhibited CRC growth in a murine xenograft model. Conclusion: These results suggest that inhibition of RPL9 expression could be an attractive option for molecular targeted therapy of colorectal cancer.

#4770

The regulation of pre-ribosomal RNA synthesis by LKB1.

Rui Jin,1 Faken Liu,1 Xiuju Liu,1 Henry Huang,1 Scott C. Wilkinson,1 Diansheng Zhong,2 Fadlo R. Khuri,1 Haian Fu,3 Adam I. Marcus,1 Yulong He,4 Wei Zhou1. 1 _Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, GA;_ 2 _Department of Medical Oncology, Tianjin Medical University General Hospital, Tianjin, China;_ 3 _Department of Pharmacology, Emory University School of Medicine, Atlanta, Atlanta, GA;_ 4 _Department of Gastrointestinal Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China_.

LKB1 is commonly thought of as a tumor suppressor gene, because its hereditary mutation is responsible for a cancer syndrome, and somatic inactivation of LKB1 is found in non-small cell lung cancer, melanoma, and cervical cancers. Unlike other tumor suppressors whose main function is to either suppress cell proliferation or promote cell death, one of the functions of LKB1 is to suppress cell proliferation in order to promote cell survival under energetic stress conditions. We recently discovered that LKB1 promotes pre-ribosomal RNA synthesis under uridine down-regulated condition. The mechanistic basis of this finding is that LKB1 activates TIF-IA-mediated pre-riobosomal RNA synthesis, partly through the control of its cellular localization. Immuno-coprecipitation study revealed that the interaction between TIF-IA and nuclear transportation machinery may be regulated by LKB1. Our finding also has important clinical implication because the suppression of de novo UTP synthesis preferentially promotes apoptosis in LKB1-inactivated cells. This unique, pro-survival function of LKB1 led us to exploit the vulnerability of LKB1-null cells in their defect in sensing intracellular UTP depletion. Such targeted agents represent a novel treatment strategy because they only induce cell killing when LKB1 is absent.

#4771

PET imaging of tissue factor using 64Cu-labeled active site-inhibited factor VII: A potential companion diagnostic for tissue factor targeted cancer therapies.

Lotte K. Kristensen,1 Carsten H. Nielsen,1 Troels E. Jeppesen,2 Mette M. Jensen,1 Jacob Madsen,2 Bo Wiinberg,3 Lars C. Petersen,3 Andreas Kjaer2. 1 _Minerva Imaging Aps, Copenhagen N, Denmark;_ 2 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet and University of Copenhagen, Copenhagen N, Denmark; _3 _Haemostasis Biology, Novo Nordisk A/S, Maaloev, Denmark_.

Introduction: The transmembrane glycoprotein tissue factor (TF) is the primary initiator of coagulation. In addition to its physiological role, TF is associated with a variety of pathophysiological processes including tumor growth, tumor angiogenesis and metastasis. Increased TF expression has been reported in 53-89% of all pancreatic adenocarcinomas and clinically correlates with advanced stage and poor survival. Non-invasive imaging of tumor TF status holds great clinical potential as a companion diagnostic for anti-cancer therapy targeting TF. The goal of our study was to develop and evaluate a PET tracer for imaging of TF expression in pancreatic cancer by utilizing the natural ligand for TF, coagulation factor VII.

Experimental procedures: Active site-inhibited factor VII (FVIIai) was conjugated to 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) followed by radiolabeling with 64Cu (64Cu-NOTA-FVIIai). Mice bearing subcutaneous pancreatic adenocarcinoma tumors (BxPC-3) were subjected to PET imaging 1, 4, 15 and 36 hours post-injection of 64Cu-NOTA-FVIIai for longitudinal assessment. The in vivo specificity of 64Cu-NOTA-FVIIai towards TF was evaluated in a competition blocking experiment and in a panel of subcutaneous pancreatic tumor models exhibiting low, medium and high TF expression. Additionally, the ability of 64Cu-NOTA-FVIIai to visualize TF expression in orthotopically implanted BxPC-3 tumors was evaluated by PET/MRI. Ex vivo biodistribution, immunohistochemistry and flow cytometry was performed to verify the in vivo imaging data.

Results: PET imaging at 1, 4, 15 and 36 hours after injection of 64Cu-NOTA-FVIIai revealed a tumor uptake of 2.3 ± 0.2, 3.7 ± 0.3, 3.4 ± 0.3 and 2.4 ± 0.3 % injected dose per gram (%ID/g), respectively. A graduate increase in image contrast (tumor to background ratio) was observed over the imaging time course with no further increase beyond 15 hours. Competition with excess unlabeled FVIIai significantly reduced tumor uptake of 64Cu-NOTA-FVIIai (p< 0.001) in subcutaneous BxPC-3 tumors, while the uptake was unaffected in other organs. The ex vivo biodistribution confirmed the data obtained by in vivo PET imaging. In pancreatic cancer models with different levels of TF expression, the tumor uptake was found to be significantly different from each other (p < 0.001). This was in agreement with the TF level evaluated by immunohistochemistry and flow cytometry. Furthermore orthotopic BxPC-3 tumors were visualized and distinguishable by PET/MRI using 64Cu-NOTA-FVIIai as the PET tracer.

Conclusions: 64Cu-NOTA-FVIIai is well suited for specific PET imaging of TF in pancreatic cancer and its uptake is related to the tumor TF expression level. The data supports further development of 64Cu-NOTA-FVIIai as a companion diagnostic and theranostic agent.

#4772

Addiction to chaperones: Investigating the role of HSP70 isoforms in cancer.

Lorna AB Suckling, Marissa Powers, Swee Sharp, Paul Workman. _Institute of Cancer Research, London, United Kingdom_.

The heat shock protein 70kDa (HSP70) family of molecular chaperones are critical for the survival of cancer cells due to their antiapoptotic activity and role in protein homeostasis. Our laboratory has previously shown that silencing two members of this family (HSP72 and HSC70) causes tumour cell specific apoptosis and also sensitises cancer cells to HSP90 inhibitors. HSP70 has therefore become an appealing therapeutic target. The human HSP70 family comprises 17 genes and the individual roles of the isoforms in cancer are poorly understood. Here we use a systematic approach to determine the importance of HSP70 isoforms, and a selection of HSP70 binding partners, for the growth of cancer cells under stress. It was hypothesised that cancer cells are more dependent on HSP70 proteins when under stressed conditions, as encountered in the tumour environment. We used RNA interference (RNAi) to determine the effect of silencing HSP70 isoforms and binding partners on cancer cell viability, as measured by CellTiter-Blue®. The siRNA screens were carried out in human colon (RKO) and breast (BT474) cancer cells under serum deprivation, oxidative stress and hypoxia. The silencing of several HSP70 genes, including the HSP70 isoform HSPA5 (also known as BiP/Grp78), were found to cause a reduction in cancer cell viability under stress. Detailed validation of hits has been carried out to confirm their importance for cancer cell survival and validated hits may provide potentially important therapeutic targets for cancer drug discovery.

#4773

Gene-targeted apoptosis as a novel therapeutic strategy for HER2-positive breast cancer.

Faye A. Rogers, Meetu Kaushik. _Yale University School of Medicine, New Haven, CT_.

The ability to target HER2-driven breast cancers using anticancer agents with mechanisms of actions that are independent of the HER2 cellular growth function, represents a powerful new tool to circumvent drug resistance experienced with traditional HER2-targeted therapies. We have discovered that manipulation of the balance between the DNA repair response and apoptotic pathways provides an alternative strategy to induce targeted apoptosis in HER2-amplified breast cancers. Our recent finding that triplex-induced DNA damage can activate apoptosis in human cells has yielded the opportunity to develop a new class of HER2-targeted therapies. Here, we have uncovered the novel finding that triplex-induced apoptosis only occurs when multiple triplex structures are formed, while NER (nucleotide excision repair)-dependent repair prevails in the presence of one or two structures. With this in mind, we have designed sequence-specific DNA-binding molecules that create chromosomal triplex structures and activate tumor-specific apoptosis through the induction of excessive DNA damage at the HER2 locus. Importantly, this only occurs in malignant cells with multiple copies of the HER2 gene. We have evaluated the efficacy of the HER2-targeted triplex-forming molecules using cell growth and apoptosis assays and demonstrate that these molecules are particularly effective in eliciting tumor specific apoptosis in HER2-amplified breast cancer cell lines. Furthermore, these molecules have the ability to suppress the growth and metastasis of human HER2-positive breast cancers in a xenograft mouse model. Moreover, we demonstrate that the NER protein, XPD mediates the survival-death decision in response to triplex-induced DNA damage via a p53-independent mechanism that involves phosphorylation of the H2AX tyrosine 142 residue, which stimulates the signaling pathway to recruit pro-apoptotic factors to the damage site. Our results establish an alternative and novel method to specifically target HER2-positive breast cancers by exploiting the cell's own DNA damage response machinery. This study introduces a new paradigm for gene-targeted drugs that can be used in the treatment of HER2-positive and drug-resistant breast cancers with minimal potential for toxicity to normal tissue.

#4774

Hypoxic bio-reduction of novel NBQ48 in treated tumor cells and the activity of cytochrome P450 reductase.

Beatriz Zayas, Vivian Lebron, Juan P. Rivera, Christian Velez, Osvaldo Cox. _Univ. Metropolitana, San Juan, PR_.

The main goal of this research was to evaluate the reduction activity of a novel Nitrobenzazolo[3,2-a]quinolinium (NBQ48) compound under hypoxic environment thru the formation of a fluorescent metabolite and further to evaluate the activity of the cytochrome P450 (CYP450) reducatase in the reduction of these compounds. To evaluate the reduction of NBQ48 and the formation of its fluorescent metabolite two (2) tumor cell lines in culture Toledo (lymphoma) and A431 (endothelial) where treated under nitrogen or argon saturated environments to create a hypoxic (low oxygen) environment. In a 15 ml conical tube 4x106 cells in RPMI 1640 media were treated with NBQ 48 (3mM) for a final volume of 5mL and incubated with argon or nitrogen saturated environment at 37ºC, 5%. Cells were exposed at different time periods (0 to 48 hours) to evaluate the effect of exposure time in the reduction of the NBQ48 by detecting the reduced fluorescent product, the amino-substituted ABQ48. In addition NBQ48 treated tumor cells were located inside a hypoxic chamber to evaluate the formation of the fluorescent metabolites indicative of a metabolically active cell. To determine if the CYP450 reductase enzyme is activated in the reduction of the NBQ towards the formation of the ABQ metabolite, an experiment containing the NBQs and the CYP reductases was designed. Four (4) samples were prepared: blank media hypoxic, NBQ experimental hypoxic, positive ABQ spiked and negative aerobic control NBQ. Samples with NBQ48 (0.4mM) were incubated for 24 hours. After the incubation period, fluorescence emissions indicating the reductive fluorescent product were measured using a Modulus fluorometer (blue filter). The results on tumor cells treated with NBQ48 under hypoxic conditions demonstrated reduction and the formation of a fluorescent product, amino-substituted ABQ48. The time dependent increase in fluorescence in comparison with non treated cells indicated that the reduction of the NBQ increased with time. Results on the determination of the activity of CYP450 in the reduction of the NBQ48 indicated that only samples containing the CYP450 showed the formation of a fluorescent species presumably, ABQ48. This study provides evidence of the reduction of NBQ48 and the formation of a fluorescent metabolite thru the activations of the CYP450 reductases. The implemented experimental design could also be used to determine activity of hypoxic tissues with clinical applications.

#4775

Astrocyte elevated gene-1(AEG-1)as a potential diagnostic/prognostic marker for prostate cancer.

Radhika Gade Andavolu,1 Catherine Stafford,1 Patrick Herling,1 Isabella Bianco,1 Jean-Luc Cardenas,1 Henry Go,1 Svetlana Rubakovic,1 Murthy V. Andavolu2. 1 _Genetic Research Institute of the Desert, Rancho Mirage, CA;_ 2 _Eisenhower Medical Center, Rancho Mirage, CA_.

AEG-1/MTDH/LYRIC was initially identified as a HIV-1-inducible gene in primary human fetal astrocytes. Astrocyte Elevated Gene-1 (AEG-1) is ubiquitously overexpressed in all or most cancers and plays a regulatory role in diverse and multiple processes of carcinogenesis. AEG1 is a multi-functional regulator of normal and abnormal physiology; it contributes to broad-spectrum resistance to various chemotherapeutics; and it was recently proposed as the first potential `pan-cancer` gene However, despite its critical relevance to carcinogenesis, until now genetic polymorphisms for this important gene have not been studied for association with cancer susceptibility. For example, though studies have reported AEG-1 gene expression in animal models and tumor tissues, no studies have been reported in Caucasians on the AEG-1 gene polymorphisms in large samples to establish an association with cancers. We report a PCR-RFLP screening protocol for AEG-1 gene polymorphism which is cost effective, simple and reproducible. This study also shows the strong association of AEG-1 gene with prostate cancer risk and metastasis.

DNA and RNA was isolated from 232 normal healthy age matched male Caucasians and 129 prostate cancer patients recruited in the study with an informed consent. AEG-1 gene polymorphism (rs2438211) was screened using PCR-RFLP method. Patients were compared with normal healthy controls and also within patient group comparisons were made between primary and metastasis cases. Genotype analysis was done using IBM SPSS 22 software and gene expression analysis was done using RT2 profiler PCR Array Data analysis version 3.5 (SABIOSCIENCES).

A strong association of AEG-1 gene polymorphism with prostate cancer (p<0.0001) was observed. A high frequency of the mutant allele was observed in patients (27%) as compared to normal healthy controls (7.9%).This study also highlights the possible effect of AEG-1 interaction with other genes ( p53, BCCIP) contributing to tumorigenesis.

### Novel Chemotherapies

#4776

Dichloro [4,40-bis(4,4,4-trifluorobutyl)-2,20-bipyridine] platinum, a novel Cisplatin analog, exhibits enhanced anticancer activity in preclinical models of upper gastrointestinal cancers.

Samhita Bapat,1 Tanmay Dichwalkar,1 Priya Pancholi,1 Byron L. Bennett,2 Vikas Sehdev1. 1 _Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY;_ 2 _Department of Chemistry, Idaho Sate University, Pocatello, ID_.

Background: Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to variable P53 status and overactive mechanisms that mediate drug resistance. Platinum based compounds like Cisplatin (CDDP) are frequently used for treatment of UGCs. However, clinical use of CDDP is limited due to development of drug resistance and dose limiting side-effects resulting in nausea, vomiting, neutropenia, thrombocytopenia and renal toxicity. In this study we investigated the anticancer potency of a novel CDDP derivative (dichloro [4,40-bis(4,4,4-trifluorobutyl)-2,20-bipyridine] platinum) (DCTF-CDDP) and compared it to CDDP in P53 wild type and mutant models of UGCs. Methods: For this study, we evaluated the effect of CDDP or its derivative (DCTF-CDDP) on AGS (P53 wild type) and FLO-1 (P53 mutant) UGC cell viability, survival, and expression of apoptotic markers. Results: The cell viability data indicated that DCTF-CDDP treatment was comparatively more effective than CDDP at inhibiting AGS (CDDP IC50: 6.3 ± 0.2 µM; DCTF IC50: 1.7 ± 0.5µM) and FLO-1 (CDDP IC50: 2.5 ± 0.68 µM; DCTF IC50: 0.5 ± 0.6µM) UGC cancer cell viability. The clonogenic cell survival assay data also showed that DCTF-CDDP was significantly (p<0.05) more potent at suppressing UGC cell survival when compared to CDDP. Similarly, the western blot data also showed that treatment with increasing concentrations (1, 2.5, and 5 µM) of CDDP and DCTF-CDDP for 24h induced higher levels of P53/P73 and cleaved PARP with DCTF-CDDP treatment in UGC cells. Conclusions: Our in vitro data indicate that DCTF-CDDP is significantly more potent at inhibiting cell viability and inducing apoptosis in P53 wild type and mutant UGC cells. Our study suggests that DCTF-CDDP could be an effective CDDP derivative that can be used to achieve better therapeutic outcome at lower doses and toxic side effects.

#4777

The role of BAG3 in human leukemia cells' response to the experimental anticancer drug laromustine.

Kevin P. Rice,1 Amanda J. Loya,1 Kayla M. Gross2. 1 _Colby College, Waterville, ME;_ 2 _Tufts University, Boston, MA_.

The experimental anticancer drug laromustine, which demonstrates moderate clinical efficacy against acute myelogenous leukemia and glioblastoma multiforme, yields two reactive electrophiles in situ. Therapeutic cytotoxicity is principally due to a 2-chloroethylating species that can cause lethal DNA crosslinks. However, the second electrophile, methyl isocyanate, triggers acute toxicity in cultured cancer cells and synergizes with the cogenerated 2-chloroethylating activity. The mechanism by which laromustine kills cancer cells likely includes apoptosis. We measured changes in transcription for 88 genes relating to this cell death pathway cultured human promyelocytic (HL-60) cells using quantitative real-time reverse transcriptase PCR. Cultured cells were treated with drug for six hours before harvesting mRNA for analysis. Of these genes, the expression of BAG3, whose encoded protein is also known as CAIR-1 or Bis, was increased by more than 300 fold in cells exposed to laromustine. By forming a complex with heat shock protein 70 (Hsp70), it is thought that BAG3 promotes anti-apoptotic activity by interfering with protein chaperones and release of cytochrome-c. We are also examining effects on the entire transcriptome upon treatment with laromustine using GeneChip Microarrays. Finally, we have created a stable BAG3 knockdown cell line using an shRNA construct via lentiviral transfection. Substantial differences in laromustine-induced cytotoxicity in differing BAG3 backgrounds would suggest a mechanistic role in a cell's defense against laromustine and possibly an improved clinical approach.

#4778

Covalent bonding of a C8-conjugated pyrrolobenzodiazepine (PBD) monomer and dimer to a terminal guanine residue of DNA duplex and hairpin fragments.

Julia Mantaj, Paul J. M. Jackson, Khondaker Miraz Jackson, David E. Thurston. _King's College London, London, United Kingdom_.

The pyrrolobenzodiazepines (PBDs) are a family of covalent-binding DNA-interactive minor-groove binding agents that are of growing interest due to their potential as stand-alone anticancer agents, and for their use as payloads in Antibody Drug Conjugates (ADCs). For example, one PBD Dimer, SJG-136 has reached Phase II evaluation in the clinic for ovarian and haematological cancers, and a number of PBD-based ADCs are now being evaluated in pre-clinical, Phase I and II clinical trials (e.g., SGN-CD19B, SGN-CD33A [vadastuximab talirine], SGN-CD70A, SGN-CD123A, SC16LD6.5 [Rova-T, rovalpituzumab tesirine] and ADCT-402).

Until now, PBDs, which bind in the minor groove of DNA and covalently bond to a guanine residue through its C2-NH2 functional group, were thought to require a three-base-pair recognition sequence prior to covalent bonding to the central guanine (with a thermodynamic preference for binding to 5'-Pu-G-Pu-3' sequences but a kinetic preference for 5'-Py-G-Py-3'; Pu = Purine, Py = Pyrimidine). Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD monomer, or one-half of a PBD Dimer, may provide stability for the adduct.

This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.

#4779

In silico design, synthesis and evaluation of a new family of C1-substituted pyrrolobenzodiazepines (PBDs).

Paul J. M. Jackson, George Procopiou, Nicolas Veillard, Julia Mantaj, K Miraz Rahman, David E. Thurston. _Femtogenix, London, United Kingdom_.

The pyrrolobenzodiazepines (PBDs) are a class of naturally occurring and synthetic DNA minor-groove binding agents. They covalently bind to the C2-amino group of guanines, and can form mono-alkylated adducts and cross-links depending on the structure of the molecule. PBDs can be extremely cytotoxic, and there are reports of IC50 values in cell lines in the high femtomolar ranges. Synthetic A-ring-linked PBD dimers have found use as payloads in antibody drug conjugates (ADCs) due to their potent cytoxicity, and a number of ADCs are now in clinical trials. The C-ring of the PBD structure has been under-exploited as a means to improve cytotoxicity, and for joining PBD units together to create novel dimers. This lack of interest in the C-ring may have been due to literature reports that related C2-linked PBD dimers are poorly DNA interactive and cytotoxic. Using our proprietary molecular dynamics (MD) protocols, we undertook a series of MD simulations to establish a rationale for the poor DNA-binding affinity of C2-linked PBD dimers. We were able to determine a number of characteristics preventing efficient DNA-interaction including (a) their shape does not follow the curvature of the DNA minor groove, (b) if a molecule is attached to DNA via one imine moiety, the other imine moiety is not oriented in an appropriate position to effect the second covalent cross-linking event, (c) if a cross-linked model is produced, significant DNA distortion is observed, suggesting that rapid repair would occur in cells, perhaps contributing to the low cytotoxicity. During this study, our models and simulations suggested that, after covalent adduct formation, C1-substituents should extend along the minor groove in an appropriate orientation to form extensive van der Waals interactions with functional groups on the minor groove floor. This was reflected in free energy of binding calculations. For example, in the case of one C2-linked dimer, calculations suggested a weak binding affinity (i.e., -42.74 kcal/mol) with the DNA sequence 5'-GCGATACTCGC-3', whereas under identical experimental conditions, an equivalent C1-linked dimer had a much higher binding affinity (i.e., -50.42 kcal/mol). Based on these promising in silico data, a small library of C1-PBD molecules was synthesised, and biophysical results indicated that they possessed significant DNA-binding affinity. HPLC-MS experiments using DNA sequences based on the STAT3 and NF-ΚB transcription factor consensus sequences showed that some library members converted up to 60% of the DNA to covalent adducts within 3 hours. Furthermore, in preliminary experiments in cell lines such as MDA-MB231, the same molecules were highly cytotoxic with IC50 values in the nanomolar region (e.g., 36 nM after 72 hour incubation for one compound). Studies are now underway to further functionalise the C1-position of PBDs to enhance cytotoxicity, and to assess their viability as novel payloads for use in ADCs.

#4780

RXDX-107 exhibits multiple mechanisms of intracellular delivery and results in extensive drug-induced interstrand crosslinks in solid tumor preclinical models.

Leenus Martin, Roopal Patel, Michael Johnson, Jerry Cao, Peter Chua, Colin Walsh, Jennifer Oliver, Pratik Multani, Robert Wild, Ralph Lin, Gary G. Li. _Ignyta, Inc, San Diego, CA_.

RXDX-107 is a dodecanol alkyl ester of bendamustine, which is then encapsulated in human serum albumin (HSA) to form nanoparticles. Bendamustine is an alkylating agent that induces interstrand DNA crosslinks (ICLs) and causes cell death via several pathways, including intrinsic apoptosis. The activity of bendamustine in the treatment of solid tumor malignancies has not been impressive, possibly due to the pharmacokinetic and limited biodistribution properties of bendamustine. RXDX-107 was designed to improve tissue biodistribution over bendamustine, which may result in superior efficacy and tolerability in patients with solid tumors. In preclinical studies, RXDX-107 displayed significant anti-tumor activity in multiple solid tumor cell lines, cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models of advanced solid tumors, such as breast, lung, and ovarian cancer.

In this study, we further evaluated the mechanism of action of RXDX-107, particularly the means of entry and accumulation of drug into tumor cells. We have developed a novel analytical method to precisely quantify both dodecanol alkyl ester of bendamustine and released bendamustine in tissue culture medium and cells. Our data demonstrate that RXDX-107 is transported into cells in three active forms. Firstly, RXDX-107 slowly releases bendamustine into the extracellular medium, and released bendamustine then enters into cells. Secondly, the dodecanol alkyl ester of bendamustine is transported into cells and causes ICLs. And lastly, by measuring macropinocytosis, we also determined that human serum albumin nanoparticles mediate the intracellular entry of RXDX-107. Each of these mechanisms results in the formation of ICLs. In addition, comet assay data demonstrate that RXDX-107 displayed stronger induction of ICLs than bendamustine, and the induced ICLs persist over 48 hours in multiple solid tumor cell lines. Taken together, our data demonstrate that RXDX-107 enters and accumulates into cells via multiple mechanisms, and causes extensive and superior ICLs compared to bendamustine in several solid tumor cell lines, thereby providing a strong rationale for further investigation of this agent in solid tumor indications.

#4781

The novel tubulin-binding 'tumor checkpoint controller' BAL101553 has anti-cancer activity alone and in combination treatments across a panel of GBM patient-derived xenografts.

Ann C. Mladek,1 Jenny L. Pokorny,2 Heidi Lane,3 Felix Bachmann,3 Mark A. Schroeder,1 Katrina K. Bakken,1 Brett L. Carlson,1 Paul A. Decker,1 Jeanette E. Eckel-Passow,1 Jann N. Sarkaria1. 1 _Mayo Clinic, Rochester, MN;_ 2 _Stanford University, Stanford, CA;_ 3 _Basilea Pharmaceutica International Ltd, Basel, Switzerland_.

Microtubule-targeting agents (MTA) have been employed in the treatment of many cancers for decades. BAL101553 is a highly soluble prodrug of BAL27862, a novel, small molecule, microtubule-depolymerizing agent that induces tumor cell death by activating the 'spindle assembly checkpoint'. Given intravenously or orally, the drug penetrates the brain and has anti-cancer activity in diverse tumor models refractory to standard MTA or radiotherapy (RT). In this study, BAL101553 was evaluated in orthotopic xenografts from 16 GBM PDX models; 7 of 16 lines demonstrated significant (p<0.01) increases in median survival with BAL101553 versus placebo (range in median survival extension 24-87%). The combination of BAL101553 with conventional therapies for GBM (RT and temozolomide (TMZ) was then evaluated in select lines. In the MGMT methylated GBM12 line, combination of RT with TMZ increased survival compared to placebo (median survival 80 days vs. 23 days, respectively; p<0.001). Extended BAL101553 monotherapy provided a short but significant extension in survival (median survival 31 days, p<0.001), while extended BAL101553 dosing during and after RT/TMZ (median survival 85 days) did not extend survival relative to RT/TMZ alone (p=0.56). In contrast, in the MGMT unmethylated GBM6 line, combination of RT and extended BAL101553 increased survival (median 90 days, p<0.001) relative to either treatment alone (median survival BAL101553 63 days; RT 69 days) or placebo (46 days). Additionally, the combination of BAL101553 with TMZ (median survival 70 days) was more effective than TMZ alone (median survival 60 days; p=0.009). Consistent with the unmethylated MGMT status, the TMZ/RT combination (median survival 66 days) was similar to RT alone (p=0.62), but the combination of extended BAL101553 with RT/TMZ (median survival 101 days; p<0.001 compared to other combination groups) was significantly more effective. To further evaluate whether BAL101553 is a true radiosensitizer, a second GBM6 study was performed. Also here, combination of RT (20Gy, 2wks) with extended BAL101553 dosing (median survival 66 days) significantly extended survival compared to RT alone (median survival 54 days; p=002). Interestingly, when BAL101553 dosing was limited to 2 weeks with RT, there was no increase in median survival (58 days; p=0.16). To evaluate effects on tumor repopulation during RT, the efficacy of an extended RT schedule (36 Gy, 6 wks) with or without 6 weeks of BAL101553 was evaluated. In this case, BAL101553 given during the RT schedule (median survival 78 days) extended median survival as compared to RT alone (61 days; p<0.001). Collectively, these data demonstrate that BAL101553 has broad single agent activity across a panel of GBM PDX models and suggests that combination with RT/TMZ therapy may provide additional benefits for survival extension.

#4782

Cisplatin induces mitochondrial damage and hippocampal neurotoxicity: a potential mechanism for chemotherapy-related cognitive impairment.

Naomi Lomeli, Jennifer Czerniawski, Kaijun Di, John Guzowski, Daniela Bota. _University of California, Irvine, Irvine, CA_.

Advances in cancer treatment, chemotherapy in particular, have substantially increased the number of long-term cancer survivors. However, these drugs often have neurotoxic effects that impair cognitive function, thereby diminishing the quality of life of millions of cancer survivors. Chemotherapy-related cognitive impairment (CRCI, chemo-brain) is commonly reported following the administration of chemotherapeutic agents and comprises a wide variety of neurological problems. Cisplatin is used to treat breast cancer and advanced ovarian cancer among other malignancies. Notably, more than 30% of advanced ovarian cancer patients develop CRCI during and after cisplatin-based chemotherapy. A plausible explanation for CRCI is that cisplatin might impair the structure and functions of neurons in brain regions involved in learning and memory, such as the hippocampus. We have recently identified mitochondrial dysfunction and increased oxidative stress as a mechanism through which cisplatin causes hippocampal cell death, and severe dendritic damage in surviving neurons. The aims of this study were to examine the effect of the antioxidant N-acetylcysteine (NAC) in mitigating cisplatin-induced hippocampal damage and assesse the effect of cisplatin on cognitive performance in a rat model.

At a high dose, cisplatin (1µM) induced ~35% increase in caspase-9 activation in primary rat hippocampal neurons, whereas at a substantially lower dose, cisplatin (0.1µM) induced non-reversible damage to dendritic spines and branches. Both doses produced severe mitochondrial respiratory deficits and significant ROS production. Delayed treatment with NAC partially mitigated neuronal apoptosis and ameliorated cisplatin induced dendritic spine loss. When administered to adult Sprague Dawley rats, cisplatin (3 mg/kg) administered for two consecutive days caused ~40% reduction in the number of dendritic spines in CA1 and CA3 hippocampal neurons.

Lastly, cognitive testing of rats treated with a chronic cisplatin regimen, revealed significant deficits in hippocampus-dependent tasks. Rats were given weekly cisplatin (5mg/kg, i.p.) or saline injections for 4 weeks and then trained in Context-Object Discrimination, 6 weeks later (n= 7,8). Cisplatin-treated rats were impaired in discriminating between the out-of-context and in-context object.

Mitochondrial dysfunction provokes free radical production, with resulting loss of dendritic spines. When administered to rats, cisplatin causes hippocampal neuronal and mitochondrial damage, as well as cognitive deficits, supporting that role of mitochondrial toxicity in the mechanisms of cisplatin-induced CRCI. Importantly the data demonstrates that these processes can be potentially mitigated with administration of the clinically available antioxidant, NAC. The effect of NAC in ameliorating cisplatin induced CRCI in rats is being evaluated.

#4783

Identification of new therapies for the treatment of BRAF/NRAS wild-type melanomas by functional screening approaches.

Marco Ranzani,1 Magali Michaut,2 Clara Alsinet,1 Vera Grinkevich,3 Kim Wong,1 Vivek Iyer,1 Nanne Aben,2 Martin Del Castillo Velasco-Herrera,1 Marcela Sjoberg,1 Mamunur Rashid,1 Graham Bignell,3 Ken Dutton-Regester,4 Antonia Pritchard,4 Daniel Vis,2 Gemma Turner,1 Ultan McDermott,3 Nicholas K. Hayward,4 Kosuke Yusa,5 Lodewyk Wessels,2 Mathew Garnett,3 David Adams1. 1 _The Wellcome Trust Sanger Institute, Experimental Cancer Genetics, Hixton, Cambridge, United Kingdom;_ 2 _The Netherlands Cancer Institute, Division of Molecular Carcinogenesis, Amsterdam, Netherlands;_ 3 _The Wellcome Trust Sanger Institute, Cancer Genome Project, Hixton, Cambridge, United Kingdom;_ 4 _QIMR Berghofer, Medical Research Institute, Brisbane, Australia;_ 5 _The Wellcome Trust Sanger Institute, Cellular Genomics, Hixton, Cambridge, United Kingdom_.

Melanoma causes 9000 deaths each year in the USA alone, more than one every hour. It represents the common tumor whose incidence has increased the most in the last 30 years. Despite promising advances, including immunotherapies, the therapeutic regimens are curative in only a fraction of patients. Implementation and combination of different therapeutic modalities is therefore required to improve patient survival.

Given the clinical efficacy of drug combinations for BRAF mutant melanomas, we performed a high-throughput drug screening to identify new drug combinations for the treatment of BRAF/NRAS wild type melanomas. Effective targeted therapies are not currently available for this sub-type of the disease, which represents up to 30% of melanomas. By viability assays we characterized the sensitivity to 240 combinations of clinically relevant drugs in a collection of 21 BRAF/NRAS wild type melanoma cell lines. We analysed 8360 survival curves and found that 16 (73%) and 5 (23%) cell lines are highly sensitive to temozolomide plus olaparib and to nilotinib plus trametinib combinations, respectively. Two independent experimental approaches validated these drug synergies.

By -omics technologies we deeply characterized the cell line profiles of mutations, copy number changes, DNA methylation, and gene and microRNA expression. We generated gene expression signatures of synergistic and resistant cell lines for the 2 drug combinations and used them to classify 374 human melanomas. Tumors significantly associated to nilotinib plus trametinib synergism signature were significantly enriched for high immune response and proliferative Jonsson's expression classes, while tumors associated to the signature of drug resistance are enriched for pigmentation class. Melanomas associated to temozolomide plus olaparib resistance signature displayed enrichment for normal class. We are currently confirming the representation of these signatures in an independent cohort of melanomas and looking for other clinical correlates. We are also validating the predictivity of these gene expression signatures in independent cohorts of melanoma cell lines. Prospectively, this may represent an approach to identify patients that could have the maximal benefit from these drug combinations.

Additionally, we have recently performed a genome-wide CRISPR/Cas9 screen to identify drug resistance genes for these 2 drug combinations. Preliminary results indicate a prominent role of tuberous sclerosis complex in the regulation of the sensitivity to nilotinib plus trametinib combination.

These results may pave the way to the development of novel patient-tailored targeted therapies for the efficient eradication of BRAF/NRAS wild type melanomas.

#4784

FGFR-selective inhibitor Debio 1347 induces tumor regressions in FGFR2-altered gastric cancer PDX models.

Brichory Franck, Anna Pokorska-Bocci, Paolo Nuciforo, Stefania Rigotti, Nathalie Lembrez, Grégoire Vuagniaux, Corinne Moulon, Anne Vaslin. _Debiopharm International SA, Lausanne, Switzerland_.

Dysregulation of the fibroblast growth factor receptor (FGFR) signaling pathway due to receptor overexpression, gene amplification, point mutations or fusions/chromosomal translocations is associated with cancer development and progression. In gastric cancer, alterations of FGFR2 are observed in a subset of patients, overexpression being more frequently observed as compared to FGFR2 amplifications. In addition, FGFR2 amplification and ERBB2, EGFR, MET and KRAS amplifications are mutually exclusive suggesting that FGFR targeted therapy would be most effective in the FGFR2-amplified subgroup of patients. Debio 1347 (CH5183284) is an oral selective FGFR inhibitor currently in clinical development. The aim of the study was to investigate the preclinical activity of Debio 1347 in patient derived xenograft (PDX) mouse models harboring diverse FGFR alterations in multiple indications, including gastric cancer.

The mouse trial was conducted in 39 different PDX models selected according to FGFR1, 2 and 3 alterations status. Debio 1347 was administered orally once daily and tumor volume was monitored for 14 consecutive days. PDX tumors were extensively characterized using FISH, RNAseq, nCounter Gene Expression and immunohistochemistry (IHC), and FGFR molecular alterations were correlated with Debio 1347 efficacy. The level of tumor heterogeneity for FGFR2 amplification and expression and its impact on drug response were also assessed.

In gastric cancer, Debio 1347 induced tumor regression in models harboring the commonly found FGFR2 amplification. Interestingly, Debio 1347 was also highly effective in models with high FGFR2 expression but without FGFR2 amplification, indicating that not only gene amplification drives the activity of Debio 1347 in this indication, but also the expression level of FGFR2.

Altogether, these data demonstrate that Debio 1347 could be highly effective in gastric cancer patients harboring FGFR2 amplification and/or high FGFR2 expression. Debio 1347 is currently investigated in a Phase I trial in selected patients harboring FGFR alterations (NCT01948297).

#4785

Signaling adaptation to FLT3 inhibition in FLT3/ITD leukemia results in a reactivation of ERK signaling that can be abrogated with MEK inhibition.

J. Kyle Bruner, Hayley Ma, Christine Pratilas, Donald Small. _Johns Hopkins University, MD_.

The use of FLT3 tyrosine kinase inhibitors (TKIs) for the treatment of FLT3 mutant acute myeloid leukemia (AML) has been explored as a promising strategy for over a decade. However, FLT3 TKIs have thus far shown limited clinical benefit in patients with FLT3/ITD AML. We hypothesized that FLT3/ITD leukemia cells exhibit mechanisms of intrinsic signaling adaptation to FLT3 TKI treatment that are associated with an incomplete biologic response. If true, combined targeted therapeutic approaches that overcome the adaptive resistance to FLT3 inhibition could be used to maximize the efficacy of anti-leukemia treatment for those expressing this mutation.

To evaluate this hypothesis, the FLT3/ITD AML cell lines Molm14 and MV4;11 were treated with FLT3 TKIs for up to 48 hours at concentrations sufficient for maximal FLT3 inhibition and downstream signaling was analyzed by immunoblotting. We identified a rebound in ERK phosphorylation discernible by six hours and continuing for the duration of treatment, despite continued drug presence. This rebound resulted in near baseline levels of phosphorylated ERK (pERK) and was coupled to a rebound in phosphorylation of elements both upstream and downstream of ERK as well as in the expression levels of ERK target genes, suggesting a global reactivation of the signaling cascade. Rebound was sensitive to a reduction in serum, suggesting a growth factor-dependent mechanism.

To explore the consequences of this adaptation, the impact of combinatory targeting of ERK activity using selective small molecule inhibitors of MEK was evaluated. When Molm14 and MV4;11 cells were treated with inhibitors of both FLT3 and MEK in combination, little to no pERK rebound was observed. Additionally, the anti-leukemia effects were more pronounced for the combination compared to either drug alone, both in vitro and in vivo. In vitro, the addition of a MEK inhibitor (PD0325901 or trametinib) to FLT3 TKI (sorafenib) treatment synergistically increased cell death and decreased cell viability. This effect was most pronounced at mid-range sorafenib combined with low dose MEK inhibitor. In vivo, the addition of low-dose PD0325901 to sorafenib treatment resulted in a significant reduction of both peripheral blood and bone marrow blasts in a transplant model (p < 0.05).

Together, these studies reveal that FLT3/ITD leukemia cells demonstrate an adaptive feedback mechanism capable of reactivating ERK signaling in response to FLT3 inhibition. This adaptation limits the efficacy of FLT3 TKI treatment and can be abrogated by the addition of a MEK inhibitor. Our data suggest that the addition of low-dose MEK inhibitor to FLT3 TKI treatment as a means to overcome signaling adaptation may improve outcomes for patients with FLT3/ITD AML.

#4786

Integrated high-throughput screen to identify treatment leads for pediatric AML.

Christina D. Drenberg,1 Anang Shelat,2 Shelley J. Orwick,3 Hiroto Inaba,2 Raul C. Ribeiro,2 Jeffrey E. Rubnitz,2 Tanja A. Gruber,2 R K. Guy,2 Sharyn D. Baker1. 1 _The Ohio State University College of Pharmacy, Columbus, OH;_ 2 _St. Jude Children's Research Hospital, Memphis, TN;_ 3 _The Ohio State University Comprehensive Cancer Center, Columbus, OH_.

While dramatic improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), some subtypes are associated with event free survival of <40%. Given that significant improvements in long-term outcome are not expected with conventional therapy alone, new therapeutic strategies are urgently needed. Therefore, we conducted a high-throughput screen (HTS) to identify novel therapeutics for the treatment of pediatric AML. We selected a panel of 8 AML cell lines representing subtypes with the poorest prognosis. An initial primary screen was performed at a single concentration (10µM) with >8000 compounds. Compounds with >50% activity (N=285) in the primary screen and known clinical candidates were prioritized for a secondary HTS performed in a dose-response manner (10 point curve, 0-10µM). Screening was performed using a 348-well plate format and a Biomek automation workstation for drug delivery. At 72 h cell viability was determined using Cell Titer Glo and automated Envision plate reader. Multiple drug classes had broad activity including: anti-malarials, cytotoxics (antimicrotubules, purine/pyrimidine antimetabolites, floxuridine, trimetrexate, topoI/II inhibitors), epigenetic modifiers (BET, DNMT and HDAC inhibitors), and agents targeting Bcl-2, Chk1, Parp, Hsp90, NF-κB, NAMPT, p53, proteasome, and Wee1. We validated 13 compounds from select drug classes (artesunate, cytarabine, gemcitabine, cabazitaxel, depsipeptide, panobinostat, vorinostat, ABT-199, RG117, bortezomib, carfilizomab, BMN673, MK-1775) using primary blast samples representing 3 high-risk groups (FLT3-ITD-positive, MLL-rearranged, FAB M7). Given the broad activity of gemcitabine and cabazitaxel across AML subtypes and the ability for repurposing in childhood AML, we further evaluated these agents alone and in combination in vitro. After 72h exposure, cabazitaxel was a potent inhibitor of viability in AML cell lines (IC50, 3-11nM) and primary blast samples (IC50, 3.4nM to >10µM); gemcitabine had similar activity (IC50, 3-62nM and 102nM to >10µM, respectively). The combination was evaluated using continuous and sequential administration schedules for up to 72h of treatment in several cell lines; simultaneous treatments were synergistic (CI, 0.2-0.45) and sequential treatments were additive to synergistic (CI, 0.82-0.95). Tolerable combination regimens were identified in NSG mice (5mg/kg cabazitaxel + 50mg/kg gemcitabine q3d x 4 weeks or q4d x 3 weeks) and efficacy studies in AML xenografts and primagrafts are ongoing. Further studies will aim to determine the underlying mechanisms of AML sensitivity to gemcitabine, cabazitaxel, and the combination. Our comprehensive approach to identify treatment leads provide advances in understanding the biology of a heterogeneous disease and development of novel treatment strategies with potential clinical benefit for pediatric AML.

#4788

Discovery of potent and selective inhibitors of ERK1/2 with a unique mechanism of action.

Yongqi Deng,1 Ahmed Samatar,1 Gerald Shipps,2 Alan Cooper,3 Brian Long,4 Donnar Carr,3 Li Xiao,3 Alan Hruza,3 Tong Wang,5 Liang Zhu,5 Yang Nan,5 Mehul Patel,5 Kiran Muppalla,6 Hugh Zhu,3 Babu Sobhana Boga,6 Xiaolei Gao6. 1 _Merck Rearch Institute, Boston, MA;_ 2 _Bill Gates Foundation, Boston, MA;_ 3 _Merck, Kenilworth, NJ;_ 4 _Merck, Boston, MA;_ 5 _Merck, Cambridge, MA;_ 6 _Merck, Rahway, NJ_.

Activation of the RAS/MAPK pathway occurs via a cascade of protein phosphorylation events which culminate in the phosphorylation & activation of ERK 1/2. Aberrant activation of this pathway has been demonstrated in several human tumor types. Inhibitors of this pathway in clinical development target upstream kinases, BRAF or MEK. Only a few ERK 1/2 has been reported. The goal of the ERK program was to develop a targeted therapy that is guided by a clear patient selection / responder identification plan based on BRAF and K/NRAS mutations.

Utilizing an affinity based high-throughput screening strategy (ALIS), we discover a novel class of small molecule ERK 1/2 inhibitors. Optimization of this chemical series led to the discovery of SCH 772984, which is highly selective ERK1/2 inhibitor, inhibiting cell proliferation selectively in tumor cell lines with an activated MAPK pathway and causes significant tumor regression in vivo in BRAF & RAS mutant xenograft models. Based on insights gained through crystallographic analysis, SCH 772984 and related compounds have a unique ERK binding mode which results in a novel dual mechanism of inhibition, blocking ERK phosphorylation by MEK as well as inhibiting ERK kinase activity. This novel mechanism enables the use of ERK phosphorylation as a target engagement biomarker. Biological differences between pathway inhibition at the level or ERK vs. inhibition of upstream kinases are being examined. The SAR and crystal structures of this series ERK inhibitors will be reported.

#4789

Drugging RET rearranged tumors through potent inhibition of RET kinase signaling.

Maximilian Riedel, Dennis Plenker, Martin L. Sos. _University of Cologne, Cologne, Germany_.

In the era of precision cancer medicine kinase fusion events involving ALK, ROS1 or RET represent therapeutically viable targets across a broad range of solid tumors (Kohno et al., Nat Med 2012; Lipson et al., Nat Med 2012; Mulligan et al., Nat Cancer Rev 2014). These rearrangements typically involve elements that allow homodimerization of the fused protein and that induce an oncogenically active conformation of the retained kinase domain of a given kinase. In thyroid cancer RET fusions have been known for decades however, a more recent identification of rearranged RET in lung cancer led to the testing of a number of drugs that target RET signaling. However, despite encouraging proof-of-principle studies involving cabozantinib as well as other known RET inhibitors to date the response rates and duration of response in lung cancer patients remain very limited (Drilon et al. Cancer Discov 2013; Drilon et al., Clin Cancer Res 2014; Gautschi et al., J Thorac Oncol 2013).

Here, we demonstrate preclinical evidence for the limited efficacy of currently available drugs in RET rearranged tumors. Furthermore, we identify a multi-kinase inhibitor that potently blocks viability and downstream signaling of RET-rearranged cells expressing the most prevalent RET rearrangements such as KIF5B-RET and CCDC6-RET. These effects were largely independent of the expression of a gatekeeper resistance mutation RET-V804M even at low nanomolar concentrations of the drug. Using CRISPR/Cas9 mediated genome editing we confirm that our inhibitor effectively blocks signaling engaged by RET kinase fusions. Interestingly, we observe reactivation of MAPK signaling as potential driver of resistance in RET rearranged cells that can be reversed through combined RET and MEK inhibition. Finally, we show that treatment of CCDC6-RET positive PDX tumor models with our multi-kinase inhibitor leads to robust tumor shrinkage and a significant reduction of the proliferation of the remaining tumor cells. Overall, our data indicate that our drug represents a potent and effective inhibitor of oncogenic signaling engaged by RET rearranged tumors.

Given the evident clinical need for effective targeted drugs against RET our results provide a strong rationale for a further optimization of our tool compound towards a clinically applicable drug. To this end the further evaluation of the relevance of resistance mechanisms such as RET-V804M gatekeeper mutations or reactivation of downstream MAPK signaling will be required.

#4790

Breast cancer stem cells: The next step in the area of personalized medicine.

Emmanuelle Charafe-Jauffret, Christophe Ginestier, Olivier Cabaud, Julien Wicinski, Arnaud Guille, Severine Garnier, Max Chaffanet, Anthony Goncalves, Francois Bertucci, Daniel Birnbaum. _CRCM, Marseille, France_.

In the developping area of personnalized medicine, targeted therapies are mainly based on genomic characterization of each tumor, and is currently proposed as promising strategies for resistant breast cancer (RBC). Despite the promises of advanced genome sequencing, many patients still fail therapy, resulting in disease progression, recurrence, and metastases. Cancer stem cells (CSCs) concept illustrates the non-genetic intrinsic resistance, recapitulates tumor heterogeneity that creates hierarchically organized tumor tissues where a subpopulation of self-renewing cancer stem cells (CSCs) sustains the long- term clonal maintenance of the neoplasm. Evidences indicate that CSCs survive many commonly employed cancer therapeutics. Patient-derived tumor xenograft (PDXs) models recapitulate tumor complexity and heterogeneity at cellular, and molecular level. We aimed to specifically address the therapeutic sensitivity in RBC, by using a PDX prospective collection, fully characterized for genomic alterations.

In this work, we aim at defining for each tumor the best therapy to target breast cancer intratumor heterogeneity, the CSC component. For that, we defined a panel of 44 FDA-approved compounds used for cancer treatment, including breast and other types of cancer, cancer stem cell drugs, chemo or targeted therapies. For each drug, we screened the differential sensitivity of the bulk tumor cells and the CSC components for 12 PDX models using an ex vivo screening approach on short term culture. To assess intra tumor heterogeneity, we set up an original dual strategy: for the bulk cells, an ex vivo assay based on IC50, and for breast CSC component a miniaturized Aldefluor assay. First, we demonstrate that bulk cells and CSCs sensitivity may be dissociated for the same drug in the same PDX models. Then, we observed that bulk cell sensitivity is often correlated to tumor genomic abnormalities. By opposite, CSC sensitivity seems not to follow the rule and displays selectivively sensitivity to specific targeted compounds belonging to Tyrosine Kinase Inhibitors family. We are exploring the pathways that sustain this selective sensitivity in the CSCs components. Then, we validated the hits predicted from ex vivo screening assays by treating different PDX models for selected drugs. As a Proof-of-concept, we have already validated one CSC targeted strategy for one PDX model.

In that work, we demonstrated that CSCs and bulks cells are not sensitive to same treatment, independently to their genomic abnormalities. This result highlights the need for differential testing on the two tumor components, proposes a dual-screening strategy to evaluate the differential drugs sensitivity, validated in PDX models. Afterall, we emphasized the importance of integrating CSC drug sensitivity in the new area of personnalized medicine currently focused on genomic-based strategies and irrespective of intra tumor heterogeneity.

#4791

Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor α and the proton-coupled folate transporter.

Mike R. Wilson,1 Manasa P. Ravindraa,2 Adrianne Wallace-Povirk,1 Lisa Polin,1 Zhanjun Hou,1 Aleem Gangjee,1 Larry H. Matherly1. 1 _Wayne State University, Detroit, MI;_ 2 _Duquesne University, Pittsburgh, PA_.

The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) α delivers folate substrates into the cell via endocytosis. FRα and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRα and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRα (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRα without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 μM) and 5-aminoimidazole-4-carboxamide (320 μM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRα and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors.

#4792

The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells.

Erica Godley,1 Ragu Kanagasabai,2 Soumendrakrishna Karmahapatra,2 Jack C. Yalowich2. 1 _Wake Forest University, Winston-Salem, NC;_ 2 _Ohio State University College of Pharmacy, Columbus, OH_.

DNA topoisomerase IIα (topo II) is a known cargo protein for exportin 1 (XPO1, CRM1) (Seminars Cancer Biol. 27: 62-73, 2014) for nuclear export. Here we utilized KPT-330, a selective inhibitor of nuclear export, to evaluate its cytotoxicity, its effects on the nuclear content of topo II in human myeloid leukemia HL60 cells and on the subsequent activity of the topo II targeting agent etoposide. Since KPT-330 is a potential michael acceptor and is known to interact with the CRM1 active site Cys528 to inhibit its activity, we first analyzed interaction of KPT-330 with glutathione (GSH)-containing cysteine. In an in vitro binding study KPT-330 was weakly bound to GSH with a Kd = 130+/-24 µM. In contrast, the natural product compound parthenolide, also a michael acceptor, bound to GSH with 30-fold greater affinity. Depletion of cellular GSH with buthionine sulfoximine (BSO) did not significantly alter the 72 hr growth inhibitory effects of KPT-330 yielding I50-values of 109 +/- 19 and 80 +/- 44 nM (p=0.133) in the absence and presence of BSO. In contrast, GSH depletion significantly enhanced the activity of parthenolide yielding I50-values of 2.59 +/- 0.16 and 1.35 +/- 0.15 µM (p<0.001) in the absence or presence of BSO. KPT-330 (50 µM) did not alter cellular GSH levels in HL60 cells. Using 3'-(p-hydroxyphenyl) fluorescein, KPT-330 was antioxidant in GSH replete and depleted HL60 cells. Together results indicate that conjugation with GSH does not play a significant role in KPT-330 activity. Less than additive cytotoxicity (trypan blue) and apoptosis (Hoescht) were observed using a single fixed ratio of KPT-330 and etoposide incubated either simultaneously or after overnight pre-incubation with KPT-330. Similarly, using the Chou and Talalay technique, KPT-330/etoposide combinations were less than additive in exponentially growing HL60 cells. The mechanism(s) for this apparent antagonism using this combination are under investigation. Using fluorescently labeled topo II antibody, nuclear content of exponentially growing HL60 cells was 85.9 +/- 2.3% of total in the absence of KPT-330 and 86.5+/- 1.8% after an 16 hr incubation with 100 nM KPT-330. In plateau phase cells there was a statistically significant decrease in nuclear topoisomerase IIα to 74.1+/-1.8% compared to exponentially growing cells (p<0.05). In these plateau phase cells, a similar 16 hr incubation with 100 nM KPT-330 resulted in a significant increase in nuclear topoisomerase IIα to 82.6+/-2.5% of total compared to controls (p<0.05). Results are in accord with the known CRM1-mediated shuttling of topo II from the nucleus only in cells approaching or in plateau phase (Exp. Cell Res. 313: 627-637, 2007). Studies are underway to evaluate etoposide-mediated DNA damage and topo II-covalent complexes in HL60 cells in various growth phases after KPT-330 treatment as a correlate with nuclear topo II residency.

#4793

Combinations containing the aza-anthracenedione pixantrone show preclinical activity in diffuse large B-cell lymphoma.

Chiara Tarantelli,1 Eugenio Gaudio,1 Ivo Kwee,1 Anastasios Stathis,2 Panteli Theocharous,3 Patrik Zintl,3 Emanuele Zucca,2 Francesco Bertoni1. 1 _Institute of Oncology Research - IOR, Bellinzona, Switzerland;_ 2 _Oncology Institute of Southern Switzerland - IOSI, Bellinzona, Switzerland;_ 3 _CTI Life Sciences, Uxbridge, United Kingdom_.

Introduction. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma and at least one quarter of patients still present with a refractory disease or relapse after first line chemotherapy. Pixantrone is an aza-anthracenedione with reduced cardiotoxicity, which has received a conditional marketing approval in the E.U. as a monotherapy for the treatment of adults with multiply relapsed or refractory aggressive B-cell non-Hodgkin lymphomas. Here, we evaluated the pre-clinical activity pixantrone as single agent and in combination with a series of additional anti-lymphoma drugs in cell lines derived from DLBCL.

Methods. DLBCL cell lines derived from activated B-cell like (ABC) DLBCL (U2932, TMD8, HBL1, RIVA, OCI-LY-3), and from germinal center B-cell (GCB) DLBCL (DOHH-2, SU-DHL-4, OCI-LY-19, KARPAS422, OCI-LY-8, OCI-LY-7) were studied. Pix was used in combination with: bendamustine, ibrutinib, idelalisib, lenalidomide, rituximab, vorinostat, bortezomib and etoposide. Due to the mechanism of action, lenalidomide, bortezomib and ibrutinib were evaluated only in ABC-DLBCL cell lines. Cell lines were exposed (72 h) to increasing doses of the compounds alone or in combination, followed by MTT assay. The Chou-Talalay combination index (CI) was estimated using the Synergy R package: CI < 0.9, synergism; CI between 0.9 1.1, an additive effect; CI >1.1, no benefit.

Results. The median IC50 of pixantrone as single agent was 200 nM among 11 DLBCL cell lines. GCB-DLBCL presented a trend for a higher sensitivity to the drug than ABC-DLBCL (P=0.05). Pixantrone was evaluated in combination with targeted agents and with other chemotherapy agents. The combination with the BTK-inhibitor ibrutinib was effective in all five ABC-DLBCL (median CI = 0.7, range 0.53-1.04): synergism in 4 and additive effect in 1. The combination with the PI3K-delta inhibitor was beneficial in 9 out of 11 DLBCL (median CI = 0.7; range 0.1-1.35): synergism in 8 and additive effect in 1. Benefit was also observed when pixantrone was combined with lenalidomide in 2/2 ABC-DLBCL (synergism and additive effect, 1 each), with the anti-CD20 monoclonal antibody rituximab in 2/5 DLBCL (synergism in both) and with the HDAC-inhibitor vorinostat in 2/4 DLBCL. An additive effect was seen when pixantrone was combined with etoposide in 2/3 DLBCL, with bendamustine in 2/4 DLBCL and with bortezomib in 1/2 ABC-DLBCL.

Conclusions. Pixantrone was very active in DLBCL cell lines as single agent and benefit was observed when the drug was combined with different additional anti-cancer agents. The combinations with the BTK inhibitor ibrutinib and with the PI3K-delta inhibitor idelalisib were the most effective and are worth of further studies at pre-clinical and clinical level.

#4794

Oligoamide-based mimics of double-stranded B-DNA as a new class of DNA topoisomerase I catalytic inhibitors.

Philippe Pourquier,1 Krzysztof Ziach,2 Céline Chollet,3 Vincent Parissi,4 Mathieu Marchivie,5 Panchami Prabhakaran,2 Partha P. Bose,2 Katta Laxmi-Reddy,2 Frédéric Godde,2 Stéphane Chaignepain,3 Jean Marie Schmitter,3 Ivan Huc2. 1 _INSERM U1194, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France;_ 2 _Université de Bordeaux, CBMN (UMR 5248), Institut Européen de Chimie et Biologie, Pessac, France;_ 3 _Université de Bordeaux, CBMN (UMR 5248), Centre de Génomique Fonctionnelle, Pessac, France;_ 4 _Université de Bordeaux, CNRS UMR5234, Laboratoire de Microbiologie Fondamentale et Pathogénicité, Bordeaux, France;_ 5 _Université de Bordeaux, CNRS - UPR 9048, Institut de Chimie de la Matière Condensée, Bordeaux, France_.

Since the discovery of Topoisomerase I as a specific target for the treatment of cancers more than 30 years ago, only two inhibitors derived from the natural compound camptothecin (CPT) have been approved in the clinic for the treatment of colon, lung and ovarian cancers: topotecan and irinotecan. The cytotoxicity of these Top1 poisons relies on their capability to stabilize covalent Top1-DNA complexes, leading to replication-mediated DNA double-strand breaks. However tumor cells develop multiple resistance mechanisms that are limiting their efficacy. Though new CPT derivatives and other Top1 poisons with various chemical structures have been developed, they share the same resistance mechanisms. Aside from conventional approaches focus on compounds with higher potency to stabilize Top1-DNA complexes, we investigated the possibility to inhibit Top1 binding to DNA and/or DNA cleavage by using DNA mimics that would act as decoys, a strategy that was never explored before. To this aim, single chain oligoamides composed by iteration of mQQ units (Q: 8-amino-2-quinoline carboxylic acid; mQ: 8-aminomethyl-2-quinoline carboxylic acid) were synthesized. These molecules fold into helices that are stabilized by electrostatic repulsions and hydrogen bonds between the amide functions and endocyclic nitrogen atoms at adjacent residues. When Q and mQ precursors are functionalized by negatively charged residues, the positions of these residues in the folded structure match the position of phosphate residues in duplex B-DNA. Because distances between residues in mQQ and QmQ units are slightly different, it is possible to define a major groove and a minor groove, as in B-DNA, with the possibility to impact on groove width by changing the positions of the substituents (Q4 or Q5 for substitution in position 4 or 5 of the quinoline monomer, respectively), clearly establishing (mQQ5)n and (mQQ4)n as potential DNA-mimics of an unprecedented kind.

We found that both (mQQ5)n and (mQQ4)n oligomers inhibited the activity of Top1-mediated relaxation of supercoiled DNA in vitro. Inhibition increased with the length of the oligoamide to reach an IC50 value (concentration inhibiting 50% of Top1-mediated DNA relaxation) around the nM range for the longest oligomer that was tested i.e. (mQQ4)16, corresponding to a double-strand DNA of 16 base pairs. By comparison, CPT had an IC50 of ~10 µM in the same conditions. Top1 inhibition was rather selective as a (mQQ4)8 oligomer moderately inhibited Top2-mediated activity and did not affect the activity of various DNA interacting enzymes such as restriction enzymes XhoI and NdeI or nucleases such as DNAse I, S1 nuclease, benzonase or Flap-endonuclease I. Because these oligomers are resistant to proteases and nucleases and can be synthesized rapidly by solid phase synthesis with the possibility to modify its substituents without altering their helicity, they represent good candidates for drug development.

#4795

Development of novel, selective, reversible inhibitors equipotent against wild-type and C481S Bruton's tyrosine kinase (BTK).

Nicolas E S Guisot, Victoria Walker, Stuart A. Best, Valentina Abet, Rose Chappell, Juliette Emmerich, Kelvin Ho, James R. Kelly, Kristina Lyons, Melanie Müller, Julienne Refuerzo, Louise Sargent, Fatima Talab, Madelene Waldron, Matilda Bingham, Mary-Ann Campbell, Caroline Phillips, Richard Armer. _Redx Pharma Plc - Redx Oncology Ltd, Liverpool, United Kingdom_.

Redx Oncology has developed novel, differentiated, reversible small molecule inhibitors of BTK, combining current best-in-class potency with improved selectivity profiles, which are suitable for oral, once daily dosing, designed to be equipotent against wild-type and C481S BTK.

BTK is a member of the src-related Tec family of cytoplasmic tyrosine kinases. BTK plays a key role in the BCR signaling pathway, which is required for the development, activation and survival of B-cells. BTK inhibitors have therefore been developed with the aim of treating B-cell malignancies that are dependent on BCR signaling, such as CLL and NHL. Ibrutinib is an irreversible BTK inhibitor that has been approved for the treatment of CLL, MCL and WM.

Irreversible and covalent reversible BTK inhibitors specifically target a cysteine residue C481 within BTK. Following treatment with ibrutinib, cases of secondary resistance have emerged in both CLL and MCL patients. Acquired mutations within BTK such as C481S, C481Y, C481R, C481F have been reported in the literature and clearly interfere with covalent drug binding. It has been predicted that the incidence of observed resistance will increase as clinical use outside clinical trials expands over time. Here, we present our reversible BTK inhibitor series demonstrating subnanomolar binding affinity for WT and C481S forms of BTK, that inhibits formation of pBTK in both WT and C481S BTK expressing cells. In addition, the compound demonstrated significant in vitro potency against a number of lymphoma cell lines including inhibition of BCR signaling and proliferation in OCI-Ly10 cells at nanomolar concentrations. To further investigate the binding nature of these compounds, PBMC wash out studies, measuring CD69 as a marker of B-cell activation, were used to highlight the reversible activity of these compounds.

Some BTK inhibitors also inhibit ITK, which plays a critical role in FcR-stimulated NK cell function that is required for ADCC. ADCC is the mechanism that anti-CD20 antibodies, such as rituximab, are believed to activate, and ibrutinib has been shown to antagonize this mechanism in vitro. As rituximab-combination chemotherapy is today's standard of care in B-cell malignancies, it would be desirable to have a BTK inhibitor with high selectivity for BTK over ITK.

At 1 µM, our lead compound is highly selective when tested against 469 kinase and did not show significant inhibition against other kinases involved in BCR signaling (e.g. Syk, Lyn). Furthermore, the compound has high selectivity versus structurally related cysteine-containing kinases (including EGFR and ITK) both in enzyme and cellular assays.

Our lead compound has a favorable in vitro safety profile and drug-like properties, displaying an improved CYP profile to competitor compounds. In vivo PK demonstrated good oral bioavailability and in vivo efficacy has been demonstrated.

#4796

BTK inhibitor BGB-3111 synergizes with lenalidomide in MCL models.

Nan Hu, Shuo Zhang, Min He, Jiye Zhang, Bin Jiang, Zhijian Sun, Lusong Luo, Lai Wang. _BeiGene Inc., Beijing, China_.

Background

BGB-3111 is a novel, irreversible, second generation BTK inhibitor with better selectivity profile and DMPK property compared to ibrutinib. It has demonstrated promising anti-tumor activities in patients with advanced B cell malignancies, including mantle cell lymphoma (MCL). Lenalidomide is an immunomodulatory agent with single-agent activity in several histologic subtypes of B-cell non-Hodgkin lymphoma (NHL). It was approved for the treatment of patients with MCL whose disease has relapsed or progressed after two prior therapies, one of which included bortezomib. In this study, we sought to investigate the combination activities of BGB-3111 and lenalidomide in MCL models.

Methods

In vitro drug efficacy was determined using MCL cell lines including REC-1, MINO and JEKO-1. Cell viability was assessed with Cell Titer Glo® assay and half maximal inhibitory concentrations (IC50) were estimated upon exposure to single agents or drug combinations. In vivo activity was assessed in REC-1 and MINO subcutaneous mouse xenograft models. Treatments were administered by oral gavage when tumors were palpable and individual body weight and tumor volume was recorded twice weekly.

Results

The in vitro combination activity of BGB-3111 and lenalidomide was tested in MCL cell lines. Strong synergy was observed in REC-1 and MINO cells, but not in JEKO-1 cells. When tested in vivo, both BGB-3111 (7.5 mg/kg, BID) and lenalidomide (30 mg/kg, QD) significantly inhibited the REC-1 xenograft tumor growth with TGI of 86% and 56%, respectively. The combination treatment resulted in 100% TGI. In MINO xenograft model, combination treatment of BGB-3111 (30 mg/kg, BID) and lenalidomide (30 mg/kg, QD) was also more efficacious than either single agent.

Conclusions

In summary, BGB-3111 and lenlidomide showed interesting in vitro and in vivo combination activity in MCL models, providing rationale for testing this combination in clinic.

#4797

The combination of ACP-196 and ACP-319 leads to increased survival in the TCL1-192 CLL mouse model.

Helena I. Mora-Jensen,1 Carsten U. Niemann,2 Michael Gulrajani,3 Fanny Krantz,3 Todd Covey,3 Brian J. Lannutti,3 Adrian Wiestner,1 Sarah EM Herman1. 1 _National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD;_ 2 _Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark;_ 3 _Acerta Pharma, Redwood City, CA_.

INTRODUCTION: Recent advances in the treatment of CLL focus on targeting the B-cell receptor (BCR) pathway, which is strongly upregulated in CLL. The BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib, both targeting the BCR pathway, are approved for clinical use based on significant survival benefit in clinical trials. In vitro studies have shown synergy for combined PI3Kδ and BTK inhibition (Griner, PNAS, 2014). Since CLL propagation relies on the microenvironment, in vivo models are needed for better testing of new drug combinations. We tested two new inhibitors of the BCR pathway, the BTK inhibitor ACP-196 and the PI3Kδ inhibitor ACP-319 as single agents and in combination using the murine TCL1-192 allograft model of aggressive, BCR driven CLL.

METHODS: TCL1-192 cells were injected into SCID mice and drug was given through the drinking water with vehicle, ACP-196, ACP-319 or a combination of the two drugs at a concentration of 0.15 mg/mL of each drug. Blood was analyzed weekly by flow cytometry and at the end of the study spleens were weighed and analyzed by flow cytometry.

RESULTS: Tumor burden in the blood significantly decreased with both single agent and combination treatment compared to vehicle, and with single agent compared to combination, P < 0.05, P < 0.0001 and P < 0.0075, respectively. Combination treatment also resulted in significantly lower spleen weights when compared to spleens from mice treated with single agents or vehicle. Both single agent and combination treatment led to decreased phosphorylation of PLCγ2, NFκB and ERK, molecules downstream in the BCR pathway, P < 0.05 compared to vehicle treated mice. Phosphorylation of PLCγ2 and NFκB was further decreased with combination treatment compared to single agent treatment. Both single agent and combination treatment greatly enhanced survival of the mice compared to vehicle treatment with a median survival from treatment start of 17 days (vehicle), 23 days (single agent) and 40 days (combination), P < 0.0001 for single agent vs to combination treatment.

CONCLUSION: We here show that a combination of ACP-196 (BTK inhibitor) and ACP-319 (PI3Kδ inhibitor) is superior to single agent treatment in the murine TCL1-192 model of aggressive, BCR-driven CLL. Survival was significantly increased by combination treatment and the tumor burden was significantly reduced compared to both single agent mice and vehicle treated mice. The on-target effects were validated by reduced phosphorylation of PLCγ2 and NFκB. Thus, evaluation of combination treatment with ACP-196 and ACP-319 in clinical trials is warranted.

#4798

Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL.

Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. _Aurigene Discovery Technologies Limited, Bangalore, India_.

Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ~30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ~90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL

Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene's compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene's lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile.

#4799

Inhibition of HER2 mutant non-small cell lung cancer using 3rd generation EGFR/HER2 inhibitors.

Jacqulyne P. Robichaux, Monique Nilsson, Bingliang Fang, John V. Heymach. _UT MD Anderson Cancer Center, Houston, TX_.

HER2 is modified in approximately 20% of lung cancers and is mutated in approximately 2-3% of non-small cell lung cancer (NSCLC). Several HER2 mutations within the tyrosine kinase domain are activating mutations, and irreversible EGFR TKIs such as afatinib are known to be effective at inhibiting HER2 phosphorylation in tumor cells expressing HER2 activating mutations. However, the clinical use of these second generation irreversible EGFR inhibitors, such as afatinib, has been limited by adverse toxicities such as skin rash and diarrhea. Our recent study in lung cancer showed that ibrutinib, a well-tolerated TKI currently FDA approved for B-cell lymphoma, is capable of inhibiting EGFR. Study by others also demonstrated that ibrutinib can inhibit HER2 phosphorylation in HER2 overexpressing breast cancer cell lines. In addition, recent studies in other cancers show that the effects of ibrutinib can be amplified in combination of with other small molecule inhibitors targeting AKT and mTOR. Therefore, we hypothesized that HER2 overexpression or activating mutations render NSCLC tumor cells sensitive to ibrutinib. To investigate this, we generated a panel of Ba/F3 cell lines expressing twelve individual clinically observed HER2 mutations and evaluated the transforming capability of the mutations as indicated by sustained cell viability following IL-3 deprivation. Common HER2 mutations such as A775insYVMA, G776del/insVV, and G776C V777insC as well as others were observed to be activating mutations. Mutant HER2 expressing Ba/F3 cells and the human HER2 mutant NSCLC cell line, H1781, were then screened against first, second, and third generation EGFR/HER2 inhibitors including ibrutinib, and cell viability was determined by the Cell Titer Glo assay. Drugs effective in growth inhibition were verified by Western blot analysis. The results showed that EGFR TKI erlotinib and the EGFR/HER2 TKI lapatinib failed to inhibit cell proliferation. However, ibrutinib and third generation EGFR/HER2 TKIs, EGF816 and AZD9291, inhibited cell proliferation and induced apoptosis at sub-micromolar concentrations. Western blotting analysis showed dose dependent decreases in phosphorylation of HER2 as well as HER2 downstream signaling molecules such as p-AKT, and p-MAPK. In order to determine if targeting downstream effectors of HER2 in combination with inhibition of the receptor would increase inhibition of cell proliferation and induction of apoptosis, we screened ibrutinib, EGF816 and AZD9291 in combination with both mTOR and AKT inhibitors. There was a modest additive effect when ibrutinib was used in combination with everolimus, an mTOR inhibitor, but there was no additive effect of EGF816 or AZD9291 was used in combination with everolimus. These results indicate that EGF816, AZD9291, or ibrutinib alone or in combination with mTOR inhibition may be effective therapeutic strategies for the treatment of HER2-driven NSCLC.

#4800

Covalent Flt3-Cys828 inhibition represents a novel therapeutic approach for the treatment of Flt3-ITD and Flt3-D835 mutant acute myeloid leukemia.

Matthias Versele,1 Burkhard Haefner,1 Berthold Wroblowski,1 Ian Stansfield,1 Laurence Mevellec,1 Ron Gilissen,1 Lars Neumann,2 Martin Augustin,2 Kris Jacobs,3 Jan Cools,3 S Barluenga,4 M Röthlingshöfer,4 G Karthikeyan,4 Ricardo Attar,5 Lieven Meerpoel,1 Nicolas Winssinger4. 1 _Janssen Oncology, Beerse, Belgium;_ 2 _Proteros Biostructures, Martinsried, Germany;_ 3 _KU Leuven Center of Human Genetics, VIB Center for the Biology of Disease, Leuven, Belgium;_ 4 _School of Chemistry and Biochemistry, NCCR Chemical Biology, University of Geneva, Geneva, Switzerland;_ 5 _Janssen Oncology, Spring House, PA_.

Inhibition of Flt3 kinase activity is a promising strategy for the treatment of Flt3 mutant acute myeloid leukemia (AML). However, clinical studies with Flt3 kinase inhibitors have shown that mutations of the gate keeper or an activation loop residue in the Flt3 catalytic domain limits the duration of response. Such mutations generally reduce kinase domain binding affinity and residence time of the kinase inhibitor, and consequently its efficacy. We therefore investigated whether covalent, irreversible binding to Flt3 could overcome some of the limitations of classic non-covalent Flt3 inhibitors. Here, we report for the first time on the high-affinity Cys828-covalent binding mode of a resorcylic acid lactone to the isolated Flt3 kinase domain using X-ray crystallography and kinetic binding assays. In a cellular context (Ba/F3-Flt3-ITD), mutation of Cys828 to Ala reduces potency (IC50) from low nM to microM, demonstrating all relevant Flt3 inhibition in cells critically depends on Cys828. Consistently, the molecule is a low nM inhibitor of both Flt3-ITD and Flt3D835Y in vitro and in BaF3 cells, translating to potent nM inhibition of Flt3-ITD driven AML cell line proliferation, with only microM antiproliferative activity in non-Flt3 driven AML and unrelated leukemia cell lines. Finally, in spite of its fast clearance when dosed to MV4-11 (Flt3-ITD AML) xenograft bearing mice, robust anti-tumor activity was observed using a once-daily treatment schedule. These data demonstrate the feasibility of covalent Flt3 inhibition, and suggest it represents an attractive novel therapeutic approach for the treatment of Flt3-driven AML.

#4802

PB1, a DDR2 inhibitor with antitumor activity in preclinical models of squamous cell carcinoma and KRAS-mutated adenocarcinoma of the lung.

Silvia Garcia-Roman,1 Miguel Angel Molina,1 José Ignacio Borrell,2 Raimon Puig de la Bellacasa,2 Daniela Morales,3 Jordi Bertran,1 Ana Gimenez,1 Niki Karachaliou,3 Rafael Rosell3. 1 _Pangaea Biotech S.L., Barcelona, Spain;_ 2 _Institut Químic de Sarrià (IQS), Barcelona, Spain;_ 3 _Instituto Oncológico Rosell, Barcelona, Spain_.

Introduction: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) activated by collagen I and identified as an oncogenic driver in squamous cell carcinoma of the lung (SCC) (1). DDR2 mutations have been reported in 4% of SCC patients and at lower frequencies in lung adenocarcinoma, gastric, breast and brain tumors (2). In addition, DDR2 is expressed during epithelial to mesenchymal transition (EMT) (3) and acts as an upstream activator of SHP2. SHP2 in turn is a key signaling effector which leads to activation of multiple signaling pathways (4). DDR2 has recently emerged as a potential therapeutic target in DDR2-mutated tumors and drugs such as dasatinib are being tested in this setting.

Results and discussion: PB1 is a small organic molecule with a simple synthesis pathway that showed strong inhibitory activity against wild type (wt) and mutated purified DDR2 protein. The compound was subsequently tested in a panel of tumor cell lines from different origins and histologies.

PB1 showed good antiproliferative activity: IC50<1 µM in (i) SCC cell lines with DDR mutations such as H2286 or H520, (ii) SCC lung tumor cell lines wt for DDR2, and (iii) some lung adenocarcinoma cell lines with KRAS mutations such as A549. Remarkably, the two cell lines tested carrying the DDR2 activating mutation I638F were extremely sensitive to PB1, with IC50 in the nanomolar range. The compound also showed good activity against cells harboring the DDR2 L239R mutation. We also generated two dasatinib-resistant cells lines from the H2286 and H520 cell lines. Cells had IC50s for dasatinib over 25 uM but remained sensitive to PB1, with IC50s of 0.6 and 5.92, respectively. Finally, in non tumor cell lines, no toxicity was observed with PB1 at concentrations tested (up to 50 µM), indicating a good therapeutic index. Western blotting was used to study the effects of PB1 on DDR2, ERK and AKT activation in H2286 and A549 cells. The compound strongly inhibited FBS-induced phosphorylation of the three proteins.

Conclusion: PB1 shows significant antitumor activity in preclinical models of mutated and wt DDR2 in SCC of the lung and some KRAS mutated adenocarcinoma cell lines. PB1 is also active against tumor cells with secondary resistance to dasatinib.

References:

1) Hammerman et al. Cancer Discovery. 2011 June; 1: 78-89.

2) Terai et al. ACS Chem Biol. 2015 Sep; Epub ahead of print.

3) Walsh et al. Matrix Biol. 2011 May; 30(4): 243-247.

4) Iwai et al. Biochem. J. (2013) 454, 501-513.

#4803

A novel class of phenylacrylonitrile-based small molecules selectively induce double-strand DNA damage in breast cancer cells.

Jennette A. Sakoff,1 Jayne Gilbert,1 Geoff De Iuliis,2 Adam McCluskey2. 1 _Calvary Mater Newcastle Hospital, Newcastle, NSW, Australia;_ 2 _The University of Newcastle, Newcastle, NSW, Australia_.

We have discovered a class of phenylacrylonitrile-based small molecules that selectively target breast cancer cell lines while having little to no effect on normal breast cancer cells or on cell lines derived from other tumour types including colon, ovarian, lung, skin, prostate and pancreatic carcinomas, neuroblastoma and glioblastoma. Indeed these molecules show more than 200-fold selectivity towards breast cancer cells compared with other tumour types, while maintaining nM potency, as determined by the MTT growth inhibition (GI50) assay with GI50 values of 0.2-2.0µM (72h exposure). Moreover, the sensitive breast cancer cell lines represent tumour types from the four main breast cancer classifications including ER+ luminal A (MCF-7, T47-D and ZR-75-1 cells), ER+ luminal B (BT-474), HER2+ (SKBR-3), and most importantly the Basal (triple negative MDA-MB-468 and BT20) classification which traditionally carries a very poor prognosis. Our novel class of molecules also retain activity in MCF-7/VP16 cells (GI50 0.2µM) which overexpresses the drug resistance ABCC1 gene. Only one breast cancer cell line has shown insensitivity, MDA-MB-231 (Basal triple negative), which unlike all of the other breast cell types has amplifying mutations in KRas and BRaf activity, a genotype found in less than 5% of breast cancer tumours. Furthermore, our novel compounds induce minimal effects on the growth of normal MCF10A breast cells.

Analysis of our most sensitive breast cancer cell line (MDA-MB-468) shows a significant increase in gammaH2AX within 12h of exposure to our lead phenylacrylonitrile molecule, indicative of DNA double strand breakage. This response is concomitant with an increase in the phosphorylation of the cell cycle check point activator CHK2. Cell cycle analysis shows an increase in the proportion of cells in the S-phase of the cell cycle within 24h of exposure, concomitant with a decrease in the proportion of cells in the G1-phase, and preceding an increase in the sub-G1 cell death population. The ability to specifically target breast tumours, while having little or no effect on normal breast cells, or other tumour types is a unique finding. Elucidating the mechanism controlling this phenomenon is now the focus of our research efforts.

#4804

Polygodial: A potential sesquiterpene dialdehyde for castration-resistant prostate cancer treatment.

Akshita P. Pillai,1 Subramanyam Dasari,1 Bhavani Patel,1 Souresh Banerjee,1 Alexander V. Kornienko,2 Gnanasekar Munirathinam1. 1 _University of Illinois, College of Medicine at Rockford, Rockford, IL;_ 2 _Texas State University, San Marcos, TX_.

Major cause for prostate cancer mortality is due to the development of castration-resistant prostate cancer (CRPC) which usually develops when prostate cancer patients undergo standard androgen deprivation therapy. Treatment options currently available for CRPC are not very efficient and have many side effects. Hence, there is an urgent need to identify effective treatment strategies for CRPC. Natural compounds have previously shown to be an effective alternative treatment strategy for cancer. Polygodial (PG) an active constituent of mountain pepper belonging to sesquiterpene dialdehyde family has shown to have several pharmacological benefits including anti-bacterial, anti-fungal, anti-inflammatory, and anti-cancer properties. The objective of this study is to determine the therapeutic effects of PG against CRPC in a pre-clinical study. Cell lines used in this study were androgen dependent LNCaP and CRPC C4-2, DU145, and PC-3 cells. MTT, soft agar, and wound healing assays were performed to evaluate the effects of PG respectively on cell survival, anchorage independent growth, and migration characteristics of prostate cancer cells. PC-3 cells were utilized to determine the anti-cancer mechanisms of PG. Cell viability assay results showed PG effectively inhibited cell proliferation of CRPC C4-2, DU145 and PC-3 cell lines as compared to androgen dependent LNCaP cells. PG treatment significantly reduced the colony growth of CRPC cells in soft agar plates as compared to controls which correlates with its inhibitory effect on anchorage independent growth of these cells. In addition, PG also reduced the migration potential of CRPC cells in a wound healing assay. Inhibition of anchorage independent growth and motility of PCa cells by PG suggests its tumor suppressive and anti-metastatic potential. Western blot analysis showed that PG treatment downregulated the expression of a key cell survival molecule, High mobility group box1 (HMGB1) while promoting PARP-1 cleavage in PC-3 cells. Interestingly, our results also showed that BiP/GRP78, a marker of endoplasmic reticulum (ER) stress was upregulated following PG treatment in PC-3 cells. These findings suggest that PG enforces its tumor suppressive action by inhibiting HMGB1 signaling axis with concurrent activation of ER stress pathway in CRPC cells. Taken together, our study shows that PG might be an effective chemotherapeutic natural compound especially for CRPC.

#4805

In vitro characterization of novel inhibitors of ROCK and MRCK kinases as anticancer agents.

Dhimant H. Desai,1 Vijay P. Kale,2 Jeremy A. Hengst,1 Taryn E. Dick,1 Ashley L. Colledge,1 Shantu G. Amin,1 Jong K. Yun1. 1 _Penn State University College of Medicine, Hershey, PA;_ 2 _Bettle Memorial Institute, Columbus, OH_.

Metastatic cancers are the second leading cause of deaths in the USA. RhoA and Cdc42 play critical roles in the regulation of plasticity of cancer cell migration/invasion and cell proliferation. ROCK1/2 and MRCKα/β are downstream kinases in the signaling pathways associated with cancer cell migration and invasion. Hence, we hypothesized that simultaneous targeting of these two kinase families would be an effective therapeutic strategy to block migration, invasion, and growth of metastatic cancers. We have identified DJ4 as a novel inhibitor of ROCK and MRCK kinases. In the cellular functional assays, DJ4 treatment significantly blocked stress fiber formation, and inhibited migration and invasion of multiple cancer cell lines in a concentration dependent manner. To study the critical functional groups required for its activity, we have modified the chemical structure of DJ4 at various functional groups and synthesized several analogs of DJ4 to perform the structural activity relationship (SAR) study for their ROCK1 inhibition. The effectiveness of these compounds were further investigated using National Cancer Institute's drug screening program in 60 human cancer cell lines representing nine different cancer types. These compounds effectively inhibit migration and invasion of multiple cancer cell types. Selected analogs were tested for their anti migration, pro-apoptotic, and anti-proliferative effects in breast cancer cells. Our studies strongly indicate that DJ4 and its analog, DJ110, are potent inhibitors of ROCK1, ROCK2, MERKα and MRCKβ. The results of our finding will be discussed.

### Targeted Therapy

#4806

Enhancing the efficacy of tosedostat through carboxylesterase induction.

Priscilla Wei Ling Hong,1 Amanda Melanie Smith,1 Lambro Angelo Johnson,2 Dorothy Loo,3 Thomas Stoll,3 Michelle M. Hill,3 Andrew S. Moore4. 1 _Childhood Leukaemia Research Laboratory, The University of Queensland Diamantina Institute, Brisbane, Australia;_ 2 _School of Medicine, The University of Queensland, Brisbane, Australia;_ 3 _Cancer Proteomics Group, The University of Queensland Diamantina Institute, Brisbane, Australia;_ 4 _Childhood Leukaemia Research Laboratory, The University of Queensland Diamantina Institute; UQ Child Health Research Centre, The University of Queensland; Oncology Service, Lady Cilento Children's Hospital, Children's Health Queensland, Brisbane, Australia_.

Malignant cells, including acute myeloid leukaemia (AML), have high protein turnover to support their accelerated cell growth. Aminopeptidases regulate proteolysis, supplying free amino acids for new protein synthesis. Inhibition of aminopeptidases depletes free amino acids, impairing new protein production, leading to impaired cell growth and proliferation. Hence, aminopeptidase inhibition is of particular interest as a therapeutic strategy for AML. Tosedostat (CHR-2797) is an aminopeptidase inhibitor which is converted to its active metabolite, CHR-79888, by intracellular carboxylesterases (CES). This active metabolite is poorly membrane permeable, resulting in intracellular accumulation. Tosedostat has shown promise as a potential therapeutic strategy for AML and is well-tolerated in adult patients. However, its efficacy in paediatric AML has not been established. We set out to investigate the use of tosedostat for paediatric AML and enhance its efficacy through CES1 induction. It is hypothesised that inducing CES will lead to increased CHR-79888 production and subsequently, increased tosedostat efficacy. The IC50 of tosedostat across 11 AML cell lines (6 paediatric and 5 adult), determined by resazurin reduction assay, was approximately 13µM. Biochemical assays demonstrated that tosedostat inhibited cellular aminopeptidase activity in a concentration-dependent manner. However, this inhibitory effect was reversible with rapid, but incomplete, enzyme activity recovery upon drug withdrawal, irrespective of initial concentration or treatment duration. Quantitative PCR and immunoblotting revealed no correlation between CES expression and sensitivity to tosedostat. Cellular CES1 activity was also measured. Benzil and Bis(4-nitrophenyl)phosphate (BNPP) were used to modulate cellular CES1 activity. Assays of CES1 enzyme kinetics demonstrated that following temporary exposure to the reversible CES inhibitor benzil, there is a rebound in CES1 activity in AML cells. Consistent with this observation, pre-treatment by benzil increased CES1 activity, especially in MV4-11 cells. Moreover, reduced cell viability with tosedostat treatment following benzil pre-treatment correlated with the induction of CES1 activity in AML cell lines. Together, these data suggest that enhanced tosedostat efficacy through induction of CES activity is a novel therapeutic approach to treat AML.

#4807

Triphenylmethanol conjugates of triptorelin as anti-cancer prodrugs.

Ryan Beni, William Boadi, Jawzah Alnakhli. _Tennessee State University, Nashville, TN_.

Triptorelin (TRP) is an anti-cancer agent used for the treatment of a wide variety of hormone-responsive cancers, such as prostate cancer. The surge of testosterone, known as a flare effect, is the major side-effect of cancer chemotherapy using TRP in a combination regiment. Improving the biological activity of TRP by increasing the cellular uptake and retention is a remedy to this end. In this study, the hydrophobicity of TRP was optimized by using an appropriate hydrophobic linker attached to tris(4-methoxyphenyl)methanol (TPM) derivatives with the expectation to improve the cellular uptake. In this regard, a number of TRP conjugates of TPM derivatives with optimized hydrophobicity were synthesized by the reaction of 2-substituted methoxy benzenes (e.g. methyl 2-methoxybenzoate, 1,2-dimethoxybenzene, methoxy-2-methylbenzene, methoxy-2-nitrobenzene, chloro-2-methxyenzene), and 1,3,5-trioxane, followed by the conjugation with TRP and decanedioic acid in the presence of HBTU/DIPEA/DIC in moderate yields. Comparative antiproliferative assays between TPM-TRP conjugates and the corresponding non-covalent physical mixtures of the TPM derivatives and TRP were performed against human acute lymphoblastic leukemia (CCRF-CEM), human ovarian adenocarcinoma (SK-OV-3), and mouse pre adipocytes (3T3-L1) cells. TPM-TRP conjugates inhibited the cell proliferation of CCRF-CEM, SK-OV-3 and 3T3-L1 cells by 55-92%, 24-73%, 37-56%, respectively, at a concentration of 10-50 µM after 24-72 h of incubation. These data suggest that TPM-TRP derivatives with optimized hydrophobicity can be used to improve the biological activity of TRP.

#4808

**Identification of a novel compound, MO2455, that induces poly(ADP-ribose) (PAR) accumulation and inhibits the growth of cancer cells** in vitro **and** in vivo **.**

Tatsu Shimoyama,1 Takeshi Sawada,1 Mari Akimoto,1 Yuka Sasaki,2 Hiroaki Fujimori,2 Yoshinobu Ishikawa,3 Tadashi Okawara,4 Tetsumi Irie,5 Takeji Takamura,6 Kenji Matsuno,7 Kengo Inoue,8 Mitsuko Masutani,9 Fumiaki Koizumi1. 1 _Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Tokyo, Japan;_ 2 _National Cancer Center Institute, Tokyo, Japan;_ 3 _University of Shizuoka, Shizuoka, Japan;_ 4 _Kumamoto Health Science University, Kumamoto, Japan;_ 5 _Kumamoto University, Kumamoto, Japan;_ 6 _Kanagawa Institute of Technology, Kanagawa, Japan;_ 7 _Kogakuin University, Tokyo, Japan;_ 8 _Fuji Pharma Valley, Shizuoka, Japan;_ 9 _Nagasaki University, Nagasaki, Japan_.

Poly (ADP-ribose) chain (PAR) formed by poly (ADP-ribose) polymerase (PARP) is catabolized mainly by poly (ADP-ribose) glycohydrolase (PARG). PARG is emerging as a therapeutic target for cancer, because its inhibition leads to cell death in some kinds of cancer cell lines.

In order to obtain small molecules that effectively inhibit PARG protein, an in-house collection of ~10,000 small molecules were screened for their ability to accumulate the PAR in A549 cells at 5 µM. We identified ~100 hit compounds. Among them, MO2282 and MO2455 induced the most significant accumulation of PAR. MO2455 is an analogue of MO2282 with improved water-solubility. MO2282 and MO2455 were evaluated in vitro for their ability to inhibit the catalytic activity of PARG. MO2282 and MO2455 showed modest inhibition activities against recombinant rat PARG. They also inhibited the growth of various kinds of cancel cell lines in vitro at an IC50 value of 0.05 - 3.0 µM and showed different spectrum of antitumor activity from conventional anticancer drugs, CDDP, ADM, PTX, and CPT-11. Next, we investigated the growth-inhibitory effect of MO2455 on A549 cells in a xenotransplanted model. A significant anti-tumor activity was observed in mice when treated with MO2455 at doses of 25 mg/kg every two days. Although treatment-related body weight loss was observed in mice treated with MO2455, body weight recovered by day 8.

In conclusion, the present data suggest that MO2455 has potential as a cancer drug with different mechanisms of action from conventional anti-cancer drugs.

#4809

Combination therapy targeting ribosome biogenesis and mRNA translation provides a novel and potent therapeutic approach to treat MYC-driven malignancy.

Jennifer R. Devlin,1 Richard J. Rebello,2 Katherine M. Hannan,3 Carleen Cullinane,4 Denis Drygin,5 Gail P. Risbridger,2 Luc Furic,2 Ross D. Hannan,3 Richard B. Pearson4. 1 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 2 _Monash University, Clayton, Australia;_ 3 _John Curtain School of Medical Research, Canberra, Australia;_ 4 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 5 _Pimera Inc, San Diego, CA_.

MYC-driven malignancies are associated with elevated rates of ribosome biogenesis and mTORC1/eIF4E-driven protein synthesis suggesting they may be vulnerable to therapeutic strategies that target the ribosome. We investigated the therapeutic efficacy of targeting multiple nodes of the network controlling the ribosome in mouse models of MYC-driven lymphoma (Eμ-Myc) and prostate cancer (HI-MYC). Simultaneous inhibition of ribosomal RNA synthesis and repression of protein translation was achieved by utilizing the novel RNA polymerase I inhibitor CX-5461 and PI3K/AKT/mTORC1 and PIM 1 signaling inhibitors.

Combined inhibition of ribosome biogenesis and function significantly improved therapeutic outcomes in lymphoma and prostate cancer models. CX-5461 and Everolimus (mTORC1 inhibitor) co-treatment more than doubled the survival of Eμ-Myc lymphoma-bearing mice. While both classes of inhibitor suppress rDNA transcription, they treat MYC-driven malignancy through distinct molecular mechanisms facilitating their combinatorial effects. In contrast to CX-5461, PI3K/AKT/mTOR pathway inhibitors did not activate a nucleolar stress response and p53-dependent apoptosis but instead induce B-lymphoma cell death via the upregulation of the BH3-only protein BMF (Devlin et al Cancer Discovery 2015 Oct 21. pii: CD-14-0673. [Epub ahead of print]). PIM kinase has been shown to regulate eIF4Edriven protein synthesis on prostate cancer cell lines. Co-treatment of HI-MYC mice with CX-5461 and the PIM kinase inhibitor CX-6258 reverted highly invasive disease to low-grade prostate intraepithelial neoplasia.

These findings demonstrate that MYC driven tumors are addicted to multiple regulatory steps associated with ribosome synthesis and function and coordinated targeting of these addictions provides an effective new therapeutic approach to treat MYC driven cancers.

#4810

Histone deacetylase 2 is a potential determinant of the sensitivity of hepatocellular carcinoma cells to sorafenib.

Hyung Seok Kim, Jung Woo Eun, Qingyu Shen, Woo Chan Shin, Hee Doo Yang, Sang Yeon Kim, Suk Woo Nam. _Catholic Univ. of Korea College of Medicine, Seoul, Republic of Korea_.

Hepatocellular carcinoma (HCC) is the fifth frequently diagnosed cancer and is the second leading cause of cancer death worldwide. Many groups have suggested possible clinical applications for liver cancer therapy but sorafenib, an orally-available kinase inhibitor, is the only standard systematic therapeutics for treatment of hepatocellular carcinoma. However, survival benefit of sorafenib is unsatisfactory due to high level heterogeneity of individual response. We previously reported that histone deacetylase 2 (HDAC2) was deregulated in HCC, and thereby contributed to liver tumorigenesis by enhancing mitotic and metastatic potential of transformed cells. Targeted-disruption of HDAC2 suppressed in vitro and in vivo tumorgigenic potential of HDAC2 in liver cancer. The goal of this study was to investigate whether both sorafenib treatment and HDAC2 inhibition elicit synergistic anti-tumor effect against HCC cells. Here, we showed that sorafenib effectively blocked ERK/MEK and AKT signaling pathway upon EGF stimulation in liver cancer cell lines. Also, we found that valproic acid (VPA), a selective inhibitor of histone deacetylase family I, and sorafenib treatment synergistically evoked liver cancer cell growth retardation. This effect was recapitulated by HDAC2 knockdown with sorafenib treatment and exhibited synergistic effect on apoptotic cell death of liver cancer cell lines, Hep3B and Huh7, compared to single treatment of sorafenib. Our data suggest that combined sorafenib treatment with HDAC2 targeting may provide more benefits toward HCC therapy providing a novel approach for future application in patients with advanced HCC.

#4811

Global demethylation with decitabine increases DNA repair and sensitizes melanoma to carboplatin.

Timothy Budden,1 Ryan J. Davey,1 Ricardo E. Vilain,2 Katie A. Ashton,1 Stephen Braye,2 Andre van der Westhuizen,3 Nikola A. Bowden1. 1 _Univ. of Newcastle, Newcastle, Australia;_ 2 _Hunter Area Pathology Service, Pathology North, Newcastle, Australia;_ 3 _Calvary Mater Hospital, Newcastle, Australia_.

Global genome repair (GGR) is critical for recognizing DNA damage by the chemotherapy agent carboplatin, and triggering apoptotic signalling. XPC is a critical gene in the GGR pathway but in melanoma XPC is not induced in response to carboplatin resulting in extreme resistance. Despite low global methylation levels across the melanoma genome, there is evidence methylation plays a role in suppression of XPC. 5-aza-2'-deoxycytidine (decitabine), is a DNA methyltransferases inhibitor used as a chemotherapy agent that results in global loss of methylation and re-expression of silenced genes. We hypothesised that restoring expression of XPC in melanoma using decitabine could overcome resistance to carboplatin. To confirm low XPC in melanoma tumour tissue, transcript and protein expression from 196 advanced primary and metastatic melanomas was quantified and compared to normal tissue and other cancer types. Melanoma cell lines with low baseline XPC were treated with pharmacological doses of decitabine or carboplatin alone, or in sequential combination. XPC transcript, apoptosis, proliferation, senescence, global and CpG island shore demethylation was quantified. XPC transcript and protein was low in a proportion of melanoma tumours and expression correlated with overall survival. Treatment of melanoma cells with carboplatin alone did not significantly induce XPC expression or increase apoptosis in the majority of melanoma cell lines. Decitabine decreased global methylation (average 44.67% decrease) and increased XPC expression (0.9-3.0 fold increase). Hotspots of demethylation after decitabine treatment in the XPC CpG island shore were detected and could be responsible for the increase in XPC expression. When carboplatin was administered following decitabine, a greater XPC induction (1.5-7.6 fold increase) occurred with significantly increased levels of apoptosis (1.6-2.2 fold increase). This was coupled with a decreased proliferation and the presence of markers of senescence. Knocking down XPC expression with siRNA significantly reduced the effects of combination treatment. The outcomes of this study are evidence that silencing of XPC by methylation is a mechanism of carboplatin resistance in melanoma and sequential combination of decitabine followed by carboplatin may be used to restore GGR function and overcome resistance.

#4812

Arginine deiminase loaded in erythrocytes: a promising formulation for L-arginine deprivation therapy in cancers.

Fabien Gay,1 Karine Aguera,1 Karine Senechal,1 Julie Bes,1 Anne-Marie Chevrier,1 Fanny Gallix,1 Christine Guicher,1 Philip Lorenzi,2 Vanessa Bourgeaux,1 Willy Berlier,1 Françoise Horand,1 Yann Godfrin1. 1 _Erytech Pharma, Lyon, France;_ 2 _MD Anderson Cancer Center, Houston, TX_.

Based on the asparaginase paradigm, several arginine-catabolizing enzymes have been developed for the treatment of arginine-dependent cancers (Wheatley, 2004). The arginine deiminase (ADI) enzyme catalyzes the hydrolysis of arginine (Arg) (Sugimura, 1990). ADI purified from Mycoplasma has a short half-life (≈4h) in the circulation and was found to be highly immunogenic (Holstberg, 2002). In order to increase half-life and to limit immunogenicity, a pegylated form of the enzyme (ADI-PEG-20) was developed. Phase I/II clinical studies in hepatocarcinoma and in advanced melanoma concluded on its limited efficacy at the tested doses. Notably, reduction of the duration of Arg depletion was linked to the emergence of ADI-PEG-20 antibodies (Ascierto, 2006). Encapsulation of ADI into red blood cells (RBC) is a promising alternative to improve the half-life and reduce the immunogenicity of the protein. Argininosuccinate synthase (ASS1) is the key enzyme in arginine biosynthesis (Haines, 2011). ASS1 expression varies in tumors and ASS1 loss is associated with poor prognosis in different cancers (Qiu, 2015). All these data strengthen the importance of selecting ASS1-negative patients for Arg-depletion based enzymatic therapy.

Using a scalable, standardized production process, we synthesized an ADI enzyme from an optimized M. arginini coding sequence. ADI was encapsulated into RBC by hypotonic dialysis (ERY-ADI) and PK-PD parameters were evaluated in CD1 mice, in comparison with free ADI. Sensitivity to ADI was assessed in vitro by measuring the cell viability of 3 cancer cell lines with different ASS1 expression levels. ASS1 expression was screened by immunohistochemistry (IHC) in a large panel of tissue microarrays (TMA) from 16 human cancer types.

Administration of ERY-ADI (5.5IU/mL) reduced mouse plasmatic Arg level to 30% of control values and led to a maintained depletion for 5 days. The same dose of free ADI strongly depleted Arg (<5µM) for 24h but concentration returned to baseline by 2.5 days. ERY-ADI at higher dose (10.4IU/mL) resulted in the complete depletion of plasmatic Arg over the 5-day period of the study. No problem of tolerability were noticed. In cell lines, sensitivity to ADI was linked to ASS1 expression: AGS cell line (ASS1-negative) was highly sensitive to ADI treatment (IC50= 0.4mIU/mL; 100% cell mortality) whereas NCI-N87 (ASS1-high) was strongly resistant (no mortality at the highest tested dose) and HL-60 (ASS1-low) was moderately sensitive (IC50= 0.3mIU/mL; 70% cell mortality). ASS1 screening in TMA revealed several cancers of interest with down-regulated ASS1 expression.

All these results highlight that arginine depletion through ADI treatment is effective against ASS1-negative cancer cells. ERY-ADI represents an innovative product with an improved efficacy for sustained Arg depletion and suitable for the treatment of ASS1 deficient cancers.

#4813

Magnetic iron oxide nanoparticles enhance anti-tumor effect of docetaxel on prostate cancer cells via ROS generation and NF-kappa B signaling.

Kanako Kojima,1 Saho Hashimoto,1 Rina Sakamaki,1 Sanai Takahashi,1 Risa Kasakura,1 Ryo Maruyama,1 Tadashi Nittami,1 Hitoshi Ishiguro,2 Hiroji Uemura,3 Masatoshi Watanabe1. 1 _Yokohama National Univ., Yokohama, Japan;_ 2 _Photocatalyst group, Kanagawa Acad. Sci. Tech., Kawasaki, Japan;_ 3 _Yokohama City University Medical Center, Yokohama, Japan_.

Chemotherapy is one of treatment options for castration-resistant prostate cancer. Especially, docetaxel-based chemotherapy has been shown to improve the quality of life in patients with the advanced disease. However, this needs to be improved because of limitations by lack of specificity, toxicity, and progression of docetaxel-resistance. Thus, combination of docetaxel with other compounds or drugs is needed to overcome these problems. Magnetic iron oxide nanoparticles (MgNPs, Fe3O4) have been investigated for nanomedicine such as drug delivery, cancer diagnosis and therapy. We have also shown that MgNPs would modify the effect of chemotherapy in prostate cancer cells in vitro. The purpose of this study was to analyze the combined effect of docetaxel and non-/carboxyl-modified magnetic nanoparticles (MgNPs/MgNPs-COOH) on a prostate cancer cell line, DU145 and clarify their mechanisms. While only MgNPs produced reactive oxygen species (ROS) and 8-OHdG, MgNPs-COOH did neither produce ROS nor 8-OHdG. The combination of docetaxel and MgNPs-COOH more effectively inhibited cancer cell growth and induced apoptosis compared with combination of docetaxel and MgNPs. Although docetaxel induced NF-kappa B expression, combination of docetaxel and MgNPs inhibited NF-kappa B expression compared with combination of MgNPs-COOH and docetaxel. However, both combinations increased p-Akt expression. These results suggest that MgNPs/MgNPs-COOH may enhance effect of docetaxel on prostate cancer cells via NF-kappa B signaling, however different factors may be involved in these phenomena, leading to new tumor ablation therapies.

#4814

**miRNA combination therapy:** In vitro **anticancer synergy between miR-34a mimic and next generation EGFR tyrosine kinase inhibitors (TKIs) in NSCLC.**

Jane Zhao, Adriana Guerrero, Kevin Kelnar, Heidi J. Peltier, Andreas G. Bader. _Mirna Therapeutics, Inc., Austin, TX_.

Background: miRNAs play a critical role in regulating key biological processes by modulating the expression of up to several hundred genes across multiple cellular pathways. miR-34a, one of the most widely studied miRNAs, is lost or expressed at reduced levels in many tumors, and normally functions as a natural tumor suppressor by down-regulating expression of >30 different oncogenes, as well as genes involved in tumor immune evasion, including PD-L1. MRX34 is a potential first-in-class liposome-encapsulated miR-34a mimic in Phase 1 study (NCT01829971) as monotherapy in patients with advanced malignancies. The ability of miR-34a to regulate the expression of key oncogenes across multiple oncogenic pathways makes MRX34 a rational candidate to combine with other anticancer therapies which are frequently subject to primary and acquired resistance in the clinic. Previous studies showed that miR-34a greatly sensitizes both EGFR wild-type and mutant NSCLC cell lines, as well as hepatocellular carcinoma cell lines, to the first generation EGFR TKI erlotinib. Here we report research combining miR-34a and the next generation EGFR TKIs afatinib (Gilotrif®) and rociletinib (CO-1686) in NSCLC cell lines.

Methods: Combination studies using single-drug ratios (~IC50 ratio of miR-34a and TKI) and multiple ratios above and below were performed in a panel of EGFR wild-type (A549, H460, H1299, H226) and EGFR mutant (H1975, HCC827 parent and HCC827 erl res) NSCLC cell lines. Cells were transfected with miR-34a and incubated 24 hrs later with afatinib or rociletinib for 72 hrs, with cellular proliferation then determined by AlamarBlue. Synergistic, additive, or antagonistic effects were determined by combination index (CI) values (based on Loewe's concept of additivity), isobolograms, and curve-shift analyses.

Results: Strong synergy was observed between miR-34a and both TKIs in all EGFR-mutant cell lines tested (CI <0.5 at effect levels ≥50%). Synergy was also observed for miR-34a + rociletinib in most EGFR wild-type cell lines (CI <0.6 at effect levels of ~50-80%), but not for miR-34a + afatinib. Best synergies overall were observed in EGFR-mutant cells with acquired erlotinib resistance. The effects were observed across a range of different dose levels and drug ratios, with maximal synergy providing a high level of inhibition (80%) at doses likely achievable in the clinic and well below those projected to be required for similar inhibition by the single agents alone.

Conclusions: Complementing previous results with miR-34a + erlotinib, the data demonstrate strongly synergistic anticancer effects between miR-34a and next generation EGFR TKIs in combination against a range of EGFR wild-type and mutant NSCLC cell lines. The results support clinical study of MRX34 + EGFR TKI combinations in patients with advanced NSCLC, including those with EGFR-mutant NSCLC that has progressed on EGFR TKI monotherapy.

#4816

**Potent inhibition of the cell proliferation and induction of apoptosis in lymphoma cells by the anthelminthic drug niclosamide:** in vitro **data.**

Junaid Ansari,1 Hazem El-Osta,1 Paula Polk,1 Jeffrey J. Aufman,2 Guillermo A. Herrera,2 James Cardelli,3 Rodney E. Shackelford,2 Glenn M. Mills,1 Magdalena L. Circu,1 Felicity N. E. Gavins,4 Reinhold Munker1. 1 _Feist-Weiller Cancer Center, LSU Health, Shreveport, LA;_ 2 _Department of Pathology, LSU Health, Shreveport, LA;_ 3 _Department of Microbiology and Immunology, LSU Health, Shreveport, LA;_ 4 _Department of Molecular and Cellular Physiology, LSU Health, Shreveport, LA_.

Background: Niclosamide, an anthelminthic drug, has demonstrated anti-cancer potential in variety of malignancies. However only a limited number of studies have been performed in lymphoma models, therefore we hypothesized that niclosamide may also have anti-cancer potential on B-cell lymphomas.

Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide and IC50 was calculated using GraphPad Prism 6.0 software. Cell viability and proliferation were assessed by CellTiter-Blue and trypan blue exclusion assays. Apoptosis was assessed by flow cytometry following Annexin-V/ propidium iodide staining. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0. Colony forming assays were performed in methylcellulose. Ultrastructural cellular changes were studied with electron microscopy. Peripheral blood mononuclear cells (PBMCs) from individuals without active cancer and from patients with different hematologic disorders, were also exposed with niclosamide.

Results: Treatment with niclosamide resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in different lymphoma cell lines including vincristine-refractory cell line. The IC50 of lymphoma cells lines is as follows: Daudi: 0.33 μM; HBL-2: 0.57 μM; KOPN-8: 0.72 μM; Ramos: 0.53 μM and SU-DHL4-VR: 0.45 μM. Niclosamide also inhibited clonal growth in semi-solid media. Gene expression changes were studied in Daudi and KOPN-8 cells treated with 2.5 μM Niclosamide for 3 and 6 hours. 96 genes were consistently overexpressed, 59 down-regulated. 10 genes involved in the tumor necrosis factor (TNF) pathway and 10 genes involving the DNA damage pathway were overexpressed. 13 out of the 59 down-regulated genes were involved in mitochondrial function. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous in KOPN-8 cells. The viability of PBMCs from 8 individuals without lymphoma was unchanged when incubated with niclosamide, whereas niclosamide showed significant cytotoxicity in a patient with mantle cell lymphoma (MCL).

Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines, including vincristine-refractory lymphoma cells, and induces apoptosis at concentrations non-toxic to PBMCs. Interestingly, niclosamide exhibited cytotoxic activity against MCL cells - a finding worth testing further in this difficult-to-treat disease. The mechanism of action of Niclosamide may involve the TNF receptor pathway, mitochondrial function and DNA damage response pathway. We plan to elucidate further specific mechanism(s) of action, and evaluate synergistic effects with other antineoplastic agents, and perform in vivo studies.

#4817

Molecular cancer therapy targeting RNA-binding protein Musashi-1.

Lan Lan,1 Hao Liu,1 Amber Smith,1 Carl Appelman,1 Jia Yu,1 Sarah Larsen,1 Rebecca Marquez,1 Xiaoqing Wu,1 Philip Gao,1 Ragul Gowthaman,1 John Karanicolas,1 Roberto De Guzman,1 Steven Rogers,2 Jeffrey Aubé,2 Kristi Neufeld,1 Liang Xu1. 1 _University of Kansas, Lawrence, KS;_ 2 _University of North Carolina, Chapel Hill, NC_.

Musashi-1 (MSI1) is an evolutionarily conserved RNA-binding protein best studied for its role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancers including colon cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively. Our hypothesis is that, small molecules disrupting MSI1-RNA binding will remove translational control of MSI1 over Numb/APC, which will in turn inhibit Notch/Wnt signaling. Previously we have shown natural product (-)-gossypol as the first small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Here we identified gossypolone (Gn), a major metabolite of gossypol, as a more potent MSI1inhibitor with more than 20-fold increase in Ki value in our fluorescence polarization binding assay. Further validation of Gn-MSI1 binding was carried out by surface plasmon resonance and nuclear magnetic resonance. In colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. However, the same concentration of Gn is less active than that of (-)-gossypol in all of the cell assays tested. In order to increase Gn bioavailability, we used the PEGylated liposome in our in vivo studies. Properties of gossypolone-liposome (Gn-lip) were evaluated by transmission electron microscopy and a Zetasizer Nano ZS90 instrument; cytotoxicity of free liposomes as compared to Gn-lip and Gn was also evaluated. To further characterize the in vivo tumor-specific accumulation of liposomes, a near-infrared (NIR) fluorescent dye DiR was used. NIR imaging demonstrated tumor-targeted delivery of DiR-loaded liposomes in DLD-1 xenografts in SCID mice. Gn-lip via tail vein injection twice weekly inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n=10). Our data suggest that Gn-lip improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhances its overall biocompatibility and drug efficacy in vivo. Our study provides a proof-of-concept to develop the Gn-lip as a novel molecular therapy for colon cancer with MSI1 overexpression.

#4818

Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus.

Riyaz M. Basha,1 Sarah F. Connelly,2 Ganji Purnachandra,3 Umesh T. Sankpal,1 Hassaan Patel,1 Jamboor K. Vishwanatha,1 Sagar Shelake,1 Leslie Tabor-Simecka1,1 Mamoru Shoji,3 Jerry Simecka W. Simecka,1 Bassel El-Rayes3. 1 _University of North Texas Health Science Center, Fort Worth, TX;_ 2 _MD Anderson Cancer Center Orlando, Orlando, FL;_ 3 _Emory University, Atlanta, GA_.

Pancreatic cancer (PC) is an aggressive malignancy. The current treatment options have limited response in addressing poor prognosis and low survival rate. Hence it is important to identify novel agents and strategies for effective treatment. Previously the combination of phytochemical, curcumin (Cur) and cyclooxygenase (COX) inhibitor celecoxib was tested for improving therapeutic efficacy in PC models. The objective of current study is to identify a combination treatment involving a low toxic small molecule and a phytochemical with anti-cancer properties to inhibit PC cell growth. Experiments were also conducted to understand potential mechanisms associated with this combination. We tested the combination of an anti-cancer non-steroidal anti-inflammatory drug (NSAID), Tolfenamic Acid (TA) and Cur using PC cell lines, L3.6 and MIA PaCa-2. Cells were treated with 5-25 µM of Cur or 25-100 µM of TA or combination of Cur (7.5 µM) and TA (50 µM). Effect on cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter-Glo kit. While the two agents showed anti-proliferative effect, Cur and TA combination caused higher growth inhibition. The cell growth inhibition was compared with two COX inhibitors, ibuprofen and celecoxib and the cardiotoxicity was assessed using cordiomyocytes (H9C2). TA showed significantly less cytotoxicity in cardiomyocytes when compared to celecoxib. The expression of transcription factors, Specificity protein1 (Sp1) and NF-kB, and an inhibitor of apoptosis family protein, survivin, were determined by Western blot analysis. The expression of NF-kB, Sp1 and survivin was decreased by combination treatment. The levels of reactive oxygen species (ROS) were also measured in flowcytometer. To evaluate the effect of these agents on apoptosis, the activity of caspase 3/7 was measured with caspase-Glo kit; apoptotic cell population was evaluated by Annexin-V staining (flow cytometry); and c-PARP expression was determined by Western blot analysis. When compared to individual agents, the combination treatment caused a significant increase in ROS levels and apoptotic markers. L3.6 and MIA PaCa-2 cells were treated with TNF-α to induce NF-kB translocation from cytoplasm to nucleus and the effect of individual (TA or Cur) and combined treatment (TA+Cur) on NF-kB translocation from cytoplasm to nucleus was evaluated by immunofluorescence. When compared to individual agents, the combination treatment caused a significant decrease in NF-kB translocation to nucleus. Cell cycle phase distribution was measured using flow cytometry. The combination treatment showed mostly DNA synthesis phase arrest; however TA caused cell cycle arrest in early phase (G0/G1). These results demonstrate that combination of Cur and TA effectively inhibits PC cell growth via inducing apoptosis and modulating cell cycle phase distribution.

#4819

Targeting the polyamine addiction of pancreatic cancers: Combination therapies and biomarkers.

Otto Phanstiel, Meenu Madan, Arjun Patel, Kristen Skruber, Deborah Altomare. _University of Central Florida, Orlando, FL_.

This project developed a combination therapy which inhibits both polyamine transport and biosynthesis in an effort to target the polyamine addiction of pancreatic cancers. Several parameters were measured in six human pancreatic cell lines (L3.6pl, Panc-1, BxPC3, AsPC-1, Capan-1 and HPNE) and one murine line (Pan02). These included the kinetics of ³H-spermidine uptake (Vmax and Km values), the 72h IC50 value for difluoromethylornithine (DFMO, an inhibitor of polyamine biosynthesis), and the ability of exogenous spermidine to rescue the viability of DFMO-treated cells. Three polyamine transport inhibitors (PTIs) were evaluated for their ability to block the uptake of exogenous spermidine into DFMO-treated pancreatic cancer cells (via EC50 values). The combination therapy (DFMO+PTI) was shown to effectively block spermidine import in vitro and improved survival times in an orthotopic mouse model. Basal c-Myc and ATP13A3 protein expression were shown to track well with polyamine transport activity (Vmax) in untreated cell lines. In the presence of DFMO, both polyamine transport activity and ATP13A3 protein expression of L3.6pl cells were increased, whereas c-Myc protein expression decreased. This suggested that ATP13A3 may be involved in the DFMO-stimulated polyamine transport response and was confirmed via siRNA experiments. Using ATP13A3 as a biomarker of polyamine transport, our results could be explained in the context of relative ATP13A3 expression and the caveolin-1 status of each cell line. Cell lines with high ATP13A3 and low Cav-1 had the highest Vmax (e.g., L3.6pl and Panc-1) and could be readily rescued from DFMO by exogenous spermidine (1 µM). In contrast, cells with low ATP13A3 and low Cav-1 expression (i.e., Capan-1 and AsPC-1) gave low Vmax values and could not be rescued from DFMO by exogenous spermidine. BxPC3 cells were found to have high ATP13A3 and high Cav-1 expression and were found to be poorly rescued by exogenous spermidine in the presence of DFMO. In summary, the combination therapy of DFMO+PTI was shown to effectively deplete pancreatic cancers of intracellular polyamines and holds great promise as a potential therapy. The discovery of ATP13A3 as a polyamine transport protein is very significant as there are few proteins identified for this pathway and this marker (along with Cav-1 status) could be used to stratify patients in ongoing DFMO trials. In this regard, the DFMO+PTI approach represents a potential 'catch-all' therapy for tumors addicted to polyamine metabolites.

#4820

**Selective CDK7 inhibitors suppress super enhancer-genes, induce massive apoptosis in acute myeloid leukemia and demonstrate durable** in vivo **efficacy.**

Shanhu Hu, Nan Ke, Yixuan Ren, Jeremy Lopez, Sofija Miljovska, David Orlando, Darby Schmidt, Michael Bradley, Kevin Sprott, Eric Olson, Christian C. Fritz, Yoon Jong Choi. _Syros Pharmaceuticals, Cambridge, MA_.

CDK7 acts bi-functionally as a CDK-activating kinase (CAK) controlling proliferation and as a transcriptional kinase phosphorylating the CTD-RNA Pol II, driving efficient transcriptional processes. CDK7 has recently emerged as an attractive gene control target in cancers addicted to oncogenic transcription factors, regulated by super-enhancers (SEs), (Kwiatkowski et al., 2014; Chipumuro et al., 2014; Christensen 2014; Wang 2015). Acute Myeloid Leukemias (AML) harbor a heterogeneous set of mutations resulting in an altered cell state and aberrant epigenetic and transcriptional control, yet lack targeted therapeutic options. Employing our SE-platform technology we reveal mechanistic insights underlying the vulnerability of AML to gene control modulation via selective CDK7 inhibition.

We describe first-in-class CDK7 inhibitors that covalently target a cysteine outside the kinase domain, resulting in sustained, highly selective inhibition. Several lead compounds exhibit significant biochemical potency (Ki = 10-60 nM and kinact = 5-13/h) and exquisite selectivity when profiled against >400 other kinases. In a cancer cell line panel, acute leukemias emerged among the most sensitive to CDK7 inhibition. Moreover, AML cell lines undergo rapid and robust apoptosis within 24 hours. This is preceded by CDK7 target engagement and concomitant loss of P-CTD-RNAPolII suggesting the primary consequence of CDK7 inhibition is the impaired transcriptional activity of CDK7. Further investigation of the transcriptional consequences of CDK7 inhibition point to a reliance on key disease relevant SE-genes (e.g. MYB, HOX10A and MYC) that are predominantly reduced.

Given the covalent mechanism of our CDK7 inhibitors and PK profile in vivo, we use short treatments in vitro with subsequent washout of free drug to model intermittent dosing regimens in vivo. We demonstrate that short drug exposures maintain a robust irreversible apoptotic response in leukemia cells. In contrast, treated non-transformed cells recover from a transient G2/M arrest followed by re-synthesis of free CDK7 and no apoptosis/cell death. We have extended these findings to in vivo experiments whereby intermittent dosing in AML patient derived xenograft models (PDX) maintains efficacy (reducing human CD45+ leukemia cells to <1%) and significant survival advantage. We have established a PD assay by measuring target engagement of CDK7 both in mouse xenografts and human blood to support: 1) establishing a PK-PD-efficacy relationship 2) quantifying target engagement during dose escalation in a Ph1 clinical trial.

In summary, we describe first-in-class CDK7 inhibitors that are potent and selective and lead to durable, complete responses in PDX models of AML. These data support the rationale for advancing one or more members of this class of compounds toward clinical development.

#4821

The WEE1 inhibitor AZD-1775 has synergic activity with trabectedin or lurbinectedin in ovarian cancer cells.

Sarah Uboldi, Laura Carrassa, Roberta Frapolli, Paolo Ubezio, Eugenio Erba, Maurizio D'Incalci. _IRCCS Istituto di Ricerche Farmacologiche Mario Negri - Milano, Milan, Italy_.

AZD-1775 is a small molecule that selectively inhibits the tyrosine kinase WEE1, with a reported antineoplastic activity in ovarian cancer. WEE1 is a tyrosine kinase key regulator of DNA damage surveillance pathways that controls G2/M transition and ensures faithful DNA replication. WEE1 is required during normal S phase to avoid deleterious DNA breakage and prevent loss of genome integrity in the absence of exogenous DNA damage.

Trabectedin is a drug approved in 2007 by EMA and 2015 by FDA for the treatment of soft tissue sarcomas and in 2009 by EMA for the treatment of ovarian cancer patients, who have relapsed after 6 months from Platinum-based therapy, in combination with pegylated liposomal doxorubicin. Lurbinectedin (PM01183) is a trabectedin analog with a similar mechanism of action and, according to ongoing clinical investigations, less toxic and thus suitable for combinations of potential clinical interest. Since both trabectedin and lurbinectedin cause a G2/M block and AZD-1775 is able to inhibit the WEE1 activity with consequent disruption of G2/M checkpoint, we hypothesized that the combined treatment with AZD-1775 could be synergistic in ovarian cancer cells.

The sensitivity of the ovarian cancer cell lines OVCAR-5, A2780 and IGROV-1 to trabectedin, lurbinectedin and AZD-1775 alone or in combination (72h of continous treatment) was evaluated by a cell proliferation assay. The cytotoxic effect induced by the simultaneous administration of trabectedin with AZD-1775 or lurbinectedin with AZD-1775 was evaluated by a combination index analysis.

The results obtained showed that the combination trabectedin with AZD-1775 or lurbinectedin with AZD-1775 is synergic in all the ovarian cancer cell lines analyzed (Combination Index <1). The combination lurbinectedin with AZD-1775 seems to be better than trabectedin with AZD-1775, in OVCAR5 cell line. The analysis of cell cycle and apoptosis are in progress in order to investigate the mechanism of the observed synergism. These positive results prompted us to study these combinations in vivo, in ovarian cancer xenografts derived from the cell lines used for the in vitro studies.

If the synergism observed in vitro will be confirmed in vivo, there will be a strong rationale to test these combinations in ovarian cancer patients.

#4822

Combination efficacy of the selective HDAC inhibitor ACY-241 and paclitaxel in solid tumor models.

Pengyu Huang, Ingrid Almeciga-Pinto, Min Yang, Simon S. Jones, Steven N. Quayle. _Acetylon Pharmaceuticals, Inc., Boston, MA_.

ACY 241 is a new, orally available and selective histone deacetylase (HDAC) 6 inhibitor in clinical development in combination with pomalidomide and dexamethasone in multiple myeloma. Like the structurally related drug ricolinostat (ACY-1215), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. One of the major effects of HDAC6 inhibition is hyperacetylation of α-tubulin, which is suggestive of potential combination activity with taxanes in cancer treatment.

Paclitaxel is a chemotherapeutic agent approved for use in the treatment of multiple solid tumor types, including breast cancer, ovarian cancer, non-small cell lung cancer and Kaposi's sarcoma (Weaver, 2014). Recent studies indicate that clinically relevant low concentrations of paclitaxel caused multipolar spindle formation resulting in aberrant mitosis and cell death, which further contributes to the anti-cancer efficacy of paclitaxel (Zasadil et al., 2014).

In cell lines, combination treatment with ACY-241 and paclitaxel resulted in enhanced inhibition of proliferation and increased cell death. The combination of ACY-241 plus paclitaxel also demonstrated enhanced efficacy in xenograft models of pancreatic and ovarian cancer. Cell-cycle analysis indicated that ACY-241 treatment in combination with paclitaxel caused synergistic inhibition of tumor cell proliferation through enhanced reduction of DNA replication in S-phase and increased induction of cell death and aneuploidy. ACY-241 treatment in combination with paclitaxel did not increase mitotic arrest but did increase the frequency of multipolar spindle formation during mitosis, leading to aberrant mitoses, abnormal nuclei, and cell death in agreement with a previous report (Zasadil et al). At the molecular level, the enhanced anti-cancer efficacy resulting from combination treatment with ACY-241 and paclitaxel in cell lines was associated with further increased hyperacetylation of α-tubulin, suggesting these agents synergistically impact the regulation of tubulin biology. Interestingly,gamma-tubulin was not always detectable at each pole of these multipolar spindles, suggesting that multipolar spindle formation may be independent of centrosome amplification and/or splitting. Additional studies investigating the association between localization of spindle proteins and multipolar spindle formation are in progress.

The enhanced efficacy of paclitaxel plus ACY-241 observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors.

#4824

Synergistic interaction of PIM kinase inhibitor, JP_11646, and cytotoxic or targeted therapies in acute leukemia models.

Krista Pundt,1 Camern Baldino,2 Justin Caserta,2 Alex Adjei,1 Kelvin Lee,1 Xu Zhu,3 Alissa Verone-Boyle,4 Frank Engler,4 Laura Pitzonka,4 Gerald Fetterly4. 1 _Roswell Park Cancer Institute, Buffalo, NY;_ 2 _Jasco Pharmaceuticals, LLC, Woburn, MA;_ 3 _University at Buffalo, Buffalo, NY;_ 4 _TransPKPD, LLC, Buffalo, NY_.

Standard therapy for acute myeloid leukemia (AML) results in significant mortality and morbidity with an overall cure rate of only 25%. Implementation of novel therapeutic options is needed in order to significantly improve patient survival and outcome. PIM serine/threonine kinases are overexpressed in hematological malignancies and regulate cell cycle, survival and drug resistance pathways—thus providing viable therapeutic targets. We hypothesized that targeted or cytotoxic therapy will enhance anti-tumor activity in combination with JP-11646, a novel PIM2 inhibitor, in preclinical AML models.

Preclinical data has shown JP-11646 exerted potent inhibition of AML cells in vitro and in vivo in subcutaneous AML xenograft models. An in vitro MTT assay was used to determine the overall anti-proliferative effects of single and combination therapies with respect to the downstream effects of PIM kinase and mechanism of action of JP-11646 in leukemia. First, the single agent efficacy in human AML (HEL, and MV(4;11)) cell lines were examined after 72 hours of exposure to JP-11646, rapamycin, daunorubicin and cytarabine. Overall potency, IC50, (0.17 µM (0.15-0.19), 0.003 µM (0.002-0.004), 0.067 µM(0.065-0.0699) and 0.31 µM (0.294-0.345), respectively) and maximal inhibition, Imax, of the various therapies was determined. Potential synergistic effects were first tested by treating AML cell lines with JP-11646 and daunorubicin, cytarabine, or rapamycin (an inhibitor of a pathway that regulates downstream molecules of the PIM kinase pathway). To quantify the synergy or interaction of JP-11646 in combination, a simple inhibitory non-competitive interaction model with an interaction parameter (Ψ) as well as a three dimensional interaction surface was employed (Ψ<1 synergism, Ψ>1 antagonism). The intensity of the interaction (Ψ) of JP-11646 with rapamycin (0.819 (0.395-0.543)), daunorubicin (0.469 (0.665-0.975)) or cytarabine (0.173 (0.118-0.229)) demonstrated synergistic effects across all combinations.

Here, we demonstrate that novel drug targets and drugs for AML treatment alone, and in combination with pre-existing therapies, may lead to improved strategies for AML treatment and these findings support future development of JP-11646 in novel combinatorial regimens for AML therapy.

#4825

A rationale for treatment of colorectal cancer with mitomycin C and crizotinib.

Avital Lev,1 Elena Shagisultanova,2 Safoora Deihimi,1 David T. Dicker,1 Joanne Xui,3 Wafik S. El-Deiry1. 1 _1Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Division of Medical Oncology, Anschutz Cancer Center, University of Colorado, Denver, CO;_ 3 _Caris Life Sciences, Phoenix, AZ_.

Colorectal cancer (CRC) is the third leading cause of cancer-related death in men and women in the United States. Microsatellite instability (MSI) is found in approximately 15% of sporadic colorectal cancers and in the majority of Lynch syndrome patients. MSI has been correlated to deficiency in the mismatch repair (MMR) genes and therefore MSI-High tumors exhibit higher mutation rates then non-MSI-High tumors. We recently reported on 26 MSI-High and 558 non-MSI-High CRCs that were profiled at Caris Life Sciences (Shagisultanova et al., 2015 ASCO Annual Meeting Abstract No. e14684 "Association of increase in BRCA2 gene mutations in microsatellite instable (MSI-H) colorectal cancer (CRC) with increased c-MET expression."). Immunohistochemistry and genomic analyses were performed in the BRCA-mutant versus BRCA wild-type MSI-High tumors. BRCA2 mutations were highly enriched (50%) in MSI-High CRC. MSI-High tumors with BRCA2 frameshift mutations had high c-MET expression. c-MET overexpression is known to be associated with aggressive metastatic CRC. We hypothesized there may be a synergistic drug interaction between drugs that are used to treat BRCA2-deficient tumors and c-MET inhibitors. We further hypothesized there may be a mechanistic link between BRCA2-deficiency, double-strand breaks following DNA damage, and c-MET overexpression. Mitomycin C (MMC) is an anti-cancer chemotherapeutic agent, which causes DNA damage by inducing double strand breaks (DSBs) through DNA cross-linking. Tumors deficient in genes encoding for proteins involved in DNA repair such as BRCA2 show hypersensitivity to MMC. Crizotinib is a small molecule inhibitor of c-MET and ALK receptor tyrosine kinases. In the present studies, we tested CRC cell lines for sensitivity to MMC plus crizotinib. CRC cell lines treated with MMC activated a DNA damage response as measured by up-regulation of γ-H2AX. Upon BRCA2 siRNA-mediated knockdown colorectal cancer cells became more sensitive to MMC shown by cleaved-PARP as a measure of apoptosis. Crizotinib inhibited the activation of c-MET in CRC cell lines treated with Hepatocyte Growth Factor (HGF). The combination treatment of colorectal cancer cells with crizotinib and MMC led to increased apoptosis (cleaved-PARP) compared to each drug alone. Combination treatment with increasing concentrations of both drugs in a CellTiter-Glo Cell Viability assay demonstrated a synergistic effect. However, we found no evidence for c-MET upregulation upon effective BRCA2 knockdown in the absence or presence of DNA damage. Although there is no mechanistic link between BRCA2 mutation and c-MET overexpression, c-MET is frequently overexpressed in CRC in general and BRCA2 is frequently mutated in CRC especially in MSI-High cases. The combination of crizotinib with MMC appeared synergistic regardless of whether CRC cell lines were MSI-High or non-MSI-High. Our results prompt the clinical testing of the combination of MMC and crizotinib in advanced CRC.

#4826

ONC212 exhibits increased cytotoxicity relative to ONC201 in a subset of human pancreatic cancer cell lines.

Avital Lev,1 Jessica Wagner,1 David T. Dicker,1 Martin Stogniew,2 Joshua E. Allen,2 Lee Schalop,2 Wolfgang Oster,2 Gary L. Olson,3 Wafik S. El-Deiry1. 1 _Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA;_ 2 _Oncoceutics, Inc., Philadelphia, PA;_ 3 _Provid Pharmaceuticals, Monmouth Junction, NJ_.

Pancreatic cancer is one of the most deadly types of cancer with five-year survival rate of ~6%. This poor prognosis is attributed to aggressive growth of the tumor cells, metastasis at an early stage and resistance to standard chemotherapy. Therefore, finding new treatments and improving outcomes of pancreatic cancer patients have been high priorities. ONC201 is a small molecule that selectively targets a broad range of tumor types tested. It is currently in phase I/II clinical trials for advanced solid, hematopoietic, and lymphoid tumors. The ONC201 analogue, ONC212, is in preclinical development for certain cancers that are not lead indications for the parent compound ONC201. We examined the efficacy of ONC201 and ONC212 in a panel of human pancreatic cancer cell lines. Among seven cell lines tested, three cell lines were relatively insensitive to ONC201, with IC50 values of 100 μM or greater. However, these cell lines showed greater sensitivity to ONC212 with IC50 values of 31-56 μM. We compared ONC201-resistant PANC-1 cells and ONC201-sensitive HPAF-II cells. Both ONC201 and ONC212 induced apoptosis in HPAF-II cells, as assessed by PARP cleavage, while PARP cleavage was detected only when PANC-1 cells were treated with ONC212 but not ONC201. We are currently evaluating the efficacy of ONC201 and ONC212 in a panel of patient-derived low passage pancreatic cancer cell lines. We found a correlation between high expression levels of RTKs (c-MET, ALK, EGFR, IGFR, HER2) and decreased sensitivity to ONC201 in pancreatic cancer cell lines. We investigated the effect of ONC201 on EGFR and IGFR, which are highly expressed in PANC-1 and found that ONC201 does not inhibit receptor activation by the specific growth factors. We are studying the effects of ONC201 and ONC212 on RTKs and their signal transduction pathways. Lastly, since aggressive drug-resistant pancreatic cancer cells express high levels of RTKs, we are developing a rationale combining ONC201/ONC212 with specific small molecule RTK inhibitors such as crizotinib or lapatinib. We are exploring these combination treatments using cell lines, organoids and PDX models to unravel specific therapy options for therapy-refractory pancreatic cancer cells. Our goal is to provide the preclinical rationale for clinical trials of ONC201 or ONC212 as mono-agents or in combination with other therapies for pancreatic cancer patients.

#4827

Co-administration of nicotinic acid (NA) enhances the therapeutic index of KPT-9274 in cancer cells.

William Senapedis, Christian Argueta, Yosef Landesman, Margaret Lee, Sharon Shacham, Erkan Balolgu. _Karyopharm Therapeutics, Inc., Newton, MA_.

We have previously described the PAK4 allosteric modulators (PAMs), KPT-7523 and KPT-9274, as potent anti-cancer small molecules. We recently found that PAMs also inhibit nicotinamide phosphoribosyl transferase (NAMPT) enzymatic activity, a protein that forms a complex with PAK4 to help regulate cytoskeletal structures, cell adhesion and migration. NAMPT and nicotinate phosphoribosyl transferase (NAPRT1) catalyze the rate limiting steps in the nicotinamide adenine dinucleotide (NAD) salvage pathways. While NAMPT is frequently overexpressed in certain cancer types, NAPRT1 is often downregulated. NAD levels can be depleted under conditions of high metabolic demand (i.e. cancer cells) when consumed by other enzymes including poly-ADP ribose polymerases (PARPs) and sirtuins (SIRTs). In addition, induction of cancer cell death by NAD depletion can be mitigated by nicotinic acid (NA) supplementation in a NAPRT1 dependent manner. The purpose of this study is to determine whether KPT-9274 co-dosed with NA can reduce potential toxicities associated with NAD depletion, while enhancing anti-neoplastic activity in cancers lacking NAPRT1.

Methods: Cyclex NAMPT colorimetric assay was used to examine NAMPT enzymatic activity in vitro. NAD/NADH-Glo and Celltiter-Glo were used to measure NAD and ATP levels, respectively. Gene expression and gene promoter methylation was determined using quantitative PCR and sequencing technologies. Western blot analysis was used to examine protein expression and protein-protein interactions.

Results: We have identified an orally bioavailable dual inhibitor of PAK4 and NAMPT, KPT-9274, which demonstrated potent anti-cancer activity in a variety of cell lines both in vitro and in vivo. Cell death induced by KPT-9274 treatment in NAPRT1+ cells (COLO 205, Z-138, THP-1, MV-4-11) can be mitigated by NA, while NAPRT1- cells (COLO 320HSR, JeKo-1, REC-1, U-2 OS, PC-3) remain sensitive to KPT-9274 + NA. A reduction of both PAK4 protein levels and cell viability is still observed in these different cell types co-dosed with KPT-9274 + NA. In preliminary toxicology studies of KPT-9274 co-administered with NA in dogs, we observed a reduction in potential toxicity (i.e. gastrointestinal and hematological effects). In vivo assessment of KPT-9274 +/- NA and NAPRT1 status is on-going.

Conclusions: Here we report that the therapeutic index of KPT-9274, the first-in-class dual inhibitor of PAK4 and NAMPT, can be enhanced when co-dosed with NA in cancers lacking NAPRT1 protein expression in vitro. Furthermore, KPT-9274 can still reduce steady state PAK4 levels and cell viability regardless of NAPRT1 status. Based on these data, co-administration of NA with KPT-9274 may be beneficial for the treatment of a wide variety of cancers and will be tested in phase 1 clinical development.

#4828

Enhanced cytotoxicity following sequential treatment with filanesib and melphalan in human myeloma cell lines.

Darla Destephanis,1 Eric J. Norris,1 Brian Turnquist,2 Saad Usmani,1 Mahrukh Ganapathi,1 Ram Ganapathi1. 1 _Carolinas Healthcare System, Charlotte, NC;_ 2 _Array Pharmaceuticals, Boulder, CO_.

As a single agent, melphalan is used as a preparative treatment prior to autologous stem cell transplant (ASCT) in patients with multiple myeloma (MM) given its myelosuppressive and anti-myeloma effects. While most patients achieve complete remission following ASCT, a majority will develop recurrent disease. Therefore, novel combinations that improve the anti-myeloma activity of melphalan may improve post-transplant outcomes. The first-in-class agent Filanesib (Fil) is a kinesin spindle protein inhibitor which induces mitotic arrest/apoptosis in proliferating cells and has demonstrated anti-myeloma activity in vitro and in vivo. In this study, we tested the effects of combination treatment with filanesib and melphalan in the human myeloma cell lines (HMCLs), U266, MM1S and NCI-H929. As single agents, melphalan and filanesib both inhibit myeloma cell proliferation as assessed by the MTS assay. However, when HMCLs were treated simultaneously with both drugs an antagonistic interaction was observed. This antagonism was in part due to cell cycle arrest in the S-phase, which prevented filanesib from downregulating the anti-apoptotic proteins, BCL-2 and MCL-1 and exerting its cytotoxic effect in the G2/M-phase.. To elucidate whether sequential treatment could enhance cytotoxicity, HMCLs were treated with either melphalan (1 hour) followed by filanesib (MelFil) for 48 hours or filanesib for 48 hours followed by 1 hour melphalan treatment (FilMel). Compared to the apoptosis observed with individual drugs, the MelFil sequence led to significantly lower apoptosis, whereas the the FilMel treatment led to enhanced apoptosis following 48 hours after drug washout. Furthermore examination of recovery of cell proliferation following seven days of recovery after drug removal revealed an increase in cell proliferation for the MelFil treatment compared to a decline in the number of viable cells for FilMel treatment. Taken together, our findings suggest that filanesib pretreatment prior to melphalan may be a clinically relevant therapeutic combination strategy. Moreover, our data underscore the importance of testing sequencing strategies for optimizing therapeutic efficacy of combination treatment.

#4829

**miRNA combination therapy:** In vitro **anticancer synergy between miR-34a mimic and cytotoxic chemotherapy (CT) in NSCLC.**

Adriana Guerrero,1 Jane Zhao,1 Xiaojie Yu,2 Alexander Pertsemlidis,2 Andreas G. Bader1. 1 _Mirna Therapeutics Inc, Austin, TX;_ 2 _University of Texas Health Science Center at San Antonio, San Antonio, TX_.

Background: miRNAs play a critical role in regulating key biological processes by modulating the expression of up to several hundred genes across multiple cellular pathways. miR-34a, one of the most widely studied miRNAs, is lost or expressed at reduced levels in many tumors, and normally functions as a natural tumor suppressor by down-regulating expression of >30 different oncogenes, as well as genes involved in tumor immune evasion, including PD-L1. MRX34 is a potential first-in-class liposome-encapsulated miR-34a mimic in Phase 1 study (NCT01829971) as monotherapy in patients with advanced malignancies. The ability of miR-34a to regulate the expression of key oncogenes across multiple oncogenic pathways makes MRX34 a rational candidate to combine with other anticancer therapies which are frequently subject to primary and acquired resistance in the clinic. Previous studies showed that miR-34a greatly sensitizes both EGFR wild-type and mutant NSCLC cell lines, as well as hepatocellular carcinoma cell lines, to the EGFR tyrosine kinase inhibitor erlotinib. Here we report research combining miR-34a and standard CT drugs in NSCLC cell lines.

Methods: Combination studies using single-drug ratios (~IC50 ratio of miR-34a and CT drug) and multiple ratios above and below were performed in a panel of NSCLC cell lines (A549, H460, H1299, H2073) with varying degrees of intrinsic resistance to CT. Cells were transfected with miR-34a and incubated 24 hrs later with cisplatin, carboplatin, paclitaxel, gemcitabine, or pemetrexed for 72 hrs, with cellular proliferation then determined by AlamarBlue. Synergistic, additive, or antagonistic effects were determined by combination index (CI) values (based on Loewe's concept of additivity), isobolograms, and curve-shift analyses.

Results: Synergistic interactions were observed between miR-34 and all CT drugs in all NSCLC cell lines tested. Synergy was observed at multiple miR-34a/cytotoxic agent dose ratios and at drug concentrations inducing 50% or greater inhibition (CI <0.6 and dose reduction index >2 at both 50% and 75% effect levels). Stronger synergy was seen in the H2073 cell line which is relatively more resistant to CT.

Conclusions: Consistent with other in vitro results with miR-34a-based combinations, the data demonstrate synergistic anticancer effects between miR-34a + CT in a range of NSCLC cell lines with varying degrees of intrinsic resistance to CT. Overall, the data support further exploration of MRX34 in combination with standard anticancer therapies, and we are evaluating the most appropriate combinations for near-term clinical trials. In addition to resistance, other critical oncogenic mechanisms present in the tumor microenvironment (eg, immune evasion, cancer stem cells, metastasis) could potentially also be mitigated to the benefit of patients by a miRNA-based approach to therapy.

#4830

Preclinical anti-tumor activity of nanoliposomal irinotecan (Nal-IRI, MM-398) + 5-FU + oxaliplatin in pancreatic cancer.

Daniel F. Gaddy,1 Helen Lee,1 Nancy Paz,1 Shannon C. Leonard,1 Ashish Kalra,1 Ninfa L. Straubinger,2 Robert M. Straubinger,2 Bryan M. Gillard,3 Michael T. Moser,3 Daryl C. Drummond,1 Stephan G. Klinz,1 Bart S. Hendriks,1 Jonathan B. Fitzgerald1. 1 _Merrimack, Cambridge, MA;_ 2 _State University of New York at Buffalo, Buffalo, NY;_ 3 _Roswell Park Cancer Institute, Buffalo, NY_.

Nanoliposomal irinotecan (Nal-IRI, MM-398) recently gained approval in combination with 5-fluorouracil/leucovorin (5-FU/LV) in post-gemcitabine metastatic pancreatic cancer based on results of the Phase 3 NAPOLI-1 trial. Nal-IRI, in combination with 5-FU/LV, improved overall survival in gemcitabine-refractory metastatic PDAC relative to 5-FU/LV alone with a well-defined and manageable toxicity profile in pretreated patients. FOLFIRINOX (5-FU/LV, irinotecan and oxaliplatin) is a chemotherapy regimen active in first-line metastatic PDAC. Herein, we evaluate the preclinical anti-tumor activity of a nal-IRI + 5-FU + oxaliplatin regimen relative to the FOLFIRINOX regimen. Using pancreatic cancer cell lines, we demonstrate enhanced cell death when nal-IRI treatment is simulated using prolonged exposure of SN-38 (the active metabolite of irinotecan) in combination with 5-FU and oxaliplatin. In cell line-derived and patient-derived xenograft models of pancreatic cancer we demonstrate improved anti-tumor activity of nal-IRI relative to exposure-matched doses of unencapsulated irinotecan. Further, nal-IRI consistently improved tumor growth inhibition and survival relative to unencapsulated irinotecan in preclinical models, both as a monotherapy and in combination with 5-FU and oxaliplatin. The addition of nal-IRI to 5-FU and/or oxaliplatin did not exacerbate the baseline toxicities of these agents, including weight loss and neutropenia, and tolerability could be further improved by delaying the administration of oxaliplatin to 1 day post-MM-398. These findings illustrate the therapeutic potential of nal-IRI in combination with 5-FU/LV and oxaliplatin and support an ongoing Phase 2 trial (NCT02551991) of this triplet regimen in first-line PDAC.

#4831

An investigation to study the role of novel rhenium compounds on onco miR's and oncogenes involved in epithelial mesenchymal transition of prostate cancer cell lines derived from African American and Caucasian patients.

Hirendra N. Banerjee,1 Jameel Joyner,1 Alexis Barfield,1 B Morris,1 D Bell,1 William Kahan,1 Monet Stevenson,1 D Crummity,1 K Prabhakaran,1 Santosh Mandal,2 Fazlul Sarkar3. 1 _Elizabeth City State University, Elizabeth City, NC;_ 2 _Morgan State University, Baltimore, MD;_ 3 _Karmanos Cancer Center, Wayne State University, Detroit, MI_.

In this research, we seek to evaluate the efficacy of novel rhenium compounds as a novel targeted agent against prostate cancer (PCa) in pre-clinical model system. Since the incidence and aggressiveness of Pca is more prevalent in African Americans (AA) patients than Caucasian Americans (CA), we first assessed the relevance of genes Lin28B, EZH2, miR-200, and let-7 expression in human PCa tissue specimens for characterizing tumor aggressiveness, since these genes are involved in the process and also epithelial-mesenchymal transition(EMT).We also assessed whether these markers are deregulated by rhenium compounds using PCa cell lines. We tested the two novel rhenium compounds ie. Rhenium pentylcarbonato complexes (RPC), fac-(CO)3(α-diimine)ReOC(O)OC5H11 (where, α-diimines are 2,2'-bipyridyl and neocuproin for anticancer properties on PCa cell lines for apoptosis and cell death, and their bioactivity for anti-proliferative, anti-migratory and antispheroid forming capabilities, respectively on EO6-AA and MDA,Pca cell lines(from AA patients) and LNCAP,PC3 (from CA patients). We also studied the expression of miR-146α in these cell lines and Pca tissue specimens, since it is reported to be overexpressed in cancer cells for tumor survival advantage, regulated by down-regulating iNOS, the nitric oxide synthesizing gene. Our initial results of Real Time PCR,Western Blotting,MTT assay,smart flare assay,flow cytometry, in situ cytochemistry( TUNEL) and spheroid forming assays showed that these drugs have cytotoxic,anti proliferative and anti spheroid forming properties,can induce apoptosis in the Pca cell lines tested and can downregulate some of the onco miR's and onco genes involved in Pca aggressiveness and EMT conversions as tested by the expression profiles of miRNAs (miR-200b, miR-200c, and let-7 family) by RT-PCR, and the protein expression of EZH2, Suz12, and Lin-28B by Western Blots. It is important to emphasize here that an estimated 40,000 men die of PCa every year in the US and each year about 60,000 men will develop castrate resistant PCa for which newer drugs are needed. Therefore, our work will contribute towards the development of a novel therapy that may translate into an effective treatment regimen against PCa and is highly relevant to the mission of both basic and translational cancer researchers.

ACKNOWLEDGEMENT: Supported by a NCI disability supplement to NIH- grant# R01CA164318 and NIH-MARC

NIGMS,grant# 5 T34 GM 100831-4.We are grateful to Dr.Li, Dr.S.Banerjee and Ms.S.Ali of Detroit Medical Center,MI,USA for their support and help in conducting this research.

#4832

Overcoming breast cancer chemoresistance and blocking metastasis by targeting RNA-binding protein HuR.

Xiaoqing Wu,1 Rebecca T. Marquez,1 Shuang Han,1 Ke Li,1 Lan Lan,1 Yuxiao Guo,1 Dan A. Dixon,2 Jeffrey Aubé,3 Danny R. Welch,2 Liang Xu1. 1 _University of Kansas, Lawrence, KS;_ 2 _University of Kansas Medical Center, Kansas City, KS;_ 3 _University of North Carolina, Chapel Hill, NC_.

Patients with metastatic breast cancer have a dismal 5-year survival rate of only 24%. Chemoresistance is another contributor to the high mortality of advanced breast cancer. The RNA-binding protein, Hu antigen R (HuR), is overexpressed at all stages of breast cancer development. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by promoting mRNA stability and translation of proteins implicated in proliferation, survival, angiogenesis, invasion, and metastasis. HuR also modulates sensitivity of breast cancer cells to chemotherapy. Taken together, HuR is an emerging target for breast cancer therapy, especially metastatic breast cancer. Using shRNA and CRISPR technologies to modulate HuR expression in breast cancer cells, we found that cells with HuR KD/KO were less invasive and more sensitive to chemotherapy. In an effort to discover small molecules that disrupt the HuR-mRNA interaction and block HuR functions in breast cancer progression, metastasis and chemoresistance, high throughput screening (HTS) was carried out using several chemical libraries (~23,000 compounds) using fluorescence polarization (FP) assay and identified several initial hits with sub-micromolar inhibitory constants (Ki). Those potential disruptors were then validated by ALPHA assay (Amplified Luminescent Proximity Homogeneous Assay), confirmed by Surface Plasmon Resonance (SPR). In cell-based assays, top hit KH-3 and its optimized analogs, specifically shortened HuR target mRNA half-lifes and decreased the level of the encoded proteins. Moreover, those compounds inhibited invasion and restored chemosensitivity. qPCR arrays focusing on specific pathways revealed that HuR inhibitors potently upregulated metastatic suppressors and downregulated genes frequently overexpressed in lung metastases. In animal studies, KH-3 not only powerfully exhibited antitumor efficacy in orthotopic xenografts, but also efficiently blocked experimental metastasis. In conclusion, we identified potent and specific small molecule disrupters of HuR-mRNA interaction for potential anti-metastatic therapy for breast cancer with HuR overexpression.

#4833

CDODA-Me augments the efficacy of chemotherapeutic agents and overcomes chemo-resistance in breast and lung cancer cells.

Ebony Nottingham, Ravi Doddapaneni, Mandip Sachdeva. _Florida Agricultural and Mechanical University, Tallahassee, FL_.

Purpose: The purpose of this research is to determine the effects of the combination of (CDODA-Me), a derivative of glycyrrhetinic acid with chemotherapeutic agents like docetaxel (DTX) and Erlotinib (a TKI inhibitor) to treat triple negative breast cancer (TNBC) and TKI resistant lung cancer.

Methods: TNBC lines MDA-MB-231 MDA-MB-468 and DTX resistant (MDA231) cells were treated in combination with CDODA-Me (nontoxic dose of 2 µM) and DTX. Wildtype (HCC827) and resistant (Erlotinib resistant) lung cancer cells were treated with Erlotinib and CDODA-Me. The cell viability of MDA-MB-231, MDA-MB-468, Docetaxel resistant (MDA231) cells and HCC 827 Erlotinib resistant (4 µM) cells in each treatment group was determined by crystal violet assay. Combination index values were calculated by isobolographic analysis. Western blot annalysis was used to investigate the influence of CDODA-Me combinations on drug resistance and key apoptotic proteins such as bcl2, survivin, SP1, SP3, and SP4. 2',7' -dichlorofluorescin diacetate (DCFDA) was used to measure reactive oxygen species (ROS) levels in all cell lines and examined by flowcytometry.

Results: Breast Cancer cells (MDA-MB-468 and MDA-MB-231) showed increased cytotoxicity with a ten fold decrease in IC50 concentration (0.3 µM DTX and 0.03 µM DTX in combination with 2 µM CDODA-Me). DTX resistant (MDA231) cells showed IC50 values comparable to wildtype cells at 0.29 µM DTX in combination with CDODA-Me. HCC827 (Erlotinib resistant) showed a three fold decrease in cytotoxicity (IC 50 values of 4.9 µM and 16.9 µM Erlotinib for the combination and Erlotinib alone respectively). The combination treatment also showed higher response in resistant cells with IC50 values comparable to wildtypes. The expression of bcl2, survivin, specific transcription factors (like SP1, SP3 and SP4) was downregulated in cells treated with CDODA-ME and DTX combination compared to CDODA-ME alone, DTX alone and control cells. Similar results were observed with CDODA-Me and Erlotinib combination in HCC827 erlotonib resistant (4 µM) cells. CDODA-Me alone treatment showed ROS levels in HCC 827 (4 µM) resistant cell lines were increased (8-fold) significantly as compared to other breast cancer cells.

Conclusion: In conclusion, CDODA-Me inhibits growth of TNBC cells and Erlotinib resistant lung cancer cells and downregulates the SP proteins and anti-apoptotic proteins. Therefore, these results indicate that CDODA-Me is a promising anticancer agent and can overcome resistance of chemotherapeutic agents against breast and lung cancer.

#4834

Preclinical analysis of the Notch gamma secretase inhibitor BMS-906024 in combination with chemotherapy in the treatment of lung adenocarcinoma.

Katherine M. Morgan,1 Francis Lee,2 Erin Michaud,2 Bruce S. Fischer,2 Sharon R. Pine1. 1 _Rutgers Cancer Institute of New Jersey, New Brunswick, NJ;_ 2 _Bristol-Myers Squibb, Princeton, NJ_.

Notch signaling is aberrantly activated in approximately one third of non-small cell lung cancer (NSCLC) cases, primarily through loss of the endogenous Notch inhibitor, Numb, or via gain-of-function mutations in the Notch1 receptor. Notch activity is associated with poor overall survival among NSCLC patients whose tumors are wildtype for TP53. Here, we characterized the interaction between BMS-906024, a clinically relevant gamma secretase inhibitor (GSI) that inhibits Notch activation, and front-line chemotherapy in preclinical models of NSCLC. MTS drug synergy assays consisting of treatment with BMS-906024, cisplatin or paclitaxel, or the combination of GSI and chemotherapy were performed on a panel of human NSCLC cell lines, most of which were derived from adenocarcinomas. Analysis of the drug effects with CalcuSyn yielded significantly lower CI values for the GSI BMS-906024 combined with paclitaxel than with cisplatin (average CI = 0.54 vs 0.85, respectively; P = 0.001). The synergy between BMS-906024 and paclitaxel was significantly greater in Kras-wildtype than Kras-mutant cells (average CI = 0.39 vs 0.68, respectively; P = 0.009), while there was no correlation with EGFR or TP53 status. Treatment of lung adenocarcinoma xenografts in NOD scid gamma mice confirmed enhanced antitumor activity for the combination treatment of BMS-906024 and paclitaxel by mechanisms currently under investigation. These results are a step toward identification of the optimal combination of the GSI BMS-906024 with standard chemotherapies, as well as potential biomarkers that could be used to predict patient response to Notch-targeted therapy.

#4835

Targeting the ATR/CHK1 axis in combination with PARP inhibition is more effective than PARP inhibition alone in BRCA mutant models.

Erin George,1 Hyoung Kim,1 Janos Tanyi,1 Ryan Ragland,1 Rugang Zhang,2 Patricia Bradford,2 Clemens Krepler,2 Katherine Nathanson,1 Brandon Wenz,1 Yiling Lu,3 Gordon Mills,3 Mark Morgan,1 Fiona Simpkins1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _The Wistar Institute, Philadelphia, PA;_ 3 _MD Anderson Cancer Center, Houston, TX_.

Introduction:

Approximately 50% of high grade serous ovarian cancers (HGSOC) have defects in genes involved in homologous recombination repair (HR). BRCA1/2 mutant HGSOCs have a deficiency in the repair of double strand DNA breaks by HR. Poly(ADP-ribose) polymerase inhibitors (PARPi) block the repair of single-stranded breaks leading to double strand DNA breaks which cannot be repaired efficiently in BRCA-deficient cancers capitalizing on synthetic lethality. PARPi have a modest clinical response of only ~40% in recurrent BRCA mutant HGSOCs. We hypothesize that PARPi alone increases reliance on other DNA repair pathways such as ATR/CHK1 in HR deficient cells, and by targeting ATR/CHK1 in combination with PARPi would be more effective in eradicating tumor growth.

Experimental Procedures:

Effects of PARP inhibitor (PARPi, Olaparib), CHK1 inhibitor (CHK1i, MK8776), and ATR inhibitor (ATRi, AZD6738) on cell cycle, survival, colony formation, genome stability were evaluated in PEO1 (BRCA2 mutant), PEO4 (BRCA wild-type), JHOS4 (BRCA1 mutant), and WO-24 (BRCA wild-type) ovarian cancer cells. A BRCA2 mutant (8945delAA) orthotopic PDX model was used to evaluate PARPi alone or in combination with CHK1/ATRi. Targeted capture massively parallel sequencing, Reverse-Phase Protein Array Analysis (RPPA) and IHC were performed on cells and xenografts to evaluate for biomarkers of response.

Results:

Monotherapy with PARPi, CHK1i, and ATRi in vitro demonstrated selectivity in mediating cell death and DNA damage in BRCA1/2 mutant cell lines (PEO1, JHOS4) compared to BRCA1/2 wild-type, platinum resistant cell lines (PEO4, WO-24). However, monotherapy only results in ~40-50% cell death in BRCA1/2 mutant cell lines. PARPi alone resulted in tumor suppression but not tumor eradication in a BRCA2 mutant PDX model. PARPi treatment resulted in an increase in ATR/CHK1 signaling in BRCA1/2 mutant cells. Treatment with ATR/CHK1i in combination with PARPi is synergistic in reducing survival of BRCA1/2 cells. Combination treatment was more effective in targeting cell cycle mediators, and promoting apoptosis. Treatment with either PARPi+ATRi or PARPi+CHK1i combinations was synergistic in causing tumor suppression but PARPi/ATRi combination caused tumor regression in a BRCA2 mutant PDX model.

Conclusions:

Strategies to optimize approaches capitalizing on synthetic lethality are needed for HR deficient HGSOC. PARPi is effective in BRCA deficient cancers but can potentially be more effective when combined with ATR/CHK1i. 

## CANCER CHEMISTRY:

### Drug Design

#4836

Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells.

Aleem Gangjee,1 Nian Tong,1 Adrianne Wallace Povrik,2 Zhanjun Hou,2 Larry H. Matherly2. 1 _Duquesne University, pittsburgh, PA;_ 2 _Wayne State University School of Medicine, Detroit, MI_.

Folates function as cofactors in one-carbon transfer reactions including de novo biosynthesis of nucleotides and play a key in DNA and RNA synthesis, epigenetic processes, cellular proliferation and survival. Cellular folate uptake is mediated by three distinct transporters. Among these, the reduced folate carrier (RFC) is the predominant folate transporter and is critical for antitumor efficacy of currently used classical antifolates such as methotrexate (MTX), pemetrexed (PMX), and raltitrexed (RTX). RFC is ubiquitously expressed in tumor cells and in normal tissues, resulting in dose-limiting toxicity with classical antifolates. We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT-expressing human tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and reduce the toxicity of clinically available antifolates which have non-selective uptake. As an extension of the SAR, we focused on a novel isosteric series of 6-substituted thieno[2,3-d]pyrimidine analogs, with a 3- or 4- carbon bridge between the thieno[2,3-d]pyrimidine and the thienoyl regioisomeric side chain. This simple isosteric replacement of pyrrole with thiophene in the scaffold, combined with varying the positions of attachment of 3- and 4-carbon bridge and the L-glutamic acid on the thiophene ring, provides compounds with different chain length, conformations and extra hydrogen bond donors and/or acceptors compared to the previously reported compounds. All the analogs (AGF 102, AGF131, AGF 271, AGF 275, AGF 276 and AGF 282) are potently inhibitory toward Chinese hamster ovary (CHO) cells (RT16) expressing human FRα (IC50s of 3.1, 0.33, 0.45, 1.1, 0.83 and 5.6 nM, respectively). All the analogs were inactive in the CHO cell line expressing RFC indicating absolute selectivity for FRα. AGF271, AGF275, and AGF282 showed modest (IC50s from 95-460 nM) with PCFT-expressing CHO cells (R2/PCFT4). AGF102, AGF 131 and AGF 271 AGF 275 AGF 276 AGF 282 were all potently inhibitory toward KB tumor cells (IC50 of 2.10, 1.70, 1.48, 1.81, 5.74 and 7.61nM, respectively). Collectively, our results suggest that AGF102, AGF 131, AGF 271, AGF 275, AGF 276 and AGF 282 are targeted antifolates with activity profiles that suggest that further preclinical studies and optimization are warranted.

#4837

Design, synthesis, and biological evaluation of thieno[2,3-d] and thieno[3,2-d]pyrimidines as dual acting RTK inhibitors and microtubule targeting antitumor agents.

Aleem Gangjee,1 Farhana Islam,1 Tasdique Mohammad Quadery,1 Susan L. Mooberry,2 Anja Bastian,3 Michael A. Ihnat3. 1 _Duquesne University, Pittsburgh, PA;_ 2 _University of Texas Health Science Center, Cancer Center & Research Center, San Antonio, TX; _3 _The University of Oklahoma College of Pharmacy, Oklahoma City, OK_.

Microtubules, attractive targets for drug development as anticancer agents, play vital roles in multiple aspects of cellular metabolism. These agents have been successfully used in the clinic for the treatment of various cancers. However, the utility of such agents are often limited by the emergence of tumor resistance due to overexpression of P-glycoprotein and/or βIII-tubulin. Tumor angiogenic mechanisms are important for tumor growth and metastasis are inhibited by antiangiogenic agents (AAs). AAs are usually cytostatic, and their utility in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of antiangiogenic agents with antitubulin agents have been particularly successful. Recently, we reported water-soluble tubulin targeting pyrrolo[2,3-d]- and pyrrolo[3,2-d]pyrimidine analogs as antitumor agents, that circumvent drug resistance. These analogs also possess RTK (EGFR, VEGFR2 and PDGFRβ) inhibitory activity. To further explore the activity of these dual RTK/tubulin inhibitors we designed isosteric thieno[2,3-d] pyrimidine and thieno[3,2-d]pyrimidine analogs which increased microtubule depolymerizing potency by 14 to 24-fold (AG95 IC50 = 7.0 ± 2.7 nM, AG370 IC50 = 7.6 ± 2.1 nM) compared with the lead compounds (AG85 IC50 = 96.6 ± 5.3 nM, AG20 IC50 = 183 ± 3.4 nM). Substitution at the 4-anilino position of both these scaffolds were also explored with various moieties to afford the 6-methoxy-tetrahydroquinoline moiety which possess the optimum balance of microtubule depolymerizing activity (AG372 IC50 = 4.3 ± 1.6 nM, AG337 IC50 = 3.4 ± 0.9 nM,) and RTK (EGFR, VEGFR2 and PDGFRβ) inhibitory activity. These two compounds demonstrated excellent inhibition for EGFR (IC50 = 12.6 ± 1.2 nM, 28.5 ± 3.9 nM), VEGFR-2 (25.2 ± 7.2 nM, 216.1 ± 18.6 nM) and PDGFRβ (IC50 = 25.6 ± 7.2 nM, 266.8 ± 35.4 nM) comparable to the standard clinically used agents. These attributes of the 4-N-substituted thieno pyrimidines provides the impetus for further development of these compounds as dual acting targeted antitumor agents.

#4838

Peptide prodrugs targeting the SH2 domains of p85 block PI3K signaling.

Pijus K. Mandal,1 Yanhua Yao,2 Anne R. Bresnick,2 Jonathan M. Backer,2 John S. McMurray1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _Albert Einstein College of Medicine, New York, NY_.

Class IA phosphoinositol-3-kinases (PI3Ks) play pivotal roles in cancer and inflammation and are targets for anti-cancer therapies. p85 was first described as a regulatory subunit of Class I PI3Ks which complexes with the p110 catalytic domain to form the heterodimeric enzyme. PI3Ks are recruited to cytokine and growth factor receptors via the SH2 domains of p85. While p85 represses the kinase activity of p110 subunits in the cytosol, binding to pTyr residues on receptors or adapters activates p110, resulting in the phosphorylation of the 3'-hydroxyl group of phosphatidylinositol-4',5'-diphosphate (PIP2) to form phosphatidylinositol-3',4',5'-triphosphate (PIP3). Membrane-associated PIP3 subsequently recruits kinases such as PDK1 and Akt to the cell membrane. PDK1 phosphorylates and activates Akt, which propagates growth and survival signals. In biochemical assays, doubly phosphorylated peptides enhance catalytic activity of p85/p110 dimers, which led to the dogma that phosphopeptides should activate PI3K in cells.

Due to the challenges of cellular entry of negatively charged phosphopeptides, blocking protein-protein interactions mediated by p85 has been largely unexplored in intact cells. We targeted the SH2 domains of p85 with phosphatase-stable, cell-permeable prodrug analogs of Y(p)VPML, a high-affinity ligand derived from Tyr751 of PDGF. To allow cell entry and to protect against phosphatase degradation, we replaced pTyr with bis-pivaloyloxymethyl esters of 4-phosphonodifluoromethylphenylalanine (F2Pmp(POM2)), which have not been reported. We synthesized Z-NMe-F2Pmp(POM2)-OPcp with the α-amino group methylated to enhance cell penetration and the carboxyl group esterified with pentachlorophenol. Z-protection was necessary for phosphonate protecting group manipulation and for stability of the Pcp ester. A series of inhibitors was prepared by coupling Z-NMe-F2Pmp(POM2)-OPcp to analogs of VPML. Hydrogenation in the presence of Ac2O gave an N-terminal acetyl prodrug. Screening for inhibition of EGF-stimulation of pAkt in serum starved MDA-MB-468 cells led to the identification of the lead inhibitor, PM-190I. In serum starved MDB-MB-231 cells, PM-190I inhibited EGF-stimulated phosphorylation of Akt with an IC50 of 5 μM. At 30 μM Akt phosphorylation was negligible. These results suggest that the phosphopeptide mimic blocks recruitment to p85/p110 dimers to EGFR or its substrates, thereby preventing PI3K pathway signaling. Further, in intact cells, a mono-phosphopeptide does not stimulate PI3K activity, which contradicts current dogma.

In addition to its role as a regulatory subunit, non-catalytic roles of p85 include complexing Rab4 and Rab5, stabilizing PTEN, invadopodium formation, cytokinesis, and nuclear trafficking of proteins such as XBP-1 and OPNi. Phosphopeptide mimics targeted to the SH2 domain of p85 have potential applications both in blocking PI3K signaling as well as in modulating non-catalytic functions.

#4839

Design of a Rofecoxib-Combretastatin hybrid drug that exerts highly potent antimicrotubule, anti-angiogenesis properties and Cox-2 inhibition both in cell culture and xenograft models.

Surendra R. Punganuru, Hanumantha Rao Madala, Constantinos Mikelis, Kalkunte S. Srivenugopal. _Texas tech University Health Sciences Center, Amarillo, TX_.

Given the molecular complexity of cancers, including the extreme heterogeneity in their genetic makeup, their plasticity, innate or acquired resistance to anticancer drugs, there is an increasing need for the development of single molecule drugs that can effectively and simultaneously impact two or more targets simultaneously triggering multiple cytocidal events in synergy to obtain superior chemosensitivities and prevent resistance. Therefore, we developed a hybrid drug by combining the Cox-2 selective NSAID rofecoxib with a trimethoxy aryl group found in combretastatin A4 (CA4) which is a potent antimicrotubule and anti-angiogenesis agent. Two of methoxy groups of the CA4, were, however, replaced with iodine in the hybrid drug named KSS-19. The structural design of KSS-19 also preserved the CA4 nucleus in the cis-configuration and prevented its isomerization to the biologically inactive trans-form. KSS-19 was highly potent in specifically killing the tumor cells of various human cancer types at 2-50 nM range. These included the colon (HT29, HCT116), breast (MDA-MB-231, SK-BR-3), lung (A549, H1299), brain (SF188, GBM10, GBM6), pancreas (MIA-PaCa-2) and fibrosarcoma (HT1080). Particularly significant was that KSS-19 was 100 time more potent than CA4 against the HT29 colon cancer cells. Various biochemical and immunocytochemical assays revealed that KSS-19 retained the microtubule disrupting effects of CA4, including microtubule depolymerization, the formation of aberrant mitotic spindles, and G2/M phase arrest. The hybrid drug also inhibited the polymerization of purified tubulins in vitro. Furthermore, KSS-19 potently inhibited the formation of tubes in three-dimensional cultures of the HUVEC (Human umbilical Vein Cell) drastically decreasing the tube length and junctions at 250 nM concentration. The cancer cell migration /invasion, determined by transwell assays were also inhibited, and the inhibition was accompanied by increased E-cadherin levels through NF-kB/Snail pathway. The basal and LPS-activated levels of Cox-2 in tumor cell lines, as determined by western blot and immunofluorescence assays were highly suppressed by KSS-9. In a mouse xenograft model using HT-29 colon cancer cells, KSS19 as a single agent (75 mg/kg) significantly inhibited the tumor growth; the intratumoral levels of Cox-2 and in vivo angiogenesis were also markedly downregulated without any organ toxicities. Higher levels of cleaved caspase-3 and PARP proteins in the tumor suggested the priming of the apoptotic pathways. In conclusion, our results indicate that KSS-19 represents a new prototype chemo drug with multiple mechanisms of action. Such hybrid anticancer drugs merit further development (supported by CPRIT grant RP130266 to KSS).

#4840

The role of the metal scaffold in the development of cytotoxic metal complexes.

Edith C. Glazer. _University of Kentucky, Lexington, KY_.

Multiple cytotoxic metal compounds are known, but structure activity relationships can be difficult to determine in these complex molecules. It is tempting to generalize the activity or off-target effects of the compounds to being related to the identity of the metal, or alternatively, to some specific "active" ligand features, but this is a misleading oversimplification that can slow the development of new therapeutics. In this presentation a systematic study of the impact of the metal center, one specific "active" ligand, and the inactive "coligands" that create the metal scaffold will be presented. There is clear evidence that each component plays a role in both desired and potentially deleterious off-target activities, providing the opportunity to adjust each of these three components independently to generate optimized potential anti-cancer agents.

#4841

Molecular targets of NOSH-naproxen (AVT-219), a dual nitric oxide and hydrogen sulfide-releasing hybrid, in a xenograft mouse model of colon cancer.

Pascale L. Duvalsaint, Mitali Chattopadhyay, Ravinder Kodela, Khosrow Kashfi. _City University of New York Medical School, New York, NY_.

Introduction: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colon cancer, but can cause serious gastrointestinal (GI), cardiovascular (CV), and renal side effects. Naproxen has a relatively favorable CV profile, but is notorious for its GI toxicity. To improve its safety profile, we developed NOSH-naproxen, where we linked nitric oxide (NO) and hydrogen sulfide (H2S) releasing moieties to the native molecule, the rational being the observations that NO and H2S may modulate some components of the local mucosal defense systems which should lead to reduced GI toxicity. Indeed, we recently reported that NOSH-naproxen displayed enhanced GI safety, with potent anti-inflammatory, analgesic, anti-pyretic, and anti-platelet properties. Here we describe the effects of NOSH-naproxen on the growth properties of several human colon cancer cell lines, and on its molecular targets in a xenograft mouse model of colon cancer.

Methods: NOSH-naproxen was synthesized by us with 1H-NMR verification. Colon cancer cell lines: HT-29, HCT 15, and SW480; Cell growth: MTT. Xenografts: Male athymic nude (NU/NU) mice (N=15) were implanted s.c. in the right flank with SW480 cells, when the tumors reached an average sizes of ~100 mm3, the mice were randomly divided into 3 groups and gavaged daily with either vehicle (0.5% CMC) or equimolar concentrations of NOSH-naproxen (100 mg/kg) or naproxen (45 mg/kg). Tumor volume was measured every three days; after 30 days the mice were sacrificed, tumors collected, weighed, photographed and stored in formalin for IHC studies.

Results: NOSH-naproxen inhibited the growth of HT-29, HCT 15, and SW480 cells with IC50 values of 90 ± 10, 100 ± 8, and 98 ± 7 nM at 24 hr, respectively. The corresponding IC50s for naproxen were 2800 ± 165, 2950 ± 215, and 3110 ± 185 nM. This represents an enhanced potency of ~30,000-fold in the 3 cell lines at 24 h. In xenografts, all the mice treated with naproxen died within 2 weeks from what appeared to be GI bleeding. However, the NOSH-naproxen treated mice did not show any overt signs of toxicity. At sacrifice, NOSH-naproxen-treated mice had a mean reduction in tumor volume of 82%, and a mean reduction in tumor mass of 55%. NOSH-naproxen inhibited growth of these cancer cell xenografts as a result of reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased number of TUNEL positive cells). NF-κB, activated in untreated xenografts was significantly inhibited by NOSH-naproxen. Other molecular targets affected by NOSH-naproxen were: induction of iNOS, significant reduction in FoxM1, and increases in p53.

Conclusions: NOSH-naproxen is more potent and efficacious than naproxen and appears to be significantly safer. It targets parameters important in determining cellular kinetics, inflammation, and signaling pathways important in the carcinogenic process.

#4844

Thymoquinone induces apoptosis through inhibition of JAK2/STAT3 signaling via production of ROS in human renal cancer Caki cells.

In Gyeong Chae, Kyung-Soo Chun. _Keimyung University, Daegu, Republic of Korea_.

Thymoquinone (TQ), a compound isolated from black seed oil (nigella sativa), has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of TQ in renal cell carcinoma (RCC) remain poorly understood. This study revealed that treatment with TQ significantly reduced the cell viability and induced apoptosis in Caki cells as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, and -3 and PARP and the inhibition of Bcl-2 and Bcl-xl expression. TQ inhibited the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) in Caki cells by blocking the phosphorylation of upstream Janus-activated kinase-2 (JAK2) kinases. Moreover, TQ attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, and D2. Since ROS is a universal entity mediating apoptosis, we examined whether TQ induced apoptosis via ROS formation. Treatment with TQ generated ROS in these renal cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine (NAC) abrogated the inhibitory effect of TQ on the JAK2/STAT3 signaling and rescued cells from TQ-induced apoptosis by blocking the induction of p53, and the cleavage of caspase-3, -9 and PARP in Caki cells. In conclusion, TQ induced apoptosis in Caki cells via generation of ROS, induction of p53, activation of caspases and inhibition of STAT3 signaling pathway.

#4845

Fraxini and mistletoe lectins inhibit the proliferation of hepatocellular carcinoma by promoting proteasomal degradation of c-Myc.

Yan Jiang, Yong Pan, Patrea R. Rhea, Richard Lee, Zhimin Lu, Peiying Yang. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Mistletoe extracts, including Fraxini, have been used to treat cancer, including hepatocellular carcinoma (HCC), in Europe. However, the mechanism(s) underlying the effect of Fraxini in HCC is(are) inconclusive which hampers its optimal use for the treatment of HCC. We previously reported that Fraxini, and two other mistletoe extracts Iscador Q and M exerted strong antiproliferative and pro-apoptotic activities in Hep3B cells through targeting c-Myc protein. In current study, we examined the efficacy of Fraxini in HCC bearing animals and further investigated the mechanism by which Fraxini down-regulates c-Myc protein. How c-Myc was regulated by Fraxini was investigated by measuring the half-life of c-Myc protein with cycloheximide chase assay, proteasomal degradation and phosphorylation of c-Myc. The antitumor efficacy of the Fraxini was tested in the Hep3B mouse xenograft model and Fraxini was administered subcutaneously. We found that Fraxini dose dependently suppressed the c-Myc protein expression, but did not affect c-Myc mRNA level in Hep3B cells, suggesting Fraxini down-regulates c-Myc through post-translational regulation. Fraxini reduced the half-life of c-Myc protein from control (40 minutes) to 27 minutes. Inhibition of 26S proteasome activity using MG-132 in Hep3B cells notably antagonized Fraxini induced down-regulation of c-Myc, suggesting that Fraxini down-regulated c-Myc by promoting its proteasome degradation. Fraxini decreased phosphorylation of S62 (stabilizing c-Myc) while slightly increased p-cMycT58 (destabilized c-Myc) in Hep3B cells. When Burkitt's lymphoma Raji cells, which are known to host T58 mutation on c-Myc and result in c-Myc stabilization, were treated with Fraxini, Fraxini (up to 20 µg/ml) exerted minimum activity in cell growth and c-Myc protein expression, augmenting that Fraxini is acting on c-Myc phosphorylation sites which resulted in down-regulation c-Myc and elicited anticancer activity in HCC. Additionally, Fraxini injected (s.c.) to mice carrying Hep3B cell xenograft tumors for 2 weeks significantly reduced the average tumor volume (71.8 ± 22.2 mm³) compared to that in the control treated mice (179.0 ± 36.3 mm³, p < 0.05) and lead to reduction of c-Myc protein in the tumor tissues. Intriguingly, mistletoe lectin, a bioactive component in Fraxini, appears to be most effective in inhibiting the proliferation of c-Myc expressing Hep3B cells compared to that of PLC/RF5 cells with limited expression of c-Myc. Mistletoe lectin (1 pg/ml) in the meantime reduced almost 90% c-Myc protein expression in Hep3B cells. In conclusion, since c-MYC gene is required for hepatocyte proliferation and liver tumorigenesis, Fraxini and mistletoe lectin through targeting c-Myc may have great therapeutic potential for patients with advanced HCC and warrant further investigation. The study is supported by the start-up fund of UT MDACC.

#4846

Chemotherapeutic and chemomodulatory effects of naturally occurring tetrahydrofuran type terpenoid.

Eman M. Atiya,1 Fahad A. Al-Abbasi,1 Mohamed-Elamir F. Hegazy,2 Ali M. El-Halawany,3 Ahmed M. Al-Abd2. 1 _King Abdulaziz University, Jeddah, Saudi Arabia;_ 2 _National Research Center, Cairo, Egypt;_ 3 _Cairo University, Cairo, Egypt_.

Naturally occurring terpenoid compounds are diverse active constituent in several plants and known of their potential anticancer activities. Camptothecin (CPT) is a topoisomerase inhibitor agent with well documented anti-neoplastic activity against several types of tumors. The purpose of this study is to investigate the potential chemotherapeutic effects of some terpenoid compounds isolated from Salvia africana lutea, Stachys aegyptiaca, Tanacetum sinaicum. Terpenoid compounds with promising anticancer profile has been further investigated for potential chemomodulatory effects to CPT against different tumor cell types. Cytotoxicity of the isolated terpenoids were assessed against human breast adenocarcinoma (MCF7), human colorectal cancer (HCT116 and LS174T) and human hepatocellular carcinoma (HepG2) cells using sulpharhodamine-B assay after cell exposure for 72 h. Cytotoxic parameters (IC50 and R-fraction) were calculated from cell viability after fitting to Emax mathematical model. The tetrahydrofuran type terpenoid, (R,E)-6-hydroxy-6-methyl-2-((2S,5R)-methyl-5-vinyltetrahydrofuran-2-yl)hept-4-en-3-one (TSS-7), isolated from Tanacetum sinaicum showed considerable cytotoxicity with IC50 ranging from 49 to 70 µM in all tested cell lines. On the other hand, IC50 of CPT ranged from 0.13 to 1.5 µM in the same cell lines under investigation. Combination index analysis (CI-value) for TSS-7 with CPT showed synergistic relationship in MCF7, HCT116 and HepG2 (CI-values were 0.17, 0.17 and 0.39 respectively). However, the same combination of TSS-7 and CPT against LS174T cells showed antagonistic relationship (CI-value of 1.2). Further investigation for apoptosis/necrosis assessment using annexin-V/PI staining was undertaken for single and combination treatment of CPT and TSS-7. In HCT116 cells, TSS-7 combination with CPT significantly increased apoptotic and necrotic cells compared to CPT treatment alone. However, in LS174T cells, TSS-7 combination with CPT significantly decreased apoptotic and necrotic cells compared to CPT treatment alone. Cell cycle analysis using DNA cytometry showed strong G2/M arrest induced by CPT treatment. Yet, TSS-7 increased cell population in S-phase and to a lesser extend in G2/M-phase which might be the reason for temporal synergism/antagonism of TSS-7 and CPT in different cell lines. In conclusion, TSS-7 could be considered promising anticancer chemomodulatory terpenoid compound which requires further molecular investigations to elucidate its sub-cellular molecular target.

#4847

Discovery, semisynthesis, and structure-activity relationship studies of hirsutinolide derivatives as new STAT3 inhibitors and anti-glioma agents.

Mingming Zhang,1 Ui Joung Youn,1 Gabriella Miklossy,2 Supakit Wongwiwatthananukit,1 James Turkson,2 Leng Chee Chang,1 Dianqing Sun1. 1 _University of Hawaii at Hilo, Hilo, HI;_ 2 _University of Hawaii Cancer Center, Honolulu, HI_.

Clinically, natural product has played a pivotal role in anticancer drug discovery and development. Cancer chemotherapy and/or radiotherapy due to glioma treatment are not ideal, frequently cause unwanted adverse effects, and justify further research and development of alternative, novel, safe, and effective agents. In our continued efforts to identify new signal transducer and activator of transcription 3 (STAT3) and antiglioma agents with enhanced efficacy and specificity, we have initiated a collaborative and multidisciplinary natural product drug discovery project and the goal was to isolate, semisynthesize, and evaluate the potential of Vernonia cinerea-derived phytochemicals as STAT3 inhibitors with therapeutic remedy of human glioma and may be even more effective than the existing ones. To date, our preliminary studies have been conducted and resulted in the discovery of a class of sesquiterpene lactone hirsutinolide series with an α,β-unsaturated-γ-lactone ring as new STAT3 inhibitors. Specifically, on the basis of encouraging biological data and to expand the existing structure activity relationship (SAR) of isolated natural hirsutinolide analogues, chemical modifications were performed using conventional Steglich esterification protocol to produce a series of semisynthetic hirsutinolide derivatives in moderate to high yields. Thus far, biological evaluation revealed that several semisynthetic analogues showed low micromolar inhibitory activities against constitutively-active STAT3 and malignant glioma phenotype. SAR showed that a bulky and lipophilic ester functionality at position 13 is essential for STAT3 inhibition as well as whole cell based anticancer activity. A lipophilic side chain at position 8 also plays an important role in anticancer activity and may contribute a detrimental effect on specificity. In addition, the methoxy group at position 1 enhances the cell-type specificity and selectivity. Finally, selected promising lead candidates also demonstrated in vivo efficacy following oral gavage delivery, inhibiting human glioma tumor growth in subcutaneous mouse xenografts. Together, natural and chemically modified hirsutinolide-type sesquiterpene lactones represent a promising natural product class of new anticancer agents for the treatment of malignant human glioma.

#4848

Discovery of ABI-231 analogs targeting the colchicine site in tubulin for advanced melanoma.

Wang Qinghui, Arnst E. Kinsie, Duane D. Miller, Wei Li. _University of Tennessee Health Science Center, Memphis, TN_.

We previously discovered a novel 2-aryl-4-benzoyl-imidazole (ABI) scaffold exerts its potent anti-proliferative effects through interacting with the colchicine binding site in tubulin. Extensive structure-activity relationship (SAR) studies of this novel scaffold showed that the best analog in this series, ABI-231, has an average IC50=5.2 nM against panels of melanoma and prostate cancer cell lines, is orally bioavailable, and strongly suppress melanoma tumors in vivo. Analyses of the potential binding pose of ABI-231 at the colchicine binding pocket suggested small substitutions to the indole moiety in ABI-231 could further increase its interactions to tubulin.

Based this hypothesis, we have established an efficient synthetic method for the synthesis of ABI-231 analogs by either rotating the indole ring or introducing substituents on the indole of ABI-231. Using this method, we synthesized and tested 27 analogs of ABI-231. Several of the compounds show excellent potency against a panel of melanoma cancer cell lines with a diverse genomic background (mutations in BRAF, TP53, CDKN2A and PTEN) that are frequently encountered in clinical metastatic melanoma tumors. Additionally, they are not substrates for the P-glycoprotein (Pgp) efflux pump and therefore strongly inhibited the multidrug resistant cell lines. The potency of the compounds was further verified by their potent growth inhibition of cancer cell colonies. Results from cell cycle analysis, apoptosis evaluation and tubulin polymerization inhibition are consistent with their expected mechanisms of action. Potential off-target analyses performed by Eurofins Cerep-Panlabs SafetyScreen 44 indicated that they are clean and does not inihibt any of the tested off-targets at 100 fold of its IC50 value in melanoma cells, suggesting the good safety profile for this compound and the probably these scaffold in general. We are currently performing in vivo efficacy studies for the most potent analogs. These compounds hold great promise as a new generation of orally available tubulin inhibitors.

#4849

Low molecular weight pyrrolobenzodiazepine (PBD) monomers have potent cytotoxicity in haematological malignancies.

David B. Corcoran,1 Thomas Lewis,2 Amrit Varsha,1 Chris Pepper,2 David E. Thurston,1 Khondaker Miraz Rahman1. 1 _King's College London, London, United Kingdom;_ 2 _Cardiff University School of Medicine, Cardiff, United Kingdom_.

The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have long been of interest as potential chemotherapeutic agents due to their ability to form covalent adducts within the minor groove of the DNA helix. The most effective synthetic modifications to PBD cores have involved the conjugation of two DNA-interactive moieties via their C8-position to create PBD dimers capable of cross-linking duplex DNA which improves cytotoxicity. Similarly, other groups have focussed on adding large substituents to the C8-position of the PBD core to improve DNA-interaction and cytotoxicity. All of these approaches tend to increase the molecular weight of the compounds, although this has not prevented their successful development to the clinic, either as stand-alone agents (e.g., SJG-136) or as a component of Antibody-Drug Conjugates (ADCs) (e.g., SGN-CD33A).

During a structure-activity relationship (SAR), we embarked on a "molecular pruning" exercise to sequentially reduce the length and bulk of the C8-substituent of a PBD monomer expecting the cytotoxicity to reduce with a decrease in length/bulk and to establish the minimal pharmacophore. Surprisingly, we found that reducing the length of the C8-substituent maintained cytotoxicity and in some cases enhanced it.

A 50 molecule library of C8-substituted PBD monomers was synthesized featuring C8-substituents of various chemical composition and length, with some containing aniline substituents. Cytotoxicity evaluation in several tumour cell types was carried out (e.g., primary CLL, JJN-3 and MDA MB 231 cell lines. Low nanomolar to high picomolar IC50 values were observed for several library members including DC-1-194 (IC50 = 4.2nM in CLL, and 0.79nM in MDA MB 231), DC-1-255 (IC50 = 9.5nM in CLL, and 1.1nM in MDA MB 231), DC-1-253 (IC50 = 8.4nM in CLL, and 10nM in JJN-3) and DC-1-275 (IC50 = 7.6nM in CLL, and 9.6nM in JJN-3). Remarkably, many of these more-active compounds had very short C8-substituents.

The observations reported here add to current understanding of the SAR of PBD monomer structures. However, there may also be significance for the future development of PBD-based therapeutic agents where there may be advantages to working with lower molecular weight molecules.

#4850

**Combination of effector mimicking with** in silico **fragment screens is an effective approach for the development of Ras inhibitors.**

Colin Fields, R. Natasha Freed, Lyuba Khavrutskii, Karen Stefanisko, Yuri Pevzner, Megan Peach, Marc Nicklaus, Nadya Tarasova. _NCI-Frederick, Frederick, MD_.

Targeting Ras proteins directly has proven to be exceptionally challenging because they lack deep pockets on the surface that can provide for effective interactions with small molecules. Several shallow pockets have been identified on Ras surface. However, their positioning does not allow for simultaneous targeting with a single small molecule. To generate high affinity Ras binders, we have combined fragment-based drug design and in silico screening of fragment libraries with mimicking of Ras effectors. We have chosen Ras effector NORE1A for mimicking because it has one of the most compact continuous Ras-binding interfaces. Peptide mimetic of the central part of the interface was chemically rigidified and optimized to expand into additional pockets adjacent to Ras's NORE1A binding interface. These included one previously described pocket (Sun et.al 2012, Maurer et al, 2012) along with two newly defined ones. In particular, we have identified a "new" pocket between helix 2 and strands 2 and 3 of Ras protein. The pocket is not highly populated and is present in only 9 out of 71 analyzed Ras structures. The apparent binding region appears disordered in seven published structures suggesting that the fold of the pocket may be environmentally regulated and is likely affected by crystallization conditions. In silico screening of specially constructed virtual libraries consisting of millions of compounds allowed for identification of several potential binders. Two fragment-like molecules have been tested for Ras binding to verify the predictions. Binding assay that utilized microscale thermophoresis revealed that: 1) both compounds bind to WT, G12D and G12V mutants of K-Ras; 2) one of the compounds has higher affinity towards GTP-bound than to GDP -loaded wild type and G12V mutant of K-Ras (KD ≈0.9 mM and 3.2 mM respectively), but binds with approximately same KD≈ 3.3 mM to GDP and GTP-bound G12D K-Ras. We also utilized for screening a novel virtual library of synthetically accessible molecules that can be prepared in one step using well established chemistries and easily obtainable reagents. This resulted in additional potential ligands for the pocket. Two compounds were prepared and proven to bind with affinities in low millimolar range. The use of the ligands identified from virtual screens allowed for the design of a NORE1A mimetic with MW< 1000. The data along with previously published studies show that in silico screens for Ras binders are challenging because of the highly flexible and allosteric nature of the protein. However, utilization of several structures rather than focusing on just one can significantly improve the prediction power of virtual screens. Computational data along with binding studies strongly suggest that a combination of in silico screening with rational optimization of effector mimetics is a promising approach for the development of clinically useful inhibitors for thus far non-druggable Ras.

#4851

Second generation 2,3-dihydroimidazo[1,2-c]quinazoline PI3K inhibitors: development of BAY 1082439, a novel balanced PI3Kα / PI3Kβ inhibitor.

William J. Scott,1 Ningshu Liu,2 Andreas Hägebarth,2 Manfred Möwes,2 Ursula Mönning,2 Ulf Bömer,2 Dominik Mumberg,2 Franz von Nussbaum,2 Michael Brands,2 Julien Lafranc2. 1 _Bayer HealthCare Pharmaceuticals, LLC, Whippany, NJ;_ 2 _Bayer HealthCare Pharmaceuticals, Berlin, Germany_.

The phosphoinositide-3 kinase (PI3K) pathway plays critical roles in cancer cell growth and survival, as well as in intrinsic and acquired resistance to both chemotherapy and targeted agents. These essential roles of PI3K in human cancer have led to the clinical development of PI3K pathway inhibitors. Due to the complexity derived from the existence of various PI3K isoforms (α, ß, γ, ∂), and their differential roles in signal transduction as well as cancer pathology, investigation of PI3K inhibitors with differential isoform activity profiles would allow potential use in novel indications. Mutation or amplification of PIK3CA and/or activation of PI3Kα (e.g., through oncogenic RTKs) are found frequently in a variety of cancers, making this isoform a prime target for anti-cancer therapy. In addition, the role of PI3Kß in PTEN-deficient tumors, as well as in acquired resistance to PI3Kα, has been described. This led to the hypothesis that development of a dual PI3Kα / PI3Kß inhibitor might provide a unique efficacy profile.

The discovery of a novel class of 2,3-dihydroimidazo[1,2]quinazoline PI3K inhibitors, and its optimization to afford the i.v. PI3Kα/∂ inhibitor copanlisib (IC50 ratio of 1:7 in biochemical assays of PI3Kα vs. PI3Kß) has been reported recently. Herein is described the structure-activity relationship (SAR) leading to potent oral 2,3-dihydroimidazo[1,2]quinazoline PI3K inhibitors with balanced PI3Kα and PI3Kß activity, and to the selection of BAY 1082439, as a clinical candidate.

BAY 1082439 has an IC50 ratio of 1:3 in biochemical assays of PI3Kα (4.9 nM) vs. PI3Kβ (15.0 nM). In addition, BAY 1082439 has unique pharmacokinetic (PK) properties with very high plasma free fractions across all species tested (33-50%), large Vss, high clearance and intermediate T1/2. Thus, BAY 1082439 represents a PI3K inhibitor with a novel pharmacological profile, warranting exploration in clinical development. BAY 1082439 is currently being studied in a phase I trial for subjects with advanced malignancies (NCT01728311).

#4852

Multivalent peptoid conjugates suppress enzalutamide-resistant prostate cancer cellular proliferation.

Yu Wang,1 Paul Levine,2 Dilani Dehigaspitiya,2 Adam Profit,2 Susan Logan,1 Keren Imberg-Kazdan,1 Kent Kirshenbaum,2 Michael Garabedian1. 1 _NYUMC, New York, NY;_ 2 _NYU, New York, NY_.

Development of resistance to latest anti-androgens treatment for advanced prostate cancer is a growing concern. New strategies to block androgen receptor (AR) function in castration resistant prostate cancer are therefore required. Here we report the characterization of a multivalent conjugate presenting two spatially defined bioactive ethisterone ligands as spatially defined pendant groups on a peptoid oligomer. The conjugate, termed Multivalent Peptoid Conjugate 6 (MPC6), suppressed the proliferation of multiple AR-expressing prostate cancer cell lines including those that failed to respond to enzalutamide and ARN509. The structure-activity relationships of MPC6 variants were evaluated, revealing that increased spacing between ethisterone moieties and changes in peptoid topology eliminated its anti-proliferative effect. Mechanistically, MPC6 blocked AR coactivator-peptide interaction, and decreased AR protein half-life. Pharmacological studies revealed that MPC6 was metabolically stable and displayed a low plasma clearance rate. Importantly, MPC6 treatment reduced tumor growth and decreased Ki67 and AR expression in mouse xenograft models of enzalutamide-resistant LNCaP-abl cells. Thus, MPC6 represents a new class of compounds with the potential to combat treatment-resistant prostate cancer.

#4853

BMVC specifically binds the major G-quadruplex structure formed in the c-MYC promoter to lower c-MYC levels.

Wenting Liu,1 Guanhui Wu,1 Clement Lin,1 Buket Onel,1 Ding Chen,1 Ta-Chau Chang,2 Danzhou Yang1. 1 _University of Arizona, Tucson, AZ;_ 2 _Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan_.

The c-MYC proto-oncogene is one of the most deregulated genes in human cancers. Transcriptional repression of c-MYC is considered as one of the attractive strategies in targeting c-MYC. Previous studies revealed that the c-MYC promoter nuclease hypersensitive element (NHE) III1 region, which regulates 80-95% of total c-MYC transcription, can form DNA G-quadruplex (G4) and that stabilization of the c-MYC G4 could repress c-MYC gene expression. We have previously determined the molecular structure of the major G-quadruplex formed in the c-MYC promoter NHE (MycG4), which is an intramolecular parallel-stranded structure. BMVC, a carbazole derivative, was designed as a fluorescent probe for recognizing telomeric G4. However, we found that BMVC binds to MycG4 with much greater affinity and specificity. Cellular studies showed that BMVC was enriched in the nucleus at 48 hr after treatment, leading to markedly reduced c-MYC expression levels. Promoter-driven luciferase assays revealed that BMVC could enhance the inhibitory effects of MycG4, suggesting BMVC can repress c-MYC expression in vivo through stabilization of MycG4. To understand the molecular interactions between BMVC and MycG4, we have determined the molecular structure of the complexes of BMVC with MycG4 by NMR. BMVC appears to bind the 5'-end of the MycG4 with very high affinity, as shown by the slow-exchange binding on the NMR timescale. The structure of the 5'-end complex of BMVC and MycG4 shows that BMVC is paired to the flanking adenine with specific hydrogen bonding interactions, while the BMVC-adenine plane stacks nicely on the 5'-end G-tetrad of MycG4. Compared to the previous MycG4 complex with a quindoline compound, the tighter and more specific binding of BMVC appears to be imparted by the novel arched shape of BMVC and specific pairing recognition of BMVC with the flanking bases of MycG4. BMVC can also bind to the 3'-end of MycG4 with lower affinity to form a second complex. However, the 3'-end complex shows much more dynamic conformation and less specific interactions. Our results indicated that the cellular targets of BMVC include not only telomeric G4, but also the c-MYC promoter G4. Because of the inherent fluorescence and novel binding mode of BMVC, our study provides useful information for future design of improved carbazole-based anticancer agents or specific cellular fluorescent probes, as well as insights into specific recognition of MycG4 by small molecules.

#4854

Bazedoxifene inhibits ESR1 somatic mutants with improved potency compared to tamoxifene and raloxifene.

Sean W. Fanning,1 Venkat Dharmarajan,2 Christopher G. Mayne,3 Weiyi Toy,4 Kathryn E. Carlson,3 Teresa A. Martin,3 Jason Nowak,2 Jerome Nwachukwu,2 David J. Hosfield,1 Emad Tajkhorshid,3 Sarat Chandarlapaty,4 Patrick Griffin,2 Yang Shen,5 John A. Katzenellenbogen,3 Geoffrey L. Greene1. 1 _University of Chicago, Chicago, IL;_ 2 _Scripps Research Institute, Jupiter, FL;_ 3 _University of Illinois, Urbana-Champaign, IL;_ 4 _Memorial Sloan-Kettering Cancer Center, New York, NY;_ 5 _Texas A &M, College Station, TX_.

Despite continued administration of antiestrogen therapies, approximately 50% of all estrogen receptor alpha (ERalpha) positive breast cancers will present new metastatic lesions. The acquisition of secondary hormone-resistant metastatic breast cancers represents a significant clinical barrier towards life-long disease free survival for the patient. Somatic mutations to the ERalpha gene (ESR1) Y537S and D538G represent a novel mechanism of acquired antiestrogen resistance because they confer hormone-free transcriptional activity and reduced selective estrogen receptor modulator (SERM) and selective estrogen receptor degrader (SERD) potency. Fulvestrant, a SERD, was the only molecule that could completely ablate mutant ERalpha activity. Unfortunately, fulvestrant possesses poor pharmacologic profiles that limit its therapeutic utility. Bazedoxifene (BZA) is a potent mixed SERM/SERD and has improved pharmacokinetics and oral bioavailability compared to fulvestrant. We show that BZA inhibits Y537S and D538G ESR1 somatic mutation transcriptional activity with a greater potency than the SERMs 4-hydroxytamoxifen (TOT) and raloxifene (RAL). Further investigations into the biophysical and structural basis for BZA action suggest that BZA increases the conformational dynamics of helix 12, a key molecular switch that governs ERalpha action resulting in SERD-like properties and improved potency against the somatic mutations compared to TOT and RAL.

#4855

Discovery of NVP-HDM201 - First disclosure of a Next-Generation Mdm2 inhibitor with superior characteristics.

Philipp Holzer, Patrick Chène, Stéphane Ferretti, Pascal Furet, Tobias Gabriel, Bjoern Gruenenfelder, Vito Guagnano, Francesco Hofmann, Joerg Kallen, Robert Mah, Keiichi Masuya, Rita Ramos, Stephan Ruetz, Caroline Rynn, Thérèse Stachyra-Valat, Stefan Stutz, Andrea Vaupel, Sébastien Jeay. _Novartis Institutes for Biomedical Research, Basel, Switzerland_.

Activation of p53 by blocking the p53-Mdm2 interaction using non-peptidic small-molecule inhibitors has been pursued for many years as a promising cancer therapeutic strategy.

We disclose the identity of NVP-HDM201, a novel, highly optimized and selective inhibitor of the p53-Mdm2 interaction. NVP-HDM201 binds to human Mdm2 protein with a sub-nanomolar Ki value, activates p53 and induces robust p53-dependent cell cycle arrest and apoptosis in human p53 wild-type tumor cells. The activity and selectivity of NVP-HDM201 have been tested and confirmed across a panel of cancer cell lines and the molecule displays desirable pharmacokinetic and pharmacodynamic profiles in animals together with excellent oral bioavailability. Application of NVP-HDM201 using various dosing schedules triggers rapid and sustained activation of p53-dependent pharmacodynamic biomarkers resulting in tumor regression in multiple xenografted models of p53 wild-type human cancers.

We report here how a promising lead series was discovered and how innovative medicinal chemistry efforts led to further optimization of the potency and physico-chemical properties, culminating in the discovery of NVP-HDM201. The superior characteristics of the compound allowed the fast progression of the compound into the clinic where NVP-HDM201 is currently in Phase 1 clinical trials both as a single agent and as a combination partner in patients pre-selected for p53 wild-type tumors.

#4856

Selective human estrogen receptor partial agonist (ShERPAs) for treatment of tamoxifen-resistant ER+ breast cancer.

Loruhama M. Delgado-Rivera. _University of Illinois at Chicago, Chicago, IL_.

Estrogen receptor positive (ER+) breast cancer represents a significant cause of death among women in the US, which is exacerbated by resistance to 1st line endocrine therapies, notably tamoxifen, which occurs in 50% of patients. Estrogen fuels growth of endocrine-dependent cancers and yet estradiol (E2) and ERα agonist, diethylstilbestrol, were used successfully in the clinic in greats cancer therapy in the 20th century. The side effects associated with these treatments are considered unacceptable, spurring the pursuit of safer, alternative therapeutic agents and the model systems to develop and optimize these agents. Three breast cancer cell lines that demonstrate resistance to tamoxifen and endocrine independence in 2D (MCF-7:5C), or 3D (T-47D:PKCα and T-47D:TAM1) cell cultures; and comparison to the parent endocrine-dependent cell lines provides a set of model systems able to test the hypothesis that a selective estrogen mimic (SEM) will provide a safe, therapeutic approach to ER+ endocrine-independent breast cancer overexpressing the PKCα biomarker. A partial agonist at ERα was selected as such a therapeutic approach: The use of partial agonists will allow us to retain the beneficial properties of E2, while attenuating the unwanted side effects. Selective human Estrogen Receptor Partial Agonists (ShERPAs) were designed, synthesized, and validated, using a combination of time-resolved FRET (TR-FRET) on ERα/SRC3 recruitment, isolated ERα affinity, and cell-based assays. ShERPAs were identified containing novel structures derived from the selective estrogen receptor modulators (SERMs) bazedoxifene and raloxifene, and their scaffolds, 3-methylindole and benzothiophene, respectively. Examples with favorable pharmacokinetic properties were validated in xenograft models of tamoxifen-resistant breast cancer.

#4857

Design and biological evaluation of substituted pyrrolo[3,2-d]pyrimidines as dual acting RTK and microtubule targeting agents.

Aleem Gangjee,1 Khushbu Shah,1 Nicholas Dybdal-Hargreaves,2 Susan Mooberry,2 Anja Bastian,3 Michael Ihnat3. 1 _Duquesne University, Pittsburgh, PA;_ 2 _UT Health Science Center, San Antonio, TX;_ 3 _The University of Oklahoma College of Pharmacy, Oklahoma, OK_.

Combination chemotherapy with antiangiogenic (cytostatic) and microtubule targeting (cytotoxic) agents (MTA) have shown significant promise and several studies with such combinations have progressed to the clinic. Single agents with both antiangiogenic activities as well as cytotoxicity would circumvent pharmacokinetic problems of multiple agents, might be effective at lower doses to alleviate toxicity, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated single agents that are novel pyrrolo[3,2-d]pyrimidine MTAs that also inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFRβ). Sixteen pyrrolo[3,2-d]pyrimidine analogs were synthesized with variations on the C-2 and the 4-anilino position. The importance of various substituents was evaluated based on their abilities to cause cellular microtubule depolymerization and inhibit proliferation of MDA-MB-435 cancer cells. Compounds with N5-Me were more potent than the N5-H analogs indicating that a conformational restriction induced by N5-Me could mimic the bioactive conformation. In addition, all C2-Cl compounds afforded 2 to 10-fold more potentIC50 values than their C2-Me counterparts as illustrated by C2-Cl analog AG336 (IC50 = 18.3 ± 5.0 nM) compared to its C2-Me analog AG85 (IC50 = 193 ± 39 nM). The most active antitubulin agents were evaluated against RTKs and demonstrated good inhibition for EGFR (IC50 = 29.5 - 91.5 nM), VEGFR-2 (182.3 - 543.6 nM) and PDGFRβ (IC50 = 204.4 - 450.2 nM), thus demonstrating dual-targeting ability. Preclinical assessment of AG165 at 75 mg/kg in mice in the NCI/ADR resistant tumor model demonstrated significant reduction of tumor volume compared to paclitaxel (10 mg/kg) treated mice [p=0.0333], indicating antitumor activity in a multidrug resistant tumor model, in addition to excellent antitumor activity in MDA-MB-435 and MDA-MB-231 xenograft models. These results demonstrate the viability of these multitargeted single agents as potential clinically active antitumor agents.

#4858

LADR™: A novel linker activated drug release technology for drug delivery.

Khalid Abu Ajaj, Stephan David Koester, Friederike Inga Nollmann, Simon Waltzer, Olga Fuchs, André Warnecke, Felix Kratz. _CytRx Corporation, Freiburg, Germany_.

Drug delivery systems in oncology for treating cancer are based on different drug release mechanisms at the tumor site including primarily hydrolytic, reductive, enzymatic and/or acid-sensitive cleavage. Drug carrier conjugates that incorporate an acid-sensitive breaking-point exploit the extra- and intracellular acidic environment of the tumor. Important requirements for acid-sensitive bonds are high stability of the carrier-bound drug in the blood circulation and an effective or sustained release of the active drug in the acidic tumor interstitium and acidic endosomes/lysosomes of tumor cells. In addition, sufficient stability of the acid-sensitive bond is a prerequisite for galenic formulation and reconstitution. As a continuation of our development of aldoxorubicin, an acid-sensitive albumin-binding drug of doxorubicin, which is being clinically assessed in advanced clinical trials (www.cytrx.com), we have set out to design a novel acid-sensitive drug release platform that is applicable to carriers such as serum proteins and antibodies.

The key components of this specific drug delivery system are linkers containing a thiol-binding maleimide group and a substituted aromatic hydrazine to form an acid-cleavable hydrazone bond with suitable carbonyl-containing drugs. For establishing and fine-tuning the pH-dependent release profiles, the aromatic moiety of the linker was substituted with a spectrum of electron-withdrawing groups, and reacted with the anthracyclines doxorubicin and nemorubicin. The resulting hydrazone derivatives were conjugated to the cysteine-34 position of albumin as a model protein. Subsequently, the drug release was determined at physiological and acidic conditions as well as in human plasma. It was discovered that by variation of the electron-withdrawing groups, the pH dependent release of the drug could be substantially varied between 1-50 h in the pH range of 4-5. The technology using these linkers was coined LADR™ (Linker Activated Drug Release) and has the additional advantage that the galenic formulation and reconstitution of LADR™-based drugs is facilitated. We have applied the LADR™ technology to several anticancer drugs such as vinblastine and gemcitabine. In summary, LADR™ is an innovative and versatile linker technology creating a platform for developing acid-sensitive drug carrier conjugates that allow the carrier-bound drug to be released in a controlled manner resulting in sustained exposure to cancer cells.

#4859

Identifying high quality, potent and selective inhibitors of ATM kinase: Discovery of AZD0156.

Kurt G. Pike. _AstraZeneca, Cambridge, United Kingdom_.

Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases (also comprising ATR, DNA-PKcs, mTOR etc.) and plays a crucial role in the cellular DNA damage response signalling activated by DNA double strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy.

Herein, we describe our efforts to identify multiple, novel series of ATM inhibitors. We will describe our optimization efforts with particular attention given to improving the potency of the compounds in cellular systems and the selectivity of the compounds over other closely related proteins (e.g. ATR, mTOR etc.). We will also describe our efforts to optimize both the physicochemical properties of the molecules as well as the pharmacokinetic profile to enable low dose, oral administration in the clinic. These efforts culminated in the discovery of the clinical candidate AZD0156, a first in class orally available ATM inhibitor. AZD0156 shows sub-nanomolar potency in cell based assays of ATM inhibition with selectivities of greater than 1000 fold over other members of the PIKK family of enzymes. AZD0156 is a permeable, highly soluble compound with excellent preclinical pharmacokinetic properties including oral bioavailability. AZD0156 shows robust efficacy in mouse xenograft models after oral administration when combined with DSB inducing agents. AZD0156 is currently undergoing early clinical assessment. 

## IMMUNOLOGY:

### Immune Modulating Agents 2

#4860

The BTK inhibitor ibrutinib modulates T cell immunity in mouse models and in differentiated human T cells.

Yujun Huang, Jeff Hsu, Mint Sirisawad, Hsu-Ping Kuo, Betty Y. Chang. _Pharmacyclics LLC, an AbbVie Company, Sunnyvale, CA_.

Ibrutinib inhibits Bruton's tyrosine kinase (BTK) and effectively treats B-cell malignancies. Ibrutinib also targets IL-2-inducible T-cell kinase (ITK) and modulates the T-cell receptor signaling pathway. Because ibrutinib enhanced antitumor immunity when combined with anti-PD-L1 in various syngeneic solid tumor and lymphoma models (Sajiv-Barfi, PNAS 2015), we sought to determine the effect of ibrutinib on T-cell function and activation. Using in vitro human primary T-cell activation assays, we found that ibrutinib did not suppress T-cell proliferation but slightly inhibited IFNγ production in CD4 and CD8 effector T cells when human peripheral blood mononuclear cells (PBMCs) were stimulated with anti-CD3 and anti-CD28 antibodies. Ibrutinib also inhibited PD-1 expression on human CD8 effector T cells. More strikingly, ibrutinib significantly suppressed naïve murine CD4 T-cell differentiation in vitro into Foxp3+ regulatory T cells (Treg) in the presence of TGF-β. Further, we evaluated the immunomodulatory effect of ibrutinib in tumor-bearing mice. In pancreatic (Pan02) and renal cell carcinoma (Renca) models, we found that single-agent ibrutinib, as well as in combination with standard-of-care agents, effectively inhibited tumor growth. In the syngeneic Renca tumor model, combination ibrutinib treatment significantly reduced CD4+Foxp3+ Treg cells both in the spleen (P < 0.001) and tumor microenvironment (P < 0.05), compared with everolimus alone. Ibrutinib also significantly reduced the percentage of PD-1hi cells among CD8 tumor-infiltrating lymphocytes in A20 lymphoma-bearing mice (P < 0.05). These data demonstrate that ibrutinib, a BTK inhibitor, can suppress immune-inhibitory factors and enhance antitumor immunity in vivo. Ibrutinib may serve as a promising immunomodulatory agent in potentiating current cancer immunotherapy in solid tumors.

#4861

Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy.

Pottayil Sasikumar, N S Sudarshan, Nagaraj Gowda, D S Samiulla, Raghuveer Ramachandra, T Chandrasekhar, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Sharad Singh, Amit Dhudashia, Nagesh Gowda, Murali Ramachandra. _Aurigene Discovery Technologies Ltd., Bangalore, India_.

Recent successes in achieving highly durable clinical responses with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have transformed the outlook for cancer therapy. While these antibody-based therapies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection, failure to show response in majority of patients and immune-related adverse events (irAEs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months may be factors contributing to irAEs observed.

We sought to discover and develop small molecule immune checkpoint antagonists capable of targeting PD-L1 and another immune checkpoint pathway. We reasoned that such therapeutic agents will be amenable for oral dosing, likely show greater response rate due to dual antagonism and allow better management of irAEs due a shorter pharmacokinetic profile.

A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the checkpoint protein(s) signaling depending upon the pockets of sequence similarity of interacting proteins. Further optimization resulted in compounds targeting PD-L1/VISTA or PD-L1/TIM-3 with desirable physico-chemical properties and exposure upon oral administration. .

The ability of compounds to disrupt specific immune checkpoint pathways was confirmed though functional studies. Identified lead compounds exhibit potent activity when tested in assays to rescue lymphocyte proliferation and effector functions inhibited by respective ligands/proteins. In a panel of functional assays, the selected lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, LAG3 and BTLA. Lead compounds exhibited sustained immune PD in vitro and in vivo suggesting that drug efficacy may extend beyond drug clearance. Lead compounds exhibited significant efficacy in syngeneic pre-clinical tumor models of melanoma, breast carcinoma and colon cancers upon once a day oral dosing. In repeated dose toxicity studies, the most advanced compound, AUPM-170, a dual antagonist of PD-L1 and VISTA, was well tolerated at >100x of the efficacious doses.

The data demonstrating the inhibition of PD-L1 and another immune checkpoint pathway (VISTA or Tim3) resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds, AUPM-170, are underway towards advancing it to the clinic.

#4862

AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model.

Stephen Shaw,1 Sascha Kristian,1 Kim Wigglesworth,2 Jenny Middleton,1 Mel Glossop,1 Giles Whalen,2 Robert Old,1 Mike Westby,1 Chris Pickford1. 1 _Agalimmune Ltd, London, United Kingdom;_ 2 _University of Massachusetts Medical School, Worcester, MA_.

Background: AGI-134 is a fully synthetic glycolipid, composed of an alpha-Gal (Galα1-3Galβ1-4GlcNAc-R) sugar epitope attached via a linker to a lipid tail. Natural antibodies to the alpha-Gal epitope are responsible for the hyperacute rejection of xenografts in humans. It is proposed that intratumorally administered AGI-134 will incorporate into the cell membranes of the tumor cells, presenting the alpha-Gal epitope for binding of anti-Gal antibodies to the tumor cells. This will initiate an immune response that attacks the injected tumor and, through uptake of immune-complexed tumor antigens by antigen presenting cells, will create a patient-specific, systemic anti-tumor response against distant metastases.

Results: We demonstrate that AGI-134 incorporates into tumor cell membranes in vitro and that the exposed alpha-Gal epitope binds anti-Gal IgG and IgM antibodies from human serum to the tumor cell surface. Using flow cytometry and a complement-dependent cytotoxicity assay we show that tumor cell opsonization with anti-Gal antibodies leads to deposition of complement proteins C3b and C5b-9, which ultimately leads to tumor cell lysis. Furthermore, we demonstrate that AGI-134-labeled tumor cells opsonized with human serum proteins are phagocytosed by professional APCs.

Using the B16-F10 melanoma model in anti-Gal producing α1,3-galactosyltransferase knockout (GT KO) mice we present data to demonstrate that AGI-134 injection into a primary tumor provides significant dose-dependent protection from the development of established distant lesions. Using GT KO mouse serum we demonstrate in vitro that deposition of complement on AGI-134-labeled mouse tumor cells is both alpha-Gal and anti-Gal dependent. In vivo, we demonstrate that the effect of AGI-134 is due to the alpha-Gal moiety by replacing it with human blood group antigens. The protection from secondary lesions conferred by AGI-134 is long lasting in the GT KO mouse melanoma model (monitored up to 90 days). Importantly, when sub-optimal concentrations of AGI-134 were tested in vivo in combination with an anti-PD-1 antibody (RMP1-14), a significant enhancement in efficacy over either of the agents administered alone was observed.

#4863

PF-06840003: a highly selective IDO-1 inhibitor that shows good in vivo efficacy in combination with immune checkpoint inhibitors.

Joseph Tumang,1 Bruno Gomes,2 Martin Wythes,1 Stefano Crosignani,2 Patrick Bingham,1 Pauline Bottemanne,2 Hélène Cannelle,2 Sandra Cauwenberghs,2 Jenny Chaplin,1 Deepak Dalvie,1 Sofie Denies,2 Coraline De Maeseneire,2 Peter Folger,1 Kim Frederix,2 Jie Guo,1 James Hardwick,1 Ken Hook,1 Katti Jessen,1 Erick Kindt,1 Marie-Claire Letellier,2 Kai-Hsin Liao,1 Wenlin Li,1 Karen Maegley,1 Reece Marillier,2 Nichol Miller,1 Brion Murray,1 Romain Pirson,2 Julie Preillon,2 Virginie Rabolli,2 Chad Ray,1 Stephanie Scales,1 Jay Srirangam,1 Jim Solowiej,1 Nicole Streiner,1 Vince Torti,1 Konstantinos Tsaparikos,1 Paolo Vicini,1 Gregory Driessens,2 Manfred Kraus1. 1 _Pfizer, San Diego, CA;_ 2 _iTEOS Therapeutics, Belgium_.

Tumors use tryptophan-catabolizing enzymes such as Indoleamine 2,3-dioxygenase-1 (IDO-1) to induce an immunosuppressive microenvironment. IDO-1 expression is upregulated in many cancers and described to be a resistance mechanism to immune checkpoint therapies. IDO-1 is induced in response to inflammatory stimuli such as IFN-γ and promotes immune tolerance through the catabolism of tryptophan and accumulation of tryptophan catabolites including kynurenine. IDO-1 activity leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As such, IDO1 is a nexus for the induction of key immunosuppressive mechanisms and represents an important immunotherapeutic target in oncology. We have identified and characterized a new IDO-1 inhibitor. PF-06840003 is a highly selective orally bioavailable IDO-1 inhibitor. PF-06840003 reversed IDO-1-induced T-cell anergy in vitro. In vivo, PF-06840003 reduced intratumoral kynurenine levels in mice by >80% and inhibited tumor growth in multiple preclinical syngeneic models in mice, in combination with immune checkpoint inhibitors. PF-0684003 has favorable predicted human pharmacokinetic properties, including a predicted t1/2 of 16-19 hours. These studies highlight the strong potential of PF-06840003 as a clinical candidate in Immuno-Oncology.

#4864

Engaging the immune system with GSK3174998, a potent anti-OX40 agonist antibody.

Carlo Toniatti,1 Niranjan Yanamandra,2 Kui Voo,1 Amin Al-Shami,1 Laura Bover,1 Peter Morley,2 Sara Brett,2 Tim Lofton,1 Jennifer Greer,1 Ningping Feng,1 Ignacio Ivan Wistuba,1 Sabyasachi Bhattacharya,2 Christopher Hopson,2 David Kilian,2 Heather Jackson,2 Paul Bojczuk,2 Mili Mandal,2 Junping Jing,2 Kevin French,2 Roopa Srinivasan,2 Axel Hoos2. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _GlaxoSmithKline, Collegeville, PA_.

Introduction: GSK3174998, a humanized IgG1 agonistic anti-OX40 monoclonal antibody (mAb) identified in collaboration between GSK and MDACC is currently in Phase I clinical development. Critical for the development of more effective cancer immunotherapy are agents that stimulate effector T cells (Teff) and inhibit the immunosuppressive function of regulatory T cells (Treg) that typically infiltrate tumors. OX40 is a tumor necrosis factor receptor superfamily member expressed on the surface of activated CD4+ and CD8+ T cells. OX40 agonism stimulates both immune effector and memory functions and attenuation of Tregs. Therefore, OX40 agonistic mAbs are ideal candidates to potentially increase the efficacy of immune-checkpoint blocking antibodies, like anti-PD1 (aPD1).

Methods: GSK3174998 has suitable cross-reactivity to cynomolgus monkey OX40 to inform directly on toxicology, pharmacokinetic and pharmacodynamic (PD) preclinical endpoints. However, to understand the antitumor efficacy of OX40 agonism in vivo, studies were performed using a surrogate mAb to murine OX40 (OX86) alone or in combination with a surrogate aPD1 antibody in A20 lymphoma and CT26 colon carcinoma syngeneic tumor models. Intratumoral (i.e. tumor infiltrating T cells) and peripheral (blood) PD biomarkers, including T cell intracellular and surface protein expression, cytokine production and gene regulation were analyzed.

Results: GSK3174998 was well tolerated in monkeys up to 100 mg/kg. In vitro T-cell activation of OX40 with GSK3174998 resulted in enhanced CD4+ and CD8+ effector T-cell proliferation, both in plate-bound as well as soluble PBMC assays. Suppression of Treg differentiation was observed with GSK3174998 as compared with an Fc-disabled mAb, which did not demonstrate these effects. In vitro GSK3174998 induced Th1 cytokine production (IFN and TNFα) and this was further enhanced by the addition of pembrolizumab. In vitro OX86 demonstrated similar characteristics to GSK3174998. In vivo OX86 induced a significant dose-dependent, durable anti-tumor response as monotherapy, which was significantly enhanced when combined with an aPD1 checkpoint inhibitor. Preclinical efficacy correlated with PD changes in several immunological markers including T-cell proliferation and activation. In silico and IHC analysis of expression of OX40 and PDL1 in human tumors was utilized to prioritize cancers most likely to respond to monotherapy and combination therapy for the first-time-in-human (FTIH) clinical study.

Conclusions: GSK3174998 is a potent anti-OX40 agonist that engages the immune system via several T-cell-mediated pathways and may further enhance the antitumor activity observed with PD1 inhibition. Preclinical studies provide a strong rationale to support the ongoing FTIH Phase I study of GSK3174998 administered alone and in combination with pembrolizumab to patients with selected advanced solid tumors.

#4865

Metformin improves multiple cytokine producing ability of exhausted peripheral CD8+ T cells of cancer patients.

Mototsugu Watanabe, Shingo Eikawa, Takenori Uehara, Yuki Kunisada, Shinichi Toyooka, Shinichiro Miyoshi, Heiichiro Udono. _Okayama University Graduate School of Medicine, Okayama, Japan_.

Background: We have recently shown that metformin, a prescribed drug for type 2 diabetes, can increase the number of CD8+ tumor infiltrating lymphocytes (CD8+ TILs) that can produce triple cytokines, thus, IFN-γ, IL-2 and TNF-α in mouse models. Increased numbers of such multifunctional CD8+ TILs have well correlated with tumor retardation, suggesting an underlying mechanism of metformin in tumor immunity. In the current study, we examined the effects of direct exposure of metformin in vitro to CD8+ T cells of cancer patients.

Methods: Peripheral blood mononuclear cells (PBMC) from cancer patients and healthy volunteers were obtained for the analysis. We cultured the PBMC with or without metformin overnight, followed by stimulation with phorbol 12-myristate 13-acetate (PMA) / ionomycin in vitro in various glucose concentrations for 6 hours. Expressions of markers for immune-check points (PD-1 and Tim-3) were examined together with accumulated intracellular cytokines, IFN-γ, IL-2 and TNF-α by flow cytometry analysis.

Results: PD-1 and Tim-3 expressions of cancer patients' PBMC were apparently higher than those of healthy volunteers'. Increased production of multiple cytokines was observed in CD8+ T cells of almost all cancer patients by metformin pretreatment, whereas no changes were detected in the samples from healthy volunteers. Moreover, deprivation of glucose clearly limited the production of cytokines on CD8 + T cells in a cancer patient. These results suggested patient' CD8 + T cells showed lower activity of glycolysis compared with a healthy volunteer and metformin might stimulate glycolysis followed by reversion of mitochondrial respiration in exhausted CD8 + T cells.

Conclusion: Metformin treatment might be useful in reversion of exhausted CD8+T cells in cancer bearing patients.

#4866

Development and characterization of novel CD40 antibody agonists for cancer immunotherapy.

Laura A. Vitale,1 Thomas O'Neill,1 Jenifer Widger,1 Andrea Crocker,1 Li-Zhen He,1 Jeffrey Weidlick,1 Karuna Sundarapandiyan,1 James Storey,2 Lawrence Thomas,2 Joel Goldstein,1 Henry C. Marsh,2 Tibor Keler1. 1 _Celldex Therapeutics, Hampton, NJ;_ 2 _Celldex Therapeutics, Needham, MA_.

CD40 is a key molecule in the regulation of immune responses and its activity can be modulated using antibodies. In particular, agonist CD40 antibodies are highly effective in preclinical tumor models either through direct interaction with CD40-expressing lymphomas, or indirectly through the activation of an adaptive anti-tumor immune response. To date, limited clinical data have been reported with strong CD40 agonist antibodies; nonetheless it seems likely that targeting this pathway will require a balance between the benefits of immune stimulation to drive anti-tumor responses, and the damage that can result from non-specific immune cell activation. We set out to develop novel human anti-CD40 antibodies with different levels of agonist activity to identify a lead candidate for systemic application.

Anti-CD40 monoclonal antibodies (mAbs) were generated by immunization of human Ig transgenic mice with recombinant and cell surface expressed human CD40. Hybridomas developed from these mice were screened using CD40 binding assays and activity on a reporter cell line engineered to express CD40 and NFκB-responsive luciferase. The variable regions of lead antibodies that displayed differential activity were cloned into vectors containing human IgG1 or IgG2 constant domains and expressed in CHO cells.

These human CD40 mAbs were further characterized by analysis of binding affinity, CD40L blocking activity, B cell and dendritic cell activation, and anti-tumor activity in xenograft tumor models. We found a wide range of activities among the CD40 mAbs that is linked to epitope specificity as well as the isotype. In general, the IgG2 isotype mAbs had greater signaling activity than their IgG1 counterparts. The lead candidate mAbs are undergoing additional testing related to functional and toxicity parameters before a final candidate is nominated for clinical development.

#4867

The CD40 agonistic antibody APX005M 'licenses' antigen presenting cells to promote tumor-specific T-cell responses.

Erin L. Filbert, Pia Björck, Xiaodong Yang, Ovidiu C. Trifan. _Apexigen Inc, San Carlos, CA_.

Modulation of co-stimulatory and co-inhibitory molecules on immune cells has become a promising approach for cancer immunotherapy. CD40 engagement on antigen presenting cells (APCs) by its ligand CD154 leads to maturation and expression of co-stimulatory molecules such as CD80, CD86, OX-40L, and 4-1BBL that are requisite for optimal antigen-specific T-cell activation, an essential component of the anti-tumor immune response. To evaluate the therapeutic potential of targeting the CD40-CD154 pathway, Apexigen has developed APX005M - a humanized IgG1 CD40 agonistic antibody that binds with high affinity to human CD40. APX005M mimics CD154, has potent CD40 agonistic activity and promotes activation of APCs. Monocyte-derived dendritic cells treated with APX005M display a mature phenotype characterized by upregulation of CD80, CD86 and HLA-DR and increased secretion of IL-12. In mixed lymphocyte reaction and viral antigen recall assays, APX005M significantly enhances proliferation of and IFN-γ secretion from both naïve and memory T cells. Since CD40-CD154 signaling is involved in the priming phase of T cell activation and acts upstream of many critical costimulatory pathways, CD40 agonistic antibodies may represent an essential component of combination immunotherapy. In support of this, we show that APX005M synergizes with checkpoint inhibitors to promote antigen-specific T-cell responses. Importantly, in an ex vivo tumor assay APX005M induces T-cell proliferation and cytokine secretion. These data demonstrate that APX005M binds to APCs and induces their activation and maturation to ultimately drive a potent tumor-specific T-cell response. T cell activation in cancer patients receiving APX005M will be assessed in current and future clinical studies.

#4868

Tyrosine kinase-dependent modulation of tumor infiltrating immune cells in melanoma.

Joël Babdor,1 Aurélie Fricot,1 Michaël Dussiot,1 Lucile Hebert,1 Patrice Dubreuil,2 Ivan Cruz Moura,1 Thiago Trovati Maciel,1 Olivier Hermine1. 1 _Laboratory of Molecular Mechanisms of Hematologic Disorders and Therapeutic Implications. INSERM U1163 - IMAGINE Institut. Paris Descartes University - Sorbonne-Paris-Cité University, Paris, France;_ 2 _INSERM CRCM - Centre de Recherche en Cancérologie de Marseille, U1068 INSERM, Institut Paoli-Calmettes, CNRS, Université Aix Marseille, Paris, France_.

Melanoma causes the greatest morbidity and mortality of all skin cancers and often harbors amplifications or activating mutations in KIT, a type III transmembrane receptor tyrosine kinase (RTK) involved in cancer cell growth, migration, invasion and metastasis. At present, no treatment that provide either sufficient response rates or a significant prolongation of overall survival are available for melanoma. In the last decade, several inhibitors of RTK (RTKi) have been developed for the treatment of cancer, however, a number of short-comings was observed. Therefore, novel TK inhibitors with improved selectivity were developed for the treatment of diseases associated with KIT activation. Masitinib (AB1010), a RTKi capable of blocking selectively KIT, is one such new drug. Using B16 melanoma subcutaneous graft on C57Bl6 mice, we demonstrate that Masitinib is able to limit tumor growth in vivo and increase mice survival. This anti-tumoral effect was not observed for other RTKi targeting KIT. Suprisingly, Masitinib has no effect on melanoma proliferation in vitro, suggesting instead that modulation on tumor environment takes place. Indeed, mast cells that accumulate in the tumors secrete several biologically active factors involved in the modulation of tumor growth. In vivo, Masitinib treatment completely abrogates mast cell maturation and migration resulting in decreased tumor invasion and down-regulation of several angiogenic factors. Using a mouse line deficient for mast cells (Wsh/Wsh), we confirmed that Masitinib effect is in part dependent of its inhibition on mast cell migration and maturation. Indeed, additional effect occurs on macrophage polarization leaning from "tolerogenic" toward "anti-tumoral" phenotype. Furthermore, in vivo macrophage depletion favored tumor promotion and reverses Masitinib effect on tumor growth. The results provided by this study suggest that Masitinib may be a useful therapy on melanoma by its dual role on tumor environment. It can block mast cell maturation and infiltration in the tumor site, avoiding the angiogenic effects of mast cells. It can revert tumor tolerent macrophage status leading to an increased inflammatory activity within the tumor microenvironment.

These effects are not observed only on melanoma, but also on other types of cancer, suggesting the cellular mechanisms triggered by Masitinib occur in various tumoral process. For instance, a phase III clinical trial with Masitinib as GIST treatment is currently ongoing.

#4869

Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK.

Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. _Rigel Pharmaceuticals, Inc., S. San Francisco, CA_.

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome.

Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK.

Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry.

Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 <100nM. Compounds were not overtly cytotoxic or antiproliferative and, importantly, do not block the activity of TLR and T cell immune effector pathways. Compounds also phenocopied the inhibition of efferocytosis observed using a MerTK blocking antibody. In vivo activity of these novel MerTK inhibitors is under investigation in PD and tumor models.

Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response.

#4870

A novel potential target for cancer immunotherapy.

Susanna Campagnoli,1 Matteo Parri,1 Alberto Grandi,1 Elisa De Camilli,2 Giuseppe Viale,2 Renata Maria Grifantini,1 Piero Pileri1. 1 _Externautics SpA, Siena, Italy;_ 2 _European Institute of Oncology, Milan, Italy_.

Suppression of the host's immune system plays a major role in cancer progression. The tumor microenvironment (TME) not only plays a pivotal role during cancer progression and metastasis but also has profound effects on therapeutic efficacy. Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clinical advances and provided a new weapon against cancer. This therapy has elicited durable clinical responses and, in a fraction of patients, long-term remissions. Several immune checkpoint inhibitors have been approved by the Food and Drug Administration (FDA), whose therapeutic effect is due to blocking the activity of proteins expressed in T lymphocytes (CTLA4, PD-1) and the interaction with surface protein expressed on tumor cells ( PD-L1 and PD-L2).

In our recent research activities we have identified 89 tumor-associated proteins by systematic immunohistochemistry analysis of tissue microarray (TMA) representing breast, colon, lung ovary and prostate cancer by using a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted proteins only marginally characterized. Among them, an interesting protein, which decorated the plasma membrane of breast cancer cells, was selected. This protein has been described to interact with the CD270/BTLA/CD160 pathway that regulates T-cell activation. It has also the capability to interfere with T-cell mediated allo-responses. Our characterization data showed that the protein is localized on the surface of several cancer cell lines. Moreover, it has the ability to stimulate cytokine production and secretion in PBMC from healthy donors. Several monoclonal antibodies have been generated in our lab against this protein and selected for the ability to bind the surface of cancer cells. Moreover, the ability of these monoclonal antibodies to interfere with the immune cells activity and to interfere with tumor growth in animal models is under evaluation. Results will be provided during the meeting.

The currently approved therapeutic targets regimens for triple negative breast cancer and ovarian cancer have limited efficacy. Therefore, the identification of other tumor-associated proteins targetable by specific mAbs is essential to improve patient survival.

#4871

**Two novel anti-PD-1 antibodies, 244C8 and 388D4, elicit** in vivo **antitumor efficacy in a lung PDX tumorgraft in immuno-humanized NSG mice.**

Felix Scheuplein, Sheila Ranganath, Bin Feng, Thomas McQuade, Lei Wang, Vikki Spaulding, Sri Vadde, Shanu Mehta, Maria Isabel Chiu, Cokey Nguyen. _Enumeral Biomedical Corporation, Cambridge, MA_.

We have previously described the identification of more than 300 anti-PD-1 monoclonal antibody candidates using Enumeral's proprietary single cell immune-profiling technology, on which our antibody discovery platform is based. Bioinformatics analysis of heavy chain CDR3 sequences show they comprise diverse families, encompassing 26 phylogenetic clades. Functional studies from cell-based, ex vivo human assays led to the discovery of two distinct lead candidates, 244C8 and 388D4. The former represents a novel class of anti-PD-1 antibodies with a potentially differentiated mechanism of action, as they appear not to compete with currently marketed antibodies for PD-1 nor do they compete with PD-L1 for PD-1 binding. Further, antibodies from the 244C8 family elicit both a higher T cell activation and an increased expression of the high affinity IL-2 receptor CD25 than currently marketed anti-PD-1 antibodies in ex vivo human cell based assays. Lead antibodies from both anti-PD-1 classes, 244C8 and 388D4, have been humanized for preclinical testing in preparation for Phase 1 clinical studies. Here we describe the in vivo efficacy testing of these two antibodies in a human PDX tumorgraft model derived from a core needle biopsy of a patient with metastatic non-small cell lung carcinoma (LG1306). Direct testing of the anti-human PD-1 antibodies was made possible by the use of immune-humanized NSG mice. Pembrolizumab, a currently marketed anti-PD-1 antibody, served as control, along with vehicle alone. Treatment with each of the humanized lead PD-1 antibodies, 388D4 and 244C8, was well tolerated at 5 mg/kg and led to significant tumor growth inhibition over a 28-day study period. Both showed equivalent efficacy to pembrolizumab with tumor growth inhibition (%TGI) at 40% and 38% respectively compared to 37% for pembrolizumab. All three treatment agents showed significant tumor growth inhibition relative to vehicle, with Student T-test p values < 0.003 at end of study assessment. These results show that, contrary to expectation, competition for binding to PD-L1 ligand by an anti-PD-1 antibody is not a pre-requisite for functional efficacy in vivo, as we observed with 244C8. In addition to the in vivo efficacy studies, we will report on post-treatment analyses of all treatment cohorts by immunohistochemistry and RNAseq of the tumor samples, immunoprofiling analysis of tumor infiltrating lymphocytes (TILs) from all treatment cohorts, as well as donor-specific differences that influence response to treatment.

#4872

Developing high affinity, soluble T cell receptors for the treatment of cancer.

Andrew Knox, Fiona Chester, Frayne Bianchi, Sarah Bailey, Lucie Bouard, Nathaniel Liddy, Giovanna Bossi, Jane Harper, Joseph Dukes, Samantha Paston, Tara Mahon, Jessie Gavarret, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Alex Powlesland, Penio Todorov, Andrew Johnson, Martin Ebner, Yvonne McGrath, Namir Hassan, Annelise Vuidepot, Bent Jakobsen. _Immunocore Ltd., Abingdon, United Kingdom_.

Immunotherapeutic strategies that drive activation of cytotoxic T cells possess significant potential to eradicate tumours. Whereas monoclonal antibodies are restricted to targeting secreted or cell surface proteins, T cell receptors (TCRs) are able to recognise a wider range of targets. This is achieved through binding to short peptide fragments derived from proteins that are degraded intracellularly and presented at the cell surface by human leukocyte antigens (HLAs). Natural cancer specific TCRs however, have weak affinities and cancer cells often develop escape mechanisms to avoid destruction by T cells.

To overcome this, we have developed Immune mobilising monoclonal TCRs Against Cancer (ImmTACs); a new class of soluble bi-specific molecules comprising affinity-enhanced, monoclonal T cell receptors (mTCRs) fused to an anti-CD3 scFv. ImmTACs target peptides presented by HLA, and through the anti-CD3 effector, re-direct cytotoxic T cells to achieve highly specific and potent tumour cell killing.

At Immunocore, we have developed an integrated in-house process for the generation of ImmTACs and here describe the critical engineering steps involved. T-cell clones that specifically recognise validated cancer antigens are isolated from peripheral blood lymphocytes and the TCR-encoding sequences are identified by RACE. To confirm antigen binding, TCR α and β chains are expressed as inclusion bodies in bacteria, co-refolded in vitro, and their binding to the target peptide:HLA tested by Surface Plasmon Resonance (SPR). The affinity of the TCR is then enhanced up to a million-fold through directed evolution, utilising phage display. Individual mutants are screened by SPR and combined to generate ImmTACs with pM affinities (KD) and binding half-lives of many hours. A range of biochemical and cellular assays are then performed to assess the potency and specificity of each ImmTAC generated.

This process has been successfully applied to produce ImmTACs for a wide range of targets, demonstrating the robustness of the platform. Our lead candidate, IMCgp100, is undergoing Phase IIa clinical trials in patients with advanced malignant melanoma. This reagent, which specifically targets the gp100 (280-288) peptide presented by HLA-A2 on melanoma cells, is well tolerated and shows very promising therapeutic potential.

#4873

ImmTACs re-direct the immune system efficiently to eradicate cancer.

Giovanna Bossi, Rupert Kenefeck, Caroline Caroline Boudousquie, Jane Harper, Joseph Dukes, Nathaniel Liddy, Samantha Paston, Tara Mahon, Peter Molloy, Malkit Sami, Emma Baston, Brian Cameron, Annelise Vuidepot, Namir Hassan, Bent K. Jakobsen. _Immunocore Ltd, Abingdon, United Kingdom_.

Immunotherapy strategies that are able to induce T cell infiltration into tumors and activate a cytotoxic T cell response have the potential to destroy the tumor. Although T cells can mediate clearance of a tumor, thymic selection and the suppressive microenvironment limit their effectiveness. To overcome poor tumor immunogenicity, we have developed a unique platform that enables the generation of ImmTACs (Immune-mobilising monoclonal TCRs Against Cancer); these are comprised of an affinity enhanced TCR specific for a cancer antigen fused to an anti-CD3 scFv. The TCR end targets and binds MHC class I/peptide complex displayed on cancerous cells while the anti-CD3 scFv end engages polyclonal T cells to mediate a potent anti-tumor response.

The most advanced ImmTAC, IMCgp100, targets the HLA-A2/gp100280-288 epitope presented by melanoma cells. IMCgp100 is currently in a Phase I/IIa clinical trial for advance malignant melanoma and is showing promising clinical efficacy in some patients. Here we report a series of in vitro experiments evaluating IMCgp100 mechanism of action. IMCgp100 is able to redirect T cells from healthy donors or from melanoma patients to destroy cancer cells and secrete a range of inflammatory cytokines and chemokines associated with T cell trafficking into tumors; some of these cytokines also induce upregulation of inhibitory pathway molecules. We show that effector memory cells in the CD8+ and CD4+ T cell compartments are very efficient in eliminating melanoma cells and in expanding upon IMCgp100 engagement. The effects of combining IMCgp100 with agents that relieve the suppression imposed by immune check-point molecules have also been investigated.

#4874

Interleukin-12/FasTI: A novel bi-functional fusion protein for cancer immunotherapy.

Xi Yang,1 Ashlee Tietje,2 Xianzhong Yu,1 Yanzhang Wei1. 1 _Clemson University, Clemson, SC;_ 2 _Southern Wesleyan University, Central, SC_.

Whereas cancer immunotherapy with cytokines demonstrates effective in activating immune response against tumor cells, one major obstacle with the use of these cytokines is their severe side effects when delivered systemically at high doses. Another challenge is that advanced tumor cells often evade immunosurveillance of the immune system and block the Fas-mediated apoptosis by down-regulating its expression. In the present study, we report the design and preliminary evaluation of the antitumor activity of a novel fusion protein:mIL-12/FasTI, consisting of mIL-12 and the transmembrane and intracellular domains of Fas. The fusion construct (pmIL-12/FasTI) was transfected into mouse lung carcinoma cell line TC-1. Stable cell clones expressing the fusion protein were established as assayed by RT-PCR and immunohistochemistry. ELISA and cell proliferation analyses demonstrated that NK cells were effectively activated by the fusion protein with increased IFN-γ production and cytotoxicity. Enhanced caspase 3 activity of the clones when co-cultured with NK cells demonstrated that apoptosis was induced through Fas/FasL signaling pathway. The bifunctional protein is clearly more effective than IL-12 alone. It, therefore, represents a promising new therapeutic agent for cancer treatment when combined with tumor cell-specific gene delivery.

#4875

Developing tumor-localized, combination immunotherapies.

Brian R. Champion, Nalini Rasiah, Sam Illingworth, Matthieu Besneux, Rochelle Lear, Darren Plumb, Prithvi Kodialbail, Alice C.N. Brown. _PsiOxus Therapeutics Ltd, Abingdon, United Kingdom_.

Building on the recent clinical successes of checkpoint inhibitor antibodies, the field of cancer immunotherapy is now focussing on combination treatment regimens to further improve efficacy benefits to patients. However, combining such systemically dosed agents is associated with a number of challenges including enhanced side effect profiles and high costs. One strategy being explored to overcome such issues is to dose the therapeutics directly into the tumor rather than systemically but many tumors will not be accessible for this type of treatment. We have developed a broadly applicable vector platform system, based on the potent chimeric oncolytic group B adenovirus enadenotucirev (EnAd), for directing the efficient local production of a combination of immunotherapeutic agents selectively within the tumor. The versatility and fidelity of the platform has been exemplified by encoding up to three separate biomolecules in the same virus, including antibodies, cytokines, chemokines and tumor-associated antigens, without altering other virus properties. A systemic clinical dosing regimen has been established for EnAd, with data directly demonstrating selective virus delivery to and protein production from colorectal and other tumor types. The advantage of this approach is that immunotherapeutics encoded in the virus can be produced locally, both in tumors that are not directly injectable and in metastases, while minimising systemic off-target effects.

A candidate virus NG-345 has been designed to produce a combination of three secreted immunomodulatory agents (human IFNα, MIP1α and Flt3L) aimed at enhancing the recruitment and activation of immune cells into tumor cell nests. We have shown that NG-345 retains the full oncolytic properties (potency and selectivity) of the parental EnAd virus, with infected human tumor cells producing high levels of all three cytokine/chemokines in the culture supernatants. Functional activity of individual encoded agents has also been demonstrated using relevant cell-based assays. EnAd is highly human-tumor selective and does not replicate, produce infectious progeny or express endogenously regulated transgenes in non-human cells. In vivo evaluation of immuno-modulatory activities of armed viruses is therefore challenging and requires the application of multiple approaches that can collectively provide informative data. In particular, studies are focusing on using surrogate candidate viruses expressing murine gene homologs in human tumor xenografts in immunodeficient mice with or without a reconstituted immune system.

#4876

Long-term high quality survival with single agent mifepristone treatment despite advanced lung cancer and advanced renal cell carcinoma - 2 case reports.

Jerome H. Check,1 Diane Check,2 Jasmine Aly,1 Carrie Wilson2. 1 _Cooper Medical School of Rowan University, Camden, NJ;_ 2 _Cooper Institute for Reproductive Hormonal Disorders, P.D., Marlton, NJ_.

The objective is to report long-term high quality survival in 2 patients with advanced cancer treated by single agent mifepristone.

Case 1: An 80 year old woman was admitted to the ICU for respiratory failure. Chest x-ray revealed many lung lesions most consistent with lung cancer with multiple lung metastases. She was subsequently admitted several more times with very low arterial pO2 levels and her serum sodium progressively decreased to 118 mmol/L. The radiologic and clinical diagnosis was probable advanced lung cancer with the syndrome of inappropriate antidiuretic hormone (SIADH) from ectopic tumor production of arginine vasopressin. She refused surgery or chemotherapy. She agreed to experimental use of 200mg daily oral mifepristone. Her pO2 returned to 99-100 mmHg with 1 month and her sodium returned to normal. Six weeks later all her lung lesions were gone by CT scan. Three and a half years after initial treatment, her PO2 remains 96-98 mmHg, she is feeling fine, and her CT-scan continues to show no tumors. Her treatment had been approved by the FDA on a compassionate basis.

Case 2 - A 60 year old male with was found to have bilateral renal cell carcinoma with metastases to local lymph nodes. He refused bilateral nephrectomy but agreed to only laparoscopic right hemi nephrectomy to remove the largest lesion, leaving the left kidney intact. He received FDA approval to use 200mg daily mifepristone because at that time there was no chemotherapy that was known to be effective. The left kidney lesions did not recede but stayed stable for 10 years. Furthermore, no new lesions appeared on bi-annual CT scan. After 10 years of therapy, he developed renal failure secondary to diabetes. At this time he underwent bilateral nephrectomy, followed by kidney transplant. 11 years from diagnosis he is doing well. Neither patient has reported any side effects to 200mg mifepristone daily despite a combined 14 years of treatment. The mechanism of action responsible for these phenomena has not been proven thus far. The proposed mechanism is that progesterone inhibits an intracytoplasmic immunomodulatory protein known as the progesterone induced blocking factor (PIBF). By inhibiting PIBF, the cellular immune cells (specifically natural killer (NK) cells) are able to attack the rapidly growing tumor cells. The FDA has approved an investigator initiated study for the use of single agent mifepristone for stage IV human non-small cell lung cancer which has progressed despite either 2 rounds of chemotherapy or 1 round of chemotherapy plus one round of biologic therapy. Mifepristone, a progesterone receptor modulator, has been demonstrated to inhibit the production of a 34-36 kDa intracytoplasmic splice variant of the 90 kDa centrosomally associated parent compound. However, this potent abortifacient failed to suppress serum PIBF levels.

#4877

Discovery of novel and potent inhibitors of indoleamine-2,3-dioxygenase (IDO1) for cancer immunotherapy.

Shengyang Liu, Taoliangshan Tao, Yue Liang, Hongli Guo, Xiaogang Ye, Zhiheng Wu, Fang Bao, Heping Yang, Yaxian Cai, Gangjing Yang, Longsheng Wang, Fang Liu, Guangliang Fu, Xiaolei Liu, Jiangang Du, Ping Qin, Yuanfei Ma, Panhu Zhu, Yajun Yu, Chen Ma, Pei Wang, Liping Zhao, Yuxun Wang, Daxin Gao, Qun Li. _Shanghai Denovo Pharmatech, Zhangjiang New Area, Shanghai, China_.

Indoleamine-2,3-dioxygenase (IDO1) is an immune regulatory enzyme that oxidizes tryptophan to kynurenine. IDO1 is overexpressed in numerous tumor cells that block T-cell activation, induce T-cell apoptosis and increase regulatory T cells, which create an environment in which tumor-specific cytotoxic T lymphocytes are no longer able to attack a patient's cancer cells. IDO1 inhibition reverses the immune suppression at the tumor site and allows the generation of an effective anticancer immune response. Preclinical and clinical studies have shown that IDO1 inhibitors increase the anticancer immune response and dramatically increase the efficacy of various therapeutic agents especially the immune checkpoint antibodies targeting PD-1, PD-L1 and CTLA-4.

Via structure-based design, we have discovered a series of novel and potent IDO1 inhibitors that demonstrate strong IDO1 on-target activity with IC50 values ranging from 10 nM to 50 nM against human IDO1 enzyme. These compounds potently inhibit IDO1 in tumor-bearing mice as well as in Hela cells with lower single-digit to lower double digit nM IC50. The lead compound selected for clinical development shows good ADME profile, low serum binding, and good efficacy in various murine animal models when dosed orally and subcutaneously alone or in combination with immune checkpoint inhibitors. Preclinical evaluation of the compound will be presented in details.

#4878

Quantitative cell-based bioassays for therapeutic development targeting immune checkpoint and co-stimulatory receptors.

Jamison Grailer, Pete Stecha, Jun Wang, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. _Promega Corporation, Madison, WI_.

Immunotherapy harnesses the immune system to fight cancer and has proven to be a very promising therapeutic strategy. Drug targets in cancer immunotherapy include both inhibitory and co-stimulatory immune receptors on T cells or NK cells, in particular. Current approaches to assay immunotherapy biologics rely on primary cells, are highly variable, and are not suitable for a quality control environment during drug development. We have developed a panel of cell-based assays using a bioluminescent reporter platform that can quantitatively determine the potencies of antibodies and ligand proteins targeting immune checkpoint receptors and co-stimulatory receptors including PD-1, CTLA-4, LAG-3, GITR, 4-1BB, OX40 and CD40. For each target, a stable cell line was generated in an immune cell background to stably express an immune checkpoint or co-stimulatory receptor and a luciferase reporter driven by a response element specifically responding to signaling induced by TCR or directly from the immune receptor. These bioassays reflect biological mode-of-action for each class of drug candidate and are able to determine the potencies for on-market biologic drugs including PD-1 antibodies pembrolizumab and nivolumab, and CTLA-4 antibody ipilimumab. The assay signals are robust, specific, and have good repeatability and linearity. Therefore they can serve as valuable tools for drug screening, QC lot release and stability studies in immunotherapy drug development.

#4879

Cancer cell-mediated signaling of TLR 2, 4, and 9 causes activation of PI3K/Akt/mTOR pathway and induces tumor cell proliferation in pancreatic cancer.

Jennifer Kreckel,1 Tanja Grimmig,1 Romana Moench,1 Christoph T. Germer,2 Martin Gasser,2 Ana Maria Waaga-Gasser1. 1 _University of Wuerzburg, Dept. of Surgery I, Molecular Oncology and Immunology, Wuerzburg, Germany;_ 2 _University of Wuerzburg, Dept. of Surgery I, Wuerzburg, Germany_.

Background: Toll like receptor (TLR) ligands are in clinical use for the immunotherapy of different cancers. They are supposed to induce an inflammatory immune response against the tumor. Interestingly, several studies showed that TLRs were expressed by cancer cells in different tumor entities and that their activation can contribute to an inflammatory microenvironment. Yet, for pancreatic cancer, TLR expression and its impact in tumor cells is only poorly understood. Therefore this study analyzed the influence of TLR2, 4, and 9 expression and activation on tumor cell signaling and proliferation in pancreatic cancer.

Methods: The expression of TLR 2, 4, and 9 was analyzed in vitro in several established as well as primary human pancreatic cancer cell lines by qRT-PCR and Western Blot. TLR stimulation was then performed in BxPC-3, MIA Paca2, and PacaDD135 cells using the TLR ligands oligodeoxyribonucleotide2006 (ODN2006), lipoteichonic acid of Staphylococcus aureus (LTA-SA) and High-Mobility Group Box 1 (HMGB1). Expression of MyD88 and pAkt was then analyzed by Western Blot. Functional analysis on proliferation (ATP assay) and cytokine expression (Luminex) was additionally performed.

Results: Expression of TLR2, 4 and/or 9 was demonstrated in all investigated human pancreatic cancer cell lines. Receptor activation by single or combined use of TLR ligands resulted in increased MyD88 and pAkt (Ser473) expression. Additionally, up-regulated anti-apoptotic protein Bcl-2 expression was found in stimulated cells, but not in the unstimulated cells. Furthermore, TLR activation resulted in the production of the interleukin(IL)-6, IL-8 and tumor necrosis factor α (TNF-α). Interestingly, tumor cell proliferation was increased within 24 and 48 hours of stimulation.

Conclusion: Our results demonstrate TLR2, 4 and/or 9 expression in all human pancreatic cancer cell lines. Additionally, our findings on TLR activation suggest chronic inflammation-mediated TLR signaling to negatively influence tumor cell apoptosis and to shift the cytokine release in pancreatic cancer towards an inflammatory microenvironment. These findings emphasize the particular role of TLR2, 4 and 9 and their activation in pancreatic cancer, outlining their relevance as potential targets for cancer therapy.

#4880

**A high-throughput siRNA screen identifies Nucleoside Diphosphate Kinase (NME3) as a novel host regulator of NF-κB signaling in response to** Salmonella **-induced activation of TLR-5.**

Caleb Gonzalez,1 Kelly Flentie,2 Brandon Kocher,2 Jayne Jayne Marasa,2 David Piwnica-Worms1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _Washington University in St. Louis, St. Louis, MO_.

Bacterial flagellin is a potent activator of NF-κB signaling, inflammation and host innate immunity. In comparison to TNFα, perhaps the most studied NF-κB-inducing ligand, flagellin elicits an overlapping, yet unique downstream transcriptional response by host cells. In contrast to the pro-oncogenic functions of TNFα, recent data indicate that flagellin may represent a novel anti-tumor ligand that acts through TLR5 and the NF-κB pathway to induce host immunity and to aid in the clearance of tumor xenografts. To begin to characterize the molecular basis for the anti-tumor effect of flagellin and to identify downstream innate signaling components of NF-κB that are responsible for the pro-inflammatory program necessary for these anti-tumor effects, we employed a high throughput screen. Utilizing HCT116 colon carcinoma cells expressing a dynamic NF-κB bioluminescent reporter and Salmonella typhimurium expressing flagellin, we performed a live cell screen of a siRNA library targeting 691 known and predicted human kinases to identify novel modulators of flagellin-induced NF-κB activation. This screen uncovered nucleoside diphosphate kinase (NME3) as a previously unrecognized, positive regulator of NF-κB signaling in response to flagellin. Targeted knockdown and overexpression assays confirmed the regulatory contribution of NME3. Furthermore, Kaplan-Meier survival analysis for breast, lung and ovarian cancer patients showed that high level expression of NME3 correlated with increased overall survival. Together, these data identify a novel pro-inflammatory role for NME3 in NF-κB signaling that may potentially be exploited to optimize cancer immunotherapy.

#4881

CD300f-regulated efferocytosis by dendritic cells and macrophages controls the initiation and resolution of experimental colitis.

Ha-Na Lee, Linjie Tian, Jacquice Davis, Mariam Quinones, Yasmine Belkaid, Konrad Krzewski, John E. Coligan. _NIAID/NIH, Rockville, MD_.

Phagocytic clearance of apoptotic cells (efferocytosis) is critical for resolution of inflammation and prevention of chronic inflammatory disorders, including inflammatory bowel diseases, and inflammation-associated cancer. CD300f (CLM-1), a cell surface receptor that recognizes phosphatidylserine exposed on apoptotic cells, has been shown to promote efferocytosis by macrophages. In the present study, we found that CD300f plays a critical role in controlling the severity and duration of dextran sulfate sodium (DSS)-induced colitis, and CD300f-expressing macrophages and dendritic cells (DC) were identified as key players controlling disease pathogenesis. Compared to Cd300f+/+ mice, Cd300f-/- mice showed increased susceptibility to DSS-induced colitis and impaired resolution of inflammation. In Cd300f-/- mice, macrophage-mediated efferocytosis was reduced, resulting in apoptotic cell accumulation in the gut mucosa. In contrast, Cd300f-/- DC engulfed apoptotic cells more efficiently than Cd300f+/+ DC. CD300f-deficient DC transfer exacerbated DSS-induced inflammation and delayed its resolution, whereas CD300f-expressing macrophage transfer attenuated DSS-induced inflammation. Hyperactive efferocytosis by CD300f-deficient gut DC resulted in excessive tumor necrosis factor-α (TNF-α) secretion, which induced secondary interferon-γ (IFN-γ) over-production, both of which impaired the resolution of colitis. TNF-α neutralization resulted in decreased levels of gut pro-inflammatory cytokines and significant disease amelioration. Collectively, these results suggest that CD300f is important for preventing chronic inflammation by enhancing macrophage efferocytosis and inhibiting TNF-α production induced by DC efferocytosis. From these findings, it is conceivable that increasing macrophage-mediated efferocytosis and suppressing DC-mediated efferocytosis through upregulation of CD300f expression would provide a new therapeutic strategy for inflammatory bowel disease patients.

#4882

Identification of potential immune targets in controlling endometrioid endometrial carcinoma metastatic progression.

Jean-Noel Billaud, Stuart Tugendreich, Debra Toburen. _QIAGEN, Redwood City, CA_.

Endometrial adenocarcinoma is a common cause of gynecological cancer death in Europe and North America. The most dominant subtype, Endometrioid Endometrial Cancer (EEC) accounts for >80% of this cancer and is estrogen-dependent. At diagnosis, 75% of women have the disease confined to the uterus, which is considered Stage One. Five-year survival for Stage One patients is 80%, however, about 15-20% develop metastasis.

Total RNA extracted from tissues obtained after surgical resection from three women at stage one EEC was subjected to RNA-sequencing. The publicly available dataset (SRP045645) was downloaded directly from the Sequence Read Archive and the FASTQ files were processed with Biomedical Genomics Workbench for secondary analysis including mapping, quantification and differential expression analysis. Through streamlined integration the data was uploaded to Ingenuity Pathway Analysis (IPA) for biological interpretation.

We show how in silico solutions developed by QIAGEN Bioinformatics enable us to analyze the biological parameters involved in EEC metastatic progression from an early stage in the three patients diagnosed at Stage One. By comparing these patients' RNA-seq results, we determined that key Canonical Pathways and other biological processes differentiated the three patients from one other. In particular, the predicted transcriptional program allowed us to visualize key upstream drivers. Three of these transcriptional drivers are immune related molecules (two cytokines: CXCL14 and GDF15 and one growth factor FGF3). They have been predicted to be potential master regulators of a Causal Network that drives increase of EEC, metastasis, epithelial-to-mesenchymal-transition (EMT), and cellular invasion in one of the three patients. Based on these results we propose these three immune molecules as possible therapeutic targets to counteract the metastatic processes in EEC.

#4883

Direct analysis of tumor immune infiltrates for "Exhausted" CD8+ T-Cells and BDCA3+ predicts responses in melanoma.

Kimberly Loo, Katy K. Tsai, Adi Nosrati, Mariela Pauli, Robert Sanchez, Miranda Broz, Lorenzo Nardo, Miguel Pampaloni, Michael Alvarado, Paul Tumeh, Alain Algazi, Michael Rosenblum, Matthew Krummel, Adil Daud. _University of California, San Francisco, San Francisco, CA_.

Background: Tumor PD-L1 expression by immunohistochemistry has limitations in predicting outcome to treatment with PD-1/PD-L-1 antibodies. We developed a novel tumor microenvironment (TME)-FACS based method to study the tumor immune microenvironment and used it in patients undergoing PD-1/PD-L-1 antibody treatment.

Methods: Multi -parameter flow cytometry was performed on freshly harvested metastatic melanoma tumor tissue (n=208) from patients prior to anti-PD-1 therapy. For T-cell analysis, CD8+ cells were sorted for PD-1, PD-L1, CTLA-4, and HLA-DR, while CD4+ cells were sorted for FoxP3, in addition to these markers. Tumor associated myloid infiltrates were gated using the markers CD45, HLA-DR, CD3, CD19, CD56, CD11c, CD11b, and CD85g. Using these markers we progressively gated the tumor immune compartment to identify dendritic cell activation markers BDCA1, BDCA3, and CD14+ TAM. Overall responses were derived from investigator reported data by Response Evaluation Criteria in Solid Tumors (RECIST). Descriptive statistics for responders vs. non-responders to anti-PD-1/PD-L1 therapy were constructed to assess the prognostic utility of these markers.

Results: TME-FACS was evaluable on 29 unique patients for T-cell analysis, and 22 patients for myloid/stromal cell analysis who were evaluable for response. A high percentage (> 25% of total CD8+ cells) of PD-1high and CTLA-4high expressing CD8\+ TILs was highly predictive of response to anti PD-1 therapy. Additionally, a high percentage (>2% of total APC HLA+ cells) of BDCA3+ dendritic cells was highly predictive of response to anti PD-1 therapy.

Conclusions: TME-FACS is a novel method to functionally define the immune microenvironment in melanoma. Together, the relative abundance of partially exhausted tumor infiltrating CD8+ T cells and stimulatory dendritic cell populations are both highly prognostic for response to anti-PD1 therapies. Combined T-cell and dendritic cell profiling describes the complex immune microenvironment of responders to anti PD-1 therapy. Our work suggests that increasing their relative frequency within tumors represents an important therapeutic goal to complement existing therapies.

#4884

Intratumoral expression of IL-12 from a dendritic cell-targeting chimeric lentiviral vector from the ZVex platform cures established tumors in multiple models and induces systemic anti-tumor responses.

Tina C. Albershardt, Anshika Bajaj, Jacob F. Archer, Rebecca S. Reeves, Andrea J. Parsons, Lisa Y. Ngo, Jan ter Meulen, Peter Berglund. _Immune Design, Seattle, WA_.

Interleukin-12 (IL-12), produced by antigen-presenting cells, plays a pivotal role in the interplay between innate and adaptive arms of the immune system. IL-12 treatment has been shown to augment cytotoxic T lymphocyte (CTL) and T-helper 1 responses and anti-tumor effects. However, its use as a systemic therapeutic agent is limited due to toxicity. Intratumoral administration of IL-12 is thus being explored as a local alternative route of administration. Such strategies involve plasmid electroporation or other methods that randomly direct cells in the tumor to express IL-12. Here, we evaluated whether targeting expression of IL-12 to intratumoral dendritic cells (DC), thus mimicking the cytokine's physiological biosynthesis and localization, would result in local and systemic immune responses and tumor control in preclinical models.

Six murine tumor models were used, including melanoma (B16F10), colon carcinoma (CT26), breast cancer (4T1), lymphoma (A20) and mastocytoma (P815). Tumors were implanted unilaterally, and for some models also bilaterally, to study systemic (abscopal) effects of therapy. A chimeric third-generation lentiviral vector from the ZVex platform, pseudotyped with the DC-tropic envelope glycoprotein of Sindbis virus, was engineered to express murine IL-12 (p35-p40) (ZVex/IL-12). ZVex/IL-12 was administered as a single intratumoral injection into palpable tumors, alone or in combination with the synthetic TLR4-agonist, glucopyranosyl lipid A (GLA). In some models, systemic anti-CTLA4 treatment was added to enhance clinical efficacy. Animals were monitored 2-3 times per week for tumor size and survival.

Greatest curative efficacy of ZVex/IL-12 was observed in the 100% lethal CT26 flank model, where all treated animals cleared their tumors and survived until end-of-study at 70 days post-challenge. In the B16 footpad and flank models, curative efficacy varied from 40% to 90%, with abscopal effects being observed after addition of anti-CTLA4. In the A20 and P815 models, efficacy varied from 30% to 60%. In the aggressive orthotopic 4T1 breast cancer model, co-administration of ZVex/IL-12 with GLA-AF resulted in significantly delayed tumor growth and increased survival time compared to either ZVex/IL-12 or GLA-AF used alone.

A single intratumoral injection of ZVex/IL-12 resulted in complete tumor regression or significant growth delay in all mouse tumor models investigated and was accompanied by significant survival benefit. The therapeutic effect could be enhanced by ad-mixing with GLA. Abscopal effects were observed in some models after treatment with ZVex/IL12 only and in others after addition of anti-CTLA4. These results point to the powerful modification of the tumor/tumor microenvironment by intratumorally expressed IL-12 by a ZVex vector and the induction of local and systemic immunity.

#4885

Intratumoral injection of G100 (TLR4 agonist glycopyranosyl lipid A) modulates tumor microenvironment and induces CD8 T cell-dependent, systemic anti-tumor immunity.

Hailing Lu, Jessica Hewitt, Jan ter Meulen. _Immune Design, Seattle, WA_.

The tumor microenvironment (TME) plays a critical role in controlling the balance between tumor progression and immune surveillance. Increased infiltration of T cells, especially CD8 T cells has been associated with a good prognosis. We hypothesized that G100, a novel synthetic TLR4 agonist, can modulate TME when directly injected into the tumor and trigger both a local and systemic effective immune response. Balb/c mice with implanted syngeneic A20 lymphoma received intratumoral (IT) injection of G100 (10 μg) or control PBS three times a week. This treatment significantly inhibited tumor growth and resulted in complete tumor regression in approximately 60% of treated mice. To investigate the effects of G100 on TME, tumors were collected after three IT G100 injections for gene expression analysis by Nanostring and immune phenotyping analysis by FACS. Out of the 770 immune response genes included in the mouse PanCancer Immune Profiling panel, 295 genes were significantly upregulated in G100 treated tumors. The upregulated genes include DC function-related genes (CD40, CD83, CD86, and Ly96) and T cell and NK cell function genes and multiple chemokines and cytokines (IL1b, IL12A, IL18, IL6, IFNγ, FcγR4, ICOS, GZMB, CCL3, CCL5, CCL7, CXCL1, CXCL2, CXCL11, CCR5, CCR6, and CCR7). T cell exhaustion markers (CTLA4 and LAG3) and CD274 (PD-L1) were also induced. G100-induced inflammation is also reflected at the cellular level as shown by increased T cells and NK cells in tumor via FACS analysis. To determine the immune cells that mediated tumor rejection, mice were selectively depleted of CD4 or CD8 T cells during G100 treatment. Results showed that the anti-tumor effect of G100 is dependent on CD8 T cells. G100-induced tumor protection was durable as mice surviving the first tumor challenge rejected a secondary tumor challenge without additional G100 treatment. Altogether, our results showed that IT G100 induces a proinflammatory cytokine and chemokine milieu that changes a "cold" tumor to a "hot" tumor, which facilitates the development of a CD8 T cell-dependent potent and durable anti-tumor effect. The induction of PD-L1 also suggests the potential synergy between G100 and checkpoint blockade therapy. These preclinical data support an on-going clinical trial of IT G100 in patients with follicular non-Hodgkin's lymphoma (NCT02501473), alone and in combination with anti-PD-1 therapy (pembrolizumab).

#4886

PEGylated recombinant hyaluronidase PH20 (PEGPH20) enhances checkpoint inhibitor efficacy in syngeneic mouse models of cancer.

Sanna Rosengren, Renee Clift, Susan J. Zimmerman, Jennifer Souratha, Benjamin J. Thompson, Barbara Blouw, Xiaoming Li, Qiping Zhao, Michael Shepard, Dan C. Maneval, Christopher D. Thanos, Curtis B. Thompson. _Halozyme Therapeutics, San Diego, CA_.

Hyaluronan (HA), a major extracellular matrix component in many solid tumors, has been proposed to contribute to tumor progression, and to play a complex role in T lymphocyte biology. Its depletion by intravenous PEGylated recombinant human hyaluronidase PH20 (PEGPH20) remodels the tumor stroma, reduces intratumoral pressure, decompresses tumor blood vessels, and facilitates tumor drug delivery. However, the impact of HA removal on intra-tumoral immune responses and the efficacy of immune checkpoint inhibitors is unknown. To evaluate checkpoint blockade efficacy with PEGPH20, two mouse tumor cell lines, CT26 (colon) and MH194 (pancreatic, derived from spontaneous tumors in KrasLSL-G12D/+Trp53LSL-R172H/+Cre mice) were transduced with hyaluronan synthase-3 (HAS3) to generate syngeneic HA-high tumor models. For anti-CTLA4 studies, parental CT26 and CT26/HAS3 cells were implanted peritibially in Balb/C mice. While treatment with anti-mouse-CTLA4 alone (clone 9D9) inhibited tumor growth in CT26 tumors (37%), PEGPH20 alone did not significantly inhibit tumor growth or increase anti-CTLA4 efficacy. In contrast, tumor growth of CT26/HAS3 tumors was inhibited to a greater extent by the combination of PEGPH20 and anti-CTLA4 (79%) (PEGPH20 treatment 24h prior to anti-CTLA4 treatment), compared to anti-CTLA4 alone (60%, p = 0.002) or PEGPH20 alone (43%, p = 0.0001). Furthermore, gene expression of markers associated with immune suppression, such as IL10 and FoxP3, was higher in CT26/HAS3 than in CT26 tumors; suggesting an association between HA content and immune suppression. To evaluate the effect of PEGPH20 on tumor growth inhibition by PD-1 blockade, MH194/HAS3 cells were implanted peritibially in C57BL/6 mice along with immortalized pancreatic stellate cells. Growth of MH194/HAS3 tumors was significantly inhibited (33%, p = 0.049) by anti-mouse-PD-L1 antibody (clone 10F.9G2), and the addition of PEGPH20 (24h prior to anti-PD-L1) to anti-PD-L1 further enhanced tumor growth inhibition (79%, p <0.0001 to both anti-PD-L1 alone and PEGPH20 alone). Similar findings were obtained with anti-mouse-PD-1 (clone RMP1-14), where the tumor growth inhibition by anti-PD-1 (33%) was further enhanced by PEGPH20 (56%, p = 0.020 and 0.017, respectively, to anti-PD-1 alone and PEGPH20 alone). At 24h following injection, the dose of PEGPH20 used (37.5 µg/kg, the human equivalent dose) removed approximately 50% of HA from tumors as shown by immunohistochemistry and HA ELISA on tumor lysates. Finally, in separate studies, PEGPH20 enhanced labelled intratumoral anti-PD-L1 accumulation (2.6 fold, p = 0.006) in a SKOV3 ovarian xenograft model engineered to express HAS2. In conclusion, in HA-high tumors, PEGPH20 reduced HA, increased anti-PD-L1 accumulation, and significantly enhanced tumor growth inhibition induced by anti-CTLA4, anti-PD-L1, and anti-PD-1 antibodies.

#4887

DNA repair mutations are associated with mutational burden and T-cell activation signature in lung adenocarcinoma.

Young Kwang Chae, Jonathan F. Anker, Massimo Cristofanilli, Aparna Kalyan, Jason Kaplan, Sunandana Chandra, Benedito Carneiro, Maria Matsangou, Cesar Santa-Maria, Francis Giles. _Northwestern University, Chicago, IL_.

DNA repair deficiencies have been linked to the generation of immunogenic neo-antigens and increased efficacy of immunotherapies.

To determine the impact of mutations in DNA repair genes, specifically those involved in homologous recombination (HR) and mismatch repair (MMR), on infiltrating immune cells in lung adenocarcinoma, we analyzed the expression of "immune metagenes" linked to specific cell types (Angelova et al. 2015) utilizing RNAseq values from TCGA (515 patients, 230 sequenced).

Tumors infiltrated by activated CD4 and CD8, all combined T cells, and all combined cell types contained a higher somatic mutation count, while tumors infiltrated by Th17, NK56 bright, and mast cells were linked to lower mutation count (Table 1, *=p<0.05). Patients with high mutational burden displayed a significant increase in infiltrating activated CD4 and CD8, effector memory CD4, all combined T cells, mature dendritic cells, and all combined cell types, as well as significantly increased mutations in HR genes, specifically BRCA2 and PALB2, and MMR genes, specifically MLH1, MSH4, MSH5, EXO1, RFC1, and RFC4. Tumors with HR mutations contained a significant 1.9-fold higher average mutation count with increased infiltrating activated CD4, neutrophils, and NKT cells, as well as decreased activated B cells. Similarly, MMR mutations were associated with a 2.7-fold increase in mutational burden and infiltration by all combined T cells. Further, the degree of alterations in DNA repair genes corresponded to escalating mutational burden, as tumors with 0, 1, or 2-3 mutated HR genes contained an average 188, 330, and 472 mutations, respectively, and those with 0, 1, or 2-3 MMR mutations contained 118, 439, and 639 mutations, respectively.

Our characterization revealed significant links between DNA repair deficiencies, mutational burden, and tumor infiltration by activated T cells, thereby identifying potential biomarkers to aid in patient selection for immunotherapy.

[J.A. and Y.C. contributed equally to this work.]

Association between tumor infiltrating immune cell types and mutational burden

---

Immune Cell Type | Average Mutation Count Fold Change | Percent of Patients with Infiltration (%)

Effector Memory CD8 T cells | 2.2 | 3.1

Memory B cells | 1.8 | 3.3

Th1 T cells | 1.5 | 3.1

Activated CD4 T cells* | 1.5* | 29.7

Any immune cell type* | 1.5* | 73.0

Any T cell* | 1.5* | 59.2

Activated CD8 T cells* | 1.4* | 26.8

NK cells | 1.4 | 3.1

Effector Memory CD4 T cells | 1.3 | 9.1

Dendritic cells | 1.3 | 3.9

T gamma-delta cells | 1.2 | 5.6

Macrophages | 1.1 | 4.3

Mature dendritic cells | 1.1 | 10.9

Activated B cells | 1.1 | 8.9

Any NK cell | 1.1 | 15.0

NK56 dim cells | 1.0 | 9.7

Immature B cells | 0.9 | 9.1

Plasmacytoid dendritic cells | 0.9 | 3.7

Regulatory T cells | 0.8 | 4.1

Th17 T cells* | 0.7* | 10.3

Monocytes | 0.7 | 5.4

NKT cells | 0.7 | 3.7

NK56 bright cells* | 0.7* | 3.9

Mast cells* | 0.6* | 7.0

#4888

Antibody blockade of semaphorin 4D breaks down stromal barriers to enhance tumoricidal immune infiltration, supporting rational immunotherapy combinations.

Elizabeth E. Evans,1 Holm Bussler,1 Sebold Torno,1 Crystal Mallow,1 Christine Reilly,1 Maria Scrivens,1 Ekaterina Klimatcheva,1 Laurie A. Winter,1 Renee Kirk,1 Alan Howell,1 Leslie Balch,1 John E. Leonard,1 Mark Paris,1 Terrence L. Fisher,1 Siwen Hu-Lieskovan,2 Antoni Ribas,3 Ernest S. Smith,1 Maurice Zauderer1. 1 _Vaccinex, Rochester, NY;_ 2 _David Geffen School of Medicine at UCLA, Los Angeles, NY;_ 3 _Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, NY_.

Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer; increased expression correlates with poor prognosis. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the immune, vascular, and nervous systems. In the tumor microenvironment (TME), SEMA4D is expressed strongly at the invasive margin and modulates the infiltration and spatial distribution of leukocytes, suppressing anti-tumor activity. Neutralization of SEMA4D was evaluated for effects on immune activity and tumor growth in preclinical models, incorporating single agent and combination treatments with other immunotherapies, including immune checkpoint blockade inhibitors. The safety and tolerability of humanized anti-SEMA4D antibody VX15/2503 was assessed in a Phase I clinical trial.

RESULTS:

SEMA4D restricts migration of myeloid cells expressing cognate PLXNB1/2 receptors, determined using trans-well migration assays and IHC of in vivo tumors. Antibody neutralization disrupted the SEMA4D gradient at the invasive margin, which correlated with recruitment of activated APCs and T lymphocytes into the TME, and significant shift toward increased Th1 cytokines (IFNg, TNFa) and CTL-recruiting CXCL9 chemokine, with concurrent reduction in Treg- and M2-macrophage promoting chemokines (CCL2, CXCL1, CCL17). Accordingly, an increase in Teff:Treg ratio (3x, p<0.005) and CTL activity (4x, p<0.0001) was observed. This orchestrated change in the tumoral immune context was associated with durable tumor rejection and immunologic memory in preclinical colon, breast, and melanoma models. Importantly, the immunomodulatory activity of anti-SEMA4D antibody can be further enhanced by combination with other immunotherapies, including immune checkpoint inhibitors and chemotherapy. Strikingly, the combination with antibody to CTLA-4 acts synergistically, with maximal increase in survival (110% tumor growth delay, p<0.01) and complete tumor regression in 100% of mice, as compared to 22% with monotherapy (p<0.01).

SEMA4D antibody treatment was well tolerated in nonclinical and clinical studies; including a Phase I multiple ascending dose trial in patients with advanced refractory solid tumors. Patients with the longest duration of treatment, 48-55 weeks, included colorectal, breast, and a papillary thyroid patient, who had a partial response by RECIST. Progression free survival strongly correlated with elevated baseline lymphocyte counts (r=0.6133), supporting an immune mediated mechanism of action for VX15/2503.

CONCLUSION:

Inhibition of SEMA4D represents a novel mechanism and therapeutic strategy to promote functional immune infiltration into the tumor and inhibit tumor progression. A phase 1b/2 trial of combination therapy with an immune checkpoint inhibitor is planned.

#4889

The highly specific CSF1R inhibitor DCC-3014 exhibits immunomodulatory and anti-invasive activities in cancer models.

Bryan D. Smith, Cynthia B. Leary, Wei-Ping Lu, Michael D. Kaufman, Daniel L. Flynn. _Deciphera Pharmaceuticals, Waltham, MA_.

The role of tumor-associated macrophages (TAMs) in promoting an invasive and immunosuppressed tumor microenvironment is well established. TAMs mediate tumor growth, angiogenesis, invasiveness, and immunosuppression through the secretion and response to a variety of factors. There are few highly specific small molecule inhibitors of CSF1R kinase in clinical development. Such specific CSF1R inhibitors are sought for use in combination with other oncology immune checkpoint inhibitors and/or chemotherapeutic agents. DCC-3014, a highly specific CSF1R inhibitor, was developed based on Deciphera's switch control inhibitor platform. Experimental procedures: DCC-3014 was evaluated in human kinase assays, including CSF1R, highly related kinases FLT3, KIT, and PDGFRa/b, and 300 additional kinases. Cellular studies included evaluation in the monocytic cell lines THP-1, MNFS-60, and a human whole blood assay. DCC-3014 was also evaluated in a human osteoclast TRAP assay. In vivo, DCC-3014 was evaluated in the murine cFOS PK/PD model. DCC-3014 was evaluated as a single agent and in combination with a murine anti-PD1 antibody in the murine syngeneic MC38 colorectal cancer model, a model characterized by high TAM infiltration. Results: DCC-3014 exhibited nanomolar (IC50 5 nM) potency for inhibition of CSF1R, sparing highly related kinases FLT3, KIT, PDGFRa, and PDGFRb, by > 100-fold, and sparing other kinases by > 1,000 fold. Cellular inhibition of CSF1R was resilient to high levels of the CSF1R ligand MCSF (10-1,000 ng/mL), due to its high residency time for binding to CSF1R and its binding mode which maintains CSF1R in a "switch off" state. DCC-3014 inhibited CSF1R in THP-1 monocytes (IC50 11 nM), MNFS-60 monocytes (IC50 4 nM), human osteoclasts (IC50 9 nM), and in a human whole blood monocyte/pERK assay (IC50 260 nM). In vivo, DCC-3014 exhibited sustained inhibition of CSF1R in the murine cFOS PK/PD model, affording 90+% inhibition at 15 mg/kg through 24 h post dose. At steady state (6 days of dosing), DCC-3014 robustly inhibited CSF1R at 3 mg/kg daily. In the MC38 colorectal cancer model, DCC-3014 (10 mg/kg daily) inhibited infiltrating TAMs, repolarized the adaptive immune cell population to an anti-tumoral profile, and depleted circulating CD16+ monocyte populations. Additionally, DCC-3014 was evaluated in combination with a murine anti-PD1 antibody and demonstrated additive effects compared to single agent cohorts. DCC-3014 exhibits optimized biopharmaceutical properties, including a favorable ADME and PK profile in preclinical studies. Conclusion: DCC-3014 is a highly specific CSF1R inhibitor and finds potential utility as a macrophage immunomodulatory agent for clinical evaluation in combination with other immune checkpoint inhibitors or chemotherapeutic agents. DCC-3014 is undergoing IND-enabling activities with a FIH study targeted for 2016.

### Immune Response Monitoring: Preclinical

#4890

Preclinical analysis of combination therapy using radiation and cellular immunotherapy.

Satoshi Wada. _Kanagawa Cancer Center, Kanagawa, Japan_.

Purpose:

To optimize the combination of ionizing radiation and cellular immunotherapy using a preclinical autochthonous model of prostate cancer.

Methods and Materials:

Transgenic mice expressing a model antigen under a prostate-specific promoter were treated using a platform that integrates cone-beam CT imaging with 3-dimensional conformal therapy. Using this technology we investigated the immunologic and therapeutic effects of combining ionizing radiation with granulocyte/macrophage colonystimulating factor-secreting cellular immunotherapy for prostate cancer in mice bearing autochthonous prostate tumors.

Results:

The combination of ionizing radiation and immunotherapy resulted in a significant decrease in pathologic tumor grade and gross tumor bulk that was not evident with either single-modality therapy. Furthermore, combinatorial therapy resulted in improved overall survival in a preventive metastasis model and in the setting of established micrometastases. Mechanistically, combined therapy resulted in an increase of the ratio of effector-to regulatory T cells for both CD4 and CD8 tumor-infiltrating lymphocytes.

Conclusions:

Our preclinical model establishes a potential role for the use of combined radiation-immunotherapy in locally advanced prostate cancer, which warrants further exploration

in a clinical setting.

#4891

A mass spectrometry-based serum test to predict outcome of treatment with nivolumab: Analysis of samples taken during therapy.

Jeffrey Weber,1 Heinrich Roder,2 Senait Asmellash,2 Krista Meyer,2 Kevin Sayers,2 Arni Steingrimsson,2 Joanna Roder2. 1 _NYU Langone Medical Center, New York, NY;_ 2 _Biodesix, Boulder, CO_.

Background. The durability of antitumor responses observed in patients treated with antibodies blocking PD-1 supports a central role for these drugs in current melanoma therapy. However, not all patients achieve a durable response, so identification of biomarkers able to aid therapeutic decision making is needed. We have recently reported on a mass-spectrometry-based test (BDX-008) able to stratify patients according to outcome following anti-PD1 therapy using pretreatment serum samples1. The test assigns a binary label of "Positive" or "Negative", denoting the likelihood of good or poor outcome, respectively, following anti-PD 1 therapy. Here we present the results of an investigation of the use of this test on samples taken during administration of immunotherapy.

Materials and Methods. Pretreatment serum samples were available from 119 patients enrolled in a study of nivolumab with or without a peptide vaccine for the treatment of advanced melanoma (NCT01176461). Of these patients, 109 provided a serum sample taken at week 7 (WK7) and 91 also provided a sample collected at week 13 (WK13). The fully locked test was performed on all samples. The association of test classifications at all three timepoints with overall survival (OS) and time-to-progression (TTP) was assessed using Kaplan-Meier methods and log-rank p values.

Results. Test classifications were obtained for 107 WK7 samples and 90 WK13 samples. The proportion of patients classified as Positive was similar across the three timepoints (61% baseline, 69% WK7, 66% WK13). Of the 90 patients with three available classifications, 56 (62%) maintained their baseline classification across all timepoints. Classification significantly stratified OS at all three timepoints (baseline: HR =0.38; WK7: HR=0.24; WK 13: HR= 0.33; log-rank p for all times < 0.001) and TTP at baseline and WK7 (HR= 0.50, log-rank p =0.001 and HR= 0.33, log-rank p <0.001, respectively). At WK13 TTP was numerically better for patients classified as Positive in the reduced set of patients (N=64) without progression before sample collection (HR = 0.58, log-rank p = 0.11). Patients changing from a Negative classification at baseline to Positive at WK7 and WK13 (N=14) had outcomes similar to those of patients maintaining an initial Positive classification (N=41).

Conclusions. These results support previous data indicating the potential clinical utility for this test as an aid to physicians in their immunotherapy treatment decisions. Further validation of the test in pretreatment and monitoring settings is ongoing.

1. J Weber et al, Pre-treatment patient selection for nivolumab benefit based on serum mass spectra, SITC 2015.

#4893

Molecular detection of ALL in the peripheral blood is more sensitive than flow cytometric analysis of the bone marrow in patients with treatment-related hypocellularity.

Hannah Smithers,1 Olivia Finney,1 Ilan Kirsch,2 Beryl Crossley,2 Michael Jensen,1 Rebecca Gardner1. 1 _Seattle Childrens Research Institute, Seattle, WA;_ 2 _Adaptive Biotechnologies, Seattle, WA_.

Measurable residual disease detection (MRD) is an important tool for early detection and monitoring relapse. Traditionally, bone marrow is collected and analyzed by flow cytometry (FC) to evaluate MRD status in acute lymphoblastic leukemia patients (ALL). More recently, highly sensitive molecular methods such as deep sequencing allow for higher sensitivity detection of MRD. Although the bone marrow is considered to be more representative of tumor burden than sampling the blood, bone marrow collection is more invasive, disruptive, and resource intensive than peripheral blood draws. ALL patients enrolled in the Seattle Children's Pediatric Leukemia Adoptive Therapy (PLAT-02) trial undergo MRD assessment pre and post CD19 CAR T cell infusion using FC analysis of a bone marrow sample. We have used high throughput sequencing (HTS) in paired blood and bone marrow samples from these patients to evaluate the complete B cell repertoire and assess MRD up to 63 days post-infusion. To-date, we have obtained 38 paired samples. Using HTS alone we have observed a significant correlation in both the frequency of total reads and the number of malignant cells per million in paired peripheral blood and bone marrow samples. 26/38 of the bone marrow samples were determined either aparticulate or hypocellular. Importantly, 8/14 of patients that were MRD negative in the bone marrow by FC were MRD positive in the blood by HTS. These data suggest, that, in cases where the bone marrow is hypocellular or aparticulate, peripheral blood samples evaluated by HTS are more sensitive than traditional flow cytometric analysis in the bone marrow for detection of MRD. The impact of B cell aplasia may be important in increasing the sensitivity of HTS in the detection of MRD. The ability to detect an ALL B cell clone in the blood through HTS has many clinical implications; blood can be sampled more often, is less of a burden to patients, and can provide earlier prediction of relapse.

#4894

Relationship of characterization of the immunological microenvironment and pathological response in advanced rectal cancer after oxaliplatin-based neoadjuvant chemotherapy.

Kentaro Sekizawa,1 Yasushi Ichikawa,2 Atsushi Ishibe,1 Hirokazu Suwa,3 Masashi Momiyama,1 Ikuma Kato,4 Mitsuyoshi Ota,3 Itaru Endo1. 1 _Department of Gastroenterological Surgery, Yokohama City University Graduate School of Medicine, Yokohama-city Kanagawa-ken, Japan;_ 2 _Department of Oncology, Yokohama City University Graduate School of Medicine, Yokohama-city Kanagawa-ken, Japan;_ 3 _Department of Surgery, Gastroenterological Center, Yokohama City University Medical Center, Yokohama-city Kanagawa-ken, Japan;_ 4 _Department of Pathology, Yokohama City University Graduate School of Medicine, Yokohama-city Kanagawa-ken, Japan_.

Background:

Characterization of the immunological microenvironment has been demonstrated to be associated with prognosis and the effect of chemotherapy of various cancers. Then it has been shown by their role several studies in 5-FU-based neoadjuvant chemoradiotherapy for rectal cancer. However, their role in neoadjuvant chemotherapy (NAC) for rectal cancer remains unknown. The objective of this study was to examine the relationship between immune reactions and the pathological response to induction oxaliplatin-based NAC in patients with advanced rectal cancer.

Methods:

A total of 65 patients with advanced rectal cancer who underwent induction oxaliplatin-based NAC followed by surgical resection were enrolled. The resected tumor specimens were counted positive cell of tumor infiltrating lymphocytes (TILs) and macrophages by using immunohistochemical staining for CD3, CD8, Foxp3, CD86, CD206. We analyzed for the relationship between the numbers of TILs or macrophages at the stroma in resected specimens and the pathological response to induction oxaliplatin-based NAC.

Results:

The numbers of Foxp3+ TILs in resected specimens were strongly correlated with pathological response. Patients with higher number of Foxp3+ TILs showed poor pathological response (p<0.001). There were no statistically significant differences between the number of CD3+, CD8+ TILs and pathological response, but patients with higher ratio of CD8+/FOXP3+ TILs was associated with good pathological response (p=0.023). No associations were observed between the numbers of CD86+, CD206+ macrophages and pathological response.

Conclusions:

In rectal cancer patients, T lymphocyte-mediated immune reactions significantly correlated with pathological response of oxaliplatin-based NAC for advanced rectal cancer. These were similar to the T lymphocyte-mediated immune responses in the neoadjuvant radiation therapy for rectal cancer.

#4895

Hodgkin lymphoma patients demonstrate evidence of chronic activation/exhaustion in circulating T cell subsets.

Catherine S. Diefenbach,1 Bruce Raphael,1 Kenneth Hymes,1 Michael Grossbard,1 Tibor Moskovits,1 David Kaminetzky,1 Meghan Mcshea,1 Peter Martin,2 Jia Ruan,2 Lina Kozhaya,3 Maryam Bonakdar,1 Cem Abidoglu,3 John Leonard,2 Derya Unutmaz3. 1 _New York Univ. School of Medicine, New York, NY;_ 2 _NY Presbyterian Weill Cornell, New York, NY;_ 3 _Jackson Labs, CT_.

Background: In Hodgkin lymphoma (HL) the malignant Hodgkin Reed-Sternberg (HRS) cells comprise only a small fraction of the total cellular tumor population, which orchestrate an inflammatory microenvironment of reactive cells that propagate a permissive milieu for HL growth. This HL-mediated immune suppression may have effects that extend beyond the tumor microenvironment. Previously we have shown that the circulating T cells of HL patients show high expression of the receptor programmed death-1 (PD-1). Here wecharacterize the systemic immune profile of 20 HL patients with newly diagnosed (ND) or relapsed (R) disease. Methods: Informed consent obtained from patients with ND (n= 14) or R (n=6) HL treated since January of 2013. Blood samples were drawn pre-treatment, and at sequential time-points during therapy. Cryopreserved PBMCs were thawed then evaluated with multi-color flow cytometry. Cells were stained with fixable viability dye (eBioscence); then stained with fluorescent-conjugated antibodies. For intracellular staining, cells were fixed and permeabilized using FOXP3 staining kit (eBioscience) then stained with intracellular antibodies. Stained cells were analyzed using LSRII flow cytometer (BD Bioscience) and FlowJo software (Tree Star). The following anti-human antibodies were used: CD3, CD4, CD8, CD25, CD160, CD45RO, CD27, HLA-DR, PD-1,FOXP3 and Helios, (Biolegend). Singlet lymphocytes were gated based on forward and side scatter properties. Dead cells were excluded based on viability dye. Patient samples were compared to normal controls matched for age and sex (n=20). Results: The median HL patient age was 38 (range: 20-90 years). Twelve of the HL subjects were male and 8 were female. All but 1 ND patient were treated with upfront ABVD. Two out of 6 R patients had prior stem cell transplant (SCT), but were not on immunosuppression. Thirteen patients (12 ND, 1 R) responded to therapy (11 CR and 2 PR); 4 patients (1 ND, 3 R) progressed on therapy or had stable disease, 2 patients do not have disease status confirmed. HL patients displayed distinct circulating immune profile compared to normal age matched controls, with increased circulating CD4 and CD8 effector cells and a decrease in CD8 central memory cells. The proportion of CD4 or CD8 effector memory T cells expressing CD160+ and PD1+ CD4 effector memory cells, were also highly significantly increased in HL patients. In contrast, a decreased percent of bona fide CD4+ regulatory T cell subset (Foxp3+Helios) was found in HL patients. Together these findings suggest that the systemic immune profile in HL patients is perturbed towards severe chronic activation/exhaustion of T cells. Conclusion: HL patients have evidence of chronic activation/exhaustion in their central memory T cells, suggesting that ineffective immune clearance of the HRS cells may be a systemic phenomenon. This underscores the rationale for immune targeted therapy in HL patients.

#4896

A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flow cytometry using cryopreserved human peripheral blood mononuclear cells.

Shigehisa Kitano,1 Makiko Yamashita,2 Hiroaki Aikawa,2 Aya Kuchiba,3 Mitsuhiro Hayashi,2 Noboru Yamamoto,1 Kenji Tamura,1 Akinobu Hamada2. 1 _National Cancer Center Hospital, Tokyo, Japan;_ 2 _National Cancer Center Research Institute, Tokyo, Japan;_ 3 _National Cancer Center, Tokyo, Japan_.

For predicting the clinical efficacy of antibody-dependent cellular cytotoxicity (ADCC) -dependent antibody therapy, analyzing the cytotoxic functions of effector cells such as NK cells against target cancer cells is thought to be necessary. The 51Cr release assay has long been the most widely used method for quantification of ADCC activity. However, the reproducibilities of these release assays are not adequate, and they do not allow evaluation of the lysis susceptibilities of distinct cell types within the target cell population. In this study, we established a novel method for evaluating cytotoxicity, which involves the detection and quantification of dead target cells using flowcytometry. CFSE (carboxyfluorescein succinimidyl ester) was used as a dye to specifically stain and thereby label the target cell population, allowing living and dead cells, as well as also target and effector cells, to be quantitatively distinguished. Furthermore, with our new approach, ADCC activity was more reproducibly, sensitively, and specifically detectable in not only freshly isolated but also in frozen human peripheral blood mononuclear cells (PBMCs) compared to the calcein-AM release assay. This assay, validated herein, is expected to be a standard assay to evaluate ADCC activity and finally contribute clinical development of ADCC dependent-antibody therapies.

#4897

First evidence of changes in the TCRβ repertoire from a cutaneous melanoma patient immunized with the CSF-470 vaccine.

Mariana Aris, María Marcela Barrio, José Mordoh. _Centro de Investigaciones Oncológicas-Fundación Cáncer, Ciudad Autónoma de Buenos Aires, Argentina_.

Introduction: A breakthrough of immunotherapy for cancer treatment has emerged, with promising results in several tumors. We have developed the allogeneic vaccine CSF-470, composed of lethally-irradiated human Cutaneous Melanoma (CM) cell lines plus BCG and GM-CSF, currently tested in a Phase II-III study in adjuvancy (NCT01729663). The aim of this work was to analyze the TCRβ repertoire in multiples samples obtained through CSF-470 vaccination from a CM patient who developed a cutaneous metastasis (Mts) at the end of the 2-year immunization protocol.

Methods: DNA from PBMC at 0, 12 and 24 months (PRE-PBMC, POST-1, POST-3) from protocol start, and from Mts-TIL (Tumor Infiltrating Lymphocytes) was obtained. TCRβ sequence and further analysis was performed with the IMMUNOSEQ platform. Patterns were defined for clone tracking: 1, present at PRE, increases during immunization; 2, absent at PRE, increases during immunization; 3, present at PRE, absent following immunization; 4, present at PRE, decreases during immunization; 5, detected only at 24 months (POST-3); 6, absent at PBMC, present in Mts-TIL.

Results: PBMC TCRβ repertoire analysis revealed an increase in clonality (2.3X) and total frequency of TOP100 clones through CSF-470 immunization. PBMC clones tracking analysis (with frequency ≥0.01%) revealed prevalence of Pattern-1 clones (54%) and Pattern-5 clones (30%). Regarding Mts-TIL, clones were distributed in Pattern-1 (23%), Pattern-3 (5%), Pattern-5 (16%), and Pattern-6 (53%). However, when analyzing TOP100 Mts-TIL clones, 60% of them corresponded to Pattern-1, 19% to Pattern-5 and only 12% to Pattern-6. Databases search revealed the presence in PBMC of shared TCRβ sequences previously reported in association to M.bovis infection. Also, some clones found in Mts-TILs matched to previously identified in other CM metastases.

Conclusions: This is the first report of the TCRβ repertoire from a CM patient immunized with CSF-470 vaccine. The main observations regarding TCRβ repertoire analysis were: 1- Vaccination induced expansion of selected pre-immunization TCR clones, which might recognize dominant Ags either from patient´s tumor or vaccine. 2- TCR clones arising after immunization might recognize dominant Ags from vaccine or derived by epitope spreading from the patient´s tumor. 3- More than 50% of total clones were exclusively present in Mts-TIL, suggesting local activation. However, most frequent Mts-TIL clones included those that were expanded exclusively by CSF-470 vaccination. 4- TCRβ repertoire comparison also allowed detection of some clones already reported in TIL from CM Mts and in memory repertoire against M.Bovis, possibly accounting BCG adjuvancy.

Finally, changes observed in the TCRβ repertoire from this patient, driven by the CSF-470 vaccine, suggest an anti-tumor immune response taking place both in the periphery and the local compartment.

#4898

Effects of chemotherapeutic agents on the tumor immune microenvironment.

Marcia P. Belvin, Shiuh-Ming Luoh, Jeanne Cheung, Erin McNamara, Rafael Cubas, Jeong Kim. _Genentech, Inc., South San Francisco, CA_.

Immunotherapeutic agents have shown dramatic success in oncology in recent years and several have been approved in melanoma and lung cancer. One strategy for extending the benefit of immunotherapy is to evaluate these agents in combination with standard of care chemotherapy, with the goal of bringing the benefit of immunotherapy into earlier lines of treatment.

We have carried out a systematic evaluation of the effects of different classes of chemotherapeutic agents (including alkylating agents, platinum based agents, and taxanes) on the tumor immune microenvironment of syngeneic murine tumor models (CT26 and MC38) in two different strains of mice (BALB/c and C57BL/6). We found that different chemotherapeutic agents caused markedly different changes in the composition and cell phenotype of the tumor immune environment. The majority of chemotherapeutic agents resulted in a mild to moderate increase in CD8+ T cells in the tumor. Some agents also altered the activation state of these CD8+ cells as measured by the level of PD-1 and IFN-gamma. A subset of the chemotherapeutic agents resulted in profound changes in the number of CD4+ cells, including regulatory T cells, as well as changes in myeloid subpopulations including monocytes, neutrophils, and macrophages. In addition to single agents studies, we carried out combination studies of selected chemo agents with simultaneous addition of anti-PD-L1. Several of these combinations caused pronounced increases in efficacy above single agent treatment. We will present the combination pharmacodynamic marker changes and efficacy of these treatments.

Our findings demonstrate that chemotherapeutic agents can stimulate the tumor immune environment, at least transiently, through multiple mechanisms. Different subsets of chemotherapeutic agents display unique changes in the tumor immune infiltrate, and may require different immunotherapeutic approaches. These findings provide a rationale for testing immunotherapy-chemotherapy combinations in the clinic.

#4899

The regulation by kinases of the expression of human major histocompatibility class I molecules.

Elliott J. Brea, Claire Oh, Eusebio Manchado, Ron Gejman, George Mo, Patrizia Mondello, Ralph Garippa, Neal Rosen, David A. Scheinberg. _Memorial Sloan Kettering Cancer Center, New York, NY_.

The major histocompatability complex (MHC) is a central receptor in the adaptive immune response and is the underlying target of several effective therapies for cancer. Druggable kinases may provide the opportunity to modulate the immune response toward MHC. However, the regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. The entire human kinome was screened using a pooled shRNA interference-based approach in a human mesothelioma cell line to uncover kinase regulators of MHC-I. Negative and positive regulators of cell surface HLA levels were discovered. A subset of highly scoring positive and negative kinase hits were subsequently validated by additional RNAi, and pharmacologic inhibitors when available. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of HLA expression in multiple cancer types. We mapped the pathways responsible for increased HLA upon kinase inhibition. Interestingly, inhibition of the MAP Kinase pathway broadly influenced expression of other components of the antigen presentation machinery. Moreover, DDR2 and MINK1 were shown to positively regulate HLA-A*02:01. This had therapeutic relevance, as shown with a therapeutic TCR mimic antibody to a MHC/peptide complex. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases.

#4900

Increased IgG antibody responses to neoepitope and native peptides containing high affinity domains for MHCI following combination cancer immunotherapy.

Tyler W. Hulett,1 Shawn Jensen,2 Christopher Dubay,2 Carlo Bifulco,2 Michael Afentoulis,2 Ashok Reddy,1 Larry David,1 Bernard A. Fox2. 1 _Oregon Health & Science University, Portland, OR; _2 _Earle A Chiles Research Inst., Portland, OR_.

The importance of anti-tumor immunity by CD8+ T cells is well defined, but antigen-specific immune monitoring for CD8+ responses across many potential tumor antigens is expensive and difficult. This difficulty is compounded by increasing evidence that every tumor has unique mutant neoepitopes. We hypothesized that new technologies in peptide printing might allow us to partially overcome this obstacle by instead characterizing antibody responses against a paired array of neoepitope and native peptides.

We designed a 15-mer peptide array of mutated and native peptides from the murine mammary carcinoma 4T1 and used it to characterize the serum IgG antibody responses to vaccination with 4T1 autophagosome-enriched vaccine (DRibbles), poly-IC adjuvant, the combination of both, or naïve animals across 4 independent experiments. This array was printed by JPT peptides and included 75 pairs of single-nucleotide polymorphic and native peptides identified by both our own whole exome sequencing and the current 4T1 literature. We also included other sequences of interest to 4T1 immunity such as the retrovirus fragment AH1. MHCI peptide predictions were robust and used the top NetMHCpan v2.8 score from all possible windowed 8,9,10, and 11-mers for H2-Kd,Ld, and Dd surrounding the mutation site in its native protein context.

The results of this work identify a linear correlation between higher predicted peptide MHCI binding affinity and the IgG antibody signals increased in combination treated groups versus controls. Linear regression analysis demonstrates that normalized antibody responses to mutated and native 4T1 peptides are more likely to be higher in sera from autophagosome-enriched vaccine plus poly-IC treated animals than naïve animals if these peptides contain higher affinity domains for MHCI (p<.0001). This correlation is not significant in the groups treated with vaccine or adjuvant alone. Animals pre-treated with this combination therapy also benefit from a significant delay in tumor growth upon challenge with 4T1 versus naïve animals (p<.001).

These results identify a previously unknown link between predicted MHCI binding affinity and the anti-tumor antibody response following combination immunotherapy - suggesting some antigen-specific interaction between B cells and CD8+ T cells that might be exploited to improve the immune monitoring of cancer therapies.

#4901

Short-course enzalutamide reveals immune activating properties in patients with biochemically recurrent prostate cancer.

Renee N. Donahue,1 Ravi A. Madan,2 Jacob Richards,1 Italia Grenga,1 Lauren M. Lepone,1 Christopher R. Heery,1 James L. Gulley,2 Jeffrey Schlom1. 1 _National Cancer Institute, Laboratory of Tumor Immunology and Biology, Bethesda, MD;_ 2 _National Cancer Institute, Genitourinary Malignancies Branch, Bethesda, MD_.

Background: Enazalutamide (enz) is an androgen receptor signaling molecule approved by the FDA for the treatment of patients with metastatic castration-resistant prostate cancer; however, trials are evaluating this agent in earlier stages of disease. Here, we have investigated the effect of enz on peripheral immunity in patients with biochemically recurrent prostate cancer enrolled in a clinical trial of enz with/without a therapeutic vaccine (NCT01875250).

Methods: Patients from the arm receiving enz alone (160 mg daily for 84 days, without ADT) were assessed in this study. Peripheral blood mononuclear cells (PBMCs) collected from 12 patients pre and post enz (days 14, 28, 84, and 100) were analyzed by flow cytometry to identify 123 immune cell subsets, including 9 standard subsets (CD4+ and CD8+ T cells, T-regulatory cells (Treg), B cells, conventional and plasmacytoid dendritic cells (cDC, pDC), natural killer cells (NK), natural killer T cells (NKT), and myeloid derived suppressor cell (MDSC)), and 114 subsets relating to maturation and function. PBMCs were also assessed for T-cell receptor excision circles (TREC) to identify recent thymic emigrants, and by microarray to determine changes in global gene expression.

Results: Treatment with enz induced several notable alterations in peripheral immune cells, suggesting that it has potential immune activating properties. These changes occurred early following treatment, and included an increase of NK cells, decreased frequencies of MDSCs with a suppressive phenotype (e.g. PDL1+ MDSC, gMDSC, and CD16+ MDSC), and decreased frequencies of both CD4+ and CD8+ T-lymphocytes expressing the immune inhibitory checkpoint molecule CTLA4. Additionally, treatment with enz increased TREC levels by >75% in 7 out of 12 patients compared to pre-therapy levels (p=0.012); naïve CD4+ and CD8+ T-lymphocytes were also elevated by >25% in those patients demonstrating the greatest increase in TREC. Gene expression analysis of PBMCs corroborated these findings, showing that enz increased activation of interferon-gamma signaling and related immune activating pathways.

Conclusions. These findings show that short-course enz has immune activating properties in cancer patients, and support the combination of enz with immunotherapy.

#4902

A mouse GITRL fusion protein drives T-cell activation and antitumor activity in preclinical mouse models of cancer.

Rebecca Leyland,1 Amanda Watkins,1 Kathy A. Mulgrew,2 Lisa Bamber,1 Natalie J. Tigue,1 Emily Dick,1 Nick Holoweckyj,2 Lesley Young,1 Michelle Morrow,1 Scott A. Hammond,2 Ching Ching Leow,2 Robert W. Wilkinson,1 Ross Stewart1. 1 _MedImmune, Cambridge, United Kingdom;_ 2 _MedImmune, Gaithersburg, MD_.

GITR is a member of the TNFR superfamily of proteins and is expressed on resting regulatory T-cells and on other T-cells following activation. Signals through GITR have been shown to drive increased T-cell activity and reduced regulatory T-cell function. In order to explore the potential of therapeutically targeting GITR in a cancer setting, we generated a mouse GITRL fusion protein (mGITRL FP) consisting of the extracellular domain of mGITRL linked to a structural domain and an IgG Fc domain. The antitumor activity and pharmacodynamic effects of this molecule were then explored in the colorectal syngeneic model of cancer (CT26). Treatment of mice with mGITRL FP mediated anti-tumor activity that was dependent on isotype and exposure. The anti-tumor activity could be attributed at least in part to the increased activation and proliferation status of CD8+ and CD4+ T-cells, as evidenced by increases in Ki67 expression and ICOS upregulation. Intratumourally we observed a significant decrease in the frequency of CD4+ T-cells (including T regs), but a corresponding increase in cytotoxic CD8+ T-cells. OX40 is another member of the TNFR superfamily, that has similar expression and functions to GITR. In order to better understand the potential differences between targeting of these two pathways, the activity and pharmacodynamic effects of the mGITRL FP were additionally compared and contrasted to those of a mOX40L FP and the observed differences will be discussed.

#4903

Multimodal therapy with a potent vaccine, metronomic cyclophosphamide and anti-PD-1 enhances immunotherapy of advanced tumors by increasing activation and clonal expansion of tumor infiltrating T cells.

Genevieve Weir, Olga Hrytsenko, Tara Quinton, Mohan Karkada, Neil L. Berinstein, Marianne Stanford, Marc Mansour. _Immunovaccine Inc., Halifax, Nova Scotia, Canada_.

Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. Using a HPV-expressing murine tumor model (C3), we found that treatment with mCPA (20 mg/kg/day PO) in combination with a DPX peptide vaccine caused selective enrichment of antigen-specific CD8+ T cells, resulting in increased immune responses detected by IFN-γ ELISPOT and in vivo cytotoxicity assay. The combination provided long term-control of tumors when initiated within one week of tumor implantation, however efficacy was limited in mice bearing advanced tumors. Antigen-specific CD8+ T cells could be detected infiltrating advanced tumors by flow cytometry, along with increased expression of PD-1 on the T cells and PD-L1 on the tumor cells, suggesting that the tumor microenvironment (TME) was mediating immune suppression through increased PD-1:PD-L1 signaling. Treatment of tumor bearing mice with vaccine, mCPA and PD-1 blockade (with anti-PD-1 or anti-PD-L1) resulted in tumor control of established tumors which were not successfully treated with antibody monotherapy. Analysis of tumor infiltrating leukocytes by flow cytometry demonstrated that anti-PD-1 treatment did not further enhance tumor infiltration with antigen-specific CD8+ T cells induced by the vaccine/ mCPA treatment. However, RT-qPCR analysis of the tumor detected an increase in expression of cytotoxic T cell gene signatures within the tumor in combination with anti-PD-1 treatment. Clonal analysis was performed of the total TCRβ sequences using gDNA extracted from the tumors. Vaccine and mCPA treatment resulted in selective expansion of clones, as the top 10 clones accounted for 35% of the total TCRβ sequences; tri-therapy including anti-PD-1 significantly enhanced the expansion of T cells within the TME so that the top 10 clones accounted for 46% of the total TCRβ sequences (p<0.05). We conclude that anti-PD-1 therapy can enhance the efficacy of vaccine immunotherapy by promoting the activity and expansion of antigen-specific T cells within the TME. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac. Assessment of T cell clonality within the tumor may be an important biomarker for immunomodulatory treatments.

#4904

The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models.

Holly K. Koblish, Michael Hansbury, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Maryanne Covington, Timothy Burn, Mark Rupar, Christine Gardiner, Thomas Condamine, Kerri Lasky, Matthew C. Stubbs, Eddy Yue, Richard Sparks, Richard Sparks, Thomas Maduskuie, Andrew P. Combs, Gregory Hollis, Reid Huber, Phillip CC Liu, Peggy Scherle. _Incyte Corporation, Wilmington, DE_.

Inhibitors of the BET family of bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate the expression of key survival and cell fate determining genes such as c-myc. In addition to their role in cancer, studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins controls the expression of pro-inflammatory cytokine genes in macrophages and is therapeutic in models of acute inflammation. These data suggest that in addition to their tumor intrinsic effects, BET inhibitors may also regulate the cytokine milieu within the tumor microenvironment and have immunomodulatory activity in cancer. To study this aspect, we evaluated INCB054329, a novel and selective BET inhibitor currently in Phase 1 trials, alone and in combination either with epacadostat, a highly selective IDO1 inhibitor, or with PD-1/PD-L1 axis blockade in syngeneic tumor models using immunocompetent animals. When used alone, INCB054329 suppressed a panel of cytokines and chemokines in a whole blood assay, confirming that INCB054329 can antagonize a pro-inflammatory response. The potency of INCB054329 in reducing the levels of these inflammatory mediators in the whole blood assay was similar to that for inhibition of c-myc, suggesting that the effects were on-target. INCB054329 was capable of inhibiting the growth of multiple syngeneic tumor models in immunocompetent mice, whereas only modest tumor growth inhibition was observed in immunodeficient mice and a lack of activity was observed in vitro, supporting the immunomodulatory activity of the compound. Because maximal in vivo tumor growth inhibition required an intact immune system, we investigated the impact of INCB054329 on various immune cell subsets, both in vitro and in vivo. Of note, increases in effector T cell populations were observed and efforts are ongoing to further characterize the tumor infiltrating immune cells following INCB054329 treatment. The mechanistic complimentarity of this novel BET inhibitor-mediated immunomodulation was also evaluated in combination with other therapeutically relevant mechanisms, including IDO1 inhibition and PD-1 axis blockade. Enhanced efficacy was observed with all INCB054329-containing regimens. These data demonstrate for the first time that BET inhibition can suppress tumor growth through both tumor-intrinsic and immune modulatory mechanisms, and support the potential of epigenetic-based, immunotherapy combinations as a novel approach to cancer therapy.

#4905

Effects of MAPK pathway inhibitors in the tumor immune microenvironment.

Marcia P. Belvin, Erin Williams, Shiuh-Ming Luoh, Jeanne Cheung, Christine Orr, Emily Chan, Peter Ebert, Ira Mellman, Jeong Kim, Mark Merchant. _Genentech, Inc., South San Francisco, CA_.

Immunotherapeutic agents have shown great promise in the clinic in recent years and this has led to their approval as single agents or as immune doublet combinations in melanoma and lung cancer. In order to increase the extent of benefit from these agents and to extend immunotherapies to additional patients, combinations are being evaluated of immunotherapeutic agents with chemotherapy and targeted agents.

Inhibitors of the mitogen-activated kinase protein kinase (MAPK) pathway, including BRAF and MEK inhibitors, have been approved in melanoma, and are being evaluated in additional indications. We evaluated the effects of MEK and ERK inhibitors on human T cells cultured in vitro as well as on the tumor microenvironment in preclinical syngeneic mouse models. In studies with human T cells in vitro, we find that MEK or ERK inhibition reduced proliferation of naïve T cells, but had little effect on the proliferation of central memory cells, suggesting a differential requirement on MAPK signaling for proliferation of these T cell subsets. In vivo, MEK and ERK inhibition resulted in an increase in CD8+ infiltration in the tumor, as well as a decrease of markers of T cell exhaustion such as PD-1 and EOMES. The combination of MEK or ERK inhibitors plus anti-PD-L1 resulted in increased CD8+ effector function as measured by increased interferon gamma (IFNg) levels. In some cases, the MEK and ERK inhibitors showed differences in their pharmacodynamic effects as single agents; however, in combination with anti-PD-L1, the effects were similar. For example, single agent MEK inhibition but not single agent ERK inhibition increased the number of CD4+ helper and regulatory T cells (Tregs) in the tumor. However, both of these CD4+ subsets, as well as CD4+ IFNg levels, were increased with MEK or ERK inhibitors combined with anti-PD-L1. Similarly, single agent MEK inhibition decreased the number of infiltrating CD11b+Ly6G+ myeloid cells whereas single agent ERK inhibition increased the number of infiltrating CD11b+Ly6C+ myeloid cells. However, in combination with anti-PD-L1, these changes in myeloid populations were less apparent.

Taken together, these data show that MAPK pathway suppression can modulate multiple immune cell subtypes within the tumor, with many of these changes expected to activate the immune infiltrate. Despite the increase in Tregs in response to ERK and MEK inhibition in combination with anti-PD-L1, these combinations were efficacious. This suggests that the other immune stimulatory effects of these treatments are sufficient to drive efficacy despite the presence of increased Tregs. Our data suggest that the combination of MAPK inhibition plus anti-PD-L1 inhibition, such as with atezolizumab, is a promising hypothesis to test in the clinic.

#4906

The selective class I HDAC inhibitor entinostat enhances the antitumor effect of PD-1 inhibition in a syngeneic orthotopic murine model of renal cell carcinoma.

Ashley R. Orillion,1 Li Shen,2 Remi Adelaiye-Ogala,1 May Elbanna,1 Sreenivasulu Chintala,1 Sreevani Arisa,1 Roberto Pili1. 1 _Indiana University - Purdue University Indianapolis, Indianapolis, IN;_ 2 _Roswell Park Cancer Institute, Buffalo, NY_.

Background: Recent advances in immunotherapy have highlighted the antitumor effects of immune checkpoint inhibition. Novel anti-PD-1/PD-L1 immunotherapies have been shown to effectively overcome tumor avoidance of immune surveillance in several tumor types including renal cell carcinoma. Our group has recently shown that the selective class I HDAC inhibitor entinostat is effective in suppressing regulatory T cells and enhancing immunotherapies in murine renal and prostate models, RENCA and Myc-Cap respectively. In this study we have evaluated the combination of entinostat with an anti-PD-1 antibody in the RENCA renal cell carcinoma model.

Methods: 32 BALB/c female mice were implanted with the syngenic, orthotopic, renal cell carcinoma mouse model, RENCA - luciferase tagged - at day -8. Treatment (8 mice /group) with anti-mouse-PD-1 (aPD-1; 10mg/kg twice a week, I.P.), entinostat (5mg/kg 5 days a week), or combination of the two was begun at day 1. Bioluminescence imaging was performed at days -1, 9 and 19 to assess the orthotopic tumor growth. End point tumor weights were taken to assess the effect of combination treatment.

Results: Analysis of tumor growth showed a reduction of bioluminescence across the three time points in the combination group as compared to the vehicle and single agent treatments. Additionally, end point analysis of tumor weights revealed an overall reduction in the size of the tumors in the entinostat/anti-mPD-1 combination group (88% inhibition) as compared to the vehicle (p=0.0002), aPD-1 alone (25% inhibition)(p=0.0181), and entinostat alone (63% inhibition)(p=0.0481) groups. Examination of the status of the infiltrating immune cells of the tumor microenvironment via flow cytometry, qRT-PCR, immunohistochemistry, and/or immunofluorescence analysis is ongoing.

Conclusions: Our preliminary results suggest that the immunomodulatory activity of the selective class I HDAC inhibitor entinostat may enhance the antitumor effect of PD-1/PD-L1 inhibition and provide the rationale for the clinical testing of this novel combination in patients with RCC.

#4907

Epigenetic treatment of ovarian cancer cells increases immune cell recruitment to the tumor microenvironment: implications for response to immune checkpoint therapy.

Meredith L. Stone, Katherine B. Chiappinelli, Huili Li, Lauren Murphy, Michael Topper, Stephen Baylin, Cynthia Zahnow. _Johns Hopkins School of Medicine, Baltimore, MD_.

Immunosuppression and relapse represent two major clinical challenges in managing patients with ovarian high-grade serous carcinoma (HGSC). Strategies to increase the host immune response or decrease immunosuppression hold promise for clinical outcome in ovarian cancer patients. Blockade of inhibitory immune checkpoint pathways with antibodies directed at the PD-1/PD-L1 pathway has demonstrated encouraging anti-tumor activity in a number of solid tumors, including a small number of HGSC patients. We hypothesize that tumors which fail to respond to checkpoint inhibition are those in which the tumor cells do not provide a sufficient positive immune stimulus needed for recruitment of immune cells. Our studies suggest that the DNA demethylating agent, azacytidine (AZA), upregulates an immune signature (AIM, Aza induced immune genes) in cancer cells and that among carcinomas, ovarian HGSC appears most immunoresponsive to AZA. We hypothesize that AIM gene expression is elevated by epigenetic treatment with AZA and/or HDAC inhibitors and promotes the recruitment of tumor infiltrating immune cells to the tumor. Moreover, immune checkpoint inhibitors (anti- PD-1) may then lead to activation of these recruited immune cells and a tumor immune response.

To test the hypothesis of whether AZA/HDACi treatment of tumor cells increases the recruitment of immune cells to the tumor microenvironment, the mouse ovarian cancer cell line, ID8, was treated ex vivo with AZA or HDACis and implanted into syngeneic C57Bl/6, immunocompetent mice. Ex vivo AZA treatment of the tumor cells led to increased recruitment of immune cells to the ascites, decreased tumor burden, and an increased sensitivity to anti-PD-1, while similar treatment with Entinostat as a single agent did not. To test the in vivo effects of these agents on both the tumor and microenvironment, mice with pre-established ID8 tumors were treated with AZA, Entinostat or Givinostat, and anti-PD-1 to test the drugs' antitumorigenic and immunogenic effects in combination and as single agents. We found that combination therapy with AZA, HDACi, and anti-PD-1 led to the most significant decrease in tumor burden, increased survival of the mice, increased numbers of activated T cells and NK cells in the ascites fluid and a decreased number of myeloid derived suppressor cells. In summary, we have clearly demonstrated that tumor cells treated with AZA effectively recruit increased numbers of immune cells to the tumor ascites. HDACis and anti-PD1 are less effective in this regard when administered as single agents, but this effect is significantly increased when used in combination with a demethylating agent. These data have implications for improving the effectiveness of epigenetic therapy and interpreting outcomes from those trials which have used only a demethylating agent or an HDACi with checkpoint inhibition.

#4908

Cancer immunotherapy with immunomodulatory anti-CD137 and anti-PD-1 monoclonal antibodies requires Batf3-dependent dendritic cells.

Alfonso R. Sánchez-Paulete,1 Francisco J. Cueto,2 María Martínez-López,2 Sara Labiano,1 Aizea Morales-Kastresana,1 Maria E. Rodríguez-Ruiz,3 Maria Jure-Kunkel,4 Arantza Azpilikueta,1 José I. Quetglas,1 David Sancho,2 Ignacio Melero1. 1 _Center for Applied Medical Research, Pamplona, Spain;_ 2 _Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain;_ 3 _Clinic University of Navarra, Pamplona, Spain;_ 4 _Bristol-Myers Squibb Company, Princeton, NJ_.

Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory monoclonal antibodies (mAbs) targeting PD-1 or CD137. Using Batf3-/- mice, which are defective for cross-presentation of cell-associated antigens, we show that Batf3-dependent dendritic cells (DCs) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3-/- mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFlt3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-OVA-derived melanomas, whereas this function was lost in Batf3-/- mice. These experiments show that cross-priming of tumor antigens by Flt3L- and Batf3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects.

#4909

The oncolytic peptide LTX-315 enhances tumor-specific immune responses and tumor regression in murine 4T1 breast cancer when combined with doxorubicin.

Meng Yu Wang,1 Alexander Kristian,1 Ketil Camilio,2 Baldur Sveinbjornsson,2 Gunhild Mari Mælandsmo,1 Gunnar Kvalheim,1 Øystein Rekdal3. 1 _Institute of Cancer Research, OuS, Oslo, Norway;_ 2 _University of Tromsø, Tromsø, Norway;_ 3 _Lytix Biopharma, Oslo, Norway_.

The oncolytic effect of LTX-315 involves perturbation of the plasma membrane and distortion of the mitochondrial membrane with subsequent release of DAMPs (Damage-Associated Molecular Pattern molecules) such as ATP, cytochrome C and HMGB1 (1,2). Also multidomain proteins from the BCL-2 family seem to be partially involved in LTX-315 mediated killing (3). In addition, LTX-315 induces the hallmarks of immunogenic cell death (4). Preclinical studies have demonstrated that LTX-315 is able to induce complete tumour regression after intra-tumoral treatment (5,6). In this study the effect of combining intratumoral administration of LTX-315 and systemic treatment with Doxorubicin was investigated in the murine 4T1 breast cancer model. The combination treatment was investigated both in s.c. tumors and in tumors established in the mammary fat pad. The 4T1 cell were labeled with mcherry reporter gene in order to assess tumor growth by whole body imaging. LTX-315 (1 mg in 50 μl) was injected intratumorally for three consecutive days. Doxorubicin (8mg/kg) was injected into the tail vein as a single dose together with the first injection of LTX-315. Tumor growth was assessed by caliper measurements and whole body luminescence. The combination of local administration of LTX-315 and systemic Doxorubicin, demonstrated significant synergy in both s.c. tumors and in tumors growing in the mammary fat pad. Furthermore, the combination induced a systemic tumour specific immune response since tumors did not develop in cured animals when re-challenged with tumor cells. The tolerable safety profile and novel mode of action of LTX-315 indicate a rationale for combining LTX-315 with other immunotherapies, targeted agents, and chemotherapy.

References:

1) Forveille et al., Cell Cycle (2015)

2) Eike et al., Oncotarget (2015)

3) Zhou et al., Oncotarget (2015)

4) Zhou et al. Cell Death & Differentiation (In rev)

5) Camilio et al., Cancer Immunol Immunother (2014)

6) Camilio et al., Oncoimmunology (2014)

#4910

**Modeling immunotherapy in** ex vivo **organ culture of non-small cell lung cancer (NSCLC).**

Jair Bar, Inbal Daniel-Meshulam, Amir Onn, Alon Ben-Nun, David Simansky, Nona Zeitlin, Nir Golan, Meirav Rokah, Ronni Ben-Avi, Iris Kamer. _Sheba Medical Ctr. Inst. of Oncology, Ramat Gan, Israel_.

PD1-PDL1 interaction is one mechanism of tumor evasion of the immune system, and inhibitors of this interaction can allow cancer cell killing by cytotoxic T cells. In NSCLC as well as in most other cancers, the clinical benefit from such inhibitors is far from universal; around 20% of tumors respond to this treatment. Response of tumors to anti-cancer agents depends on interactions of epithelial tumor cells and the microenvironment, including stromal cells such as fibroblasts, immune cells and extracellular matrix. Studying cell signaling and drug-sensitivity of cancer should take into consideration the different compartments of an individual tumor. Regarding immunotherapy, manipulating regulators of PDL1 expression might augment the activity of these drugs, or possibly be active as an immunotherapy on its own. Specifically, little is known about the impact of chemotherapy, radiotherapy or targeted agents on the expression and activity of the PD1-PDL1 signaling in human cancer. Combining such treatments with immunotherapy is a potentially promising approach that is currently investigated clinically. However, the number of potential combinations is vast, and no valid and convenient experimental model exists to test candidate treatments and combinations. Experimental procedures: Ex vivo organ cultures (EVOC) were directly established from fresh NSCLC tissues, as a model that recapitulates real tumor and its microenvironment, including immune cells. Tissue elements were mechanistically dispersed to cell clumps (30-100 cells per clump), or cut to one cubic mm pieces and placed in culture. LDH release was used as a surrogate of cell death. Samples were analyzed by formalin fixation and paraffin embedment, sectioning and hematoxilin and eosin visualization of cells. PDL1 mRNA and protein levels were measured by RT-PCR and western blots. Results: Cell viability of NSCLC EVOC is maintained over a time window of at least 4-7 days. Cytotoxic drugs evoke cell death. PDL1 mRNA and protein levels are elevated in NSCLC EVOC in response to inflammation signals as Interferon gamma. Glucocorticoid steroidal drugs causes reduction in PDL1 mRNA and protein in NSCLC EVOC. Cisplatin treatment causes elevation in PDL1 protein. Variability in basal and induced PDL1 protein levels was detected in response to inflammation signals in EVOCs generated from different patients. Conclusions: Our results indicate the feasibility of EVOC for NSCLC and the potential to use it as a model to study the impact of immunotherapy agents, alone or in combination with other therapeutic tools such as chemotherapy or radiotherapy.

#4911

SOCS-1 inhibits tumor growth by enhancing T cell mediated antitumor immunity related to PD-L1.

Satoshi Nakagawa,1 Satoshi Serada,2 Satoko Matsuzaki,1 Yutaka Ueda,1 Kiyoshi Yoshino,1 Minoru Fujimoto,2 Tadashi Kimura,1 Tetsuji Naka2. 1 _Osaka University, Suita,Osaka, Japan;_ 2 _National Institute of Biomedical Innovation, Ibaraki,Osaka, Japan_.

[Background]

In many solid cancers, constitutive activation of JAK/ signal transducers and activators of transcription (STAT) signaling pathway is associated with poor prognosis. Constitutive JAK/STAT signaling enhances the expression of cyclins and anti-apoptosis proteins, leads to the increased tumor cell proliferation and survival. Furthermore, one of the immune checkpoint molecules programmed cell death 1 ligand 1 (PD-L1), which is involved in the suppression of T cell mediated antitumor immunity, is induced by STATs. Suppressor of cytokine signaling (SOCS)-1 is a negative feedback molecule, which is induced by JAK/STAT signaling and inhibits the JAK activity, resulted in the suppression of JAK/STAT signaling. This study aimed to reveal the antitumor effects by SOCS-1 in vitro and in vivo.

[METHODS]

Five human OC cell lines (OVCAR-3, SKOV-3, RMG-1, A2780, ES-2) and 4 murine cancer cell lines (B16F10, LLC, 4T1, CT26) were assessed. SOCS-1 or LacZ were overexpressed by adenoviral vector. Anti-proliferative effect was assessed by WST-8 assay. Female BALB/c mice were injected with CT26 for subcutaneous xenograft experiments. AdSOCS-1 or AdLacZ was intra-tumorally administered every other day and PD-L1 expression in tumor cells and activation levels of tumor infiltrated T cells were analyzed by flow cytometry.

[RESULT]

Overexpression of SOCS-1 inhibited proliferation of all cancer cell lines. Three OC (OVCAR-3, SKOV-3, ES2) and all murine cancer constitutively expressed PD-L1. Expression levels of PD-L1 in OVCAR-3 and CT26 cells were downregulated about 30% compared with control by overexpressed SOCS-1 in vitro. In CT26 allografted model, SOCS-1 treatment significantly inhibited tumor growth (62.2±5.2%, P<0.01)and suppressed expression of PD-L1 on CT26 cells in the tumor (65.2±8.4%, P<0.05). Granzyme B and CD107a expression of intratumorally infiltrated CD8 T cells were increased more than 50% (P<0.05) in the AdSOCS-1 injected group compared with AdLacZ injected group. PD-L1 Fc fusion protein significantly inhibited antitumor effect of SOCS-1.

[Conclusion]

SOCS-1 mediated inhibition of JAK/STAT signaling shows not only direct antitumor effect against tumor cells but also enhances T cell mediated anti-tumor immunity in vivo by downregulating the expression of PD-L1 on tumor cells and preventing the interaction of PD-1/ PD-L1.

#4912

Boosting immunotherapy via stealthy nanoparticle-mediated photothermal therapy.

Hongwei Chen, Xin Luan, Nicholas Stevers, Kanokwan Sansanaphongpricha, Joseph Burnett, Hayley Paholak, Duxin Sun. _University of Michigan, Ann Arbor, MI_.

Despite T-cell based immunotherapy having shown promising efficacy in various advanced cancers, only a small subset of patients can benefit from this strategy. Boosting cancer immunotherapy via an effective strategy to increase response rate in cancer patients is imperatively desired. Combining checkpoint inhibitor-based immunotherapy with translational nanoparticle-mediated photothermal therapy (PTT) may provide a solution. Using a murine breast cancer model, we demonstrate that an enhanced tumor-specific immune response is achieved by combining CTLA-4 inhibitor with PTT mediated by stealthy and biodegradable iron oxide nanoparticles (IONPs). Our data shows that the biodegradable IONPs coated with an antifouling polymer grants stealth to enhance tumor accumulation (5.3% of the injection dose) following systemic administration (20 mg Fe/Kg mouse body weight), and such MRI imaging capable IONPs are effective nanomediators for PTT under the near-infrared light (885 nm) irradiation in vivo. Using an orthotopic 4T1 mouse xenograft model we demonstrate that IONP-mediated PTT (0.5 Watts, 10 minute irradiation at 24 h and 48 h post IONP administration) plus anti-CTLA-4 antibody (60 µg/mouse for each injection, systemic administration, 3 doses) could completely eliminate 5−6 mm-sized tumors, while control treatments including IONP-mediated PTT alone, anti-CTLA-4 antibody alone, and mock-treatment fail to do so. The dramatic differences between single agents and combination therapy suggest that the observed response is synergistic. Furthermore, our data shows that mice treated with PTT, alone or in combinations, can significantly inhibit distal tumor growth, when a secondary inoculation is performed at a distal site one day prior to primary tumor treatment to mimic metastases. Eight days following primary therapy all distal tumors form in both control and anti-CTLA-4 treated mice with no significant difference in size. However, at the same time in mice treated with either IONP-mediated PTT plus anti-CTLA-4 antibody or IONP-mediated PTT only against primary tumors 50% of secondary tumors do not form and those which do are significantly smaller than those of control treated mice. Further, the duration of response was significantly longer for combination therapy. We speculate that this enhanced anticancer efficacy is a product of simultaneously increasing intratumoral T-cell trafficking toward primary tumor and enhanced T-cell activation in situ triggered by released tumor-specific antigens during PTT. In conclusion, our work demonstrates that T-cell-based cancer immunotherapy to eliminate both primary and metastatic cancer cells can be enhanced when combined with PTT mediated by systemic administered IONPs. The current work will pioneer a translational nanomedicine-based approach to boost T-cell cancer immunotherapy.

#4913

Engineering dendritic cell-based vaccines as targeted immunotherapy against medulloblastoma, neuroblastoma, and Ewing sarcoma.

Stacey Zahler, Wen Luo, Janet Ayello, Mitchell S. Cairo. _New York Medical College, Vahalla, NY_.

Medulloblastoma (MB), Neuroblastoma (NB), and Ewing Sarcoma (ES) are malignant pediatric neuroectodermal solid tumors. Current treatments are highly intensive but toxic and subsets of patients such as metastatic and/or relapsed patients have a dismal outcome (Esiashvili N et al, 2008; Leavey PJ et al, 2008; Bernstein ML et al, 2006). Targeted treatments with reduced side effects are urgently needed. Immunotherapy is a promising approach to inducing tumor control. Among various strategies of immunotherapy, dendritic cell-based vaccines have shown promising preliminary activity in the setting of minimal residual disease (Lasky JL et al, 2013; Westers TM et al, 2011; Shumway NM et al, 2009). To evaluate the feasibility of dendritic cell based vaccine in pediatric solid tumors, we incubated HLA A02+ human peripheral blood mononuclear cells (PBMC) with monocyte-derived autologous dendritic cells (DCs) pulsed with or without whole tumor cell lysates in interferon-γINFγELISPOT assays. We demonstrated that the number of spots detected in test wells (Daoy: 141.6±3, SKPNDW: 233.3±33.5, EWS502: 157.6±22.3, TC71: 249.6±24.5) is significantly (p<0.05) greater than control wells (PBS: 42±2), suggesting successful tumor antigen presenting of DCs and specific immune response from PBMC. To test T-cell responses, we isolated CD8+ T cells from PBMC and incubated them with tumor cell lysate pulsed mature DCs. Interestingly, we observed a similar trend in activation but fewer number of spots (PBS: 27±9, Daoy: 52.6±14.5, SKPNDW: 76.6±30.1, EWS502: 44.6±8.3, TC71: 68.6±19) in ELISPOT assays. Further analysis using HLA-DR blocking antibody (L243) showed that CD4+ T cell function is required for the responsive activity of PBMC because the number of spots was significantly (p=0.03) reduced in test wells with the antibody (45.5±4.9) compared to that in control wells (352.5±17.6). These data indicate that both CD4+ and CD8+ T cells are required for the T-cell immune response to tumor antigens in whole tumor cell lysate presented by DCs. We next tested in vitro cytotoxic activity of DC-activated PBMCs to MB, NB, and ES cells by DELFIA cytotoxicity assay. PBMCs incubated with tumor lysate pulsed DCs induced significantly increased cell lysis compared to control PBMCs in Daoy (78±4.8% vs. 46.5±3.5% at E:T=10:1, p=0.019), SKPNDW (80±14.1% vs. 15±2.3% at E:T=10:1, p=0.016), and TC71 (82.5±3.5% vs. 39.5±13.4% at E:T=20:1, p=0.036). Taken together, our proof-of-principle studies demonstrated the efficacy of DC-based vaccines using whole tumor cell lysate as antigens in pediatric neuroectodermal solid tumors. Further investigation in the preclinical setting in a xenograft NSG model is warranted.

#4914

Auristatin-based antibody drug conjugates activate multiple ER stress response pathways resulting in immunogenic cell death and amplified T-cell responses.

Anthony Cao, Ryan Heiser, Che-Leung Law, Shyra J. Gardai. _Seattle Genetics, Bothell, WA_.

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate (ADC) directed against CD30. This ADC consists of monoclonal antibodies conjugated to the auristatin monomethyl auristatin E (MMAE) a microtubule-disrupting agent. ADC antitumor activity is thought to be primarily the result of intracellular payload release leading to mitotic arrest and apoptotic cell death. We have recently demonstrated that brentuximab vedotin induces surface expression of calreticulin and HSP70 as well as activation of the transcription factor ATF6 which are classical hallmarks of immunogenic cell death (ICD) associated with unfolded protein/ER stress response. In this study, we provide evidence that additional ER stress response pathways are activated.

Treatment of CD30+ Hodgkin lymphoma (HL) cells or xenografts with brentuximab vedotin resulted in the phosphorylation of the ER-associated, unfolded-protein response transmembrane protein inositol-requiring transmembrane kinase/endonuclease 1 (IRE1). Activation of IRE1 was confirmed by the increase in phosphorylation of the downstream effector Jun N-terminal kinase (JNK). Kinetics of tubulin disruption, ER stress induction, evidence of ICD, mitotic arrest, and apoptosis was assessed. Onset of ICD occurred concurrently with induction of ER stress response pathways including induction of pIRE1 and pJNK. Interestingly, induction of ER stress response and ICD preceded mitotic arrest and caspase activation. These results indicate that brentuximab vedotin-mediated disruption of the microtubule network contributes to ER stress, which maybe an apoptotic mechanism mediated by auristatin-ADCs in addition to mitotic arrest at G2/M phase of the cell cycle. Induction of ICD may also be a class effect of the auristatin-based ADCs, as the small molecule payload MMAE, was able to activate IRE1 and JNK and induced surface expression of calrecutilin on target cells.

Previously, brentuximab vedotin-killed HL cells were shown to induce innate immune cell activation in vitro and in vivo. Addition of ADC-killed tumor-like cells to autologous PBMCs results in the expansion of INFγ-secreting cytotoxic T-cells. Via induction of ER stress, tumor cells killed by auristatin-based ADCs may initiate an antitumor immune response and provides a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens.

#4915

Evaluation of the immune response following treatment with anti-CTLA-4 antibody, radiation therapy or the combination in a murine model of breast cancer.

Maryland R. Franklin, Matt Thayer, David Draper, Dan Saims, Scott Wise. _Molecular Imaging, Ann Arbor, MI_.

While breast cancers are considered poorly immunogenic, several approaches utilizing immunotherapies are being undertaken in the clinic to evaluate their potential for improving outcomes. Radiation therapy (RT) is a highly utilized clinical treatment modality in breast cancer, is known to modify the tumor microenvironment and has been shown to potentially synergize with immunotherapies. To evaluate this potential synergy preclinically, we utilized the 4T1 murine mammary carcinoma model to investigate the immune response following treatment with either the 9H10 clone of an anti-CTLA-4 antibody (9H10), RT (8Gy, QDx3) or the combination of the two. Tumors were established in the mammary fat pad and treatments initiated when tumors reached a mean volume of 63-72mm3. We found that both monotherapies resulted in a period of stable disease followed by tumor regrowth. The combination did not demonstrate added benefit over either single agent treatment. As the 4T1 model is known to be highly metastatic, with tumors seen in lung, liver and lymph nodes, we closely examined each mouse at the time of euthanasia for evidence of metastatic disease. None of the treatments effectively reduced metastasis. To attempt to gain a better mechanistic understanding around the lack of added benefit of the combination, we profiled different immune cell subsets in the tumor by flow cytometry. We found that within the tumor infiltrating lymphocytes (TILs), 9H10 antibody and RT treatment resulted in an increased percentage of CD4+ T cells. Conversely a decreased percentage of CD8+ T cells was observed. However, the greatest effect exerted by 9H10 and RT was on the myeloid derived suppressive cell (MDSC) population. Although RT slightly decreased the percentage of MDSC within tumors and 9H10 had no effect, the two treatments synergized to reduce the number of tumor-infiltrating MDSC. Interestingly, a similar synergistic effect was observed when we analyzed the expression of the proliferation marker Ki-67 within CD8+ T cells. We also found that RT, and to a lesser extent 9H10, increased the percentage of CD45+ cells expressing the T cell inhibitory receptors PD1, PD-L1, and CTLA-4. No additive effect on the expression of these receptors was observed with combined therapy. Taken together, our results suggest that anti-CTLA-4 treatment combined with RT triggers both pro- and anti-tumor signaling pathways thus providing a possible explanation for the marginal anti-tumor responses we observed with these therapies in this disease model. Furthermore, this model could be very beneficial to evaluate other agents either in combination with 9H10 or RT or as a triple combination with both 9H10 and RT.

#4916

Improved statistical approaches that account for pseudoprogression in preclinical studies of RRx-001 with immunotherapies.

Nacer E. Abrouk,1 Jan Scicinski,2 Bryan Oronsky,2 Susan J. Knox,3 Donna Peehl,4 Shoucheng Ning,3 Gary R. Fanger2. 1 _Innovexe, Inc, Mountain View, CA;_ 2 _EpicentRx, Inc, Mountain View, CA;_ 3 _Stanford University, Department of Radiation Oncology, Stanford, CA;_ 4 _Stanford University, Department of Urology, Stanford, CA_.

Background and Objective

Immuno-oncology agents are associated with apparent tumor growth due to immune infiltration leading, in some cases, to misinterpretation of radiological scans that can result in premature discontinuation of effective therapeutic agents. In the clinic, this resulted in the introduction of modified immune-related response criteria. Preclinically, in the absence of radiology or immunohistochemistry, traditional tumor growth delay may similarly lead to misinterpretation of progression even in the context of a true response. More powerful statistical strategies are therefore required to distinguish between true and pseudoprogression.

RRx-001 is a novel immuno-epigenetic agent in Phase 2 with promising single agent and combination activity that is associated with clinical pseudo progression in some cases. Previously, we tested the hypothesis that RRx-001 + anti-PD-L1 would increase the complete response rate in a myeloma preclinical model. Time-to-event statistical analyses methods that account for the study design features and capture the longitudinal and panoramic aspect of pseudo progression were used to test superiority of the combination in lieu of traditional, mean-based mixed effects modeling approaches that did not show statistical significance. Nonparametric p-values for the difference of cumulative incidence rates of time to ≥50% tumor growth reduction and its associated restricted mean survival times are illustrated.

Methods and Results

Analysis of the raw data growth curves from a study comparing RRx-001, anti-PD-L1 alone and in combination in mice with J558L myeloma tumors showed an initial increase followed by a reduction in size suggesting a pseudoprogressive response. In light of this observation, the study-end probability of tumor volume reduction (≥50%) was chosen as a suitable endpoint. The difference in the Kaplan-Meier estimate for this endpoint between the combination and anti PD-L1 alone was 50% (95% CI: 10.8%, 89.2%). The two-sided nonparametric (Cox model) p-value was 0.0124, a statistically significant finding in favor of the combination group as compared to the PD-L1 group alone.

Conclusion

Tumor flare or pseudo progression describes a transient increase in tumor volume due to immune cell infiltration and inflammation/edema, which resolves over time. Pseudo progression is visualizable in the clinic with radiological scans; however, pre-clinically it is difficult, if not impossible, to distinguish between pseudo and true progression with traditional statistical methods. Kaplan-Meier description of time-to-volume reduction (≥50%) coupled with Cox's proportional hazards model follows the data longitudinally and therefore permits an analysis of immune infiltration resolution, and make it an improved method for analysis of pre-clinical experiments with immuno-oncology agents. 

## CLINICAL RESEARCH:

### Biomarkers for Gastrointestinal Cancer

#4917

High expression of miR-181c in tumor tissue as a recurrence marker of Dukes B colorectal cancer.

Yoshikatsu Koga,1 Nobuyoshi Yamazaki,1 Hirokazu Taniguchi,2 Motohiro Kojima,1 Yukihide Kanemitsu,2 Norio Saito,3 Yasuhiro Matsumura1. 1 _National Cancer Ctr., Kashiwa, Japan;_ 2 _National Cancer Ctr. Hosp., Tokyo, Japan;_ 3 _National Cancer Ctr. Hosp. East, Kashiwa, Japan_.

Background & Aims: A standard treatment for Dukes B colorectal cancer (CRC) which invades deeply through the muscularis propria without lymph node metastasis and distant metastasis is surgical resection without any additional chemotherapy. However, tumor recurrence is occurred in approximately 20% of these patients after curative resection. To date, there were no biomarkers for detecting the high risk group for tumor recurrence of Dukes B CRC. In the present study, miRNAs extracted from both frozen tissues and formalin-fixed paraffin-embedded (FFPE) tissues of CRC were investigated for a candidate for detecting the high risk group in Dukes B CRC.

Methods: Patients with histologically confirmed as Dukes B CRC were enrolled; 10 patients (5 patients with recurrence and 5 patients without recurrence) for preliminary study, 80 patients for training cohort, and 57 patients for validation cohort. First, comprehensive analysis of miRNAs was performed to select candidate miRNAs associated with recurrence using a high-sensitivity miRNA array chip in the preliminary study. Next, the candidate miRNAs were analyzed by real-time RT-PCR using miRNA extracted from frozen tissue and FFPE tissue in the training cohort. Finally, miR-181c was assessed by real-time RT-PCR using miRNA extracted from FFPE tissue in the validation cohort.

Results: In the preliminary study, 15 miRNAs were expressed significantly higher in the cancer tissue with recurrence than that without recurrence (P < 0.05) and selected as candidates for further study. Regarding let-7a, -7d, -7e, miR-23c, -26b, -128a, -151-5p, and -181c, tumor recurrence in the high expression group of these miRNAs was observed significantly higher than that in the low expression group in the training cohort using frozen tissue (P < 0.05). According to multivariate analysis including pathological factors, the high expression of miR-181c was detected as an independent predictive factor for recurrence (P = 0.001, OR: 9.43, 95%CI: 2.57-34.48). The same result was observed in the training cohort using FFPE tissue (P = 0.003, OR: 7.46, 95%CI: 1.97-28.57). In the validation cohort using the FFPE tissue, the recurrence rates of Dukes B CRC patients with high miR-181c expression and low miR-181c expression were 4.5% and 38.5%, respectively (P < 0.001).

Conclusions: miR-181c expression in CRC tissue (both frozen and FFPE) may be a useful predictive biomarker for tumor recurrence of Dukes B CRC.

#4918

Emerging prognostic biomarkers in colorectal cancer: HURP and ZEB1.

Logan Fair, Ingrid Espinoza, Xu Zhang, Abdelouahid Elkhattouti, Tangeng Ma, Joy King, Elizabeth Tarsi, Richard Whitlock, Vijay Kannuthurai, Ryan Jimenez, Tara Craft, Mary Graichen, Sharon Lobert, Roy Duhe, Charulochana Subramony, Christopher Lahr, Christian R. Gomez. _University of Mississippi Medical Center, Jackson, MS_.

The current diagnosis of colorectal cancer (CRC) is based on tumor-node-metastasis staging. This classification system has weaknesses due to different genetic and epigenetic backgrounds. Biological markers would improve early detection and guide clinicians in subsequent treatment decisions. Stem-like molecules have the potential to identify patients at high risk of developing aggressive carcinomas. Herein, Hepatoma Up-Regulate Protein (HURP) and Zinc finger E-box binding homeobox 1 (ZEB1) are assessed as potential prognostic biomarkers in CRC. Tumors (N=21) obtained from colectomies and 5 µm formalin-fixed paraffin-embedded blocks were sectioned and immunostained for HURP and ZEB1. For certain cases (N=8), flash frozen tissues from tumor and adjacent normal colonic tissue were used to extract total RNA, synthesize cDNA, and perform RT-PCR. Presence of tumor cells in analyzed tissue sections was assessed by a pathologist. HURP mRNA expression was 4-fold higher (p=0.0005) in tumor tissue relative to adjacent normal tissue. Furthermore, an increase in HURP expression was evident in all tumor samples relative to their respective normal tissue control with an average 3-fold increase (p=0.001). Immunohistochemical analysis (IHC) of HURP revealed expression in all analyzed specimens. Although the stroma was weakly stained for HURP in some cases, all tumors were immunostained for this protein (average intensity score = ++, p<0.0001). These results suggest HURP's potential as a tumor marker in CRC. ZEB1 mRNA expression in tumors did not differ from that observed in normal adjacent tissue; moreover, a tendency of mRNA to a decrease (n.s.) was observed. IHC for ZEB1 showed immunostaining in all analyzed cases. Weak staining was observed in both tumor and stroma (average intensity score = ++, n.s.). Further stratification of patients by race revealed that in Caucasian-American (CA) patients, staining of ZEB1 was less likely to be associated with disease progression or death after at least 2 years of follow-up. Interestingly, samples obtained from African-American (AA) individuals, known to have worse CRC outcomes, had an association between ZEB1 presence and poor survival regardless of the immunostaining intensity. These findings suggest that IHC localization of ZEB1 may represent a marker of a poor CRC prognosis, particularly in AA patients. However, one must be careful not to over-interpret these results as a Fischer's Exact test revealed a non-significant p value = 0.387. Overall, we have identified two stem cell-like molecules, HURP and ZEB1, with potential as markers of aggressiveness in CRC. Our findings also provide evidence of a possible biological basis for ethnicity-related differences with regards to stemness and regulatory factors of CRC aggressiveness. Future goals include collecting additional samples and expanding the project to examine other potential prognostic markers through IHC analysis of tissue microarrays.

#4919

Evaluating the gene expression of frozen tissue-derived prognostic signatures in FFPE colorectal cancer samples.

Jing Zhu,1 Natasha G. Deane,1 Keeli B. Lewis,1 Chandrasekhar Padmanabhan,1 Mary K. Washington,1 Kristen K. Ciombor,2 Cynthia Timmers,2 Richard M. Goldberg,2 R. Daniel Beauchamp,1 Xi Chen3. 1 _Vanderbilt University, Nashville, TN;_ 2 _The Ohio State University Comprehensive Cancer Center, Columbus, OH;_ 3 _University of Miami, Miami, FL_.

OBJECTIVE: Defining molecular features that can predict the recurrence of colorectal cancer (CRC) for stage II and stage III patients remains a challenging problem in cancer research. Most available clinical samples are Formalin Fixed and Paraffin Embedded (FFPE). Nanostring nCounter® and Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA) are the two platforms marketed for high throughput measurement of mRNA expression from FFPE tissue samples. In this study, to identify an optimal platform for the gene expression profiling of FFPE CRC samples, we compared the expression of genes that make up published frozen tissue-derived prognostic signatures measured by these two platforms. METHODS: FFPE primary tumor tissue blocks were identified from 500 patients with stage II or III CRC and complete pathological information. We removed samples with neoadjuvant therapy, inadequate clinical follow-up or tumor specimens, samples with failure of RNA extraction and highly degraded samples. We identified 194 eligible FFPE-derived CRC primary tumors with a 7.4-year mean follow up. To measure the gene expression of the 194 samples, we designed a custom nCounter codeset using 536 gene elements from multiple published frozen tissue-derived prognostic signatures for CRC. We also performed gene expression profiling using the HTA platform on a subset of 84 of the 194 samples. For the nCounter data, samples with low total average count or with more than 20% genes having lower average count than the synthetic negative control genes were omitted to ensure adequate data quality. RESULTS: In total, for CRC samples from 42 patients, the gene expression data of 516 common genes measured by both platforms were of sufficient quality for comparative analysis. Based on HTA platform-derived data, we found that (1) 36 and 97 of the 516 common genes are significantly associated with overall survival (OS) and disease-free survival (DFS) at the single gene level (FDR < 0.05), respectively; and (2) two of the nine reported multi-gene signatures, for which sufficient information from the original papers enabled such evaluation, can divide patients into high-risk and low-risk groups with significantly different DFS (p ≤ 0.05). Based on nCounter platform-derived data, no individual gene was found to be significantly associated with survival at the single gene level (FDR < 0.05), but one of the nine published multi-gene signatures can divide patients into two groups with significantly different OS (p ≤ 0.05). Our results also showed moderate correlations between paired FFPE samples measured by nCounter and HTA platforms (the median correlation coefficient is 0.52). CONCLUSION: Our data demonstrate that while both platforms may identify gene or gene set expression differences associated with survival outcomes, the HTA appears to provide a more robust gene expression analysis dataset when using genes selected from published gene signatures.

#4920

Evaluation of a diagnostic blood test for colorectal cancer screening and as a triage tool to stratify patients for colonoscopy.

Leah J. Cosgrove,1 Kathy Surinya,1 Kim YC Fung,2 Ilka Priebe,1 Mike Buckley,2 Tim Adams,3 Bruce Tabor,2 Leanne Purins,1 Trevor Lockett,2 Hugo Leroux,4 Peter Gibbs,5 Jiangqin Wei,1 Ed Nice,6 Tony Burgess,5 Michelle Thomas,7 James Moore,7 Raj Singh,8 Andrew Ruszkiewicz9. 1 _CSIRO, Adelaide, Australia;_ 2 _CSIRO, North Ryde, Sydney, Australia;_ 3 _CSIRO, Melbourne, Australia;_ 4 _CSIRO, Brisbane, Australia;_ 5 _WEHI, Melbourne, Australia;_ 6 _Monash University, Melbourne, Australia;_ 7 _Royal Adelaide Hospital, Adelaide, Australia;_ 8 _University of Adelaide, Adelaide, Australia;_ 9 _University of SA, Adelaide, Australia_.

Worldwide, colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death, with an annual incidence in 2012 of 1.4 million CRC cases and an annual mortality around 700,000 [1]. Fortunately the majority of colorectal cancer (CRC) cases are preventable by early detection and removal by colonoscopy or surgery. Currently in Australia, the only widely used non-invasive screening test for CRC is the immunological faecal occult blood test (iFOBT), which has been shown to reduce CRC incidence and mortality by 15-25% [2]. There are however significant issues that limit its effectiveness as a diagnostic screening tool, such as a poor positive predictive value of 5.3% for suspected cancer and low participation rate of approximately 33.5% in 2013, as seen in the Australian National Bowel Cancer Screening Program (NBCSP)[2]. Evidence indicates a robust, blood-based, diagnostic assay would increase screening participation and compliance. In our systematic analysis of 68 individual biomarkers using logistic regression strategies, we have identified a unique 3 protein biomarker panel blood test consisting of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2)[3]. This panel not only differentiates between CRC and normal patient sera with 95% specificity and 73% sensitivity in a single measurement (c.f. FOBT: 35-50 sensitivity [4]) but early stage disease. Currently, we are prospectively collecting a new cohort of over 1,000 blood samples from volunteers that have undergone colonoscopy, that include CRC patients, controls and other pathologies (IBD, other cancers) to specifically determine the performance of this blood test in asymptomatic and surveillance screening populations. We will also evaluate directly the accuracy and performance of our biomarker panel, as a diagnostic test suitable for CRC screening versus the iFOBT used in the NBCSP, as an intermediary test in the triage of individuals with positive iFOBT result for need and urgency to have a colonoscopy.

1. Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F. GLOBOCAN 2012 v1.1, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11

2. National Bowel Cancer Screening Program: monitoring report 2012-2013. AIHW & Australian Government Department of Health and Ageing, 2014.

3. Fung KYC et al 2015. Blood-based protein biomarker panel for the detection of colorectal cancer. PloS one. 2015;10(3):e0120425.

4. Morikawa, T et al., A comparison of the immunochemical fecal occult blood test and total colonoscopy in the asymptomatic population. Gastroenterology, 2005. 129(2): p. 422-8.

#4921

Plasma proteomics for the discovery of biomarkers of incisional hernia in colorectal cancer patients in the ColoCare Study.

Jürgen Böhm,1 Frank Pianka,2 Nina Stüttgen,2 Biljana Gigic,3 Yuzheng Zhang,4 Petra Schrotz-King,3 Nina Habermann,5 Alexis Ulrich,2 Martin Schneider,2 Paul Lampe,4 Markus Diener,2 Cornelia M. Ulrich1. 1 _Huntsman Cancer Institute, Salt Lake City, UT;_ 2 _Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany;_ 3 _National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 4 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 5 _European Molecular Biology Laboratory, Heidelberg, Germany_.

Background

Incisional hernia is the most common long-term complication after laparotomy for colorectal cancer resection with an incidence of 9-20%. Predisposing factors have been identified. Nevertheless, there are limited approaches to precisely predict individual risk, creating a need for predictive biomarkers of incisional hernia development.

Methods

We utilized pre-operative plasma samples of patients with newly diagnosed colorectal cancer [n=72; (stage I-IV)] from the ColoCare Study in Heidelberg, Germany, who underwent laparoscopic tumor resection between 2010 and 2013. Patient questionnaires and telephone-interviews were used to assess the incidence of an incisional hernia, and demographic and clinical-surgical data were abstracted from medical records. 21 patients with incisional hernia occurrence were matched with 51 patients without an incisional hernia (=controls) by gender, age, and BMI with at least 18 months follow-up. To assess predictive markers of incisional hernia risk we screened the plasma proteome for 3061 proteins using a well-validated antibody microarray test. Paired t-tests were used to compare protein levels between cases and controls. A gene-set-enrichment analysis (Gene Ontology and KEGG) was applied to test for differences in signaling pathways between the two groups.

Results

The proteome screen identified 27 proteins that showed elevated or reduced plasma levels in the hernia group compared to the control group (nominal p-values <0.05). Most of these proteins were connected to cell adhesion and inflammation, e.g. the encoding genes CCL21, IL12A, EPCAM and CDH3. The gene-set-enrichment analysis identified several pathways of extracellular matrix receptor interaction or cell proliferation that differed significantly between the hernia and control group.

Conclusion

To date no predictive blood-based biomarkers of incisional hernia risk are available. We discovered several candidate protein biomarkers which, after validation in further studies, could be incorporated into a multifactorial risk model to guide clinical decision making. This could enable the initiation of prophylactic treatments such as a mesh implantation and individualized patient recommendations to prevent incisional hernia occurrence in high-risk patients.

#4922

Chronic diseases and age >65 years may cause false positive results in colorectal cancer screening with the blood-based Sept9 methylation assay.

Mai-Britt W. Ørntoft,1 Hans Jørgen Nielsen,2 Torben Falck Ørntoft,1 Claus Lindbjerg1. 1 _Department of Molecular Medicine, Aarhus University Hospital Skejby, Aarhus, Denmark;_ 2 _Department of Surgical Gastroenterology 360, Hvidovre Hospital, University of Copenhagen, Hvidovre, Denmark_.

The aim of this study was to investigate whether the performance of the colorectal cancer (CRC) screening marker Sept9 was influenced by comorbidities and/or demographic characteristics. Annually, CRC is diagnosed in >1.4 million subjects worldwide and incidence is increasing. Much research is therefore directed at CRC screening, which has proven to reduce cancer-related mortality. The Sept9 DNA-methylation assay is among the most well studied blood-based epigentic screening markers. However, currently we have only limited knowledge of how comorbidities and/or demographic characteristics influence assay performance, as this has not been part of earlier study objectives. Using a retrospective nested case-control study design fulfilling the REMARK criteria, we studied plasma from 149 cancers and 150 controls selected from a well-characterized cohort of 4698 subjects referred for diagnostic colonoscopy due to CRC-related symptoms. The cohort had a wide variety of comorbidities. Cases and controls were matched on age and gender, and cases were further stratified by tumor-site and tumor-stage. Plasma Sept9 levels were assessed using a commercially available qMSP based assay in the most specific setting; the cut-off value was set at 2 positive out of 3 replicates (2/3 algorithm). STATA V.12.1 was used for statistical analysis. Fishers exact test and logistic regression were used to estimate the association between Sept9 results and CRC. With the specific 2/3 test algorithm, clinical sensitivity for CRC stages I-IV was 17%, 74%, 63%, and 86%, and the overall sensitivity 59% (95% CI, 64-80%) and specificity 95% (95% CI, 91-98%), respectively. Sensitivity for adenomas was 0% (95% CI, 0-16%). Age >65 was significantly associated with both increased false positive ((p<0.05) and false negative results (p=0.007). Arthritis was associated with a higher false negative rate (p=0.07) and arteriosclerosis was significantly associated with a higher false positive rate (p=0.007). Diabetes was associated with Sept9 positivity with an OR of 4.7 (95% CI 1.5-14.5). In a multivariate logistic regression, adjusting for the associated comorbidities and age >65, the OR for a positive Sept9 test to be associated with CRC increased from 21 (95% CI 9-47) to 117 (95% CI 26-528). The results indicate that the Sept9 assay is not only affected by CRC stage, but by several comorbidities and age >65 as well; these are factors commonly associated with CRC screening populations. The findings are of importance not only if the Sept9 assay is used as a single marker for CRC screening: it may also have a wider impact, as it is likely that such factors may affect other blood-based methylation markers as well. We hypothesize that an increased plasma input might increase assay sensitivity and that multiplexing of several biomarkers may diminish false positive results in blood-based screening with methylation specific markers.

#4923

Expression of class III beta-tubulin (TUBB3) in 3580 colorectal cancers (CRCs) and correlation with clinico-pathological and molecular features.

Joanne Xiu,1 Sandeep Reddy,1 Wafik S. El-Deiry2. 1 _Caris Life Sciences, Phoenix, AZ;_ 2 _Fox Chase Cancer Center, Philadelphia, PA_.

Class III beta-tubulin plays a crucial role in intracellular transport, meiosis and mitosis. High expression of TUBB3 has been shown to associate with poor prognosis and taxane resistance in various cancer types. CRC is known to be generally resistant to taxane therapy. We investigated expression of TUBB3 in 3580 CRCs and made correlations with clinicopathological and molecular parameters. 3580 CRC samples were evaluated by tumor profiling (Caris Life Sciences, Phoenix, AZ). Tests included Sanger or next generation sequencing (NGS), protein expression by immunohistochemistry (IHC) and gene amplification by in situ hybridization (ISH). TUBB3 expression was evaluated by immunohistochemistry (Ab: POLY, Covance) and expression higher than 2+, 30% was scored as positive. TUBB3 positive expression was observed in 37% (1320/3580) of the complete CRC cohort, specifically 27% in mucinous histology (164/617) and 17% in signet ring histology (29/171). While the expression was not associated with average patient age (59 years old) or gender (53% vs. 50% male), TUBB3 was more frequently expressed in tumors that originated from the left colon (370/1016 or 36%) than from the right colon (235/790 or 30%, p=0.003). In the 1847 tumors taken from metastatic sites, 40% (746) overexpressed TUBB3 while in 1629 CRCs taken from primary tumors, only 34% (547) overexpressed TUBB3 (p<0.0001). Interestingly, in tumors that overexpressed TUBB3, 65% also overexpressed cMET (828/1275) and 33% also overexpressed TLE3 (431/1299). This compares to 50% cMET expression (1096/2207, p<0.0001) and 23% TLE3 expression (517/2225, p<0.0001) in tumors that were negative for TUBB3. Microsatellite instability detected by fragment analysis was more prevalent in the TUBB3-negative cohort than the TUBB3-positive cohort (7.4% or 77/1046 vs. 2.9% or 16/558, p=0.0002). Mutations in APC (62% vs. 56%, p=0.0003) and KRAS (55% vs. 46%, p<0.0001) were more frequent in TUBB3-positive tumors, while GNAS (2% vs. 5%, p<0.0001) and SMAD4 (10.1% vs. 14.6%, p=0.0005) mutations were significantly more frequent in tumors that were negative for TUBB3 expression. Thus, high expression of TUBB3 was found in 37% of CRCs, and was significantly associated with tumors that originated from the left colon and with tumors taken from metastatic sites. Distinct biomarker features detected by IHC and sequencing suggest that TUBB3 expression may carry theranostic importance which warrants further investigation in clinical trials.

#4924

Retrospective evaluation of a system model of the BCL2 family of proteins as a predictive and prognostic biomarker for the clinical outcome of stage II-IV colorectal cancer patients.

Andreas U. Lindner,1 Manuela Salvucci,1 Mattia Cremona,2 Naser Monsefi,1 Sarah Curry,2 Clare Morgan,2 Alexa Resler,1 Robert O'Byrne,1 Orna Bacon,2 Michael Stuehler,1 Lorna Flanagan,1 Richard Wilson,3 Patrick G. Johnston,3 Manuel Salto-Tellez,3 Sophie Camilleri-Broët,4 Deborah A. McNamara,2 Bryan T. Hennessy,2 Elaine W. Kay,2 Pierre Laurent-Puig,4 Sandra Van Schaeybroeck,3 Jochen H.M. Prehn1. 1 _Royal College of Surgeons in Ireland, Dublin, Ireland;_ 2 _Beaumont Hospital, Dublin, Ireland;_ 3 _Queen's University Belfast, Belfast, United Kingdom;_ 4 _Université Paris Descartes, Paris, France_.

With 1.4 million new cases every year, colorectal cancer (CRC) is the fourth most common cancer worldwide [Globocan 2012, WHO]. Despite therapeutic advances and improvements in overall care, TNM staging remains the best prognostic indicator for CRC patients' clinical outcomes and is pivotal for deciding on use of adjuvant chemotherapy after resection of the tumour. Adjuvant chemotherapy is not recommended for many stage II patients and mostly high-risk patients receive chemotherapy. However, there is a lack of robust biomarkers for identifying patient response to chemotherapy, recurrence and mortality risk.

We developed a system model of the BCL2 family of proteins (DR_MOMP) to assess the sensitivity of cells to genotoxic stress and to induce apoptosis triggered by chemotherapy. It calculates the stress dose required to induce mitochondrial outer membrane permeabilization (MOMP) based on absolute protein levels and the interaction of pro- and anti-apoptotic BCL2 family proteins. Cells predicted to require a high stress dose showed decreased cell death rates after being exposed to 5FU and Oxaliplatin. Profiles of BAK, BAX, BCL2, BCL(X)L and MCL1 were determined by Reverse Phase Protein Array (RPPA) technology in FFPE primary tumours collected from two distinct cohorts: stage III CRC patients who underwent adjuvant 5FU-based chemotherapy (n = 128), and stage II CRC patients from a completed clinical trial with patients randomised to adjuvant 5FU-based chemotherapy or observation only (n = 138). Protein profiles were inputted into DR_MOMP to determine chemotherapy sensitivity and to classify patients into high- or low risk categories. Findings were validated on the TCGA COAD cohort using both protein (RPPA) and mRNA (SeqV2 RSEM) expression data.

Stage II patients classified as high-risk by DR_MOMP and randomised to observation only had approximately 2-fold increased risk of death from CRC compared to those classified as low-risk or received chemotherapy (HR 2.4; 95% CI 1.2-4.8; p-value = 0.0199). Among stage III patients treated with FOLFOX, those classified as high- versus low-risk had a more than 10-fold increased risk of death from CRC (HR 10.6; 95% CI 2.4-46.3; p-value < 0.0001). We validated this finding in 261 stage II-IV patients of the TCGA COAD cohort (HR 10.6; 95% CI 1.2-12.5; p-value = 0.0125). DR_MOMP predicted mortality risk independent of TNM staging and KRAS mutation status.

Our system delivers a novel predictive and prognostic biomarker that could be combined with TNM staging when assessing initial risk and subsequent clinical management of CRC patients.

#4925

Plasma levels of cartilage oligomeric matrix protein (COMP), are significantly increased in patients with colorectal cancer.

Hmc Shantha Kumara,1 Hiromichi Miyagaki,2 Xiaohong Yan,1 Linda Njoh,1 Vesna Cekic,1 Nipa D. Gandhi,1 Melissa M. Alvarez-Downing,1 Richard L. Whelan1. 1 _Mount Sinai Roosevelt Hospital, Department of Surgery, Division of Colorectal Surgery, New York, NY;_ 2 _Department of Surgery,Saiseikai Senri Hospital 1-1-6, Tsukumodai, Suita,, Osaka, Japan_.

Introduction: Cartilage Oligomeric Matrix Protein (COMP) is a member of the thrombospondin family of extracellular calcium-binding proteins that plays role in the assembly of extracellular matrix. COMP maintains the structural integrity of cartilage through its interaction with a number of extracellular matrix proteins. COMP has a unique binding site for Vitamin D, indicating that it may also participate in storage and delivery of cell-signaling molecules. An expression array analysis of colorectal cancer samples vs normal tissue has noted overexpression of COMP mRNA. This study was undertaken to assess preoperative blood levels of COMP in the setting of colorectal cancer(CRC). Our hypothesis is that plasma levels of COMP may be elevated due to tumor overexpression and that this protein may have potential as a diagnostic marker for colorectal cancer.

Method: Patients (pts) undergoing elective colorectal resection for CRC or benign colonic pathology (BCP) that had been prospectively enrolled in an IRB approved tissue and data bank for whom adequate PreOp plasma samples were available were studied. Demographic, clinical, and pathologic data were collected. Plasma COMP levels were determined via ELISA in duplicate and are reported as median +95%CI (ng/ml). Tissue expression levels were determined in paired tumor and normal tissue samples of a subpopulation of study pts by QRT-PCR .The candidacy of COMP as a diagnostic marker for CRC was validated by the receiver operating characteristic (ROC) curve and by the area under the ROC curve (AUC) results. The Wilcoxon/Kruskal-Wallis test was used for statistical analysis, (significance p<0.05).

Results: A total of 151 CRC (73%colon, 27%rectal) and 73 BCP pats (Adenoma 21%, diverticulitis 57%, other 22%) were studied. The male/female ratios were similar. The CRC stage distribution was: Stage 1, 22%; Stage 2, 32%; Stage 3, 31%; and Stage 4, 15%. The median plasma COMP levels were significantly higher in the CRC pts (209.2,CI: 187.8,234) vs. the BCP pats(129.6,CI: 110.7,149.2; P=< 0.0001). The plasma COMP levels were significantly higher in Stage III pts. (p=0.001) and in Stage II+III pats. (p=0.03) compared to Stage I pts. The AUC value for the ROC curve was 0.781 (sensitivity 53%, specificity 93%). Increased expression of COMP was noted in 59% of the CRC tissue samples tested (10/17) vs. paired normal tissue samples.

Conclusion: The CRC median plasma COMP level was 61% higher than the median BCP result and the Stage 3 median level 50% higher than the median Stage I level. In general, plasma COMP levels increased with advancing cancer stage. The AUC results suggest COMP may have value as a CRC prognostic marker, perhaps, in combination with other protein markers to increase the sensitivity. Tissue expression analysis suggests that the most likely source of the added COMP in the plasma is the tumor itself. Further study with larger populations of control and CRC pts is warranted.

#4926

Clinical significance of SNORA21, an H/ACA box snoRNA, as a metastasis predicting and prognostic biomarker in colorectal cancer.

Kazuhiro Yoshida,1 Shusuke Toden,1 Wenhao Weng,1 Takeshi Nagasaka,2 Toshiyoshi Fujiwara,2 Ajay Goel1. 1 _Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX;_ 2 _Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan_.

Background: Colorectal cancer (CRC) remains a prevalent malignancy worldwide with a high mortality rate. Over the last decade, the roles of several types of noncoding RNAs in oncogenesis have been clarified. Emerging evidence indicates that not only microRNAs, but small nucleolar RNAs (snoRNAs) appear to be dysregulated in various malignancies and they play a critical role in modulating cell transformation, tumor progression and metastasis. Functionally, cancer associated snoRNAs are subdivided into four major categories based on their oncogenic functions: 1) an independent function from imprinting gene in cancer initiation and progression 2) Activation of human telomerase 3) Inhibition of ribosomal maturation 4) Act directly as oncogene or tumor suppressor. Despite these exciting evidences, the role of snoRNAs in CRC remains limited and their clinical significance remains largely unexplored in this malignancy.

Aims: We aimed to clarify the clinical relevance of snoRNAs in tumors and evaluate their prognostic potential in CRC. Furthermore, we investigated the functional role of snoRNAs in CRC tumorigenesis.

Methods: Initially, we screened for the most commonly differentially expressed snoRNAs from publicly available microarray and RNA-sequence databases for other malignancies. Three clinical cohorts (a total 345 CRC tissues) were then used to select/validate the candidate snoRNAs. The prognostic potential of the candidate snoRNAs were evaluated and the association with clinicopathological features were assessed. CRISPR/Cas9 system was used to knockdown the target snoRNA in multiple CRC cell lines to determine the functional role of the target snoRNA in a series of in vitro assays.

Results: In the discovery phase, we identified four differentially overexpressed snoRNAs in human cancers. We then confirmed overexpression of three snoRNAs (SNORA21, SNORA34 and SNORD66) in CRC compared to adjacent normal tissues. Of these three snoRNAs, high SNORA21 expression in tumors resulted in poor prognosis (p=0.0086, Log-rank test). In an independent validation cohort, we confirmed high SNORA21 expression results in poor overall survival (p=0.0060), especially in stage IV CRC (p=0.0272). Clinicopathological analysis showed positive association between SNORA21 and vascular invasion and metastasis (p=0.030 and 0.011, by Kruskal-Wallis test and Mann-Whitney U test, respectively) indicating that SNORA21 could be involved in metastasis. Moreover, multivariate analysis revealed that increased SNORA21 expression was an independent prognostic factor for overall survival. Using CRISPR/Cas9 stable SNORA21 knocked-down cell lines, we demonstrated that SNORA21 suppression resulted in reduced cell proliferation and colony forming capacity.

Conclusion: We firstly identified SNORA21 as a novel oncogene which could also be a potential prognostic marker in patients with CRC.

#4927

Identification of novel candidate of driver genes on chromosome 7 in colorectal cancer.

Kuniaki Sato,1 Qingjiang Hu,1 Shinya Kidogami,1 Yushi Ogawa,1 Tomoko Saito,1 Sho Nambara,1 Hisateru Komatsu,1 Hidenari Hirata,1 Shotaro Sakimura,1 Ryutaro Uchi,1 Naoki Hayashi,1 Tomohiro Iguchi,1 Hidetoshi Eguchi,1 Shuhei Ito,1 Takaaki Masuda,1 Takashi Nakagawa,2 Koshi Mimori1. 1 _Kyushu University Beppu Hospital, Beppu City, Oita Prefecture, Japan;_ 2 _Kyushu University, Fukuoka City, Fukuoka Prefecture, Japan_.

Background

Colorectal cancer(CRC) is one of the most common types of cancer worldwide. Its high mortality rate is serious problems. Hence it is urgently necessary to identify novel molecular target to improve it. Gain of chromosome 7 is frequently found in CRC, and it has been considered to harbour driver genes that promote tumorigenesis or tumor progression by the gain of function. The aim of this study is to identify novel candidate driver genes on chromosome 7 and to clarify the clinical significance of their expression in CRC.

Material and Methods

1. We have selected the candidate genes which satisfied the following criteria using CRC data from The Cancer Genome Atlas (TCGA), 1) their DNA copy number and the mRNA expression is positively correlated with each other, 2) overexpressed in tumor tissues compared to normal tissues.

2. We measured the mRNA expression of the candidate genes in 125 surgically-resected primary CRC tissues and the paired normal colon tissues in our hospital by quantitative RT-PCR. Difference of mRNA expression between CRC tissues and normal colon tissues was analyzed by Mann Whitney U-test.

3. Survival analysis between high and low expression group of the candidate genes was performed by Kaplan-Meier method. Correlation between the mRNA expression of the candidate genes and clinicopathological factors were analyzed by Fisher's exact test.

4. We performed Gene Set Enrichment Analysis (GSEA) in CRC data from TCGA to elucidate the correlation between the candidate genes and gene sets that are associated with tumorigenesis or tumor progression.

Results

We focused on phosphoserine phosphatase(PSPH), the rate-limiting enzyme in serine biosynthesis pathway(SSP), as a novel candidate driver gene on chromosome 7 in CRC. PSPH expression was significantly higher in CRC tissues than in normal colon tissues (p<0.005). PSPH expression positively correlated with depth of invasion (p<0.05), venous invasion (p<0.05), and distant metastasis (p<0.05). The high PSPH expression group had a significantly poorer prognosis than the low expression group (p<0.001). On multivariate analysis, high PSPH expression was an independent prognostic factor affecting 5-years OS (p<0.005) with hazard ratios (95% CI) of 4.85(1.77-16.2) among conventional clinicopathological factors. The high PSPH expression group in TCGA data set also had poorer prognosis than the low expression group (p<0.05). GSEA showed that PSPH expression was positively correlated with expression of gene set of c-Myc-downstream.

Conclusions

We identified PSPH as a promising candidate driver gene on chromosome 7. Moreover, PSPH expression was a prognostic factor in CRC. It has been reported that c-Myc upregulates expression of SSP enzymes including PSPH. Therefore, PSPH is suggested to be involved in tumorigenesis or tumor progression as a downstream molecule of c-Myc in CRC. PSPH should be an important gene on chromosome 7 to promote the progression of CRC.

#4928

MiR expression profiles can predict response to systemic treatment in patients with advanced colorectal cancer.

Dennis Poel,1 Maarten Neerincx,1 Daoud L.S. Sie,1 Nicole C.T. van Grieken,1 R Shankaraiah,1 F. S.W. van der Wolf,2 J. H.T.M van Waesberghe,2 J. D. Burggraaf,3 Paul P. Eijk,1 Bauke Ylstra,1 Cees Verhoef,4 Mark A. van de Wiel,2 Henk M.W. Verheul,1 Tineke E. Buffart1. 1 _VUmc/CCA, Amsterdam, Netherlands;_ 2 _VUmc, Amsterdam, Netherlands;_ 3 _Spaarne Hospital, Hoofddorp, Netherlands;_ 4 _Erasmus Medical Center Cancer Institute, Rotterdam, Netherlands_.

Background and aim: Patients with advanced colorectal cancer (mCRC) are commonly treated with systemic treatment consisting of fluoropyrimidine-based regimens being ineffective in 20-25% of the patients. Currently, selection criteria for patients to predict who will respond to this treatment is lacking. The aim of this study is to identify which patients will respond to first line fluoropyrimidine-based treatment based using microRNA (miR) expression profiles in order to avoid ineffective treatment.

Material and methods: Total RNA was isolated from 88 fresh frozen colorectal cancer tissue samples consisting of ≥ 70% tumor cells, collected from patients with mCRC. MiR expression profiles were generated by next generation sequencing using the Illumina High Seq 2000 platform. Of all patients clinical and pathological data, including treatment response based on RECIST criteria, were collected. Class prediction and miR selection were performed using the GRidge package in R. Penalized selection and internal cross validation were used to select miRs predictive for treatment response.

RESULTS: Next generation sequencing resulted in a mean of 10.087.107 (range 6.114.932 to 74.313.067) reads per sample corresponding to 2567 unique mature miR sequences, including 457 novel candidate and 2110 known miRs sequences (miRbase version 19). Penalized regression analysis on tumor specific miRs identified an expression profile which was predictive for clinical benefit (defined as response and stable disease) from first line treatment.

Conclusion: With miR profiling of CRC tissue samples response prediction to first line fluoropyrimidine-based treatment in patients with mCRC is possible. We foresee that selection of treatment using miR expression profiling will avoid unnecessary treatment related toxicity and improve outcome for patients with mCRC

#4929

UbcH10 may represent a potential marker of gastric carcinoma.

Mengxuan Yang,1 Yingying Qu,2 Gang Hu,2 Shiwei Tu,2 RL Shi,1 XB Wu,1 ZQ Hu,1 QM Zhang,1 SQ Liu,1 GF Pan,1 Ziping Zhang,1 He Zhou2. 1 _Shanghai Minhang Central Hospital, Shanghai, China;_ 2 _Shanghai ChemPartner Co. Ltd, Shanghai, China_.

Gastric cancer is a fatal disease with limited early diagnostic methods available. There is an urgent need to find more effective targets for early diagnosis and therapeutics. UbcH10 is an ubiquitin-conjugating enzyme with a high expression reported in some cancers. Several gastric tumor cell lines with high or low expression of UbcH10 were exploited to study the role of UbcH10 in gastric cancer. Knocking down of UbcH10 expression using siRNA in high expressing gastric cancer cell lines resulted in reduced proliferation, increased cisplatin-induced apoptosis and reduced serum-induced ERK, Akt and p38 phosphorylation signaling. In agreement, overexpression of UbcH10 expression in low-expression gastric cancer cell lines led to enhanced cell proliferation, resistance to cisplatin-induced apoptosis. Most importantly, IHC analyses showed that the UbcH10 protein expressed at a high level in patient gastric cancer tissues, but not in adjacent mesenchyme tissues. These data suggest that UbcH10 may promote gastric cancer growth and can serve as a biomarker for diagnosis or target for new therapeutics in gastric cancer.

#4930

Ki-67 serves as a prognostic marker in early gastric cancer, but not in advanced gastric cancer.

Won Sup Lee, Gyung Hyuck Ko, Se-Il Go, Jeong-Hee Lee, Sang-Ho Jeong, Young-Joon Lee, Soon Chan Hong, Woo Song Ha. _Gyeongsang National Univ. Hospital, Jinju, Republic of Korea_.

Ki-67 protein is a cellular marker for proliferation. The role of Ki-67 as a prognostic factor has not been established in gastric cancer. We previously demonstrated that CD44v plays an important role in EThe present study was performed toinvestigate the significance of Ki-67 expression as a biomarker in early gastric cancer (EGC), but not advanced gastric cancer (AGC). With the study, we foundthat there is correlation between CD44v and Ki67. Here, evaluated the clinical significance with tissue microarray of gastric cancer by performing immunohistochemical staining for Ki-67. Its clinical significance was analyzed with adjustment via the propensity score-matching. The median follow-up duration was 72 months (range: 3 - 120 months). Ki-67 positive group showed worse prognosis than Ki-67-negative group in EGC (5-YSR, 78.9% vs. 92.0%, p =0.018), but not in advanced gastric cancer (AGC) (5-YSR, 58.5% vs. 59.2%, p =0.951). Interestingly, in the patients with well-differentiated histology, prognosis for Ki-67-positive group was considerably worse than that for Ki-67-negative group (5-YSR, 67.0% vs. 94.4%, p = 0.012), but not in those with moderately differentiated (p = 0.504) and poorly-differentiated histology (p = 0.905). In this cohort, there was a strong correlation between the proportion of EGC and well-differentiated histology (r = 0.215, p = 0.002). Multivariate analysis also revealed that the positive-Ki- 67 expression serve as a poor prognostic factor in EGC (HR 4.346, 95% CI 1.397 - 13.515, p = 0.011), but not in all the patients (p = 0.171). With bootstrap resampling, we internally validated these results (p = 0.011). In conclusion, this study suggests that Ki-67 expression may be a good biomarker for prognosis prediction for EGC, but not for AGC. [This study was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare & Family Affairs, Republic of Korea.(0820050).]

#4931

Validation of Trastuzumab immunohistochemistry as a predictive and prognostic biomarker in gastric cancer patients.

Jiwon Koh,1 Soo Kyung Nam,2 Younwoo Lee,3 Chan-Young Ock,4 Jin Won Kim,5 Keun-Wook Lee,5 Do-Youn Oh,4 Do Joong Park,6 Hyung-Ho Kim,6 Keon-Wook Kang,7 Woo Ho Kim,1 Ho-Young Lee,3 Hye Seung Lee2. 1 _Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Pathology, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 3 _Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 4 _Department of Internal Medicine, Seoul National University Hospital, Seoul, Republic of Korea;_ 5 _Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 6 _Department of Surgery, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea;_ 7 _Department of Nuclear Medicine, Seoul National University Hospital, Seoul, Republic of Korea_.

Although HER2 immunohistochemistry (IHC) is widely used to assess the HER2 status, not all patients could be clarified with HER2 IHC positivity for Trastuzumab treatment. Here, we introduce a novel IHC method utilizing Trastuzumab itself; compared to conventional HER2 IHC, the new method evaluates the extracellular epitope which is the target of action for Trastuzumab. The purpose of this study is to test whether this new method has better performance in predicting treatment outcome of Trastuzumab therapy, and whether it is the significant prognostic factor in consecutive gastric cancer patients.

From two individual institutions, 37 and 32 GC patients treated with Trastuzumab were enrolled respectively, the former as the training cohort and the latter for validation, and progression free survivals (PFS) were compared according to the results of Trastuzumab IHC. To validate the prognostic significance of Trastuzumab IHC in consecutive GC, we constructed tissue microarrays (TMA) from 536 consecutive cases and Trastuzumab IHC, HER2 IHC, and HER2 silver in situ hybridization (SISH) were performed.

In training cohort (n=37), all cases were HER2 IHC 2+ (n=11) or 3+ (n=26), but 2+ in 3 cases (8.1%) and 3+ in 10 cases (27.0%) by Trastuzumab IHC. Similar results were observed in validation cohort, 17 of 32 cases (53.1%) were 2+ or 3+ by Trastuzumab IHC. Trastuzumab ≥2+ group in the training cohort had significantly better PFS than Trastuzumab <2+ group (p=0.016), and survival analysis of PFS in validation cohort and combined cohort also showed favorable PFS in Trastuzumab ≥2+ group (p=0.031 and 0.001, respectively). By Cox regression analysis in training cohort, Trastuzumab IHC ≥2+ was proved to be the independent predictive factor for PFS in patients receiving Trastuzumab. Of 536 consecutive cases of GC, 23 (4.3%) were HER2 IHC 3+, and among these patients, only 5 (21.7%) were also 3+ by Trastuzumab IHC. Disease specific survival (DSS) and overall survival (OS) in Trastuzumab IHC ≥2+ group were shorter compared to Trastuzumab IHC <2 group (p=0.015 and 0.063, respectively), while both HER2 IHC and HER2 SISH failed to discriminate significant survival differences between subgroups.

In this study, we demonstrated that Trastuzumab IHC is a significant predictive factor for PFS in Trastuzumab-treated GC patients, and poor prognostic factor of DSS in consecutive GC, even better than conventional HER2 IHC and HER2 SISH. We believe that Trastuzumab IHC is a very promising, relatively easy-to-implement, novel method to assess HER2 status in routine clinical practice.

#4932

Stratification markers for the risk of recurrence after curative resection of stage II or III gastric cancer and potential clinical applications.

Takashi Oshima,1 Yayoi Kimura,2 Kentaro Sakamaki,3 Yohei Miyagi,4 Takaki Yoshikawa,5 Yasushi Rino,6 Kazuhiro Sentani,7 Naohide Oue,7 Wataru Yasui,7 Toshio Imada,8 Munetaka Munetaka1. 1 _Depertment of Surgery, Yokohama City University, Yokohama, Japan;_ 2 _Advanced Medical Research Center Yokohama City University, Yokohama, Japan;_ 3 _Depertment of Biostatistics, Yokohama City University, Yokohama, Japan;_ 4 _Kanagawa Cancer Center Research Institute, Yokohama, Japan;_ 5 _Depertment of Gastrointestinal Surgery, Kanagawa Cancer Center, Yokohama, Japan;_ 6 _Depertment of Surgery, Yokohama City Univrsity, Yokohama, Japan;_ 7 _Department of Molecular Pathology, Hiroshima University, Hiroshima, Japan;_ 8 _Saiseikai Yokohamashi Nanbu Hospital, Yokohama, Japan_.

Objective: Standard treatment for stage II or III gastric cancer is curative resection followed by adjuvant chemotherapy. Treatment outcomes are expected to be further improved by individualized therapy based on biomarker analysis. We extracted mRNA and protein from frozen specimens of gastric cancer and analyzed markers for stratifying the risk of recurrence after curative resection of stage II or III gastric cancer. We report our currently available findings.

Methods: Candidate markers for stratification of recurrence risk were identified by quantitative real-time polymerase chain reaction (Q-PCR) at the gene level and liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the protein level. The study group for quantitative real-time PCR comprised 255 patients with stage II or III gastric cancer (training set, 145 patients; validation set, 110 patients). The relative expression levels of 127 genes were measured in gastric cancer tissue. Genes identified be independent predictors of poor outcomes in the training set were verified in the validation set. The study group for exploratory proteomics analysis LC-MS/MS comprised 24 patients with T4a Nx M0, Stage III gastric cancer (recurrence and death, 12 patients; relapse-free survival >5 years, 12 patients). Proteins with max fold change >2 and Anova (P) <0.001 were searched.

Results: On Q-PCR, 5 genes; SPARC, HER2, CXCR4, INHBA, and VSNL1 were identified as candidate markers. On exploratory proteomics analysis by LC-MS-MS, 4 proteins were identified as candidate markers for recurrence risk stratification.

Conclusions: Promising candidate markers for stratification of the risk of recurrence after curative resection of stage II or III gastric cancer were identified at the gene and protein levels. These candidate markers are now being analyzed by tissue microarray, using specimens from about 500 patients each at two high-volume centers, with the goal of developing clinical applications.

#4933

In Situ analysis of FGFR2 RNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients.

Yasutoshi Kuboki,1 Christoph A. Schatz,2 Sabine Jabusch,2 Karl Koechert,2 Janine Feng,3 Sabine Wittemer-Rump,2 Karl Ziegelbauer,2 Thomas Krahn,2 Akiko Nagatsuma,1 Atsushi Ochiai1. 1 _National Cancer Center Hospital East, Tokyo, Japan;_ 2 _Bayer Pharma AG, Berlin, Germany;_ 3 _Ventana Medical Systems, Inc, Tuscon, AZ_.

Gastric cancer is the 3rd most common cause of cancer-related mortality worldwide, thus new treatment options are urgently needed. In gastric cancer, over-expression of the tumor-promoting tyrosine kinase receptor fibroblast growth factor receptor 2 (FGFR2) has been described, representing a potential therapeutic target. FGFR2 expression in gastric cancer has been reported to be heterogeneous. Most methods for detection of RNA in FFPE (formalin fixed paraffin embedded) do not provide spatial resolution for assessment of tumor heterogeneity. Here we used a novel RNA in situ technique called RNAscope. The technology allows visual investigation of FGFR2 transcription on FFPE slides. Samples from 1036 gastric adenocarcinoma patients who underwent surgery in National Cancer Center Hospital East (Chiba, Japan) were assembled in tissue micro-arrays and analyzed by RNAscope. A total of 578 samples were also analyzed by DISH to determine gene amplification. Using these tissue based methods we were able to assess the localization and heterogeneity of both FGFR2 gene amplification and RNA expression. Strong FGFR2 gene amplification (FGFR2:CEN10 >10) was detected in 2% of 578 samples. Moderate FGFR2 gene amplification (FGFR2:CEN10 between 2 and 10) was seen in 8% of the samples. High FGFR2 RNA expression (score 4+) was seen in 4% of 718 evaluable samples. Gene amplification and RNA expression were closely correlated. All samples with dense clusters of score 4+ RNA showed FGFR2:CEN10 ratios >10. The identical tumor areas showed high gene amplification and RNA expression. FGFR2 RNA and gene amplification was highly heterogeneous in the tissue. Only 0.4% of the samples showed homogeneous FGFR2 expression in >80% of the tumor cells. High RNA expression intensity was associated with a more homogeneous expression pattern compared to moderate FGFR2 expression. In early stage I/II gastric cancer samples with score 4+ RNA expression are mostly of the differentiated type. RNA score 4+ patients of grade III/IV gastric cancer were mostly of undifferentiated histology. In a subset of 195 samples with available data cMet IHC 3+ and Her2 positivity status were not mutually exclusive with regards to FGFR2 RNA Score 4+ expression. In a multivariant analysis FGFR2 RNA expression was associated with patient outcome. Gastric cancer patients with score 4+ RNA expression had a shorter progression free survival compared to patients with score 0-3. Interestingly, there was a trend for patients with homogeneous score 3+ RNA expression, but not heterogeneous score 3+ expression, to show shorter PFS and OS suggesting that patients with high intensity or homogeneous FGFR2 RNA expression may define a subgroup of patients with gastric cancer. Patients with homogenous or strong focal FGFR2 expression might therefore be candidates for FGFR2 directed therapies in gastric cancer.

#4934

Detection of HBV-host junction DNA sequences in urine of patients with hepatocellular carcinoma.

Selena Lin,1 Evan R. Trauger,2 Benjamin P. Song,2 Ling Lan,3 Patrick M. Jongeneel,4 Emilie G. C. Thompson,2 Malcolm C. Hoffman,2 Surbhi Jain,4 Ting-Tsung Chang,5 Timothy M. Block,2 Wei Song,4 Ying-Hsiu Su2. 1 _Drexel University College of Medicine, Doylestown, PA;_ 2 _Baruch S. Blumberg Institute, Doylestown, PA;_ 3 _The People's Hospital, Zhengzhou University, Zhengzhou, Henan Province, China;_ 4 _JBS Science, Inc., Doylestown, PA;_ 5 _National Cheng Kung University Medical College, Tainan, Taiwan_.

Human urine has been shown to contain DNA from circulation. In this study, we aim to provide unambiguous evidence that urine contains hepatocellular carcinoma (HCC)-derived DNA, and thus can be used for detecting HCC-associated DNA markers. We used novel hepatitis B virus (HBV)-host junction sequences (HBV-JSs), created by viral integration in the human genome, as unique markers to track the HBV-integrated DNA from tumor tissues to their corresponding urine samples. An HBV DR1-2 enriched Next-Generation sequencing (NGS) assay was developed to identify the major HBV-JSs in 15 HBV-HCC tissues. Upon validation of the major HBV-JSs by Sanger sequencing, a short-amplicon PCR assay tailed for each HBV-JS was developed to test the junctions' existence in the corresponding urine DNA. 15 major HBV-JSs were identified by the HBV DR1-2 enriched NGS assay from 15 HBV related HCC (HBV-HCC) tissues, 13 of which were validated by Sanger sequencing. By using HBV-JS specific short-amplicon junction PCR assays, we detected and confirmed six of nine HBV-JSs for which there were matching urine samples. Urine contains detectable major HBV-JSs derived from HCC, and thus can be used for liquid biopsies to study not only the complexities of HBV-JS species during chronic HBV infection and carcinogenesis, but also other HCC-related DNA modifications for the early detection of HCC and disease management.

#4935

Targeted proteomic analysis of hepatocellular carcinoma and its histologic mimickers.

Fabiola Cecchi,1 Nam Ku,2 Hanlin Wang,2 Adele Blackler,1 Todd Hembrough,1 Shahrooz Rabizadeh,3 Patrick Soon-Shiong,3 Jiaoti Huang2. 1 _NantOmics, Rockville, MD;_ 2 _UCLA, CA;_ 3 _NantOmics, CA_.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Well differentiated HCC can sometimes be extremely difficult to be distinguished from other hepatocellular mass lesions such as hepatocellular adenoma and dysplastic liver nodule due to considerable morphologic overlaps. Immunohistochemical markers may have a limited utility, especially on core biopsy.

Choice of treatment depends on the extent and location of the cancer, and the overall health of the patient. For patients who are healthy enough to undergo surgery and who have early-stage cancer confined to the liver, treatment may involve hepatic resection; however many patients develop a cancer recurrence, which is the main cause of death in long-term evaluations. Recurrence rates after treatment with resection are high, highlighting the importance of finding effective adjuvant treatments and markers that aid in determining differences between hepatocellular lesions.

To explore differences between hepatocellular lesions that could be indicators of tumor behavior, we used targeted proteomic analysis to assess different protein biomarkers in formalin-fixed paraffin-embedded (FFPE) HCC tumor tissue

Twenty-two FFPE HCC tissue blocks were obtained and a pathologist marked a minimum 8mm2 of tumor area. Following laser microdissection, proteins were extracted using the Liquid-Tissue® process and subjected to selected reaction monitoring mass spectrometry to quantify the amounts of 30 different targeted proteins.

As anticipated, well differentiated HCC, hepatocellular adenoma and dysplastic nodule expressed high rates of P-glycoprotein and the majority expressed multi-drug resistance gene protein (MDR1). Such markers may account in part for the chemotherapy refractory nature of HCC. All 22 patients expressed high levels of hENT1 and lacked expression of RRM1, indicating that gemcitabine-based therapy would be an appropriate choice. All 22 patients lacked expression of marker of sensitivity to anthracycline (TOPO2A) and the majority of patients did not express a marker of resistance to platinum therapy (ERCC1).

Of the 22 patients whose tumors expressed EGFR, 5 had expression above the 75%ile and would thus be eligible for EGFR small molecule inhibitor therapy. Dysplastic liver nodule patients did not expressed significant level of EGFR. Further, multiplex-targeted proteomics discovered patients expressing cMET and IDO1, which indicate eligibility for clinical trials of targeted therapies or immunotherapies. 60% of dysplastic liver nodule patients did not expressed cMET at any level

This study retrospectively evaluates HCC patients in an attempt to identify predictors of tumor behavior. Proteomic and genomic screening should be performed to identify differences between various hepatocellular lesions. Prospective evaluation of molecular and genetic profile is warranted in HCC patients to be distinguished from other hepatocellular mass lesions.

#4936

The function role of Brf1 in alcohol-induced human and mouse hepatocellular carcinoma*.

Shuping Zhong. _USC, Los Angeles, CA_.

Brf1 (TFIIB-related factor 1) specifically regulates the transcription of Pol III genes (RNA polymerase III-dependent genes), including tRNAs and 5S rRNAs, which are elevated in both transformed and tumor cells, suggesting that they play a crucial role in tumorigenesis. Our recent studies have demonstrated that alcohol consumption increases Brf1 expression and Pol III gene transcription to promote tumor development. However, it remains to be investigated that Brf1 is expressed in human cancers. At the present studies, we have determined the significance of Brf1 overexpression in human hepatocellular carcinoma (HCC) and the impaction of repressing Brf1 expression in alcohol-promoted cell transformation. Biopsies of human HCC, liver tumor samples of mice and cell lines of normal and tumor liver were utilized to determine alteration of Brf1 expression using cytological and molecular biological approaches. The result indicates that Brf1 expression is increased in human cases of HCC. High expression of Brf1 displays shorter overall survival period. Levels of Brf1 and Pol III gene transcription in HCC patients with alcohol consumption show an additional increase, compared with the cases of non-HCC with or without alcohol intake. Induction of Brf1 and Pol III genes by ethanol in hepatoma cells is higher than in non-tumor cells. Ethanol increases the rate of cell transformation. Repression of Brf1 inhibits alcohol-promoted cell transformation. Together, these studies demonstrate that Brf1 is overexpressed in HCC cases and liver tumor cell lines. High level of Brf1 in human HCC is correlated with short survival time. Alcohol consumption enhances Brf1 expression to promote cell transformation. Brf1 is a new biomarker of diagnosis and prognosis of HCC.

*: The project is supported by NIH grants: AA017288, AA021114 and AA02324 to Shuping Zhong

#4937

Atm expression predicts ABT-888 and CPT-11 sensitivity in gastric cancer cells by mediating p53 independent regulation of cell cycle and apoptosis.

Vinod Vijayasubhash, Shihui Tan, Fui Leng Yan, Natalia Liem, Wei Peng Yong. _National University of Singapore, Singapore, Singapore_.

Background: Identification of synthetically lethal cellular targets and synergistic drug combinations are important in cancer chemo therapy as they help to overcome treatment resistance and increase efficacy. The Ataxia Telangiectasia Mutated (ATM) kinase is a nuclear protein that plays a major role in the initiation of DNA repair signalling and cell cycle check points during DNA damage. Although ATM was shown to be associated with poor prognosis in gastric cancer, its potential implication as a predictive biomarker for cancer chemotherapy remains unexplored. The present study evaluated ATM induced synthetic lethality and its role in sensitization of gastric cancer cells to PARP and TOP1 inhibitors, ABT-888 and CPT-11 respectively.

Methods: ATM expression in a panel of 10 gastric cancer cells were detected by immunohistochemistry and the IC50 against ABT-888 and CPT-11 was determined by MTS cell proliferation assay. The synergistic effect of ABT-888 and CPT-11 in gastric cancer cells was determined by cell cycle and DNA damage assays. ATM expression was knocked down using siRNA and its effect in P53 induced cell cycle regulation and apoptosis was determined by immunoblot analysis.

Results and Conclusion: ATM deficiency was found to be associated with enhanced sensitivity to ABT-888 and CPT-11 monotherapy, hence suggesting a mechanism of synthetic lethality. Cells with high ATM expression showed reduced sensitivity to monotherapy, however showed a higher synergistic effect towards ABT-888 and CPT-11 combinational therapy that predisposed cells to enhanced drug sensitivity. Furthermore, ATM expression was shown to play a major role in cellular homeostasis by regulating cell cycle progression and apoptosis in a P53 independent manner. The present study highlights the clinical utility of ATM expression as a predictive marker for sensitivity of gastric cancer cell to PARP and TOP1 inhibition and provides a deeper mechanistic insight into ATM dependent regulation of cellular processes.

#4938

Molecular mechanisms of intrinsic resistance to taxanes in gastric cancer.

Giuseppe Galletti, Chao Zhang, Kyle Cleveland, Doron Betel, Manish Shah, Paraskevi Giannakakou. _Weill Cornell Medical College of Cornell University, New York, NY_.

Gastric cancer (GC) is histologically divided into intestinal (INT) and diffuse (DIF) clinical subtypes. Even though these two GC groups are structurally and biologically different, this classification is not used to inform choice of treatment.

Taxanes (paclitaxel, docetaxel (DTX) and cabazitaxel) are widely used for cancer treatment and for GC specifically the TAX-325 study revealed therapeutic benefit when DTX was added to the standard chemotherapy regimen. Despite these improvements, however, patients often exhibit intrinsic or acquired resistance to DTX adversely affecting patient survival. Yet, the molecular basis of clinical drug resistance remains poorly elucidated posing a major barrier for the effective treatment of GC patients.

We performed a post-hoc analysis of the TAX-325 study to examine the potential influence of GC subtypes in clinical response to DTX. We classified randomized patients as diffuse or non-diffuse histology and correlated histology with clinical outcomes using a Cox proportional hazards model. Non-diffuse GC showed a significant improvement in overall survival with the addition of DTX (12.1 v 8.8 mo, p=0.002), whereas diffuse histology was not associated with an improvement in survival (8.3 v 8.5 mo, p=0.66). To investigate the molecular mechanism of GC DIF subtype resistance to taxanes, we used a panel of 12 GC cell lines representative of both subtypes (4 intestinal subtype, 8 diffuse subtype). DTX cytotoxicity assays revealed that similarly to what we observed clinically, 63% (5/8) of the DIF GC cell lines were resistant (IC50 > 600 nM) to DTX compared to 25% (1/4) of the INT GC cell lines. Further functional studies revealed that there was minimal DTX drug-target engagement in the DIF GC cells, assessed by confocal microscopy of the microtubule (MT) network and tubulin polymerization assays. These results suggested that DTX interaction with its target, MT, was impaired in the DIF GC cell lines. To rule out multi-drug resistance (MDR) as potential cause of intrinsic DTX resistance in these cells we performed flow cytometric evaluation of P-glycoprotein and found that all of the DIF GC cell lines were negative. Additionally, drug accumulation studies with C-14 radiolabeled DTX revealed that the drug accumulated intracellularly in all of GC cell lines in our panel.

Next generation sequencing of our panel of untreated or DTX-treated GC cell lines revealed 84 significantly differentially expressed genes in drug-sensitive cell lines but no changes in resistant cells. Our analysis showed a significant enrichment and a transcriptional co-regulation after treatment of genes encoding for kinesins, motor proteins associated with MT, in DTX-sensitive cells but not in DTX-resistant cells.

These studies will provide novel insights into mechanism of drug resistance and sensitivity and will ultimately allow us to design more effective targeted therapies to overcome chemo-resistance and eventually prolong patient survival.

#4939

Evaluating a novel biomarkers set for postoperative outcome in patients with gastrointestinal cancer.

Kunizaki Masaki,1 Shigekazu Hidaka,1 Tetsurou Tominaga,1 Yoichi Furukawa,2 Takeshi Nagayasu1. 1 _Univ. of Nagasaki, Nagasaki, Japan;_ 2 _Univ. of Tokyo, Tokyo, Japan_.

Gastrointestinal cancer is the leading cause of cancer deaths in developed countries. The incidence of gastroinetestinal cancer has been increasing dramatically in Japan and has attracted worldwide attention. Despite the introduction of new postoperative chemotherapy regimens and the progress in endoscopic surgery techniques, overall survival is still poor for most advanced gastrointestinal cancer patients. The prognosis of gastrointestinal cancer patients is based on the presence of lymph node metastasis and the depth of tumour cell invasion. Usually, these parameters can be determined by microscopic examination of tissue sections from the primary neoplasm and lymph nodes. However, it is not always possible to establish a prognosis based only on the histopathological examination of cancer specimens. Therefore, there is continuing interest in prognostic factors that can permit more accurate patient stratification, improve clinical decision-making and possibly contribute to more rational study analysis. In the past few years, much progress has been made towards a better understanding of the molecular mechanisms of gastrointestinal cancer. Mutations and overexpression of p53 are common characteristics of various solid tumors. The overexpression of mutant p53 protein has been found to induce the formation of serum p53 IgG antibodies in gastrointestinal cancer patients. In addition, recent studies have revealed that the inflammation-based scoring is a useful tool for predicting postoperative outcome in gastrointestinal cancer patients. Therefore, in this study, we retrospectively reviewed a database of patients who had undergone elective surgery for gastrointestinal cancer at the Division of Surgical Oncology, Nagasaki University Hospital , and that was conducted to evaluate the usefulness of the combination of such biomarkers (CEA, CA19-9, GPS, s-p53ab etc.) for prediction of postoperative mortality in gastroinetestinal cancer patients.

#4940

Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas.

Max Jacobsen,1 George Sandusky,1 Kavita Shah2. 1 _Indiana University School of Medicine, Indianapolis, IN;_ 2 _Purdue University Center for Cancer Research, West Lafayette, IN_.

Pancreatic cancer is the 4th most common cause of cancer deaths in this country. Close to 50,000 deaths occur every year. This is due to the fact that most patients are not diagnosed until presentation at a stage IV. In a recent study, it is estimated that more than 10 years go by between the first genetic mutation and the formation of invasive cancer. More mutations occur in metastatic lesions after an additional 6 years. Both SNAIL and TWIST have gained more attention as biomarkers because that contribute to E-cadherin dysregulation that allows the tumor cells to invade. In this study we evaluated 17 cases of pancreatic ductal adenocarcinoma using twist-1 antibody to evaluate tumor cell expression in these cases. All paraffin blocks were retrieved from the IU pathology department files via an IRB approved protocol. The unstained slides were stained with primary antibody Twist-1 using the DAKO Flex system. Control sections were treated with an isotype control using the same concentration as primary antibody to verify the staining specificity. The Aperio whole slide system was used to image the slides. Computer-assisted morphometric analysis of digital images was carried out using the Aperio Image scope software using the positive pixel algorithm. The algorithm was used to quantify the Twist-1 positivity of normal pancreas, and pancreatic ductal adenocarcinoma. This algorithm is the only FDA approved algorithm for human clinical trials. The immunostaining of Twist-1 was found only in the ductal epithelial carcinoma cells in the pancreas. There was no staining in the stroma in any of the normal pancreas and ductal pancreatic carcinoma cases. We analyzed 5 normal pancreata, and 12 pancreatic ductal adenocarcinomas. The data from the image analysis was 3.38% expression in normal pancreata and 11.44% expression in the pancreatic ductal adenocarcinoma. In summary, the image analysis data demonstrates the overexpression of Twist-1 in stage IV pancreatic adenocarcinomas compared to the normal pancreas. This suggests that this marker may be a predictor of tumor invasiveness and metastasis.

#4941

Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia.

Qing Li,1 Hao Wang,2 George Zogopoulos,1 Qin Shao,1 Jun-Li Liu,1 Zu-Hua Gao1. 1 _McGill University Health Center, MONTREAL, Quebec, Canada;_ 2 _Qingdao Municipal Hospital, China_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types, with a 5-year survival rate <5%. Tumor specific biomarkers that can aid in early detection and treatment monitoring are urgently needed in order to improve the outcomes. Human Reg family proteins (Reg1A, Reg1B, Reg3A, Reg3G and Reg4) are a group of calcium-dependent (C-type) lectins that promote cell growth and respond to inflammatory stimulants. We examined their associations with pancreatic ductal adenocarcinoma (PDAC). In patients with PDAC, the serum levels of Reg1A and 1B were significantly elevated (p=0.0003 and 0.0018), which correlated with TNM stages of the patients. By using immunohistochemistry (IHC), the expression of Reg1A, Reg1B and Reg4 was observed in the malignant glandular epithelium of pancreatic intraepithelial neoplasia (PanIN) and invasive PDAC. The intensity of Reg1A and Reg1B staining positively correlated with the histological grades of PanIN. The serum levels and tissue expression of Reg1A and Reg1B were negatively correlated with the differentiation grades of invasive PDAC. Additionally, patchy expression of Reg1A andReg3A/G was also observed in areas with acinar-to-ductal metaplasia (ADM) adjacent to cancer cells. Our data suggests that the changes in Reg proteins were closely associated with the carcinogenesis of PDAC. Reg1A and 1B proteins showed specificity for pancreatic ductal differentiation and could be used as clinical markers for early detection of PDAC, monitoring therapeutic response and predicting tumor recurrence after therapy and tumor metastasis. The expression of Reg family proteins in both ADM and PanIN cells supports the notion that ADM could be an alternative mechanism of PDAC carcinogenesis.

#4942

Towards a screening blood test for esophageal adenocarcinoma.

Alok K. Shah,1 Virendra Joshi,2 Kim-Anh Le Cao,1 David C. Whiteman,3 Andrew P. Barbour,1 Michelle M. Hill1. 1 _The University of Queensland, Brisbane, Australia;_ 2 _Ochsner Health Systems, New Orleans, LA;_ 3 _QIMR Berghofer Institute of Medical Research, Brisbane, Australia_.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has been rapidly increasing globally. The majority of EAC cases are diagnosed at late stages leading to poor 5-year survival of <20%. Monitoring programs for patients with the metaplastic condition, Barrett's esophagus (BE) involves endoscopy with biopsy. However, the cost-effectiveness is limited by the low rate of progression to cancer and expense of the method. Our research program aims to improve the cost-effectiveness of screening by developing a blood test to select patients for endoscopy/biopsy. Using our integrated lectin magnetic bead array (LeMBA) glycoprotein biomarker pipeline [1, 2] with Australian cohorts for the discovery and qualification phases, we identified serum lectin-glycoprotein biomarker candidates which can distinguish EAC from BE and healthy controls [3]. Here, we report the performance of these biomarker candidates in an independent patient cohort from the Ochsner Health System, New Orleans, USA.

APPROACH & METHODOLOGY: Serum samples were collected from consenting patients undergoing endoscopy (Control - 16, BE - 13, and EAC - 10). The control group consisted of all patients not classified as BE with or without dysplasia, or EAC. LeMBA-MRM-MS was performed using 4 different lectins with different glycan specificities; AAL (Fucoseα1-2, -3, -6 linked glycan), EPHA (bisecting GlcNAc), JAC (Galα1-6GalNAc and Galβ1-3GalNAc), NPL (Manα1-6Man). Statistical analysis was performed using Shiny MixOmics as previously described [3].

RESULTS & DISCUSSION: Several lectin-protein candidate biomarkers cross-validated in the USA cohort. Most notably, differentially glycosylated complement component C9 is highly diagnostic in both cohorts, achieving area under the receiver operating curve (AUROC) of 0.74 (AAL-C9) to 0.90 (JAC-C9) as a single marker. Complement C9 binding to all four monitored lectins were increased 1.4 to 1.8 fold in EAC, compared to either BE or controls. Another consistent biomarker is gelsolin, with its binding to EPHA and NPL lectin significantly decreased in EAC, compared to either BE or controls. Hence, glycosylated C9 and gelsolin show promise as potential serum biomarkers for EAC screening, and will be further evaluated in prospective cohorts.

REFERENCES: [1] Loo et al., J Proteome Res 9, 5496-5500 (2010); [2] Choi et al., Electrophoresis 32, 3564-3575 (2011); [3] Shah et al., Molecular & Cellular Proteomics 14:3023-39 (2015).

#4943

Detection of specific KRAS mutation type, Gly12Asp (GGT>GAT), in EUS-guided fine needle aspiration cytology (EUS-FNAC) identifies pancreatic cancer patients with poor prognosis.

Marek Minarik,1 Tereza Halkova,1 Barbora Belsanova,1 Bohus Bunganic,2 Miroslav Zavoral,2 Lucie Benesova1. 1 _Genomac Research Institute, Prague, Czech Republic;_ 2 _Department of Internal Medicine, 1st Medical Faculty of Charles University, Military University Hospital, Prague, Czech Republic_.

Background

Although the disease progression of pancreatic ductal adenocarcinoma (PDAC) is very rapid with median survival typically around 3 to 6 months, there are rare cases of patients remaining on therapy for longer periods of time. Early estimation of survival prognosis would allow for rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is a great interest in finding prognostic markers that can be used for patient stratification. KRAS, often mutated in PDAC, has been a frequent subject of studies in molecular pancreatic cancer research. It was reported early on that KRAS mutation detected in pancreatic mass itself does not show any prognostic value (1). In this work we have investigated role of various KRAS mutation types on the prognosis of pancreatic cancer patients. Similar to other types of solid tumors it is expected that different KRAS mutations will result in slightly different clinical outcome (2,3)

Methods

A total of 118 pacients with PDAC clinically confirmed by EUS-FNAC were included in the study. DNA was extracted from FNAB slides following a standard cytology evaluation to ensure adequacy (viability, quantity) and to mark tumor cell fraction. Kaplan-Meier survival curves were calculated for individual KRAS mutation types.

Results summary

KRAS mutation was detected in 90% of specimes (106/ 118). All mutations were localized at exon 2, codon 12 with Gly12Asp (GGT>GAT) as the most frequent at 45% (48/106), followed by other types including Gly12Val (GGT>GTT) at 31% (33/106), Gly12Arg (GGT>CGT)at 17% (18/106), Gly12Cys (GGT/TGT) at 5% (5/106) and Gly12Ser (GGT/AGT) at 1% (1/106). Overall survival for patients with the Gly12Asp mutation was only 107 days, compared to 198 days for the Gly12Val, 286 days for Gly12Arg and 206 days for Gly12Cys (P=0.01, long-rank test). Survival of patients with the Gly12Asp was less than a half when compared to all of the other mutation types combined (107 days vs. 218 days, P=0.002, long-rank test).

Conclusions

The overall survival of PDAC patients correlates with the type of KRAS mutation present in the tumor. The most frequent Gly12Asp (GGT>GAT) mutation confers the worst prognosis resulting in a reduction of survival typically by 3 - 6 months. KRAS testing from EUS-FNAC slides presents a useful tool for stratification of PDAC patients. Supported by IGA MZ grant no. NT13638.

Literature:

1) Salek C et al. Anticancer Res. 2009, 29, 1803-1810

2) Garassino MC, Ann Oncol, 2011, 22, 235-237

3) Fiala O et al., Cancer Genet., 2013, 206, 26-31.

#4944

Detection of functional P-selectin ligands expressed on colon cancer tissue using a novel flow-based assay.

Eric W. Martin,1 Ramiro Malgor,1 Vicente A. Resto,2 Douglas J. Goetz,1 Monica M. Burdick1. 1 _Ohio University, Athens, OH;_ 2 _UTMB, Galveston, TX_.

The presence of functional P-selectin ligands is well-documented for human colon cancer cell lines, but not in situ on human colon carcinoma tissue. Presently, immunostaining with antibodies is used to detect critical components of selectin ligands, e.g., sialofucosylated moieties. However, this static biochemical tissue analysis (SBTA) cannot ascertain if a potential selectin ligand is able to mediate (rolling) adhesion. Due to the immense difficulty in detecting functional selectin ligands using traditional methods, we have developed a flow-based assay known as dynamic biochemical tissue analysis (DBTA) for detecting functional selectin ligands expressed on human tissue. DBTA using P-selectin microspheres was performed on colon cancer tissue sections from multiple cases, in conjunction with SBTA using P-selectin, antibodies against purported selectin ligand carbohydrate moieties sLeX and sLeA (HECA-452, CSLEX-1, and KM-231), and antibodies against peptide structures of putative P-selectin ligands (CD24, CD44, and PSGL-1). Examination of serial sections, in the same regions of tissue displaying DBTA probe adhesion, revealed significant detection inconsistencies with SBTA. Subsequently, due to the well-documented force-dependency of selectin ligands, DBTA was conducted with microspheres coated with either HECA-452, CSLEX-1, or KM-231 to determine the effect of applied force on the detection capabilities of these antibodies. Analysis of signet ring cell colon carcinoma tissue revealed microspheres coated with HECA-452, CSLEX-1, or KM-231 antibodies all displayed significantly lower amounts of adhesion than the DBTA P-selectin microspheres. Interestingly, although microspheres coated with these antibodies adhered to signet ring cell carcinoma tissue, these DBTA probes did not interact with all regions of tissue that displayed adhesion with P-selectin microspheres. Specificity of interaction was validated using corresponding isotype control coated microspheres. Taken together, these results show DBTA with P-selectin coated microspheres is able to unequivocally detect functional P-selectin ligands, in contrast to SBTA (immunostaining) and DBTA using microspheres coated with antibodies. In summary, DBTA using P-selectin coated microspheres is able to detect functional P-selectin ligands expressed on colon cancer tissue, data that may provide valuable diagnostic and prognostic information for malignant tumors.

### Biomarkers for Genitourinary Cancers: Prostate

#4947

**Branched chain RNA** in situ **hybridization for the detection of androgen receptor splice variants within archival prostate cancer tissue.**

Philip J. Saylor, Richard J. Lee, Kshitij S. Arora, David T. Ting, Vikram Deshpande, Miguel N. Rivera, Rong Hu, Chin-Lee Wu, David T. Miyamoto. _Massachusetts General Hospital, Boston, MA_.

Introduction:

Androgen receptor (AR) mRNA splice variants have emerged as predictive biomarkers for response to AR targeted therapies. There are no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. In this pilot study, we developed an RNA ISH assay to detect the constitutively active AR-V7 splice variant, and we correlated AR-V7 expression with clinical outcomes after first-line androgen deprivation therapy (ADT) for metastatic prostate cancer.

Methods:

We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes (Affymetrix). Automated ISH assays were performed using ViewRNA eZl Detection Kit on the BOND RX IHC and ISH Staining System. A semiquantitative scoring method was used to score the RNA ISH signal in FFPE tissue.

We analyzed AR-V7 and full length AR within two prostate cancer cohorts that we hypothesized to represent "low likelihood" and "high likelihood" to have detectable AR-V7. "Low likelihood" men had undergone prostatectomy for Gleason 6 adenocarcinoma, and "high likelihood" men had metastatic castration resistant prostate cancer with tumor tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide). We then analyzed two additional cohorts who experienced a "sustained response" (>2.5 years; n=13) or "brief response" (<9 months; n=9) to first-line ADT for metastatic prostate cancer and for whom baseline tumor tissue was available.

Results:

"High likelihood" cohort samples had detectable AR-V7 with a score of 1+ to 3+ in all samples (n=12). "Low likelihood" cohort samples had no detectable AR-V7 (n=10). Given the apparent binary distinction of AR-V7 signal among the above groups, we analyzed pre-treatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naive metastatic disease. Detectable AR-V7 was more common among "brief response" samples (6 of 9) than among "sustained response" samples (4 of 13) (not significant; P = 0.19). Patients without detectable AR-V7 RNA had significantly longer overall survival (OS, logrank P = 0.044), with a non-significant trend toward superior progression-free survival (PFS, logrank P = 0.055).

Conclusions:

Within an institutional cohort, the RNA ISH assay identified AR-V7 in all tested samples of high clinical likelihood for the splice variant RNA and no tested samples of low clinical likelihood. AR-V7 RNA was detectable in some pretreatment samples, and its presence was associated with significantly shorter overall survival. The potential prognostic and predictive utility of AR-V7 status as determined by RNA ISH from conventionally prepared FFPE tissue warrants further study in larger cohorts.

#4948

miR-375 induces docetaxel resistance in prostate cancer by targeting SEC23A and YAP1.

Yuan Wang, Rachel Lieberman, Jing Pan, Qi Zhang, Meijun Du, Ming You, Liang Wang. _Medical College of Wisconsin, Milwaukee, WI_.

Background: Castration-resistant prostate cancer (CRPC) patients are commonly treated with the chemotherapy drug, docetaxel. We previously reported that elevated miR-375 was significantly associated with poor overall survival of CRPC patients. However, the biological role and mechanism of action of miR-375 in CRPC chemotherapy remain unknown. In this study, we evaluated if miR-375 induced chemo-resistance through regulating target genes associated with multiple drug resistance.

Methods: We first compared miR-375 expression level between prostate cancer tissues and normal prostate tissues using TCGA prostate cancer data. To examine the role of miR-375 in docetaxel sensitivity, we then transfected miR-375 using a pre-miRNA lentiviral vector and examined the effect of the exogenously overexpressed miR-375 on cell growth in two prostate cancer cell lines, DU145 and PC3. To determine the effect of overexpressed miR-375 on tumor growth and chemo-resistance in vivo, we subcutaneously injected cells with miR-375 overexpression into mice. Lastly, we utilized qRT-PCR and western blot assay to examine two miR-375 target genes, SEC23A and YAP1, for their expression changes after miR-375 transfection.

Results: By comparing 495 tumor tissues and 52 normal tissues, we found that miR-375 was significantly overexpressed in prostate cancer tissues (8.45 fold increase, p value=1.98E-23). Docetaxel treatment induced higher expression of miR-375 with 5.83 and 3.02 fold increases in DU145 and PC3, respectively. Interestingly, miR-375 appeared to play a contradictory role in determining prostate cancer fate. We found that miR-375 overexpression caused cell proliferation inhibition, cell cycle arrest and cell apoptosis. However, we also found that the elevated miR-375 significantly reduced cell sensitivity to docetaxel treatment in vitro, evidenced by less apoptotic cells and higher cell proliferation. Mouse models also showed that the increased miR-375 expression was resistant to docetaxel treatment, demonstrated by greater tumor weight and sizes in miR-375 transfected cells than in empty vector controls. In addition, previous studies in other laboratories have shown that SEC23A and YAP1 are miR-375 target genes and are able to reduce cancer cell growth. We therefore examined expression levels of the two genes and observed significant expression reduction of both proteins and mRNAs in the miR-375 transfected prostate cancer cell lines. TCGA dataset analysis further confirmed the negative correlations between miR-375 and the two target genes (r= -0.62 and -0.56 for SEC23A and YAP1, respectively; p<0.0001).

Conclusions: miR-375 may confer chemo-resistance to docetaxel treatment during prostate cancer chemotherapy through directly targeting SEC23A and YAP1. Our study implies that miR-375 or its target genes, SEC23A or YAP1 might serve as potential diagnostic and/or therapeutic targets to overcome chemo-resistance in CRPC treatment.

#4949

18q copy number variation in primary prostate tumors as a predictor of long-term outcome.

Keith A. Ashcraft, Teresa L. Johnson-Pais, Jonathan A.L Gelfond, Javier Hernandez, Ian M. Thompson, Robin J. Leach. _University of Texas Health Science Center San Antonio, San Antonio, TX_.

The goal of this study is to use primary prostate tumors with long-term outcome to identify regions of copy number variation (CNV) on 18q along with other known loci to determine which regions correlate with poor prognosis.

Most prostate cancers (PCa) grow slowly and remain indolent, yet some become aggressive and metastasize. Clinical decision-making requires prognostic markers that can be utilized at the time of diagnosis to determine which tumors will become aggressive. Previous studies have shown a correlation between alterations on the long arm of chromosome 18 (18q) and metastatic PCa. Prostatectomy specimens were collected and banked in the University of Texas Health Science Center San Antonio Genitourinary tissue biorepository. Subjects were categorized as No Evidence of Disease recurrence (NED) or those who developed Metastases (MET) with a minimum of five years follow-up. Tumor foci were marked after H&E staining. Using Agilent's SureDesign software, a custom comparative genomic hybridization (CGH) array was created containing high-density tiling of 18q, as well as 15 genes located elsewhere in the genome that have been implicated in PCa prognosis. Tumor DNA was isolated and analyzed for CNV using standard CGH methodology and the Agilent Cytogenomics software. Chromosome-wide (18q) and genome-wide statistical significance of the relative CNV anomaly prevalence (NED vs. MET) was determined with permutation testing.

Thirty-five primary prostate tumors were analyzed; 17 were classified as NED and 18 as MET. Two significant regions of copy number gains were found on 18q. One gain at 18q21.31 was found only in MET samples while another gain at 18q11.2 was found in NED samples. Additionally, confirmatory gains and losses in the regions containing 15 PCa related genes were found, only three of which were statistically different between the two sets.

Our arrayCGH results implicate DNA gain of copy at 18q21.31 as being predictive of poor outcome. MicroRNA-122, which lies within this region, has been shown to have increased expression in prostate tumors with higher Gleason score and has been implicated in breast cancer metastasis. Our data suggests it may be an important prognostic marker for aggressive PCa. The 18q11.2 region found to be amplified in NED samples warrants further study to identify genes in this region that may predict good outcome. Of the 15 gene regions included, MYC and Cyclin D1 regions both exhibit gain of copy number in primary tumors with poor outcome, in line with other reports that these gains that these are early events in PCa progression.

#4950

Collagen, type I, alpha 1 (COL1A1): a potential urinary biomarker that can distinguish between benign prostate hyperplasia and localized prostate cancer.

Andrej Jedinak,1 Camille Vuichoud,2 Andrew El-Hayek,3 Kevin R. Loughlin,4 Marsha A. Moses1. 1 _Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 2 _Boston Children's Hospital; Brigham and Women's Hospital, Boston, MA;_ 3 _Boston Children's Hospital, Boston, MA;_ 4 _Brigham and Women's Hospital, Harvard Medical School, Boston, MA_.

Benign prostate hyperplasia (BPH) is the most frequent benign disease among men worldwide and its incidence increases with age. Prostate cancer (PCa) and BPH share similar symptoms and an elevated serum prostate-specific antigen (PSA) can be observed with either benign or malignant growth of the prostate. It is the demographic overlap of BPH and PCa, and the lack of discrimination between these two prostate diseases by PSA, that defines the diagnostic dilemma faced by clinicians when treating prostate disease. The goal of the current study was to identify novel non-invasive, biomarkers that can distinguish between BPH and PCa. We utilized iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry techniques to sensitively and accurately identify the urinary proteome of men with BPH vs. men with PCa. We then performed functional enrichment analysis and pathways enrichment analysis of these proteins using Ingenuity Pathway Analysis (IPA) tools to determine differentially expressed pathways and functions in PCa as compared to BPH. Network analysis identified differences in a number of signaling molecules including ERK1/2, TGFβ, PI3K, p38 MAPK and NFκB, with a high degree of interactivity. Protein expression was validated by immunoblot analyses using monospecific antibodies. Here, we focused on collagen, type I, alpha 1 (COL1A1), one of a number of extracellular matrix-associated proteins detected. We found that COL1A1 was significantly (P < 0.001) elevated in the urine of patients diagnosed with early or localized PCa but not in the urine of men with BPH. Complementary studies were conducted on human BPH and PCa cells. Our data suggest that COL1A1 may represent a novel non-invasive biomarker that can discriminate between BPH and early PCa. (Supported by The Ellison Foundation)

#4951

FGFRL1 in prostate cancer progression.

Lan Yu,1 Andrew Erickson,2 Mervi Toriseva,1 Teresa Elo,1 Johanna Tuomela,1 Heikki Seikkula,3 Martti Nurmi,3 Peter Boström,3 Tuomas Mirtti,4 Kalle Alanen,5 Markku Kallajoki,5 Matthias Nees,6 Pirkko Härkönen1. 1 _Institute of Biomedicine, Cell Biology and Anatomy, University of Turku, Turku, Finland;_ 2 _Institute for Molecular Medicine Finland, Helsinki, Finland;_ 3 _Department of Urology, Turku University Hospital, Turku, Finland;_ 4 _Department of Pathology, Helsinki University Hospital (HUSLAB) and Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland;_ 5 _Department of Pathology, University of Turku, Turku, Finland;_ 6 _Institute of Biomedicine, Cell Biology and Anatomy, University of Turku, and Turku Centre for Biotechnology, University of Turku, Turku, Turku, Finland_.

Prostate cancer (PCa) is a disease with high incidence, however, many PCa patients are over-diagnosed and over-treated. Molecular characterization of PCa provides a valid approach to stratify patients, and thus reduce overtreatment. Fibroblast growth factors and their receptor (FGF/FGFR) signaling pathways are involved in various cellular functions such as proliferation, differentiation, migration, and apoptosis of prostate cancer cells. Dysregulated and constitutively activated FGF/FGFR pathways have been shown to be involved in the initiation and progression of prostate cancer. Fibroblast growth factor receptor like 1 (FGFRL1, FGFR5) is the most recently identified member of the FGFR family. FGFRL1 binds several FGFs but its short intracellular part lacks a tyrosine kinase domain. Therefore, the extracellular domain has been suggested to act as a dominant negative regulator of other FGFRs. However, cellular functions of FGFRL1 remain poorly understood. Aberrant FGFRL1 expression has been reported in ovarian, bladder, colon, and other cancers. In silico data analysis indicated altered FGFRL1 mRNA expression in 17% of PCa cases. To date, there have been no systematic studies of FGFRL1 expression and function in prostate and PCa. We studied FGFRL1 expression and function in PCa cell lines, xenografts and in human PCa specimens. FGRL1 was knocked-down in PC-3M cells, by shRNAs, which showed reduced growth as nude mouse xenografts when compared to control. This argues against a dominant negative function. To study FGFRL1 in human PCas, we collected formalin-fixed paraffin-embedded samples from PCa patients undergoing radical prostatectomy (n=243), metastatic PCa (n=36) and castration-resistant PCa (n=21). Samples were prepared for mRNA analysis, and immunohistochemistry. The overall levels of FGFRL1 immunostaining were significantly increased in PCa compared to normal tissue. In normal tissue, FGFRL1 immunostaining localized to the cell membrane and to a lesser extent to the cytoplasm and nuclei. In PCa, protein expression of FGFRL1 was decreased at the cell membrane, while expression in the cytoplasm and nucleus were increased. Low membrane and high nuclear immunostaining of FGFRL1 correlated with high Gleason grade. High nuclear FGFRL1 also correlated with high levels of preoperative serum PSA and an increased proportion of tumors with positive surgical margins. Our results suggest that FGFRL1 may play an active role in PCa cells and in tumor progression and can possibly be used to assess PCa prognosis.

#4952

Co-expression of pAKT and MYC is associated with aggressive disease in human prostate cancer.

Nelma Pértega-Gomes,1 Svitlana Tyekucheva,1 Kathryn Penney,2 Giorgia Zadra,1 Lorelei A. Mucci,2 Massimo Loda1. 1 _Dana Farber Cancer Institute, Boston, MA;_ 2 _Harvard School of Public Health, Boston, MA_.

Introduction: AKT and MYC are amongst the most prevalent oncogenes in prostate cancer (PCa). MYC cooperates with activated AKT in pre-clinical, genetically engineered in vivo models accelerating disease progression and increasing resistance to therapy. However, the clinical relevance of phosphoAKT/MYC co-expression in human prostate tumors remains unknown.

Experimental Procedures: A prospective cohort (Physicians' Health Study and Health Professionals Follow-up Study) of 578 men with PCa with a mean 13-year follow up was used. Protein expression of MYC and phosphoAKT (Serine 473) was characterized by immunohistochemistry on tissue microarrays. pAKT and MYC expression was analyzed using the Cri Vectra/InForm multispectral analysis platform. The InForm software package was trained to segment PCa regions only (i.e., no stromal regions were included in the analysis). We used quantile normalization to adjust for the potential batch effects of protein expression staining across each of the tissue microarrays using the limma package R. The continuous score for pAKT and MYC were divided into quartiles and assigned a value of 1, 2, 3 or 4 for low (1), moderate (2 or 3) and high staining (4). Spearman correlations between pAKT, MYC and the clinico-pathological data were calculated.

Results: High pAKT and High MYC were co-expressed in 10% of patients. (Quartile 4). Also 10% were negative or weakly positive for both pAKT and MYC (Quartile 1). Only 3% of the High pAKT patients had weak or no expression of MYC and 4% of High_MYC patients had Low pAKT expression. The majority of patients were distributed in the Quartiles 2 and 3 (73%). Importantly, only High pAKT and High pMYC co-expressors were associated with tumor stage (pT3 or pT4) (p=0.03), high Gleason Score (p=0.021) and presence of metastatic disease (p=0.011). GEP analysis showed that the same patients exhibited up-regulation of genes involved in cell invasion/migration capacity and stromal remodeling.

Conclusions: Co-expression of AKT and MYC is associated poor clinical outcome in PCa patients. Gene expression profiling suggest that concomitant activation of AKT and MYC results in the activation of pathways associated with an invasive phenotype.

#4953

Whole-exome sequencing of single circulating tumor cells according to epithelial-mesenchymal marker expression in metastatic prostate cancer.

Vincent Faugeroux,1 Céline Lefebvre,1 Emma Pailler,1 Valérie Pierron,2 Fanny Billiot,1 Charles Marcaillou,3 Philippe Vielh,1 Semih Dogan,1 Philippe Rameau,1 Maud Ngocamus,1 Jean Charles Soria,1 Karim Fizazi,1 Yohann Loriot,1 Sylvia Julien,2 Françoise Farace1. 1 _Institut Gustave Roussy Inserm U981, Villejuif, France;_ 2 _IPSEN Innovation, Les Ulis, France;_ 3 _Integragen, Evry, France_.

Molecular characterization of metastatic castration-resistant prostate cancer (mCRPC) is limited by tumor tissue availability. The analysis of circulating tumor cells (CTCs) offers an attractive non invasive surrogate option to analyze molecular alterations. We report whole exome sequencing (WES) of CTCs at the single cell level in 11 mCRPC patients. We examined single somatic nucleotide variant (sSNV) shared between matched metastatic tumor sample and CTCs and sSNV specific to CTCs.

Blood samples were drawn from 11 patients enrolled in the clinical program MOSCATO (2011-A00841-40). CTC enrichment, detection and single cell isolation were performed using three methods to obtain pools of 1-10 CTCs. The first method used ISET filtration, immunofluorescent staining (CD45, pan-cytokeratin, EpCAM, Vimentin and Hoechst 33342) on filters and laser microdissection of single CTCs; the second combined CellSearch and the VyCap puncher system; the third used RosetteSep enrichment, immunofluorescent staining and isolation by cell sorting. Whole Genome Amplification (WGA) was performed using the Ampli1 kit. WGA quality was assessed by qPCR of 7 genes located on different regions of the genome. WES was performed by preparation of a genomic DNA bank, Agilent capture and sequencing on the Illumina HiSeq 2000 platform. Data were aligned to the human genome reference hg19. GATK Haplotype Caller enabled identification of germline polymorphisms from each patient in normal DNA, metastatic sample and CTCs in order to consider WGA induced bias. The detection of sSNV in tumor biopsies and CTCs was assessed with Mutect and IndelGenotyper respectively.

189 WGAs of CTC pools were performed. A first round of WES showed that at least 3 well amplified genes were required to obtain a coverage of at least 50% at 10X depth sequencing. 34 pools of phenotypically different CTCs from 7 patients were selected and sequenced. Mean coverage of 51% was obtained at a sequencing depth of 10X. Allelic drop out was lower for CTC pools containing 5 to 10 cells. 17/34 (50%) CTC samples (4 patients) had shared sSNV with the paired tumor sample (range 0.35%-68%). Epithelial CTCs had more shared sSNV with metastatic biopsies than CTCs of other phenotypes but shared sSNV were also detected in large Cytokeratin-Vimentin- CTC. Shared sSNV in cancer genes between epithelial CTC pools, but not in the paired biopsy, were present in 2 patients.

We report WES of CTC pools harboring distinct EMT marker phenotypes is possible with the use of 3 different approaches to enrich, detect and isolate CTCs. The detection of shared sSNV between CTC pools and corresponding biopsy could validate the use of CTCs as a liquid biopsy. The finding of sSNV specific to CTCs could offer additional data on tumor heterogeneity. Ongoing work examining if sSNV detected in phenotypically different CTCs converge to similar signaling pathways will be presented.

#4954

Nuclear localized AR-V7 protein as a predictive biomarker for treatment selection in metastatic castration resistant prostate cancer (mCRPC).

Howard I. Scher,1 David Lu,2 Nicole A. Schreiber,1 Jessica Louw,2 Ryon P. Graf,2 Ann Johnson,2 Adam Jendrisak,2 Glenn Heller,1 Richard Bambury,1 Herbert A. Vargas Alverez,1 Brigit McLaughlin,1 Justin Wahl,2 Stephanie Greene,2 Martin Fleisher,1 Ryan Dittamore2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Epic Sciences, Inc., San Diego, CA_.

Background: A critical decision in the management of patients (pts) with mCRPC is when to administer an androgen receptor signaling (ARS) directed or a taxane therapy. The detection of AR-V7 mRNA in CTCs has been shown to predict for resistance to ARS,but not to taxane chemotherapy. We evaluated the relationship between AR-V7 protein expression and localization on CTCs to treatment outcomes in a separate, larger cohort as a predictive biomarker for clinical decision making.

Methods: 193 prospectively collected blood samples from 161 unique pts with progressive mCRPC about to start an ARS or taxane therapy were evaluated with an Epic Sciences CTC immunoflorescent assay that assesses CTC AR-V7 protein expression and localization in individual

cells. Associations between the presence AR-V7(+) CTCs pre-therapy and anti-tumor effects post-therapy included prostate-specific antigen (PSA) changes, radiographic progression free survival (rPFS), time on therapy, and overall survival (OS).

Results: 130 pre-ARS inhibitor and 63 pre-taxane samples were assessed of which 191 (99%) were evaluable. AR-V7(+) CTCs were found in 34 (18%) samples including 3% of the 1st, 18% of the 2nd and 31% of the 3rd+ line baseline pre-therapy samples. Patients with AR-V7 positive CTCs in a pre-ARS sample showed no PSA response and had shorter rPFS, time on therapy, and OS than those without AR-V7(+) CTCs. PSA resistance to ARS was also seen in 65 of 112 (58%) of the AR-V7(-) samples. There was no association between PSA response, rPFS, and time on therapy between AR-V7(+) and AR-V7(-) pts treated with taxane therapy. In a multivariate model adjusting for age, type of therapy, line of therapy, and pre-therapy LDH, Hgb, and presence of visceral metastasis, AR-V7(+) pts had a superior OS on taxane therapy relative to ARS (HR: 0.242, CI: 0.103 to 0.569, p = 0.0350). Conclusions: The results validate the expression of the AR-V7 protein in the nucleus of CTCs in men with mCRPC as a treatment specific biomarker that predicts resistance to ARS inhibitor therapy and separately, clinical benefit with taxane therapy over ARS-directed therapy in a clinical practice setting. Continued examination of this biomarker in prospective studies will further determine its clinical utility.

#4955

Isolation and characterization of circulating tumor cells (CTCs) in breast and prostate cancer: Comparison of Harpoon CTC assay performance with the CellSearch CTC Test.

Mark Connelly,1 David Chianese,1 Carrie Morano,1 Thai Bui,1 Shemeeakah Powell,1 Noel Ngoubilly,1 Renouard Sanders,1 Tom Barber,2 Ravi Kapur,3 Shyamala Maheswaran,4 Mehmet Toner,2 Daniel Haber4. 1 _Janssen Diagnostics, LLC, Huntingdon Valley, PA;_ 2 _Massachusetts General Hospital, Boston, MA;_ 3 _Harvard Medical School, Boston, MA;_ 4 _Massachusetts General Hospital and Harvard Medical School, Boston, MA_.

Circulating tumor cells (CTCs) can provide important information about a patient's prognosis. Currently, the CELLSEARCH® CTC Test (hereafter referred to as CS) is the only US Food and Drug Administration-cleared assay for isolating and enumerating CTCs. This technology uses a CTC antigen-dependent method to isolate CTCs that may limit its ability to detect CTCs with low or no epithelial cell adhesion molecule (EpCAM) expression. We evaluated the Harpoon CTC Isolator and Chip, which uses an antigen-independent, negative depletion method to enrich CTCs from patient blood samples. As a result of the negative depletion method, the final Harpoon Product (ie, CTC-enriched cell suspension) contains CTCs that express cytokeratin (CK) and EpCAM (canonical CTCs) as well as those with low or no expression of these antigens (non-canonical CTCs). After isolation, CTCs were spun onto microscope slides and imaged using a Vectra Multispectral Imaging System. We obtained performance data on an initial prototype Harpoon assay (Harpoon + Vectra [Hrp+Vec]) and compared CTC enumeration between Hrp+Vec and CS using blood samples from 36 breast and prostate cancer patients. From 7.5 mLs of blood, the Hrp+Vec assay recovered 79.0% to 93.5% of canonical EpCAM+/CK+ CTCs recovered by CS. The Hrp+Vec assay also successfully detected non-canonical CTCs (EpCAM-/CK+ or EpCAM+/CK-) in these samples. At the CS clinical cutoff for prognostic value, where ≥5 CTCs is associated with poor prognosis, the Hrp+Vec assay was 97% concordant with CS. Hrp+Vec also detected CTCs in 12.5% (2/16) more patient samples than CS. Finally, a consensus analysis of all Vectra scan results (including both canonical and non-canonical CTCs) demonstrated that the Hrp+Vec assay detected between 90.5% and 108.9% of the number of CTCs as identified by CS. These results indicate that, when compared with CS, the prototype Hrp+Vec assay detected CTCs with similar efficiency and identified more samples as CTC-positive in these breast and prostate cancer patients. Importantly, the Hrp+Vec assay did enrich non-canonical CTCs thought to be missed by CS, and ongoing efforts are aimed to further characterize these cell populations.

#4956

Droplet digital PCR in validating copy number variations in hereditary prostate cancer families from Louisiana.

Kirsten Wood,1 Ratish Gambhira,2 Elisa Ledet,2 Oliver Sartor,2 Diptasri Mandal1. 1 _Louisiana State University Health Sciences Center, New Orleans, LA;_ 2 _Tulane Cancer Center, New Orleans, LA_.

Prostate cancer (PCa) is the second leading cause of death in American men, and approximately 27,540 men will die from the disease in the US this year. Louisiana has one of the highest rates of PCa in the country, with approximately 4,000 new cases each year. Family history is one of the most prominent predictors of PCa risk, and approximately 10% of PCa cases are attributable to inheritable genetic factors. However, identifying PCa genetic susceptibility has been extremely difficult due to genetic heterogeneity. Genomic copy number variations (CNVs) have been detected in prostate tumors and several other cancers, but changes in copy number have not been studied extensively in germ-line DNAs from hereditary PCa (HPC) cases. The goal of the present study is to validate the presence of CNVs using a cutting edge technology Droplet Digital Polymerase Chain Reaction (ddPCR). ddPCR provides absolute quantification of target DNA copies and can measure small fold differences in target DNA copy numbers. We hypothesize that ddPCR will precisely validate the CNVs identified using array comparative genomic hybridization (aCGH) technology.

In our preliminary study, we have identified CNVs in germline DNAs from 7 HPC cases from 7 Louisiana HPC families of European ancestry by using aCGH technology. The regions identified were 2q22, 11q22, and 16q23, and these regions were found to be duplicated in all HPC cases analyzed. In addition, a 68 kb duplication was observed at 2q14 in all 7 HPC cases of European ancestry, and this region was further identified by using linkage analysis in 4 of the 7 HPC families (heterogeneity LOD-score = 1.94). In order to validate the duplication observed at 2q14 (BIN1), we performed ddPCR. Each ddPCR reaction uses duplex TaqMan assay reagents for the target region and the reference gene. Analysis of ddPCR data is conducted using QuantaSoft Analysis software. From this study we found that several of our HPC cases had germline copy number values of 2.2 or higher in the BIN1 region, suggesting that these samples may have partial gene amplification at the 2q14 region rather than duplication of the entire gene. These findings suggest that ddPCR is capable of providing absolute quantification of copy number differences. Using the recently launched custom design ddPCR Copy Number assays for our specific regions of interest, we will perform analysis on additional CNV regions in affected as well as in other unaffected family members from these HPC families. The absolute quantification of copy number differences using ddPCR will contribute to the impending need of cancer biomarker identification in the era of precision medicine, and will allow us to facilitate prostate cancer screening in HPC families.

#4957

Keratin 13 drives the dissemination of bone and brain metastases of human prostate and breast cancer cells.

Lijuan Yin,1 Qinlong Li,2 Jen-Ming Huang,1 Michael Lewis,3 Isla P. Garraway,4 Quanlin Li,1 Hong Bu,5 Leland W. K Chung,1 Haiyen E. Zhau1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _Fourth Military Medical University, Xian, China;_ 3 _West Los Angeles VA Medical Center, Los Angeles, CA;_ 4 _David Geffen School of Medicine at UCLA, Los Angeles, CA;_ 5 _West China Hospital, Sichuan University, China_.

Introduction and Objective: Bone and brain metastases are prevalent in human prostate and breast cancer patients. Keratin 13 (KRT13), a member of the intermediate filament proteins and an epithelial stem cell differentiation marker, induces cancer cell migration and progression toward increased malignant state. The objectives of this study are: 1) to determine KRT13 expression in human prostate and breast cancer and its clinical correlation; 2) to determine the functional cell signaling role of KRT13 in prostate and breast cancer cells using gene transfer technology.

Methods and Results: We evaluated KRT13 protein expression in primary prostate cancer tissues from 44 cases with well-documented overall survival by quantitative quantum dot immuno-labelling. We found that KRT13 expression in primary prostate cancer correlated with patient overall survival and bone metastasis (p<0.05). We next evaluated KRT13 protein expression in 36 prostate and 58 breast cancer tissue specimens by immunohistochesmistry (IHC) and found that KRT13 is overexpressed in breast cancer bone (5/8) and brain (7/10) metastases and prostate cancer bone (6/12) metastasis specimens (p<0.05). KRT13 expression in primary prostate and breast cancer specimens is associated with decreased patient overall survival based on our data as well as publicly available Oncomine gene expression datasets. To elucidate the mechanisms by which KRT13 promotes cancer invasion, migration and metastasis, we genetically overexpressed KRT13 in human prostate CWR22 Rv1 and LNCaP, and human breast MCF7 and MCF10A cells, with respectively high or low malignant potential. KRT13-overexpressing prostate and breast cancer cells increase differential expression of genes associated with epithelial to mesenchymal transition (EMT; E-Cadherin and Vimentin), stemness (Nanog), neuromimicry (Chromagranin A and Synaptophysin), osteomimicry (RANKL) related genes as analyzed by qRT-PCR, western blot, and IHC. KRT13 also promotes increased cancer cell proliferation, migration and invasion in vitro, and bone and brain metastases in vivo.

Conclusion: These results suggest that KRT13 drives prostate and breast cancer bone and brain metastases via osteomimicry and neuromimicry associated genes resulting in downstream cell signaling network convergence between EMT, stemness, cell growth, and survival pathways.

Funded in part by NCI-P01-CA098912

#4958

Immunobiomarkers: novel autoantibody panel comprising oncogenic ERG, C-MYC, AMACR and HERV-K Gag for the detection of prostate cancer.

Anshu Rastogi,1 Amina M. Ali,2 Sreedatta Banerjee,1 Lakshmi Ravindranath,1 Gyorgy Petrovics,1 Shyh-Han Tan,1 Jennifer Cullen,1 Yongmei Chen,1 Denise Young,1 Isabell A. Sesterhenn,3 Jacob Kagan,4 Sudhir Srivastava,4 David G. McLeod,1 Shiv Srivastava,1 Alagarsamy Srinivasan1. 1 _Uniformed Services University of the Health Sciences & Walter Reed National Military Medical Center, Rockville, MD; _2 _Walter Reed National Military Medical Center, Bethesda, MD;_ 3 _Joint Pathology Center, Silver Spring, MD;_ 4 _National Cancer Institute, Bethesda, MD_.

INTRODUCTION: Biomarkers for early detection as well as disease stratification are a major area of focus in prostate cancer (CaP) research. Autoantibodies against a large array of tumor associated antigens (TAAs) have been noted in a diverse number of cancers, including CaP. Since autoantibodies represent humoral responses elicited in response to TAAs expressed in tumor cells, it is likely that autoantibodies could serve as diagnostic/prognostic markers for CaP. Hence, the present study addresses the following: i) Are autoantibodies against ERG, an oncogene frequently overexpressed in CaP, present in the sera of patients?; ii) Does a multiplex autoantibody panel containing ERG, AMACR, C-MYC, and HERV-K Gag improve the detection of CaP?

METHODS: Sera from 50 CaP patients positive for ERG expression and 50 patients negative for ERG expression, along with 50 healthy control subjects were analyzed. Purified recombinant protein for ERG, AMACR, and C-MYC as well as synthetic peptides corresponding to epitopes in ERG and HERV-K Gag were used in an enzyme-linked immunosorbent assay (ELISA) developed in our laboratory to detect autoantibodies in the sera of CaP patients. In addition, a luciferase immunoprecipitation assay (LIPS), involving a chimeric luciferase-ERG protein, was used as an independent assay to test the presence of anti-ERG autoantibodies.

RESULTS: Analysis of patient sera by ELISA revealed significantly higher detection of autoantibodies against ERG (P = 0.0001), AMACR (P < 0.0001), C-MYC (P = 0.0013), and HERV-K Gag (P < 0.0001) proteins in CaP cases compared to healthy controls. The specificity of anti-ERG autoantibodies was supported by a competitive ELISA assay. Further, the LIPS assay was also able to detect anti-ERG autoantibodies in patient sera. In addition, a multiplex panel assay involving ERG, AMACR, and HERV-K Gag greatly improved the detection of true positive CaP cases (AUC = 0.792).

CONCLUSIONS: Here we demonstrated that autoantibodies against ERG are present in the sera of prostate cancer patients through ELISA analysis. In addition, utilizing a panel of genes for autoantibody detection, consisting of ERG, AMACR, and HERV-K Gag, showed high specificity for CaP cases. These promising findings suggest the diagnostic potential of autoantibodies and similar autoantibody panels in enhancing CaP diagnosis.

Source of Funding: Center for Prostate Disease Research, Uniformed Services University Grant HU0001-10-2-0002, NCI/EDRN Grant ACN12011-001-0, and the National Cancer Institute Grant R01CA162383.

#4959

Multi-level analysis of prostate cancer circulating tumor cells allowing IHC-based identification, 6-parameter fluorescence phenotyping, and individual cell molecular analysis.

Daniel Campton,1 Rachel Needham,1 Josh Nordberg,1 Arturo Ramirez,1 Nick Drovetto,1 Alisa Clein,2 Daniel E. Sabath,2 Jackie Stilwell,1 Eric Kaldjian1. 1 _RareCyte, Inc., Seattle, WA;_ 2 _University of Washington, Seattle, WA_.

Background. Analysis of circulating tumor cells (CTC) allows non-invasive investigation of prostate cancer biology and response to treatment. The primary level of analysis is the CTC count, which has been demonstrated to be prognostic of outcome. Deeper characterization of CTC phenotype and pertinent biomarkers can confirm cancer lineage and identify drug targets or drug-resistance markers. Single cell analysis of individual CTCs can provide genomic insight into cancer heterogeneity. RareCyte has developed AccuCyte® - CyteFinder® (AC-CF), an integrated technology platform for highly sensitive visual identification and retrieval of rare cells in blood by both immunohistochemistry (IHC) and immunofluorescence (IF) staining. Recently we have developed technology allowing 6-marker assays for broader phenotypic analysis.

Methods. Normal human whole blood samples were spiked with prostate cancer lines as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced prostate cancer were collected under an IRB-approved protocol. Blood was processed using AccuCyte and the nucleated cell fraction was collected and spread onto microscope slides. Slides were stained on an automated stainer using (1) an IHC assay for cytokeratin, (2) a standard 4-wavelength IF assay (DAPI, CD45, cytokeratin and EpCAM) or (3) a novel 6-parameter IF assay using SYTOX-Orange (nuclear stain), cytokeratin, EpCAM, androgen receptor (AR), prostate-specific membrane antigen (PSMA) and CD45. An assay for AR variant 7 (ARv7) was applied to samples with mCTCs with the ARv7 splice variant. Percent recovery of IHC-stained slides (by blinded pathologist review) was compared to IF-stained slides (by CyteFinder image analysis). Individual IHC-stained CTCs were retrieved after on-slide visual identification and re-visualized after dispensing for confirmation. Whole genome amplification (WGA) of retrieved cells was performed, followed by X- and Y-chromosome gene-specific PCR.

Results. There was strong linear correlation between IF and IHC counts of mCTCs over a range of ~25 - 100 cells/mL (R2 = 0.99). The 6-parameter IF assay was successfully applied to mCTC and clinical samples. AR and PSMA were co-expressed in the majority of epithelial-marker positive clinical CTCs. The ARv7 assay identified mCTCs that express the splice variant. Individual IHC-stained mCTCs spiked into female donor blood were demonstrated to be male after WGA and PCR.

Conclusions. Light microscope identification of IHC cytokeratin-positive mCTCs approximated IF identification. 6-parameter phenotyping of prostate cancer CTCs is feasible and allows identification of lineage-specific markers. IHC-stained cells can be individually retrieved from slides for genome amplification and molecular analysis.

#4960

Molecular characterization of in-vivo isolated EpCAM-positive circulating tumor cells in high-risk prostate cancer patients.

Athina N. Markou,1 Marifili Lazaridou,1 Panagiotis Paraskevopoulos,1 Shukun Chen,2 Thomas Kroneis,2 Monika Swierczewska,3 Joanna Budna,3 Andra Kuske,4 Tobias M. Gorges,4 Maciej Zabel,3 Peter Sedlmayr,2 Catherine Alix-Panabieres,5 Klaus Pantel,4 Evi S. Lianidou1. 1 _Analysis of Circulating Tumor Cells Lab, Department of Chemistry, University of Athens, Athens, Greece;_ 2 _Institute for Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, Graz, Austria;_ 3 _Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland;_ 4 _Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;_ 5 _University Institute for Clinical Research (IURC), Laboratory of Rare Human Circulating Cells, University Medical Centre of Montpellier Saint-Eloi Hospital, EA2415, Montpellier, France_.

Introduction: CTC have been verified as prognostic markers for disease progression in various cancer types. The main aim of the EU project "CTC-SCAN" is to validate the number of CTC isolated from patient's blood as a prognostic marker for relapse in high-risk prostate cancer patients treated with primary radical prostatectomy or radiotherapy. In this study, we present our results on gene expression profiling of CTCs that were isolated, using the CellCollector, a novel clinical device designed for the in vivo isolation of EpCAM-positive CTCs.

Patients and methods: We first developed and validated 3 multiplex and 3 single-plex highly sensitive RT-qPCR assays amplifying:a)Epithelial markers:CK-19,EpCAM,E-CAD & PBGD, b)Stem cell markers:PSCA,ALDH1,CD133& HPRT, c) EMT markers: TWIST, VIM, N-CAD and B2M and d)PSA, e)TMPRSS2-ERG fusion, f)Plastin-3. 62 patients and 36 healthy volunteers participated in the study. After in vivo isolation, total RNA was extracted from captured cells,followed by cDNA synthesis. RT-qPCR was performed for the molecular characterization of captured cells. In all cases, peripheral blood was also collected for CTC analysis by CellSearch and the EPISPOT.

Results: The findings of our study are summarized in Table 1. Briefly, in 13/15(87%) samples, in which at least one cell was detected by CellSearch, we detected the expression of at least one gene. In 28/47 samples, negative by CellSearch, we detected the expression of several genes by the developed RT-qPCR assays. In 9/14 samples that were exclusively found to be positive by EPISPOT for PSA immunospots, at least one of the analyzed genes was also expressed.

Conclusions: In vivo isolation of CTC in combination with a downstream molecular analysis is minimally invasive, and in combination with high specific and sensitive RT-qPCR assays for CTC detection and molecular characterization seems very promising. Comparison studies with the CellSearch and the EPISPOT have shown a higher sensitivity, but a poor agreement. | |

in vivo(n=18) | in vitro(n=18)

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

GENE | POSITIVE(%) | POSITIVE(%) | POSITIVE(%)

CK-19 | 20(32%) | 1(0.5%) | 0(0%)

E-CAD | 0(0%) | 0(0%) | 0(0%)

EpCAM | 16(26%) | 1(0.5%) | 0(0%)

CD133 | 0(0%) | 0(0%) | 0(0%)

ALDH1 | 11(18%) | 0(0%) | 0(0%)

PSCA | 0(0%) | 0(0%) | 0(0%)

VIMENTIN | 10(16%) | 0(0%) | 0(0%)

TWIST | 15(24%) | 0(0%) | 0(0%)

N-CAD | 11(18%) | 0(0%) | 0(0%)

PLASTIN-3 | 6(10%) | 0(0%) | 0(0%)

PSA | 6(10%) | 0(0%) | 0(0%)

TMPRSS:ERG | 0(0%) | 0(0%) | 0(0%)

CELLSEARCH | |

|

(cut-off>1cells/ml) | 15(24%) | |

0(0%)

EPISPOT | |

|

|

15(24%) | |

0(0%)

#4961

Molecular heterogeneity in diverse prostate cancer circulating tumor cell subsets.

Jamie M. Sperger, Erika Heninger, Jennifer L. Schehr, Haley E.F. Allen, Anupama Singh, Joshua M. Lang. _University of Wisconsin, Madison, WI_.

Purpose:

Circulating tumor cell (CTC) enumeration or Androgen Receptor (AR) splice variant 7 expression from Epithelial Cell Adhesion Molecule (EpCAM) positive CTCs are predictive biomarkers for patients with prostate cancer. Recent reports suggest subpopulations of CTCs with decreased EpCAM expression may have greater invasive capacity or drug-resistant potential. These include cells with mesenchymal phenotypes that express N-cadherin, or MUC1, or stem cell populations that express CD133. Integrated molecular analysis across different CTC subpopulations has not been performed. We sought to compare the molecular profile of different populations of CTCs from the blood of patients with prostate cancer.

Methods:

A multiparametric flow cytometry assay was used to identify different populations of CTCs from patients with prostate cancer. We then employed an integrated CTC capture and analysis technology known as the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform to in parallel or sequentially capture CTC subsets based on differential cell surface marker expression. Target populations of CTCs include those expressing EpCAM, N-Cadherin, MUC1 or CD133. mRNA was extracted on chip for comparison of gene expression profiles for the AR, multiple AR splice variants, downstream targets in the canonical AR signaling pathway

Results

Flow cytometry results showed both intra- and inter-patient heterogeneity in populations of CTCs from patients with prostate cancer. Using flow cytometry we are able to identify CTCs at numbers comparable.to those isolated in the VERSA. We identified putative tumor cells (AR+Cytokeratin+CD45-) that co-expressed Epcam with mesenchymal or stem cells markers. Patients differed in cell surface marker compositon with ranges of 0-40% Epcam+/N-Cad+, 10-27% Epcam-/Ncad+ and 5-10% Epcam +/Ncad-. Based on this data, we captured CTCs using both parallel and serial capture from the same blood sample iwth different capture antibodies including EpCAM, N-Cadherin and CD133. We identified full length androgen receptor gene expression from CTCs captured with EpCAM and N-Cadherin from the same patient sample. The gene expression patterns of the Epcam-/N-cadherin+ CTCs varies across patients with differential expression of PSMA, PAP and PSA.

Conclusions

Prior reports have identified different populations of CTCs in patients with advanced prostate cancer. Using flow cytometry and a CTC capture methodology, we identified significant heterogeneity in CTC sub-populations. We confirmed expression of the full length Androgen Receptor with gene expression analysis across subtypes. This heterogeneity in CTCs may represent populations of cells from different metastatic sites or with different invasive potential.

#4962

Very-small-nuclear circulating tumor cell (vsnCTC) as a putative biomarker for visceral metastasis in metastatic castration-resistant prostate cancer (mCRPC).

Jie-Fu Chen,1 Hao Ho,2 Elisabeth Hodara,1 Alexander Ureno,1 Ann Go,1 Elizabeth Kaufman,1 Margarit Sievert,1 Daniel J. Luthringer,1 Jiaoti Huang,2 Leland Chung,1 Zunfu Ke,2 Ker-Chau Li,2 Hsian-Rong Tseng,2 Edwin M. Posadas1. 1 _Cedars-Sinai Medical Center, Los Angeles, CA;_ 2 _University of California, Los Angeles, Los Angeles, CA_.

Introduction and Objective: Patients with metastatic castration-resistant prostate cancer (mCRPC) who develop visceral metastases (VM) have poorer clinical outcomes in comparison to those without VM. Currently, VM are discovered late in the clinical course of mCRPC-VM and this aggressive natural history typically culminates in organ failure. There are no existing tests that identify men at risk for VM other than radiography. Our team performed circulating tumor cell (CTC) enumeration using NanoVelcro CTC Assay on prostate cancer patients across the spectrum of metastatic states: no metastasis, non-visceral metastasis, and VM. We identified an association between the presence of very-small-nuclear CTCs (vsnCTCs, DAPI+/Cytokeratin+/CD45- with nuclear size < 8.5µm) and VM. Serial enumeration studies suggested the emergence of vsnCTCs occurred before the radiographic detection of VM, and the change of vsnCTC counts reflected the patients' disease progression. We then hypothesized that presence of vsnCTC signals the presence of VM and has predictive and prognostic value with respect to VM.

Methods: We identified mCRPC patients who had progressed through next generation hormonal maneuvers such as abiraterone, enzalutamide, or an equivalent drug. Serial blood specimens were used for vsnCTC enumeration using NanoVelcro CTC Assay as previously published. The vsnCTC counts were related to the presence and development of VM (evaluated by radiography) as well as the response to anti-cancer treatment.

Results: Blood specimens were identified from 28 patients who met the eligibility criteria; 15/28 patients presented with VM and 13/28 had bone-only disease at their first CTC enumeration. Five out of 13 non-VM patients developed VM during follow-up, and vsnCTCs were detected 86-196 days prior to radiographic detection of VM (true positive); 4/13 had vsnCTCs detected but no VM was found by the time of analysis (false positive). None of the vsnCTC(-) patients developed VM. vsnCTCs were detected in 20/20 VM patients compared to 4/8 non-VM patients. Reduction of vsnCTC count occurred at initiation of anti-cancer treatment; transition from vsnCTC(-) to vsnCTC(+) was seen prior to progression under the treatment. Of the patients who have VM, 14 passed away at the time of this abstract submission including all the patients converting from non-VM to VM during the time of follow-up. Two out of 8 non-VM patients passed away including one patient who had vsnCTCs detected around 6 months prior to death.

Conclusions: vsnCTCs are associated with the presence of VM. The vsnCTC is a potential biomarker for predicting the development of VM and monitoring the treatment response in mCRPC. Transition from vsnCTC(-) to vsnCTC(+) was associated with the development of VM and progression under the treatment.

#4963

A distinct AMP-activated protein kinase phosphorylation site is associated with metastasis and castration-resistance in prostate cancer.

Melissa A. Babcook,1 Mahmut Akgul,2 Seunghee Margevicius,3 Gregory T. MacLennan,2 Pingfu Fu,3 Robert Abouassaly,2 Sanjay Gupta3. 1 _University of Toledo College of Medicine, Toledo, OH;_ 2 _University Hospitals Case Medical Center, Cleveland, OH;_ 3 _Case Western Reserve University, Cleveland, OH_.

AMP-activated protein kinase (AMPK) is a critical regulator of cellular metabolism and plays important role in the development and progression of several human diseases including cancer. We have previously shown that AMPKα activity is significantly inhibited by Ser-485/491 phosphorylation in cell culture and in vivo models of metastatic and castration-resistant prostate cancer. To determine potential translatability, we investigated AMPKα-Ser-485/491 phosphorylation status in clinical prostate cancer specimens and its correlation with metastasis and progression to castration-resistance. Metastasis and matched primary tumor specimens from 45 metastatic prostate cancer patients, 31.1% of which were post-androgen deprivation therapy failure (castration-resistant prostate cancer), and primary tumor specimens from 30 non-metastatic, hormone-dependent prostate cancer patients with undetectable PSA for a mean of 97.1 ± 17.5 months were obtained from the University Hospitals Case Medical Center Pathology Archive. Slides were cut from paraffin blocks, immunostained for p-Ser-485/491 AMPKα and total AMPKα, analyzed by a pathologist, and statistical significance determined by T-test, Kruskal-Wallis test, Fisher's exact test, or Chi-Square test. Mean pre-diagnostic biopsy PSA was 6.5 ng/mL (range 1.20-25.3 ng/mL) in non-metastatic patients and 501.9 ng/mL (range 1.82-3200 ng/mL) in metastatic patients (p < 0.0001). Mean total Gleason score of primary tumors was 6.5 in the non-metastatic group and 8.2 in the metastatic group (p < 0.0001); primary prostate tumor volume (mean ± S.D.) was 22.1 ± 19.5% in the non-metastatic group and 68.1 ± 28.7% in the metastatic group (p < 0.0001). The intensity of overall p-Ser-485/491 AMPKα staining and the number of p-Ser-485/491 AMPKα positively-stained cells was significantly increased in primary prostate tumors (p = 0.0003 and p = 0.0008, respectively) and metastases (p = 0.0011 and p = 0.0062, respectively) of metastatic prostate cancer patients when compared to staining of primary tumor specimens from non-metastatic prostate cancer patients. Further, the intensity of overall p-Ser-485/491 AMPKα staining increased 2.21-fold (p = 0.0050) and the number of p-Ser-485/491 AMPKα positively-stained cells increased 1.77-fold (p = 0.0041) in castration-resistant metastatic prostate cancer specimens compared to androgen-dependent prostate cancer specimens. Therefore, AMPKα Ser-485/491 phosphorylation is associated with metastasis and castration-resistance in clinical prostate cancer and may be a novel target for treatment.

#4964

The isolation of prostate cancer specific circulating tumor cells in the blood of patients with metastatic castration-resistant prostate cancer.

Gerit Theil,1 Stefanie Schmidt,1 Kersten Fischer,1 Klaus Lücke,2 Paolo Fornara1. 1 _Martin Luther University Medical School Halle, Halle/ Saale, Germany;_ 2 _GILUPI-GMbH, Potsdam, Germany_.

Background:

Circulating tumor cells (CTC) consist of a heterogeneous population of very rare cells of the primary tumor or its own metastases. A growing amount of evidence has shown that subpopulations of carcinoma cells undergo epithelial-to-mesenchymal transitions (EMT), resulting in their increased motility which facilitates their intravasation into the blood circulation. Gold standard of CTC-isolation is the CellSearchSystem based on epithelial cell adhesion molecule (EpCAM) enrichment, though it is known that EpCAM is downregulated in al advanced prostate cancers and in all CTCs. Furthermore these CTC isolation techniques are not able to isolate organ specific CTCs. The aim of this pilot study is the development of a prostate cancer specific functionalized wire, which detects prostate cancer specific circulating tumor cells (PCTC) in metastatic PCa patients. The CTC detection rate will be compared between an EpCAM functionalized wire and a PCa specific functionalized wire.

Methods:

We combined 4 antibodies against Prostate specific membrane antigen (PSMA), Prostate specific antigen (PSA), Prostate specific stem cell antigen (PSCA) and epithelial cell adhesion molecule (EpCAM). Evaluations of these antibodies were performed by immunofluorescence analysis of the human prostate cancer cell lines PC3 and LNCaP. PCa specific functionalization of the wire was determined with spiking experiments. Blood samples from healthy donors were spiked with PC-3 and LNCaP cells to test the wire for cell-binding. Afterwards blood of 15 metastatic castration-resistant prostate cancer (CRPC) patients with metastatic progression, documented by prostate-specific antigen or radiologic criteria, was investigated. These samples were simultaneously analyzed in the flow system with two different functionalized wires. The captured CTC and PCTC were identified by immunofluorescence staining using pan cytokeratin and Hoechst-33258 positive signals as well as CD45 negative criteria.

Results: The spiking experiments indicate that a sensitive ex vivo isolation with different functionalized wires is possible. In summary, PCTC counts ranged from 0-122 per PCTC (median 9) and CTC counts ranged from 0-22 per CTC (median 3). Our data shows that a more sensitive isolation of PCTC´s is possible using prostate cancer specific functionalized wire compared to EpCAM functionalized wire (p ≤ 0,001). The CTC isolation sensitivity was about 86% for PCa-functionalized wire and about 73% for EpCAM functionalized wire.

Conclusions: PCTC can be isolated with prostate cancer-specific functionalization of the wire. Simultaneously CTC that underwent EMT can be isolated. These organ specific subpopulations might have a prognostic effect on the clinical outcome and on the development of personalized medicine. This proof of concept shows how important it is to optimize the EpCam based CTC-detection.

#4965

Analytic validation and clinical qualification of a novel immunohistochemical assay for AR-V7 protein expression in metastatic prostate cancer.

Daniel Nava Rodrigues,1 Jon Welti,1 Adam Sharp,1 Shihua Sun,2 Ruth Riisnaes,1 Ines Figueiredo,1 Zafeiris Zafeiriou,1 Pasquale Rescigno,1 Johann S. de Bono,1 Stephen R. Plymate2. 1 _Institute of Cancer Research and the Royal Marsden Hospital NHS Trust, Sutton, United Kingdom;_ 2 _Department of Medicine, University of Washington, WA_.

BACKGROUND:

The androgen-receptor splice variant 7 (AR-V7) has been implicated in the development of castration resistant prostate cancer (CRPC) and resistance to current therapies including enzalutamide and abiraterone. AR-V7 mRNA expression in circulating tumour cells of patients with CRPC correlated with treatment resistance. However, the importance of AR-V7 has been questioned in light of low AR-V7 mRNA levels relative to the full-length androgen receptor in CRPC and it is critically important to develop validated assays that confirm AR-V7 protein levels and its clinical importance in patients with CRPC.

METHODS:

Following validation of a monoclonal antibody, immunohistochemical analysis of nuclear AR-V7 (alongside a nuclear AR N-terminal domain antibody; AR-NTD) was performed in a patient cohort identified with matched therapy-naive hormone-sensitive primary prostate cancer (HSPC) and CRPC. We determined the levels of nuclear AR-V7 as patients progressed from HSPC to CRPC. We also determined if AR-V7 expression levels associated with overall survival from time of CRPC biopsy.

RESULTS:

In our patient cohort (n=39), nuclear AR-V7 (p=<0.0001) and nuclear AR-NTD (p=0.0006) increased significantly as patients progressed from HSPC to CRPC. Lower nuclear AR-V7 expression was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (18.7 vs 9.6 months; HR 0.36 [95% CI 0.17-0.62]; p=0.002), 50th (13.0 vs 9.8 months; HR 0.60 [95% CI 0.29-1.07]; p=0.09) or 75th (6.0 vs 11.5 months; HR 0.31 [95% CI 0.04-0.34]; p=0.0004) percentile of AR-V7 expression. Similarly, a lower nuclear AR-V7 to nuclear AR-NTD ratio was associated with improved overall survival from time of CRPC biopsy in patient groups divided by the 25th (17.8 vs 9.1 months; HR 0.42 [95% CI 0.22-0.78], p=0.01), 50th (14.0 vs 8.8 months; HR 0.43 [95% CI 0.17-0.69], p=0.005) and 75th (11.5 vs 7.3 months; HR 0.39 [95% CI 0.09-0.69], p=0.009) percentile. Nuclear AR-NTD did not associate with overall survival from CRPC biopsy.

CONCLUSION:

We provide first evidence that expression of nuclear AR-V7 protein not only increases with emerging treatment resistance in CRPC but also is associated with overall survival from time of CRPC biopsy. These data support AR-V7 protein being key to CRPC progression and that agents targeting AR splice variants will be important to improve patient outcome in CRPC.

#4966

High-definition single cell analysis (HD-SCA) reveals enrichment in androgen receptor (AR) expression in tumor cell clusters in bone marrow and blood of metastatic castration-resistant prostate cancer (mCRPC) patients.

Amado J. Zurita,1 Anders Carlsson,2 Patricia Troncoso,1 James Hicks,2 Christopher J. Logothetis,1 Peter Kuhn2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _University of Southern California, Los Angeles, CA_.

Introduction. The analysis of tumor cells in the circulation (CTC) provides an opportunity for non-invasive characterization of mCRPC and monitoring of therapy response, but it is the bone where prostate cancer cells almost consistently migrate to, expand and adapt to therapy. We adapted the HD-CTC assay to bone marrow aspirates (BMA) to discern features most significant to the tumorigenicity and the dynamics of metastatic progression in this disease, and to allow for serial comparison of tumor cells in the circulatory and bone compartments. Since available experimental data suggests that tumor cells organized in clusters are more important contributors to metastasis than single CTC, we evaluated their presence and characteristics in the 2 compartments.

Methods. Peripheral blood and BMA obtained through the posterior iliac crest were synchronously collected from individual mCRPC patients (n=89) while progressing on therapy, and tumor cells in them were detected and characterized using the HD-SCA high-content platform. This flexible non-enrichment-based approach allows for quantification of cell morphometric and protein expression parameters and provides easy access to individual cells and cell clusters for single cell genomics and proteomics analysis. Candidate tumor cells based on cell size and morphology were presented and manually classified as DAPI and cytokeratin-positive and CD45-negative. Clusters were defined as two or more tumor cells in direct contact. AR protein expression and sub-cellular localization were also examined for each tumor cell and tumor cell cluster.

Results. Metastatic tumor cells were detected in 24/61 (39%) available BMAs. A greater number of tumor cells were organized in clusters, and clusters were generally larger, in BMAs than in blood (50% vs. 33%, and 20% vs. 5% with 6 or more cells, respectively). In 14 patient-matched and synchronously collected blood and BMA specimens with at least 1 tumor cell, we found 10 (71%) with clusters in the BMA (13-357 clusters/case, with the exception of 1 case that had 1 cluster), while only 3 (21%) had CTC clusters (2-4 clusters/case) (p = 0.02). The 4 cases that had no clusters in the marrow did not have any clusters in the blood. Patients with larger cluster size did significantly worse both in terms of progression-free (p=0.002) and overall survival (p=0.02). AR expression in individual cells was significantly higher in those forming part of cell clusters than in single cells, and was positively correlated with cluster size, both in blood and BMAs.

Conclusions. Tumor cell clusters are frequent in the circulation and especially in the bone marrow of mCRPC patients whose disease has progressed to therapy. In this setting, the increasing AR expression observed in tumor cell clusters is of probable biological relevance.

#4967

Label-free collection of prostate circulating tumor cells using microfluidic Vortex technology.

Edward Pao,1 Corinne Renier,2 Clementine Lemaire,2 James Che,1 Melissa Matsumoto Di Carlo,1 Melanie Triboulet,3 Sandy Srivinas,3 Stefanie S. Jeffrey,3 Rajan P. Kulkarni,4 Matthew Rettig,5 Elodie Sollier,2 Dino Di Carlo1. 1 _UCLA Bioengineering, Los Angeles, CA;_ 2 _Vortex Biosciences Inc, Menlo Park, CA;_ 3 _Stanford University School of Medicine, Stanford, CA;_ 4 _UCLA Medical Center, Los Angeles, CA;_ 5 _UCLA, David Geffen School of Medicine, Los Angeles, CA_.

BACKGROUND

Prostate cancer is among the most common cancers in men worldwide. Better markers than Prostate Specific Antigen (PSA) are still needed for the detection and monitoring of disease progression. Circulating Tumor Cells (CTCs) are shed into the blood stream from primary tumor(s) and may play key roles in the metastatic process. Liquid biopsies have emerged as a promising approach, with a correlation between the CTC numbers and patient prognosis for prostate cancer. CTCs have also been shown to enable early detection of recurrence, and could be potential candidates for guiding cancer therapy in real-time [1]. Current CTC enrichment technologies, including immuno-affinity and size-based filtration methods, have focused on high capture efficiency with sometimes tedious sample preparation and overall low purity.

METHOD

Here, we describe the use of the microfluidic Vortex Chip [2] for rapid and size-based isolation of CTCs from the blood of 23 patients with advanced prostate cancer, and 10 healthy donors; 5 being <30 years old, 5 age-matched with the patient cohort. Requiring no upstream sample preparation, blood was diluted 10-fold and processed through the highly parallelized Vortex Chip at 8 mL/min (800 µL/min whole blood). Larger cells (predominantly CTCs) were captured in microscale vortices produced on the Chip, released into a small volume and collected off-chip for CK, PSA, CD45 and DAPI immunostaining and enumeration.

RESULTS

Preliminary work with LNCaP prostate cancer cells spiked in blood showed a 29% capture efficiency and 50% purity. In vitro cell assays confirmed that cells enriched with Vortex chip were alive and proliferating for up to 7 days. For 23 patient samples, CTCs were captured (0.5 - 20 CTCs/mL) with high purity (3.6 - 72.3%), in less than 1H, without prior sample preparation. 11.5% of the cells collected were CK and PSA-negative, but some were identified as undergoing epithelial-mesenchymal transition (EMT) following staining for vimentin and N-cadherin. Few atypical cells were also isolated from age-matched healthy donors (0.7 - 2.8 CTCs/mL), while none was detected in younger healthy donors. Using a threshold calculated from the age-matched healthy donors (3.31 CTCs/mL = mean + 2CV), 70% of the patients were characterized as "positive for CTCs". No correlation was found between CTC counts and elevated PSA level.

CONCLUSION

These results demonstrate the ability to rapidly collect pure populations of CTCs in metastatic prostate cancer, independent of surface marker expression, without prior sample preparation. Future studies will use chips with optimized capture performance, sample recycling, and will include CTC molecular analysis by targeted panel sequencing. A larger cohort of healthy donors is also being examined to determine a statistically-robust CTC baseline for this size-based capture approach.

[1] Scher Hi, et al., J. Clin. Oncol. 2015

[2] Sollier E, et al., Lab Chip 2014

#4968

"Moonlighting Functions" of glycolytic enzymes relate to human prostate cancer invasion.

Jiaquan Yu,1 Ashley M. Weichmann,1 Alexandria Craig,1 Wei Huang,1 Dawn R. Church,1 Farideh Mehraein,1 Laurie L. Parker,2 David J. Beebe,1 George Wilding,3 Hirak S. Basu1. 1 _University of Wisconsin Comp. Cancer Center, Madison, WI;_ 2 _University of Minnesota, Minneapolis, MN;_ 3 _MD Anderson Cancer Center, Houston, TX_.

Background: Castrate-resistant prostate cancer (CRPC) is the second leading cause of cancer deaths among US men. Currently, a large number of small and low grade prostate cancers (PCa) are being diagnosed. Only a few of them will metastasize and become lethal. A clinical method to distinguish aggressive from the indolent tumors is warranted. An enhanced glucose metabolism (Warbürg effect) and invasion of cells from their organs of origin and metastasis to distant organs are two characteristics that are ubiquitous in most solid tumors. In the last few years, the non-glycolytic activities ("moonlighting functions") of glycolytic enzymes have been investigated in relation to cancer cell invasion. We propose that oxidatively stressed PCa cells, as a countermeasure, redirect some of the glycolytic enzymes to their moonlighting functions.

Methods: We used a novel microfluidic device that separates the invading from the non-invading PCa cells based on their migratory characteristics in a 3D collagen matrix. We performed ICC analysis of the separated cells for oxidative stress generating enzyme spermidine/spermine N1 acetyl transferase (SSAT) and glycolytic enzymes aldolase, GAPDH and F-actin levels. We also carried out proteomic analysis of aldolase and GAPDH levels and oxidation state and metabolomic analysis for enzymatic activities of those two enzymes.

Results: Under identical condition, LNCaP cells show minimum invasion, whereas between 30-40% of C4-2 cells invade more than 0.8 mm in 16 hours. ICC assay of C4-2 cells shows that most migratory C4-2 cells have SSAT overexpression, but less than 5% stationary cells show this effect. Proteomic analysis shows that androgen treatment that increases overall oxidative stress in both LNCaP and C4-2 cells actually decreases oxidation status of GAPDH in LNCaP cells and increases that in C4-2 cells. Anti-androgen enzalutamide reverses the androgen effect in both cell lines. The glycolytic activity of GAPDH decreases with an increase in protein oxidation. Our metabolomic data further confirmed an increase in GAPDH activity in LNCaP and a decrease in C4-2 cells after androgen treatment and the reversal with enzalutamide as expected from its oxidation status.

Discussion: SSAT expression and a consequent increase in oxidative stress are related to invasion of CRPC. Oxidation of certain glycolytic enzymes in CRPC cells may divert them to their moonlighting function related to cellular invasion and metastasis. These functions may be monitored in patient biopsies and/or prostatectomy tissues for PCa prognosis. Application of the microfluidic method for the separation of invading from the non-invading cells and proteogenomic and metabolomic analysis of these cells isolated from patient prostatectomy tissues are currently being standardized.

#4969

Exosomal RNA based liquid biopsy detection of androgen receptor variant 7 in plasma from prostate cancer patients.

James Hurley,1 Vincent O'Neill,1 Graham Brock,1 Javed Siddiqui,2 Rohit Malik,2 Arul Chinnaiyan,2 Johan Skog1. 1 _Exosome Diagnostics, Cambridge, MA;_ 2 _University of Michigan, Ann Arbor, MI_.

Introduction

Androgenic signaling is a critical part in the growth and survival of prostate cancer (PCa), especially in advanced, metastatic or recurrent disease. Consequently, the androgen receptor (AR) is a target for hormone therapies. Full-length androgen receptor mRNA is derived from 8 exons and encodes an N-terminal transactivation domain (exons 1-2), a DNA-binding domain (exons 2-3) and C-terminal ligand binding domain (exons 4-8). However, AR variants have been identified in which the ligand binding domain is removed by splicing between exons 1-3 and intronic, cryptic exons. One of these variants, ARv7, is highly expressed in patients and cultured PCa cells, which demonstrate resistance to hormone therapy treatment. To avoid ineffective procedures, the development of a liquid biopsy test for ARv7 expression would be a useful and non-invasive means of determining treatment strategies for PCa patients.

Methods

We have developed a diagnostic test to detect the presence of full-length androgen receptor and ARv7 transcripts in plasma samples taken from patients with advanced prostate cancer. The ExoDx™ ARv7 assay comprises a column-based isolation step yielding total exosomal RNA from 1-4 mls of patient plasma, followed by detection of both full-length androgen and the v7 receptor variants, using qPCR. Assay quality is monitored by inclusion of internal and external controls. Following validation, using both synthetic spikes and human samples, we monitored AR expression in plasma samples from treatment-resistant PCa patients. The data was analyzed and aligned with patient's response data.

Results

Here, we present proof of concept data for the ExoDX ARv7 assay. Using this novel diagnostic test, we were able to detect ARv7 variant mRNA in exosomal RNA isolated from patient plasma. We determined the profile of ARv7/FL transcripts in a cohort of prostate cancer patients with both high sensitivity and specificity, and observed concordance of the qPCR-based plasma results.

Conclusions

Liquid biopsies represent a low-risk, non-invasive and viable approach to testing for biomarkers in prostate cancer patients. Here, we demonstrate the capability of our validated diagnostic test to determine the presence of AR alternate transcripts in plasma. Since this analyte is the result of alternative splicing, it would not be detected in cell-free DNA alone. Monitoring plasma levels of ARv7 transcripts could enable effective personalized treatment for patients with advanced prostate cancer and has clear clinical application.

#4970

Phosphorylation of the intrinsically disordered cancer/testis antigen PAGE4 by HIPK1 and CLK2 in prostate cancer.

Luciane T. Kagohara,1 Steven Mooney,1 Yihong Chen,2 John Orban,2 Prakash Kulkarni,2 Robert W. Veltri1. 1 _Johns Hopkins University, Baltimore, MD;_ 2 _University of Maryland, Baltimore, MD_.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered cancer/testis antigen that is typically restricted to the normal testis but is aberrantly expressed in prostate cancer (PCa). Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the fetal prostate and is also a stress-response protein that is up-regulated in response to cellular stress. PAGE4 interacts with c-Jun and plays an important role in the development and pathology of the prostate gland. HIPK1, a component of the stress-response pathway, and CLK2, a dual specificity kinase are kinases that phosphorylate PAGE4 at predominantly at T51 and at multiple S/T residues, respectively. While phosphorylation at T51 is critical in potentiating c-Jun, the functional significance of the multisite phosphorylation by CLK2 is not known. The aim of this study was to determine the expression levels of HIPK1 and CLK2 in PCa cell lines, and in a set of 80 paired cases of PCa and benign adjacent tissue. Protein levels of HIPK1 and CLK2 were evaluated in cell lines and tissue samples by Western Blotting (WB) and immunohistochemistry (IHC), respectively. WB was performed with whole cell lysates from BPH1, DU145, LNCAP and PC3 cells using a fluorescence approach. IHC was performed with TMAs containing paired tumor and benign samples and after scanning (Aperio ScanScope), data were quantified using the ImageScope software. WB showed that HIPK1 is expressed in all cell lines selected with no significant changes in expression levels, while CLK2 was expressed only in BPH1 and LNCAP, suggesting this protein is expressed in cells displaying a less aggressive phenotype. Protein expression analysis by IHC in PCa and benign samples showed that CLK2 is more commonly expressed in PCa than HIPK1 when compared to non-cancer cases. CLK2 expression was positive in 81.7% of the cancer cases and HIPK1 was expressed in 72.2%. When compared to benign cases these differences are significant with p<0.0001. To further evaluate the importance of these two proteins as biomarkers for PCa, we performed ROC curve analysis to verify if the expression profile could be used to separate cancer from non-cancer cases. Both, CLK2 and HIPK1 can discriminate PCa from the benign tissue (AUC=0.81 and 0.70, respectively). CLK2 as a biomarker for PCa is more specific than HIPK1, with specificity of 78.4% for the first vs. 64.4% for the last. Since CLK2 is expressed in cell lines with a less aggressive phenotype, we evaluated whether CLK2 expression correlates with Gleason score (GS) in an attempt to determine their utility as outcome predictors. However, no association was observed with GS and CLK2 and HIPK1 expression patterns. In summary, our results suggest that CLK2 may be a potential biomarker for PCa.

#4971

Quantitative image analysis of androgen receptor (AR) and tubulin biomarker profiles in circulating tumor cells (CTCs) from metastatic castration resistant prostate cancer (mCRPC) patients.

Daniel Worroll, Giuseppe Galletti, David M. Nanus, Scott T. Tagawa, Paraskevi Giannakakou. _Weill Cornell Medicine, New York, NY_.

Background: CTCs represent a real-time, liquid biopsy for analyses of a variety of molecular biomarkers that offer clinically relevant information. In prostate cancer, the AR pathway controls tumor cell growth and survival and is an important therapeutic target. AR becomes biologically active upon translocation from the cytoplasm to the nucleus in a microtubule dependent fashion, resulting in the activation of numerous genes. Real-time analysis of AR and tubulin status in CTCs may provide insight to therapy response in mCRPC patients.

Methods: Androgen sensitive human prostate cancer cells, LNCaP, were treated with and without a synthetic androgen (R1881; 10 nM, 1 hr) and docetaxel, spiked into healthy donor blood, and captured using a prostate specific microfluidic device (GEDI chip), following the same protocol that we use with patient samples. Cells were fixed and stained (N-terminal AR, CD45, CK, DAPI, and tyr-tubulin) directly on the device and imaged using high resolution multiplex confocal microscopy. AR and tubulin status were analyzed using a quantitative image analysis algorithm. Nuclear AR percentage was calculated by integrating fluorescence intensity within regions defining the entire cell and nucleus. H-Score was calculated by multiplying the nuclear AR percentage by the normalized total cell AR intensity. Tubulin status and microtubule bundling were assessed using a variety of quantitative parameters including measurements of tubulin fluorescence intensity and distribution within the cell.

Results: LNCaP cells in the presence of synthetic androgen exhibited higher nuclear AR percentage (p=0.0004) and H-score (p=0.003). Docetaxel treatment led to lower nuclear AR percentage (p=0.04) and lower H-Score (p=0.03). Cells treated with docetaxel exhibited higher tubulin intensity range (p=0.008) and standard deviation (p=0.03), consistent with docetaxel's mechanism of action. We applied these methods of quantitative image analyses to CTCs isolated from a small cohort of mCRPC patients. Single CTC image analysis revealed heterogeneity in AR status. Quantitative evidence for microtubule bundling was observed in patients receiving docetaxel chemotherapy.

Conclusions: We have developed a high throughput quantitative image analysis algorithm to interrogate mCRPC specific biomarkers, such as AR and tubulin status, in single CTCs. These methods of quantitative image analyses will be prospectively validated with CTCs from mCRPC patients followed longitudinally over the course of treatment with AR inhibitors and taxanes, yielding patient specific, clinically relevant information that can guide physicians' strategies for the clinical management of cancer.

#4972

Detection of PSMA on circulating tumor cells from blood of prostate cancer patients using anti-PSMA Centyrin.

Bhavesh Vaidya,1 Peter Vulfson,2 Renouard Sanders,2 Donna Klein,1 Shalom Goldberg,1 Steve Jacobs,1 Chandra Rao1. 1 _Janssen R &D, Spring House, PA; _2 _Janssen R &D, Huntingdon Valley, PA_.

Prostate specific membrane antigen (PSMA), a transmembrane glycoprotein, is a clinically validated marker of prostate cancer. PSMA is overexpressed in primary and metastatic prostate cancer when compared to normal prostate tissue which makes it an attractive target for immunotherapy. Several antibodies are being considered to target PSMA for the development of an anticancer drug. The expression levels of PSMA on tumor cells may play a role in response to treatment with anti PSMA drugs. Therefore, it is important to monitor PSMA expression on tumor cells before and during treatment. We used CellSearch® CTC assay to determine the presence of PSMA on circulating tumor cells (CTCs) using anti PSMA Centyrin as a biomarker. Centyrins are a class of alternative scaffold molecules which are proteins of small size (~ 10 kDa). They are engineered to bind to target molecules with high specificity and sensitivity. The anti PSMA Centyrin was tested for specificity and sensitivity using tissue cultured tumor cell lines spiked into normal healthy blood and then compared to anti PSMA antibody. The anti PSMA Centyrin showed 100% positivity with the high PSMA expressing cell line (LNCap), 25% positivity with the low PSMA expressing cell line (22Rv1) and 0% positivity with the PSMA negative cell line (PC3-9). The anti PSMA Centyrin was further tested with prostate patient blood samples to determine the expression levels of PSMA on CTCs. The patient samples which contain CTCs are positive for anti PSMA Centyrin and the percentage of CTCs positive for PSMA ranged from 25-100%. Thus, PSMA expression on CTCs can be determined using CellSearch® system and anti PSMA Centyrin as a biomarker in a clinical trial.

#4973

The role of cholesteryl esters in progression and racial disparity of prostate cancer.

Xinchun Zhou,1 Timera Brown,2 Janice Lage,1 Jinghe Mao2. 1 _University of Mississippi Medical Center, Jackson, MS;_ 2 _Tougaloo College, Tougaloo, MS_.

Background Prostate cancer (PCa) is featured by uncertainty of future outcomes at the time of diagnosis, development of castration resistant prostate cancer (CRPC), and racial disparity in morbidity and mortality, which is especially prominent between African American (AA) and Caucasian American (CA). This study is aimed to investigate whether accumulation of cholesteryl esters (CE) in PCa is a key biological process that determines progression potential, and serves as a common mechanism underlying these features.

Methods The methods used in this study include: 1) ESI/MS-MS on 47 fresh-frozen prostatic tissues for global lipid profiling; 2) Real-time PCR on 16 fresh-frozen prostatic tissues for the expression level of genes related to the pathogenesis of prostate cancer and metabolism of cholesteryl esters; and 3) immunohistochemistry on 165 formalin-fixed and paraffin embedded prostatic tissues for the expression level of ACAT1 and LAL.

Results We found that 1) prostatic concentration of total lipids was significantly higher in PCa than benign prostatic tissues (BPT), with a PCa to BPT (P/B) ratio of 2.3, p=0.013; 2) when total lipids were stratified into groups of neutral lipids and polar lipids, only neutral lipids are significantly higher in PCa than BPT (P/B ratio: 3.1, p=0.02); 3) after neutral lipids were further stratified into class of triglycerides, diglycerides, free fatty acids (FFA) and CEs, total CEs are most sensitive in differentiation of PCa from BPT (P/B ratio: 5.8, p=0.025); 4) 17 out of 31 prostatic individual CE species are significantly higher in PCa than in BPT all the studied populations; and 5) intriguingly, 14 of these 17 prostatic CE individual species remain significantly different between PCa and BPT in AA population, whereas only 3 of them remain significantly different between PCa and BPT in the CA population. The real-time PCR results indicated that the expression levels of genes Pten, LIPA, and ABCA1 are obviously lower in high grade than in low grade PCa (not obviously for ACAT1). The expression levels of proteins ACAT1 and LAL (LIPA gene product) are reversely correlated with the progression of PCa: in prostate cancer, ACAT1 is highly expressed, but LAL is not expressed. In contrary, in benign ACAT1 is not expressed, but LAL is highly expressed. Such a reverse correlation is especially prominent in AA than CA population.

Conclusion and Significance Accumulation of cholesterol esters correlates with the progression and racial disparity of PCa. Down regulation of LIPA gene and its product LAL could play major role in CE accumulation. Thus prostatic CE and its individual species could serve as racially specific biomarkers in diagnosis and in differentiation of indolent from aggressive cases of PCa, and LAL could be potential therapeutic targets for treatment of advanced prostate cancer, including castration resistant prostate cancer.

#4974

Using serological proteomics analysis to identify anti-nucleophosmin 1 autoantibody in sera from prostate cancer patients as potential diagnositic and prognostic biomarker.

jitian li,1 Liping Dai,1 Ningjing Lei,1 Mengtao Xing,1 Carlos A. Casiano,2 Jianying Zhang1. 1 _The University of Texas at El Paso, El Paso, TX;_ 2 _Loma Linda University School of Medicine, Loma Linda, CA_.

Prostate specific antigen (PSA) level test in blood has been widely implemented for the early detection and prediction of prostate cancer (PCa). However, lack of specificity led to overdiagnosis, following by unnecessary biopsies and overtreatment. Therefore, novel serological biomarkers are urgently needed to complement PSA by enhancing its sensitivity and specificity in the diagnosis of PCa. Autoantibodies against intracellular tumor associated antigens (TAAs) are commonly found in various types of human cancers. Their utility in providing insights into tumor biology and as potential cancer biomarkers is well documented. Previous studies have demonstrated that sera from patients with PCa contain autoantibodies that react with TAAs. In this study, we identified and characterized nucleophosmin 1 (NPM1) with 33 kDa molecular weight as an autoantigen by using serological proteomics analysis (SERPA) approach. In the further validation study, it was observed that the level of anti-NPM1 in sera from patients with PCa was significantly higher than that from benign prostatic hyperplasia (BPH) and healthy individuals and receiver operating characteristic (ROC) curve analysis showed high diagnostic value for PCa (area under the curve (AUC):0.805). Interestingly, when considering anti-NPM1 combined with PSA level in sera from PCa patients at the early stage as well as BPH, 100% PCa patients could be identified correctly, while 69.2% patients with BPH who has elevated PSA level were found anti-NPM1 negative. The data suggested that anti-NPM1 can elicit humoral immune response in PCa and might be a potential biomarker for the immunodiagnosis of PCa, and it would be a supplementary of PSA examination to distinguish PCa from BPH.

Key words: prostate cancer; nucleophosmin 1 (NPM1); tumor-associated antigens (TAAs); autoantibodies; serological proteomics analysis (SERPA)

#4975

Detection and characterization of circulating and disseminated tumor cells in patients with bladder and prostate cancer.

James E. Verdone, Emma E. van der Toom, Kenneth J. Pienta, Michael A. Gorin. _Johns Hopkins School of Medicine, Baltimore, MD_.

Introduction: Increasing evidence suggests that cancer cells display dynamic molecular changes in response to systemic therapy. Circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in bone marrow represent a readily available source of cancer cells with which to measure this dynamic process. To date, many strategies to isolate and characterize these cells have been described. These techniques, however, have been limited by the requirement for positive or negative selection thus resulting in cell losses. Herein we report a method to detect and characterize CTCs and DTCs in matched peripheral blood and bone marrow samples without need for a selection step.

Methods: Following blood or bone marrow aspirate collection, nucleated cells were extracted using a modified density-media centrifugation technique. Nucleated cells were spread on positively charged slides and stained with epithelial markers (Pan-cytokeratin and EpCAM) and a leukocyte marker CD45. High throughput scanning microscopy was performed followed by image processing and subsequent ranking for probability of being a cancer cell. Candidate cancer cells were defined as events which were positive for PanCK and/or EpCAM but negative for CD45. Candidate cells were independently confirmed with high resolution microscopy.

Results: Cytokeratin-positive, EpCAM-positive DTCs and CTCs were readily detectable in patients with clinically-localized bladder and prostate cancer without need for positive or negative selection.

Conclusions: Future work aims to incorporate disease specific makers to confirm that the observed cells truly represent cells of either bladder or prostate origin. This method offers the flexibility to preserve samples for downstream molecular assays including single cell isolation and sequencing. All told, this assay provides a novel means to detect and characterize CTCs and DTCs without positive or negative selection methods.

#4976

Monitoring CHD1 during prostate cancer progression.

Gunther Boysen,1 Daniel Nava Rodrigues,1 Joaquin Mateo,1 Rossitza Christova,1 Ruth Riisnaes,1 Mateus Crespo,1 Theresa MacDonald,2 Susana Miranda,1 Ines Figueiredo,1 Veronica Gil,1 Sara Aziz,1 Adam Sharp,1 Niven Mehra,1 Pasquale Rescigno,1 Juan M. Mosquera,2 Christopher E. Barbieri,2 Mark A. Rubin,2 Johann S. de Bono1. 1 _Institute of Cancer Research, Sutton, Surrey, United Kingdom;_ 2 _Weill Cornell Medical College, New York, NY_.

Adenocarcinomas of the prostate consist of a collection of malignancies with distinct molecular underpinnings. Linking molecular background to clinical behaviour, i.e. tumour progression and response to therapy has not yet been fully established. This is in large measure due to a paucity of well-annotated patient cohorts, which cover the evolution from localized, hormone naïve to metastatic castration resistant prostate cancer. Deletions in the gene Chromodomain Helicase DNA Binding Protein 1 (CHD1) are among the most frequent genomic alterations in prostate cancer. CHD1 is a ATPase-dependent helicase mediating a variety of biological processes by modifying chromatin structure. In hormone sensitive prostate cancer, loss of CHD1 has been associated with increased genomic instability and aggressiveness. Here we monitor CHD1 status during the progression from hormone sensitive (HSPC) to castration resistant prostate cancer (CRPC). To molecularly stratify this patient subgroup we correlate CHD1 status with markers of prostate cancer including AR, ERG, PTEN and Ki67. Finally, we analyze the impact of alterations in CHD1 on outcome and response to therapy of CRPC patients. We retrospectively identified 52 patients of whom HSPC and CRPC tissue was available for immunohistochemical analysis of CHD1, ERG, PTEN, AR and Ki67 as well as for CHD1 fluorescence in situ hybridization. Relationship with outcome was analyzed using univariate Cox regression and log-rank analyses. By using Fluorescence In Situ Hybridization (FISH) and immunohistochemistry (IHC) in a cohort of 52 men who developed CRPC, we show that the frequency of loss of CHD1 does not change during disease progression (7.7% in HSPC vs 9.6% in CRPC). Homozygously deleted tumors demonstrate a tendency towards poorer prognosis but this observation lacks statistical power in our cohort. Interestingly, in patients that show CHD1 protein expression in their hormone naïve sample, a decrease of expression in the matched CRPC sample correlates with poorer overall survival (OS) (p=0.03). Although preclinical data suggest a role for CHD1 in androgen receptor (AR) activity, CHD1 expression did not correlate with AR protein level. Additionally, Ki67 expression did not show correlation with CHD1 levels of expression. Further molecular stratification confirms a known mutual exclusive relationship with the expression of the ERG transcription factor (p=0.048). Clinically, although decreased CHD1 protein expression during disease progression correlated with worse outcome (overall survival (p=0.03) and survival from diagnosis of CRPC (p=0.02)), no significant impact on survival after docetaxel or abiraterone treatment was measured.Here we monitor CHD1 deletion during prostate cancer progression. Our results show that this genomic marker defines a distinct molecular subgroup of prostate cancer. In addition, loss of CHD1 protein expression during disease progression is associated with poorer clinical outcome. 

### Immune Modulation from Non-Immunotherapy and Antibodies: Clinical

#4977

Immunogenic chemotherapy synergize PD-1 blockade by enhancing dendritic cells infiltration in triple-negative breast cancer (TNBC).

Xiangliang Yuan, Yi Xiao, Wenling Kuo, Dihua Yu. _The University of Texas M. D. Anderson Cancer Center, Houston, TX_.

Targeting immune checkpoints such as programmed cell death protein 1 (PD1), programmed cell death 1 ligand 1 (PDL1) has achieved noteworthy benefit in multiple cancers. However, their efficacy remains unsatisfactory in triple-negative breast cancer (TNBC). Breast cancer is not highly mutagenic and immune checkpoints blockade therapies for TNBC is not as effective as for melanoma or lung cancer with high mutation rates. Combination of different modalities offers an opportunity to enhance their effect. We studied the synergistic effect of cancer chemotherapy and immunotherapy in TNBC mouse model. Here we report that the combination of Doxisome (liposomal encapsulated formulation of Doxorubicin) and anti-PD-1 antibody suppresses tumor growth, reduces lung metastasis and improves survival of mice bearing 4T1-triple negative breast cancer. Moreover, the combination of Doxisome and anti-PD-1 was able to overcome low dose Doxisome resistance in mouse models of TNBC. The success of anticancer chemotherapy is linked to a durable immune response of targeting tumor. We present evidence that the synergistic therapeutic activity of Doxisome with anti-PD1 blockade results in enhanced T cell anti-tumor immune responses. Moreover, the synergetic effect is due to increased dendritic cells (DCs) infiltration in tumor microenvironment, which internalize tumor antigens and subsequently activate tumor-reactive T cells. In TNBC mouse model, by combining anti-PD1 checkpoint blockade while shifting the immune response towards CD4+ Th1 with Doxisome, immune tolerance to TNBC tumor can be overcome. Enhancing antigen presenting cells (APCs, e.g. DCs) infiltration by immunogenic chemotherapy and T cell activation by anti-PD1 blockade increase therapy response in TNBC. These results provide rationale for several newly planned clinical trials combining immunogenic chemotherapy with PD-1 or PD-L1 blockade in breast cancer and exploration of such a combination in others solid tumors.

#4978

T cell mediated immunity after combination therapy with intralesional PV-10 and co-inhibitory blockade in a melanoma model.

Amy M. Weber, Hao Liu, Krithika N. Kodumudi, Amod A. Sarnaik, Shari Pilon-Thomas. _H.Lee Moffitt Cancer Center, Tampa, FL_.

PV-10 is a 10% solution of Rose Bengal, a xanthene dye that was originally developed for ophthalmic use and later used in liver function studies. PV-10 was formulated for intralesional (IL) injection and is currently being investigated as a novel cancer therapeutic. In murine studies, we have previously shown that intralesional (IL) injection of PV-10 leads to a regression of both injected tumors and untreated bystander tumors. We have also shown that combination therapy of IL PV-10 with blockade of PD-1 and PDL-1 leads to increased anti-tumor immunity. In this study, we have examined the role played by specific immune cell populations in eliciting this response in a murine melanoma model. We first investigated the antigen specificity of CD8+ T cells in the spleens of mice treated with the combination therapy of IL PV-10 and systemic co-inhibitory blockade. We found that splenocytes from mice treated with the combination of IL PV-10 and anti PD-1 antibody have an increased mean percentage of OVA antigen-specific CD8+ T cells (5.77%) compared to single treatment with anti PD-1 antibody (3.8%) or IL PV-10 (3.60%) alone in OVA-expressing B16 tumor bearing mice. To investigate the role of T cell subsets in mediating an immune response, OVA-expressing B16 tumor bearing mice were treated with IL PV-10 followed by intraperitoneal injection of anti-PD-1 antibody. In addition, mice were given either 2.43 antibody to deplete CD8\+ T cells, GK1.5 antibody to deplete CD4+ T cells, or PC61 to deplete regulatory T cells (Tregs). We found that depletion of CD4\+ T cells in combination with IL PV-10 and anti-PD-1 antibody treatment resulted in an enhanced anti-tumor effect, with an average tumor size of 52.8 mm2 on day 25 compared to the control group (173.7 mm2), and depletion of Tregs resulted in an even greater anti-tumor effect, with an average tumor size of 1.2 mm2 on day 25 (p<0.05). In contrast, mice treated with the CD8+ depleting antibody exhibited diminished anti-tumor immunity compared to the control group, with an average tumor size of 200 mm2 on day 25. Together, these studies indicate that the effect of combination therapy with IL PV-10 and co-inhibitory blockade is mediated by CD8+ T cells, and that depletion of both CD4+ T cells and CD25+ Tregs significantly enhances anti-tumor immunity in a melanoma model.

#4979

Cabozantinib eradicates advanced murine prostate cancer by activating neutrophil-mediated anti-tumor innate immunity.

Akash Patnaik,1 Kenneth D. Swanson,1 Eva Csizmadia,2 Marina P. Gehring,2 Katja Helenius,3 Athalia R. Pyzer,2 Lily C. Wang,4 Jesse Novak,5 Olivier Elemento,4 Thomas B. Thornley,2 John M. Asara,1 John G. Clohessy,1 Kathy Kelly,6 Pier Paolo Pandolfi,1 Jacalyn M. Rosenblatt,1 David E. Avigan,1 Huihui Ye,1 Sabina Signoretti,7 Steven P. Balk,1 Lewis C. Cantley4. 1 _Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA;_ 2 _Beth Israel Deaconess Medical Center, Boston, MA;_ 3 _Massachusetts Institute of Technology, Cambridge, MA;_ 4 _Weill Cornell Medical College, New York, NY;_ 5 _Brigham & Woman's Hospital, Boston, MA; _6 _National Institutes of Health, Boston, MA;_ 7 _Brigham and Women's Hospital/Harvard Medical School, Boston, MA_.

Several kinase inhibitors targeting aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in cancer clinical trial patients across several malignancies. Here we show that cabozantinib rapidly eradicates invasive, poorly-differentiated PTEN/p53 deficient murine prostate cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells, including CXCL12 and HMGB1, resulting in robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor, plerixafor. Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anti-tumor innate immune response, resulting in rapid tumor clearance.

#4980

Upregulation of PD-L1 expression by cisplatin in esophageal squamous cell carcinoma cells is independent of interferon/JAK/STAT pathway.

Ta-Chen Huang, Kuan-Ling Lin, Ann-Lii Cheng, Chih-Hung Hsu. _National Taiwan University Hospital, Taipei, Taiwan_.

Introduction

Blockade of immune check points, especially the program cell death 1 (PD-1) and its major ligand PD-L1 signaling, has become an important mode of therapy against cancers. The upregulation of PD-L1 expression in cancer cells is an important mechanism contributing to immune evasion of cancers. The PD-L1 upregulation in cancer cells could be mediated by intrinsic genetic or signaling alterations, or could be induced in response to specific cytokines, in particular interferon (IFN)-gamma, produced by tumor-infiltrating immune cells. Whether chemotherapy per se has direct impact on PD-L1 expression in cancer cells is currently unclear.

Materials and Methods

Four esophageal squamous cell carcinoma (ESCC) cell lines, TE5, KYSE70, KYSE270, KYSE510, were included. The expression of PD-L1 in ESCC cells, treated with various concentrations of cisplatin, were evaluated by flow cytometry, Western blotting, and qRT-PCR. To study the potential signaling pathways involved in the PD-L1 upregulation induced by cisplatin, we evaluated the nuclear expression of p-STAT1 and IRF1 (for IFN/JAK/STAT pathway), and p65 and p50 (for NF-kB pathway). Finally, we used BAY-11-7082, an irreversible inhibitor of IKKα, to validate the significance of NF- kB pathway in mediating the PD-L1 upregulation induced by cisplatin.

Results

Cisplatin at the doses of 5~ 10 μM increased the proportions of PD-L1-expressing cells detected by flow cytometry in all tested ESCC cells. The folds of increase were 19.29±2.27, 10.13±8.42, 23.19±10.12, 6.20±0.81 for TE5, KYSE70, KYSE270, and KYSE510 treated with 10 μM of cisplatin, respectively. The kinetics of this cisplatin- induced PD-L1 expression, evaluated in TE5 cells, was: after exposure to cisplatin for 24 hours, the PD-L1 expression peaked at 48 hours, and remained elevated at 144 hours after drug exposure. In multiple ESCC cells, exposure to cisplatin did not increase the expression of p-STAT1 and IRF1, suggesting that IFN/JAK/STAT pathway did not involve in the PD-L1 upregulation induced by cisplatin. In co-culture experiments with cisplatin-treated TE5 cells or with condition medium from cisplatin-treated TE5 cells, we confirmed that the PD-L1 upregulation in ESCC cells was not through paracrine manners. In TE5 cells, cisplatin treatment induced nuclear translocation of p65, suggesting that NF-kB pathway was activated. The cotreatment of BAY11-7082 could partially abrogate the PD-L1 upregulation induced by cisplatin in TE5 cells.

Conclusion

PD-L1 expression in ESCC cancer cells can be up-regulated by cisplatin. Our data indicated that this activation is not mediated through the IFN/JAK/STAT pathway, but may involve in activation of NF-κB signaling. (The study was supported by the grant NTUH 104-M2891 and the grant MOST 104-2314-B-002-111)

#4981

Cisplatin and anti-CD70 therapy: Ideal partners in crime against NSCLC.

Julie Jacobs, Vanessa Deschoolmeester, Karen Zwaenepoel, Christian Rolfo, Filip Lardon, Evelien Smits, Patrick Pauwels. _University of Antwerp, Wilrijk, Belgium_.

Introduction:

Upon interaction of CD70 with its receptor CD27, the NFκB pathway is activated, leading to proliferation and survival. Therefore, CD70 expression is tightly regulated and only transiently expressed on cells of the lymphoid lineage. Constitutive expression of CD70 by tumor cells can facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Previously, we have detected CD70 positivity in non-small cell lung cancer (NSCLC) specimens, comprising patients that lack other targeted treatment options. This CD70 expression can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Furthermore, anti-CD70 therapy has the exciting potential to enhance antitumor immune responses by blocking Treg proliferation. In this study, we have focused on the combination of anti-CD70 therapy with chemotherapeutic agents, to increase tumor cytotoxicity.

Material and Methods:

The cytotoxic effect of cisplatin was evaluated on a panel of five NSCLC cell lines (CRL-2868, CRL-5908, CRL-5883, LUDLU-1, A549), differing in their genetic aberrations, histological subtype and expression of CD70. Based on these cytotoxicity experiments 3.5µM, 7µM and 13µM were considered low, medium and high concentrations of cisplatin respectively. Using flow cytometry, we tested the effects of cisplatin on the expression of membranous CD70 at different time points (1h, 6h, 24h, 48h). Next, we evaluated whether an induction in CD70 expression could increase the ADCC potential of anti-CD70 therapy using the xCELLigence RTCA technology. Finally, we assessed the immune stimulatory potential of this combination strategy by analysis of Treg proliferation as well as cytokine production.

Results:

Flow cytometric analysis demonstrated the highest induction of CD70 expression, 24 hours after treatment with 7µM cisplatin, reaching significance in 4/5 cell lines. Next, we assessed whether this increase in CD70 expression could intensify the ADCC effect of anti-CD70 therapy. CRL-5883, a CD70- cell line bearing no response to anti-CD70 monotherapy, showed a significant inhibition of proliferation after sequential administration of cisplatin and anti-CD70 therapy. Also, in A549 (weak CD70+) and CRL-5908 (strong CD70+), the combination of anti-CD70 therapy and cisplatin resulted in a significant decrease in cell survival compared to monotherapy. Also our immune data point towards a favorable effect of the combination.

Conclusion: Our preliminary data show that the combination of anti-CD70 therapy with medium doses of cisplatin significantly increases tumor specific cytotoxicity of the drug, compared to single treatment regimens. This combination strategy has the potential to reduce drug-related side effects and improve general outcome of NSCLC, still the most lethal type of cancer worldwide.

#4982

BNC105 induces tumor micro-environment changes which enhance the efficacy of checkpoint inhibitor therapy in preclinical models.

Daniel J. Inglis, Donna M. Beaumont, Annabell F. Leske, Michaela A. Scherer, Tina C. Lavranos. _Bionomics Ltd., Thebarton, Australia_.

BNC105 is a Phase II stage drug that specifically targets tubulin causing rapid depolymerisation. The therapeutic window of BNC105 has been shown to be superior compared to similar compounds in its class with highly specific action on tumor vasculature and minimal off target activity. BNC105 shows evidence of strong anti-cancer efficacy in vitro and in animal models not only through destruction of tumour microvasculature but also through strong suppression of cancer cell proliferation. Complementary studies in models of chronic lymphocytic leukaemia have shown that BNC105 also activates pro-apoptotic proteins, which mediate cancer cell death. Recent studies have been conducted to assess changes in the tumour microenvironment that make BNC105 amenable to combination with immune checkpoint inhibitors.

Strong synergy with BNC105 has been seen with anti-PD-1 (97%TGI in combination compared to 40% with BNC105 and 74% with anti-PD-1 as monotherapies) and anti-CTLA-4 (70%TGI in combination compared to 27% with BNC105 and 14% with anti-CTLA-4 as monotherapies) using syngeneic MC38 and CT26 colorectal murine tumor models respectively. We hypothesised that the rapid BNC105 induced tumor microvasculature destruction and onset of tumor hypoxia and necrosis leads to an efflux of tumor antigens being presented to the immune system. This has the potential to elicit an immune response against tumors with a lower mutational load than those that would otherwise activate the immune system using checkpoint inhibitors alone. An increase in tumoral IFNgamma can be associated with the presence of antigen-stimulated lymphocytes within the tumor. IFNgamma levels, CD3+ and CD8+ cells were assessed in tumors treated with BNC105 to elucidate the changes in the recruitment of T cell populations within the tumor microenvironment. A more rapid rate of maturation of dendritic cells upon presentation of antigen is also a potential mechanism associated with changes in the tubulin content of the dendritic cell.

The elucidation of the mechanism whereby the disruption of micro-vasculature caused by BNC105 further validates the complementary functional role of BNC105 in immune activation with checkpoint inhibitors. These findings support the investigation of combining BNC105 with immune checkpoint inhibitors with the view of progressing such combinations to clinical evaluation.

#4983

Aminoflavone acts as an immunomodulator of tumor growth in a breast cancer mouse model.

Mariana Alejandra Callero, Cristina Elisa Rodriguez, Aldana Sólimo, Elisa Bal de Kier Joffé, Andrea Loaiza Pérez. _Angel H. Roffo Inst. of Oncology, Buenos Aires, Argentina_.

Aminoflavone (AFP464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER+). AFP464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor growth rate in an estrogen dependent, tamoxifen-sensitive mammary cancer murine model (M05) syngeneic in BALB/c developed in our animal facility. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, it is our aim to investigate whether AFP464 modulates the immune response in our mouse model. Cytometric analyses of splenic cells obtained from AFP464-treated and control animals, at three different times after treatment (13, 21 and 41 days), revealed that MDSCs were lower in animals treated with AFP464 than in the vehicle control group. On the contrary (On the other hand) no differences in macrophages, NK, CD8+ T, and CD4+ T cells were observed between these two groups. We also found a diminished quantity of MDSCs in the inflammatory infiltrate, isolated from tumors treated with AFP464. According to these results, we also observed a higher cytotoxic activity of splenic cells from the AFP464-treated group compared with the control one (13 days: 1,50 ± 0,12 vs 0,74 ± 0,18; 21 days: 2,4 ± 0,3 vs 1,16 ± 0,2; 41 days: 2,97 ± 0,2. vs 1,07 ± 0,2 p ˂ 0,05). After an adoptive transfer of splenic cells to mice bearing M05 tumors, we observed that tumors were significantly smaller in mice that received splenic cells from AFP464-treated mice compared with controls (p ˂ 0,05) . Finally, we also investigated the effects of AFP464 on metalloproteinase, arginase and iNOS activities in peritoneal macrophages. We found a time-dependent modulation: at 13 day after treatment AFP464 induces an anti-tumor M1 profile, which was gradually reverted to a pro-tumoral M2 profile, at 41 days after treatment. These data suggest that AFP464 modulates the immune response in order to collaborate with its anti-tumor activity and places the immune system as a novel target for this anti-cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens.

#4984

Chemotherapy induces a coordinate autoantibody and T-cell response against tumor-associated antigens in pancreatic cancer patients.

Francesco Novelli, Giorgia Mandili, Emanuela Mazza, Sonia Bulfamante Bulfamante, Daniele Giordano, Laura Follia, Paola Cappello. _University of Turin, Turin, Italy_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most difficult cancer to treat, both for lack of effective screening method and for resistance phenomenon. At present surgical resection is the only potentially curative option, but the absence of early symptoms or clinical-pathological markers results in diagnosis at a late, inoperable stage in mostly of cases. Once diagnosed, a number of chemotherapy (CTX), radiation and combination therapy regimens are usually used to treat patients, but responses remain poor. However, chemotherapy is able to enhance tumor immunogenicity. Thus more immunogenic tumor associated antigens (TAA) can be induced by CTX and targeted by passive or active immunotherapy. We have previously identified a number of TAAs that are recognized by autoantibodies in PDAC patients. One of these, alpha-enolase (ENO1), was found to have good antigenic capability and immunization with DNA coding for ENO1 effectively enhanced the survival of genetically engineered mice that develop spontaneously PDAC. To discover TAAs that might be selected for immunotherapy, we have analyzed autoantibody response in 19 PDAC patients' sera before and after CTX. The reactivity of PDAC patients' sera obtained was analyzed on 2-dimensional gel electrophoresis proteome map of CF-PAC1 pancreatic cancer cell line and TAAs recognized by IgG autoantibodies were identified by mass spectrometry. The antibody response against 25 proteins was induced or up-regulated after CTX. Among CTX-induced TAA identified, only four, namely ENO1, FUBP1, CK8 and G6PDH were found able to ex novo induce or up-regulate IgG response in more than seven patients were selected and their ability to activate proliferation of autologous PBMC was evaluated following in vitro stimulation with the corresponding recombinant proteins. Notably, mostly of autologous T cells obtained after CXT displayed an increased proliferation in response to at least one or more of the four selected TAA after in vitro stimulation compared to autologous T cells obtained before CTX. Experiments are in progress to validate these CTX- induced TAA in a mouse model of PDAC and to evaluate the therapeutic value of the combination of CTX with the immunotherapy against these TAAs.

#4985

Radiation potentiates systemic anti-tumor immunity unleashed by a novel TLR9 agonist (SD-101).

Karsten Pilones, Jeffrey Kraynak, Sandra Demaria, Encouse Golden, Silvia Formenti. _Weill Cornell Medicine, New York, NY_.

Background: SD-101 is a CpG oligonucleotide-based TLR9 agonist that engages its target within early and late endosomes of plasmacytoid dendritic cells (pDCs), resulting in DC activation, robust IFN-α responses, and priming of tumor-specific T-cells. We have previously shown that local radiotherapy (RT) may also act as an immune adjuvant and thereby contribute to immune-mediated tumor rejection (J Natl Cancer Inst, 2013: 105:256-265). We hypothesized that the effects of SD-101 may be potentiated by RT and trigger immune-mediated abscopal responses, outside the irradiated field (Int. J. Radiat. Oncol. Biol. Phys., 58:862-870).

Methods: BALB/c mice were inoculated s.c. with syngeneic TSA mammary tumor cells in 2 sites (primary [ipsilateral flank] and secondary [contralateral flank]) to detect abscopal responses. When tumors reached an average of 5 mm in diameter, mice were randomly assigned to 1 out of 4 treatment groups: control (untreated), SD-101, RT, or SD-101+RT. RT was delivered to primary tumors (8 Gy x3 fractions) in 3 consecutive days; secondary tumors were completely shielded from RT. SD-101 was injected into primary tumors (50 μg) three times per week starting on the last day of RT. Mice were followed for tumor growth in both the primary and secondary tumors. In a parallel experiment, mice were euthanized 10 days after initiation of therapy for immune analysis.

Results: Significant growth delays (p <0.05) were observed in the primary tumors of mice treated with monotherapy (RT or SD-101), as compared to the primary tumors of the untreated controls. In contrast, no significant growth delays were observed in the secondary tumors of these mice, when compared to the secondary tumors of their untreated counterparts. The combination of SD-101+RT, however, effectively controlled the primary tumors (100% of mice exhibited complete tumor rejection) of treated mice, while significant growth delays were also observed in the secondary tumors (p <0.05), when compared to the secondary tumors of mice from the other groups. SD-101 alone or in combination with RT, induced intratumoral enrichment of CD8α+ DCs (3.02% of CD11c+ cells in SD-101-treated mice vs 1.08% in untreated mice), whereby these CD8α+ DCs preferentially expressed high levels of activation markers (CD40, CD80, and CD86). Yet, mice treated with SD-101+RT, when compared to the other groups, also demonstrated an expansion of tumor-reactive T-cells (CD8+/PD-1+) both locally and systemically (J Clin Investig 2014 124(5):2246-2259).

Conclusions: Our studies support the rational use of RT as a powerful therapeutic partner to potentiate the anti-tumor effects of SD-101.

#4986

Regulation of radiation-induced in situ tumor vaccination by TGFβ superfamily members.

Claire I. Vanpouille-Box, Silvia C. Formenti, Sandra Demaria. _Weill Cornell Medical College, New York, NY_.

Transforming Growth Factor-beta (TGFβ) and activin A (actA) are members of the TGFβ superfamily with overlapping as well as distinct functions in many processes including regulation of inflammation and immunity. Similar to TGFβ, actA has been shown to promote the conversion of CD4+CD25- T cells into induced regulatory T cells (iTregs), and to potentiate iTregs generation by TGFβ.

Many cancer cells produce actA, and we have recently found that levels of actA released in vitro by breast cancer cells are enhanced by radiotherapy (RT). Interestingly, prolonged exposure to TGFβ inhibitors also resulted in enhanced actA release, consistent with a compensatory mechanism described in development (Carvalho et al., J Cell Sci, 2007). Here we tested the hypothesis that actA plays a role in regulating anti-tumor immunity induced by local tumor RT and TGFβ blockade in vivo (Vanpouille-Box et al., Cancer Res 2015).

4T1 derivatives with conditional actA knockdown (4T1shActA) or non-silencing control (4T1shNS) were engineered using inducible doxycycline (DOX) plasmids and injected s.c. in BALB/c mice (day 0). ActA gene knockdown (KD) was induced by DOX at day 8. TGFβ neutralizing 1D11 or isotype control antibodies were given i.p. every other day starting on day 12. RT was delivered to the primary tumor in 6Gy fractions on five consecutive days starting at day 13. Mice were followed for tumor growth and survival or euthanized at day 22 or 28 for analysis.

Consistent with in vitro data, ActA gene expression was upregulated at day 28 in 4T1 tumors of mice after prolonged TGFβ blockade by 1D11. Neither 1D11 nor ActA gene KD by themselves inhibited tumor growth. However, each intervention significantly improved tumor control achieved by RT. ActA KD in mice treated with RT+1D11 prevented tumor recurrence and improved survival (RT+1D11 vs RT+1D11+shActA p=0.026; RT+shActA vs RT+1D11+shActA p=0.0008). Interestingly, blockade of TGFβ or actA resulted in increased intratumoral Tregs (Control: 11.6%; 1D11: 26.2%, shActA: 21%) and markedly enhanced the increase in Tregs seen with RT alone (RT: 15.7%; RT+1D11: 27.5%; RT+shActA: 30.3%). In marked contrast, when both TGFβ and actA were inhibited Tregs significantly decreased in both non-irradiated (1D11+shActA: 13.6%) and irradiated tumors (RT+1D11+shActA: 7.9% of Tregs). IFNγ production by CD8+ T cells in response to the tumor-specific AH1 peptide was significantly higher in RT+1D11+shActA treated mice compared to RT+1D11 (p=0.045) and RT+shActA (p=0.0147). Additionally, in RT+1D11+shActA mice expression of the activation marker CD69 was markedly increased in intra-tumoral CD8+ PD-1+ T cells (MFI Control: 187; 1D11: 139.5; shActA: 162; 1D11+shActA: 111; RT: 138.5; RT+1D11: 379; RT+shActA: 182; RT+1D11+shActA: 498.5).

Overall, data indicate that TGFβ and actA regulate Tregs numbers in tumors and show a complex interaction with RT. Combined blockade of TGFβ and actA during RT may be required to improve in situ vaccination by RT.

#4987

Role of the PD-1/PDL-1 pathway in resistance of patients with metastatic breast cancer to treatment with radiotherapy and TGFβ neutralization.

Sandra Demaria,1 Claire Vanpouille-Box,1 Rachael E. Hawtin,2 Christie Fanton,2 Andy Conroy,2 Erik Evensen,2 Neha Dixit,2 Alan Barber,2 Dorthe Schaue,3 William H. McBride,3 Mary Helen Barcellos-Hoff,4 Silvia C. Formenti1. 1 _Weill Cornell Medical College, New York, NY;_ 2 _Nodality, South San Francisco, CA;_ 3 _UCLA, Los Angeles, CA;_ 4 _New York University School of Medicine, New York, NY_.

Experimental data and clinical observations indicate that local radiation therapy (RT) converts the irradiated tumor into an in situ vaccine and improves responses to immunotherapy. Preclinical breast cancer models show that TGFb neutralization was required to achieve CD8+ T cell priming specific for multiple tumor antigens, and rejection of the irradiated tumor and non-irradiated metastases (Vanpouille-Box et al., Cancer Res 2015). Tumor response and survival were improved by PD-1 blockade. We explored the role of the PD-1/PDL-1 pathway in resistance to combined RT + TGFβ neutralizing antibody (fresolimumab) in metastatic breast cancer patients (pts).

22 pts were treated in a prospective trial at NYU and UCLA (NCT01401062, supported by DOD MTA BC100481). Pts were randomly assigned to 2 doses of fresolimumab (freso) (1mg/kg and 10 mg/kg, q 3 weeks). Image guided RT was delivered to one metastasis on weeks 2 and 7, 7.5 GyX3. 1 pt achieved objective response, with 28% reduction of tumor burden. The response lasted 11 months, anthracycline-induced acute myelogenous leukemia developed. Pts randomized to the higher freso dose had a hazard ratio of 2.17 (95% CI: 0.753-6.272) for risk of death at 1 year follow up. Currently, at a median follow up of 2 years, 20/22 pts have died.

To explore reasons for the limited response, peripheral blood mononuclear cells collected at baseline, 5 and 15 weeks into treatment, were analyzed. Responses to survivin, as a tumor antigen, were assessed using tetramers. Single cell network profiling (SCNP) was used to evaluate expression of 6 immunomodulatory receptors plus immune signaling in T cells collected from 7 healthy donors (HD) and 15 breast cancer pts (6 at 1mg/kg, 9 at 10mg/kg fresolimumab). In vitro activation of p-AKT and p-Erk was quantified following in vitro T cell receptor (TCR) modulation with anti-CD3/αnti-CD28.

6/12 HLA-A2.1+ pts had pre-existing survivin-specific CD8+ T cells, and 3 showed an increase over time. 2 pts who were negative generated modest responses after RT and 10 mg/kg freso. Prior to treatment, PDL-1 expression was increased in monocytes (p=0.01) and CD4+ T cells (p=0.037) compared to HD. Elevated PD-1 (p=0.054) and OX-40 (p=0.014) expression were identified on CD4+ T cells of pts. Upon TCR modulation, CD4+ and CD8+ T cells from pts showed reduced signaling through p-AKT and, to a lesser extent, p-Erk, compared to HD. Signaling was lower in PD-1+ vs PD-1- CD8+ and CD4+ T cells. Addition of anti-PD-1 pembrolizumab partially restored TCR-mediated signaling through p-AKT and p-Erk in PD-1+ but not PD-1- T cells.

Overall, data indicate impaired T cell signaling in PD-1+ T cells of metastatic breast cancer pts, which may explain the inability to respond to RT + TGFβ blockade. In vitro addition of anti-PD-1 improved T cell signaling through p-AKT and p-Erk. Together with the preclinical data, this supports adding anti-PD-1 to the combination of RT +TGFβ blockade.

#4988

Reprogramming the immunologic microenvironment through radiation and Axl targeting.

Todd A. Aguilera, Marjan R. Rafat, Laura Castellini, Mihalis S. Kariolis, Rie vonEbyen, Edward E. Graves, Amato J. Giaccia. _Stanford University, Stanford, CA_.

There is increasing evidence that hypofractionated high dose ionizing radiation (RT) can enhance antitumor immune responses in many cancers. In some cases the combination of RT and checkpoint immunotherapy can enhance antitumor immune responses. Here, we developed a model to study the genetic, microenvironmental, and immunologic factors of immune mediated antitumor responses after radiation. Two different tumor clones from the same parental transgenic PyMT mammary carcinoma model reveal similar growth characteristics but different responses to RT primarily due to an antitumor immune response. The responsive Py117 tumors increase expression of PD-L1 after RT and the antitumor response is enhanced with immune checkpoint antibodies (Ab) targeting PD-1 and CTLA-4. However the unresponsive Py8119 tumors do not respond to radiation or combination immunotherapy. Key differences in the microenvironment include greater infiltration of suppressive macrophages, lower MHCI on tumor cells, and low T cell infiltrates. Upon profiling differences in the tumors we found that Axl a receptor tyrosine kinase of the TAM family associated with invasion and metastasis in a broad range of tumors was overexpressed on the surface of Py8119 cells but not Py117. We genetically knocked out Axl in Py8119 cells using CRISPR/Cas technology and found greater tumor latency and enhanced radiosensitivity in the mouse but not in culture. The effects are largely immune mediated as the RT response was diminished in nude mice. On analysis of the tumor cells we found that MHCI is significantly increased and secretion of a number of chemokines is diminished after Axl targeting. In the tumor microenvironment there was significant decrease in macrophages, increase in myeloid dendritic cells, and resulting influx of T cells by 10 days after RT. These data suggest that Axl may not only mediate invasion and metastasis but can influence immunoserveilance and response to therapy through suppression of antigen presentation and supporting a tumor promoting immune microenvironment through chemokine signaling.

#4989

Toxicity profile of contemporaneous PD-1 inhibitor immunotherapy and radiotherapy.

Tyler J. Wilhite,1 Ravi A. Chandra,2 Tracy A. Balboni,3 Allison Taylor,3 Ayal A. Aizer,3 Alexander Spektor,3 Patrick A. Ott,3 F. Stephen Hodi,3 Jonathan D. Schoenfeld3. 1 _Harvard Medical School, Boston, MA;_ 2 _Harvard Radiation Oncology Program, Boston, MA;_ 3 _Dana-Farber / Harvard Cancer Center, Boston, MA_.

Purpose/Objectives: PD-1 inhibitors are approved for treatment of metastatic melanoma and non-small cell lung cancer (NSCLC) and under active investigation for treatment of metastatic renal cell carcinoma (RCC). Prior studies suggest that the majority of patients with these malignancies are likely to receive palliative radiotherapy at some point during their care. Growing preclinical and clinical evidence suggests that if delivered optimally, radiotherapy and immunotherapy may synergize. However, there is currently limited data on the safety of contemporaneous treatment with PD-1 inhibitors and radiotherapy and how this combination affects the risk of immune-related or radiotherapy-related adverse events. We aimed to evaluate toxicity in patients with metastatic melanoma, RCC, and NSCLC following contemporaneous treatment with PD-1 inhibitors and radiotherapy.

Materials/Methods: We retrospectively reviewed records from patients with metastatic melanoma, RCC, and NSCLC cancer treated with PD-1 inhibitors and palliative radiotherapy. To account for delayed immune/radiation-mediated effects, patients were included who received radiotherapy either within 75 days prior to a PD-1 inhibitor or within 75 days after. Patient demographics, treatment parameters, and immune-related (ir-AE) and radiotherapy-related adverse events were recorded using CTCAE v4.0.

Results: Fifteen patients (7 melanoma, 4 RCC, 4 NSCLC) underwent 23 courses of palliative radiotherapy and a median of 8 doses of PD-1 inhibition between 2011-2014. Median follow up from date of metastatic diagnosis was 32 months. Radiotherapy courses included both brain (n=6)/non-brain (n=17) and fraction sizes ranging from 3-8 Gy (total doses 8-36 Gy). Radiation was given 19-43 days prior to PD-1 inhibition (n=7) or 30-75 days afterwards (n=19). One patient completed a course of radiotherapy the same day as receiving PD-1 inhibition. Any grade immune-related adverse events were observed in 3 patients (20%). Only 1 of those 3 patients who had received radiation to the skull base was thought to have ir-AEs likely attributable to the PD-1 inhibitor (Grade 2 pericarditis, anemia, and rash). Radiation-related adverse events were observed in 1 patient (7%), who developed moderate mucositis and dysphagia following submandibular radiation to a dose of 30 Gy. No patients developed pneumonitis, despite 7 patients (47%) receiving radiation with fields that partially irradiated the lung.

Conclusions: In this early clinical experience, the contemporaneous receipt of PD-1 inhibitors and palliative radiotherapy did not seem to confer an increased risk of toxicity beyond the expected level associated with each independently. Thus, prospective studies to explore combined treatment with these modalities would likely be safe if undertaken.

#4990

Sequential tracking of PD-L1 expression and RAD50 induction in CTCs and circulating stromal cells of lung cancer patients during treatment with radiotherapy.

Daniel L. Adams,1 Martin J. Edelman,2 Penny Fang,3 Wen Jiang,3 Jianzhong He,3 Ting Xu,3 Hui Gao,3 James M. Reuben,3 Yawei Qiao,3 Steven Hahn,3 Ritsuko Komaki,3 Zhongxing Liao,3 Cha-Mei Tang,4 Steven H. Lin3. 1 _Creatv MicroTech, Inc., Monmouth Junction, NJ;_ 2 _University of Maryland School of Medicine, Baltimore, MD;_ 3 _MD Anderson Cancer Center, Houston, TX;_ 4 _Creatv MicroTech, Inc., Rockville, MD_.

Background: PD-L1 expression can be induced by radiotherapy (RT) and may be an immune escape mechanism after RT, and has the potential to predict for responsiveness to immune checkpoint blockade immunotherapy. However, repetitive biopsies for monitoring PD-L1 expression in tumor and/or stroma are not feasible. Sequential assessment of PD-L1 expression on cancer associated circulating cells during treatment may be a way to measure the efficacy of combining RT and immunotherapy. RAD50 is a DNA repair gene that can be used in tracking tumor cell response to radiation. We therefore evaluated PD-L1 expression and RAD50 induction in CTCs and Cancer Associated Macrophage-Like Cells (CAMLs) in lung cancer patients (pts) before and during RT to track expression changes of these markers.

Methods: Twenty-nine pts with stage I-IV lung cancer were included in this prospective pilot study. Four pts received radiation therapy for stage I disease and 25 other pts received chemoradiation for stage II-IV disease. A baseline blood sample (7.5 ml) was drawn prior to the start of RT (T0), and a second blood sample was drawn at a treatment visit during RT (T1) for a total of 58 samples. Blood was processed using CellSieve™ microfiltration (Creatv MicroTech), stained for cytokeratin 8, 18 & 19 and CD45, and imaged. Using the QUASR (Quench, Underivatize, Amine-Strip and Restain) technique to remove fluoresce signal, all cells were restained for RAD50-AlexaFluor550 and PD-L1-AlexaFluor 488, along with DAPI nuclear stain. The RAD50 foci were quantified within nuclear regions and PD-L1 pixel intensity measured and grouped into 4 IHC groups: 0-negative (pixel average 0-215), 1-low (pixel average 216-300), 2-medium (pixel average 301-750), and 3-high (pixel average 751+).

Results: There was at least one cytokeratin positive cell (i.e. CTC or CAMLs) found in all but 2 samples (97%). CTCs were found in 69% of T0 and 72% of T1 samples, and CAMLs in 79% of T0 and 97% of T1 samples. PD-L1 expression ranged from 34-2552 pixel intensity, with an average of 285 at T0 and 525 at T1 (p=0.07). Interestingly, 9 pts had no PD-L1 expression at T0 but an increase to 2 to 3+ at T1, 12 pts with low/no PD-L1 expression remained low at T1, and 8 pts had high PD-L1 expression that remained high or decreased at T1. RAD50 foci ranged from 0-16 per cell, with an average of 0.56 at T0 that increased to 4.68 at T1 (p<0.001) during radiotherapy. There was no correlation between RAD50 induction and PD-L1 expression.

Conclusions PD-L1 expression and RAD 50 foci were quantifiable in both CTCs and CAMLs, and a correlative response to radiotherapy +/- chemotherapy was quantified. This data suggests that sequential tracking of CTCs and immune-related cells from the primary lung tumor is feasible using microfiltration and may potentially serve as a predictive biomarker for cancer therapy sensitivity.

#4991

Immunologic analyses of uveal melanoma patients treated with radiation therapy.

Christopher A. Barker, Jasmine H. Francis, Robin K. Kuriakose, Ming Lian, Brian P. Marr, David H. Abramson. _Memorial Sloan Kettering Cancer Center, New York, NY_.

Early stage uveal melanoma is frequently treated by radiation therapy (RT) alone. Preclinical studies suggest the efficacy of RT in melanoma is dependent on the immune system. We studied the immune system of patients with uveal melanoma before and after RT, hypothesizing there would be associations between these parameters and clinical outcomes. With institutional review board permission, patient, tumor, peripheral blood count characteristics were extracted from medical records of 299 patients with clinically localized uveal melanoma undergoing RT. Similar data was collected for 16 patients who presented with metastatic uveal melanoma at initial diagnosis. A subset of 8 patients treated with RT for uveal melanoma underwent peripheral blood sampling before, one and three months after RT on a prospective biospecimen collection protocol. Flow cytometry was performed to characterize leukocyte populations before and after RT. Serum was analyzed for cytokines and antibody titers before and after RT. Associations between neutrophil-to-lymphocyte ratio (NLR), leukocyte subpopulations, cytokines and antibody titers with tumor response to RT and survival were investigated. NLR was significantly lower in patients presenting with clinically localized uveal melanoma (mean 2.7), compared to those presenting with metastatic uveal melanoma at initial diagnosis (mean 3.4, p<0.05). Among patients with clinically localized uveal melanoma who underwent RT, a multivariable Cox regression model found that age, tumor stage, and NLR were all significantly associated with overall survival. Changes in several leukocyte populations were observed after RT, with the overall CD8 / regulatory T cell ratio increasing by 27% one month after RT. Increases in several cytokines were observed, with C reactive protein and granulocyte-macrophage colony stimulating factor increasing 2-3 fold in the first month after RT. One of eight patients seroconverted to a positive Melan-A antibody titer three months after RT; this was associated with one of the largest reductions in tumor size observed (>60%). In conclusion, several immune system parameters of patients with uveal melanoma treated with RT appeared to change after treatment, and may be associated with tumor response and treatment outcome, including survival. Further study on the development of distant metastasis in an expanded cohort of patients is planned. Additional investigations into the capacity of RT to modulate the immune system of cancer patients are warranted.

#4992

Development of a novel radiation-induced targeted immunotherapy strategy through oligonucleotide aptamer conjugation.

Ana Paula Benaduce, Randall Brenneman, Diana Cardero, Brett Schrand, Eli Gilboa, Adrian Ishkanian. _University of Miami, Miami, FL_.

Monoclonal antibodies (mAbs) produce dramatic anti-tumor responses in multiple solid tumors. Unfortunately, systemic administration can result in end organ retention and dose-limiting toxicities. Radiotherapy (RT) is a standard-of-care treatment modality that induces direct tumor cell kill through DNA damage. While studies assessing the synergy of RT-induced tumor cell kill with mAbs are ongoing, an additional unexploited effect of RT is the induction of stress response products in the tumor microenvironment, such as vascular endothelial growth factor (VEGF). Oligonucleotide aptamers are short pieces of nucleic acid that exhibit little to no immunogenicity compared to mAbs, making them ideal delivery vehicles for therapeutic mAbs. The purpose of this study was to assess the feasibility of a novel strategy for targeted tumor delivery of existing immunotherapeutic mAbs through conjugation to ligands that bind to radiation induced products.

An antibody recognizing the costimulatory molecule 41BB was covalently modified with a 21-mer ssDNA oligonucleotide linker sequence that serves as an anchor site for the VEGF aptamer synthesized with a 3' extension. Annealing of complementary strands formed a 41BB mAb-VEGF aptamer conjugate. First, VEGF aptamer conjugation to modified 41BB mAb was verified by polyacrylamide gel electrophoresis analysis which revealed the expected size shift of the 41BB mAb-VEGF aptamer conjugate versus the unannealed controls. Conjugation was then verified using flow cytometry. Recombinant mouse 41BB-Fc was immobilized on Protein A Dynabeads and 41BB mAb conjugated with 5' biotinylated VEGF aptamer. 41BB mAb-VEGF aptamer conjugate retention on Dynabeads was confirmed by using streptavidin-PE 5' biotin of aptamer attached to mAb in conjugate binding epitope target.

Functionality of 41BB mAb-VEGF aptamer conjugate was confirmed in vitro using a CD8+ T cell costimulation assay. CD8+ T cells were isolated from 8 week old female C57BL6/J splenocytes, and exposed to sub-optimal levels of CD3 mAb. 41BB mAb-VEGF aptamer conjugate, unconjugated 41BB mAb and isotype control were added to activated cells. Elevated levels of interferon gamma were observed and equivalent in conjugated and unconjugated 41BB mAbs. These results indicate that conjugation of 41BB mAb-VEGF aptamer is feasible and that conjugation does not affect 41BB mAb functionality. Use of this novel targeting approach to improve the therapeutic index of immunotherapeutic mAbs requires validation in murine tumor models.

#4993

Novel fully human anti-GD2 monoclonal antibodies with potent therapeutic activity against neuroblastoma, sarcoma and melanoma.

Govind Ragupathi,1 Xiaohong Wu,1 Ritsuko Sawada,2 Philip O. Livingston,2 Wolfgang W. Scholz2. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _MabVax Therapeutics Inc,, San Diego, CA_.

Background: Gangliosides such as GD2, GD3 and GM2 are promising targets for antibody-mediated cancer therapy since they are expressed at high levels on the surface of several cancers, including neuroblastomas, sarcomas and melanomas. Anti-GD2 monoclonal antibodies have shown promising clinical outcomes in neuroblastoma and a chimeric anti-GD2 antibody (Unituxin™) was recently approved by FDA. However, murine-derived antibodies discovered so far show adverse effects that limit their clinical utility. Fully human antibodies derived from immunized patients might be able to overcome these limitations, since epitope specificity has evolved in the human tissue environment. Here we describe the characterization and functional evaluation of two potential development candidates.

Methods: PBMCs from patients immunized with GD2 lactone-keyhole limpet hemocyanin (GD2L-KLH) conjugate vaccine were utilized to isolate fully human monoclonal antibodies (humAbs). HumAbs were selected for 1) specificity against GD2 initially by ELISA and cell surface binding by flow cytometry 2) by glycan array and affinity assays and 3) by effector functions in complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC) assays. The therapeutic potential of lead antibodies was further evaluated in xenograft models in SCID mice using GD2 positive CHLA255 neuroblastoma as well as with TC71 and SaoS2 sarcoma cell lines.

Results: Antibodies that bind specific to purified GD2 by ELISA were expressed as recombinant antibodies in CHO cells. Many recovered antibodies showed cross-reactivity with several gangliosides in ELISA assays. Two antibodies with high specificity for GD2, 1B7 and 31F9 were selected for further studies. Binding to native antigen expressed on the cell surface of different cell lines was confirmed by FACS analysis. 31F9 was monospecific for GD2 (affinity by Surface Plasmon Resonance was ~4 nM) while 1B7 showed dual specificity for GD2 and GM2 with affinities of ~1 nM for GD2 and ~ 370 nM for GM2, respectively. These two antibodies induced CDC ranged between 35% and 80%, and ADCC with human PBMCs between 40% and 65% on CHLA255, TC71 and SaoS2 cell lines. Survival following IV or SC challenge with SaoS2 and TC71 cells (respectively) was at least doubled following six IP treatments with 200 mcg of either 1B7 or 31F9 twice weekly. Inhibition of CHLA255 tumor growth was less dramatic.

Conclusions: Vaccinated patients produce antigen-specific IgG antibodies that were further analyzed on the molecular level. Antibodies with single and dual specificity (GD2 and GM2) were recovered and shown to be very active in functional assays. Based on these favorable in vitro and in vivo studies, 1B7 and 31F9 humAbs merit further development efforts to evaluate potential utility for treatment of GD2 positive cancers.

#4994

SEA-CD40: from bench to bedside.

Shyra J. Gardai, Haley Neff-LaFord, Angela Epp, Jing Yang, Thomas Manley, Che-Leung Law. _Seattle Genetics, Bothell, WA_.

SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent immune stimulatory activity. CD40 receptor ligation induces multiple pathways; to pave the road for identification of a specific activity signature in the clinical setting, in vitro preclinical assays were developed to monitor the immune modulatory activity of SEA-CD40. Human PBMCs stimulated with increasing concentrations of SEA-CD40 were assessed for immune changes including cytokine production, cellular activation, and modulation of cellular subsets. SEA-CD40 PBMC stimulation elicited a unique set of cytokines including MIP-1β, MCP-1, and IL-8. In addition to inducing cytokines, specific immune cell changes were also observed including up-regulation of stimulatory molecules on monocyte/ macrophages, activation of NK cells, and changes in cellular subsets such as deletion of B-cells. While some of these changes were common across the other CD40 therapeutic antibodies being tested in the clinic, SEA-CD40-specific changes were identified. These changes included a reduction in the immune dampening cytokine IL-10, induction of Th1 CXCR3 positive cells, and reduction of T-regulatory cells, all potentially contributing to an antitumor immune response.

The specific in vitro SEA-CD40 signature was also observed in vivo in cynomolgus monkeys. SEA-CD40 treatment induced the same signature cytokines observed in vitro and elicited the same cellular changes including depletion of B-cells, and activation of CD8+ T-cells. A CD40 receptor occupancy assay was also created to correlate receptor engagement with activity. Interestingly, while SEA-CD40 is rapidly cleared from plasma, it is detectable on the surface of antigen-presenting cells for up to 3 weeks.

The initial starting dose for SEA-CD40 clinical trials was calculated using the minimal anticipated biological effect level (MABEL). SEA-CD40 cytokine induction was the most sensitive preclinical marker of biologic activity and was, therefore, used for MABEL dose calculation. In the ongoing phase 1 First-In-Human clinical trial, this preclinical SEA-CD40 activity signature is being monitored in adult patients with advanced solid tumors (study NCT02376699). Establishing a clear immune biomarker strategy from pre-clinical research to clinical trials is vital for tracking the activity of our immuno-oncology drugs in patients and for identifying a safe and efficacious regimen.

#4995

anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer.

John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. _Emergent BioSolutions Inc., Seattle, WA_.

Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC.

Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume.

Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival.

Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies.

#4996

Targeting FGFR4 with monoclonal antibodies as therapeutic agents for the treatment of rhabdomyosarcoma.

Sivasubramanian Baskar. _National Cancer Institute, Bethesda, MD_.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with two major subtypes, embryonal (ERMS) and alveolar (ARMS), and current treatment modalities have yielded event free 5-year survival in only 30% of the patients with high-risk disease. Therefore, there is a need for novel strategies to identify and validate clinically relevant targets and therapeutics for the treatment of RMS. The fibroblast growth factor receptor 4 (FGFR4) is a very attractive therapeutic target because: 1) the FGFR4 gene is over expressed in RMS, 2) it is directly transcriptionally induced by the PAX3-FOXO1 fusion oncogene found in ARMS, 3) it is crucial for survival, proliferation, metastasis and drug resistance, 4) activating mutations in the kinase domain lead to aggressive growth and poor survival in patients with ERMS and 5) genetic or pharmacologic inhibition of FGFR4 signaling inhibited tumor growth in vitro and in vivo. Most normal human tissues do not express FGFR4 protein as determined by immunohistochemistry and electrochemiluminesence assay. Based these, we hypothesize that monoclonal antibodies (mAbs) targeting FGFR4 and their derivatives can serve as potential therapeutic agents for the treatment of RMS.

We have developed a total of 15 mAbs against human FGFR4 protein from rabbit and mouse (by hybridoma technology), and from human immunoglobulin libraries (by recombinant DNA technology). These mAbs specifically react with human FGFR4 protein in an ELISA and not to the other members of the FGFR family. Flow cytometer analysis demonstrated that they specifically bound to cell surface FGFR4 protein in RMS cell lines although the FGFR4 expression levels was variable in both ARMS and ERMS cell lines. Furthermore, cell surface FGFR4 mediated internalization of the bound antibody upon incubation at 37oC for as little as 30 min and maximum internalization was observed at 2 h. Two of the mouse mAbs tested were able to mediate specific cytotoxicity in FGFR4-expressing cell lines in the presence of an anti-mouse secondary antibody conjugated to cytotoxic drugs (2oADCs), e.g. monomethyl auristatin F (MMAF) or a duocarmycin derivative (DMDM). Dose titration of the anti-FGFR4 mAbs revealed maximum killing at 100 pM indicating their robust activity in vitro, and both ARMS and ERMS cell lines were susceptible to the mAb-mediated cytotoxicity. Ongoing investigations include generation of FGFR4-targeting direct ADCs and evaluation of their efficacy in vivo. Together, these observations support the contention that anti-FGFR4 mAb can be used as a vehicle to deliver a cytotoxic payload in the form of small molecule drugs and toxins. Thus, FGFR-ADCs may serve as potential therapeutic agents for the treatment of patients with RMS, as well other FGFR4-positive cancers including breast, liver, prostate and lung.

#4997

Novel anti-PD-L1/IL-15 bifunctional immunotherapeutics potentiates antitumor immunity.

Yan Wu,1 Zhaojing Zhong,1 Stella Martomo,1 Dan Lu,1 Haifan Zhang,1 Zhanna Polonskaya,1 Xenia Luna,1 Zhikai Zhang,1 Zhun Wang,2 Leo Liu,2 Jeegar Patel,1 James Tonra,1 Henry Li,2 Larry Witte,1 Sam Waksal,1 Zhenping Zhu1. 1 _Kadmon Corporation, New York, NY;_ 2 _Crown Bioscience, Inc., Santa Clara, CA_.

Target-based immunocytokine approaches have been reported to be efficacious in the control of tumor growth in preclinical and clinical studies. By targeting cytokines-activated immune effectors to local tumor sites, an antibody-based immunocytokine is able to achieve antitumor immunity in the tumor microenvironment while reducing cytokines-mediated systemic side effects. Recently, immune checkpoint antagonists to PD-1/PD-L1 have shown success in certain clinical settings across multiple cancer types. However, the full potential of the checkpoint inhibitor is limited due to impaired overall antitumor immunity. It is therefore desirable to develop immunotherapeutics with the capacity of simultaneously inhibiting immunosuppressive pathways and stimulating immune effector cells to potentiate innate and adaptive immune responses against tumor growth. To this end, we generated a bifunctional fusion protein, KD033, composed of an antibody specific for PD-L1 and IL-15 as a novel immunocytokine for achieving better immunotherapeutic efficacy against tumors. Previously, we demonstrated that KD033 has an enhanced immunological activity and stronger antitumor efficacy in some syngeneic mouse tumor models in comparison to single agents. In the present report, we show that the mechanisms of actions of the bifunctional protein in the enhancement of antitumor immune responses results from an increase in Th1 cytokine secretion, the expansion and cytotoxicity of CD8 T-cells and NK cells and a decrease in immunosuppressive cells, i.e. regulatory T cells and myeloid derived suppressive cells in a number of preclinical experimental models. In the preclinical studies, KD033 regimens, with the unique immunological properties, led to stronger anti-tumor efficacy in controlling primary tumor growth and prolonging the survival of tumor bearing mice in a number of mouse tumor models including PDX and GEMM tumor models. Importantly, the PD-L1-targeted IL-15 bifunctional protein had significantly less cytokine-related toxicity when compared to non-targeted full IgG antibody-IL-15 fusion protein in vivo. These results further elucidate the capacity of targeting IL-15-stimulated innate and adaptive immune effector cells into tumor microenvironment, thereby effectively controlling tumor progression while having minimized adverse effect in vivo. These encouraging preclinical results of the novel immunotherapeutics suggest further advancement of this innovative therapeutic candidate towards clinical development for cancer treatment.

#4998

Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer.

Satoko Matsuzaki,1 Kosuke Hiramatsu,1 Satoshi Serada,2 Satoshi nakagawa,1 Shinya Matsuzaki,1 Yutaka Ueda,1 Minoru Fujimoto,2 Kiyoshi Yoshino,1 Tadashi Kimura,1 Tetsuji Naka2. 1 _Osaka university, Suita-city, Japan;_ 2 _Laboratory of immune signal, national institutes of biomedical innovation, health and nutrition, Ibaraki-city, Japan_.

Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens.

Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times.

Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p<0.01). In adhesion assay, we observed significant inhibition of cell adhesion in LSR knockdown cells. These results suggested that LSR is related to cell proliferation and adhesion. A significant anti-tumour effect with 78% was observed in the anti-LSR mAb antibody administrated group compared with control IgG antibody administrated group after 25 days (p <0.001). Furthermore, in intraperitoneal xenograft model, anti-LSR antibody administration group significantly decreased signal intensities compared with the control antibody administration group at day 22 after implantation.

Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer.

#4999

Cellular mechanism of action of bispecific T-cell engager (BiTE®) antibody constructs targeting Folate Receptor 1.

Julie M. Bailis,1 Jessica N. Orf,1 Alexander Sternjak,2 Brian Belmontes,3 Ryan Case,1 Natalia Grinberg,1 Wesley Chang,1 Beth Hinkle,3 Matthias Friedrich2. 1 _Amgen, Inc., South San Francisco, CA;_ 2 _Amgen, Inc., Munich, Germany;_ 3 _Amgen, Inc., Thousand Oaks, CA_.

Bispecific T-cell engaging (BiTE®) antibody constructs connect T cells to target-positive cells to induce cytotoxicity. Clinical proof of concept was achieved in B cell malignancies with the anti-CD19 BiTE® Blincyto®, and several BiTE® antibody constructs are currently in development for solid tumor indications. With the goal of developing a BiTE® for ovarian cancer, we focused on targeting the Folate Receptor (FOLR1). FOLR1 mRNA and protein are highly expressed in >80% of ovarian tumor samples. The FOLR1 protein localizes to the cell membrane of ovarian tumor epithelial cells, whereas in normal tissues such as kidney tubules expression is restricted to the apical cell layer. We generated human FOLR1-BITE® antibody constructs that cross react to cynomolgus monkey and here present the preclinical characterization of these molecules. FOLR1-BiTE® antibody constructs had low nanomolar binding affinity for FOLR1 but did not bind to other FOLR family members. The FOLR1-BiTE® antibody constructs recognized diverse epitopes of the FOLR1 protein. In vitro, binding of FOLR1-BiTE® to CD3 on T cells and FOLR1 on tumor cells led to upregulation of CD25, CD69 and GITR on T cells, as well as release of cytokines including TNF, IFNγ, IL-6 and MCP-1. T cell activation by FOLR1-BiTE® was strictly target cell-dependent. Using time lapse imaging, we demonstrated that T cells were recruited to cancer cells expressing FOLR1 to form a cytolytic synapse and induce target cell lysis. Cytotoxicity mediated by FOLR1-BiTE® antibody constructs occurred even at very low target expression levels (1000 receptors per cell), and was not impacted by the presence of soluble receptor at levels found in ovarian cancer patients. FOLR1-BiTE® antibody constructs also mediated redirected lysis of cell lines that were resistant to standard of care chemotherapy. The in vivo activity of the FOLR1-BiTE® was explored in mouse xenograft studies where immunocompromised mice were implanted with mixtures of human T cells and human cancer cell lines. Daily intraperitoneal administration of the BiTE® inhibited tumor growth, even though the serum half-life of the BiTE® was just a few hours.

#5000

Immunotherapy with anti-PSMA x anti-CD3 bispecific antibody stimulates potent killing of a human prostate cancer cell line and target-mediated T cell activation in monkeys: A potential therapy for prostate cancer.

Seung Y. Chu, Erik W. K. Pong, Rumana Rashid, Hsing Chen, Emily W. Chan, Sheryl Phung, Nancy A. Endo, Maria C. Ardila, Christine Bonzon, Irene W. L. Leung, Umesh S. Muchhal, Gregory L. Moore, Matthew J. Bernett, John R. Desjarlais, David E. Szymkowski. _Xencor, Inc., Monrovia, CA_.

PSMA (Prostate-Specific Membrane Antigen) is a promising therapeutic target in prostate cancer. Exploiting the high and selective expression of PSMA in the prostate, capromab and J591 antibodies are used as imaging or therapeutic agents, and antibody-toxin conjugates such as MLN2704 are being developed. Such antibodies do not stimulate T cell-mediated killing of prostate cancer cells; however, promising clinical data with T cell-recruiting bispecific agents and chimeric antigen receptor (CAR) T cells have created new immunotherapy paradigms that may also have potential in prostate cancer.

We designed long-acting humanized bispecific antibodies that coengage PSMA+ cells and CD3+ T cells to stimulate redirected T cell-mediated cytotoxicity (RTCC) of prostate cancer cells. Unlike other bispecific formats, our antibodies possess an Fc domain and form stable heterodimers that are easily manufactured. Binding to Fcγ receptors was also abolished (reducing the potential for nonselective T cell activation), yet binding to human FcRn was preserved to maintain long serum half-life.

We screened several anti-PSMA x anti-CD3 bispecific antibodies in vitro, and selected XmAb14484 based on its potent (< 1ng/ml) stimulation of human T cell killing of the LNCaP prostate tumor cell line. RTCC activity required PSMA binding, because a non-specific control (XENP13245, anti-RSV x anti-CD3) was inactive. XmAb14484 crossreacts with monkey but not mouse targets; therefore, we evaluated its effects in cynomolgus monkeys. Peripheral blood cells do not express PSMA, so we were unable to monitor target cell depletion. However, T cell engagement of target cells induces distinct effects on peripheral T cells, which can be used as surrogate markers for activity in the prostate. After treatment with 0.03 mg/kg XmAb14484, CD4+ and CD8+ T cells were rapidly activated (quantified by CD69 upregulation) and redistributed from the periphery. Serum cytokines including IL-6 and TNF were also strongly induced. T cell and cytokine responses returned to baseline within 2-3 days. In marked contrast, a 100-fold higher dose (3 mg/kg) of the anti-RSV x anti-CD3 control antibody induced minimal effects, demonstrating that PSMA+ target cells are required for T cell activation by XmAb14484.

In summary, the pharmacologic activities of XmAb14484 on human cells and in monkeys support its clinical assessment in prostate cancer. We also demonstrate that T cell effects readily measured in peripheral blood (T cell redistribution, activation and cytokine induction) are useful surrogate markers of target-specific engagement.

#5001

Quantitative systems pharmacology modeling and analysis provides biological insights into anti-PD-1 dosing and predicts optimal PD-1 x TIM-3 therapeutic properties for bispecifics and fixed dose combinations in immuno-oncology.

Joshua Apgar,1 Jamie Wong,2 Ryan Phennicie,2 Robert Mabry,2 Tatiana Novobrantseva,2 Michael Briskin,2 John M. Burke1. 1 _Applied BioMath LLC, Winchester, MA;_ 2 _Jounce Therapeutics, Cambridge, MA_.

The goal of this collaboration was to enable early quantitative thought experiments and risk assessments by developing and interrogating a quantitative systems pharmacology (QSP) model of the co-modulation inhibitory receptors PD-1 and TIM-3 in immuno-oncology. The QSP model was to: (1) provide predictions of best-in-class profiles for a PD-1 and TIM-3 fixed dose combination (FDC) and dual antagonist platforms, and (2) provide biological insights.

The QSP model was based on first principles as a system of elementary mass-action, mechanistic PKPD, ordinary differential equations. The model parameters and reactions were based on biophysics, and are interpretable. The model reactions include protein synthesis and elimination, ligand-receptor and drug-target formation and turnover, and drug administration and first order clearance. There were four versions of the model: PD-1 monospecific, TIM-3 monospecific, PD-1 x TIM-3 bispecific and fixed dose combination (FDC) targeting PD-1 and TIM-3. The monospecific models were then benchmarked against published data such that model parameter values were set to known values and unknown parameters were estimated. Once benchmarked, the FDC and bispecific models were analyzed by systematically investigating how tuning the model parameters (e.g., affinity, avidity, dose, half-life, target expression, etc.) impacted target inhibition, and to simulate patient variability.

The model predictions were in good agreement with published clinical data from Nivolumab and Pembrolizumab, providing a hypothesis for the apparent inconsistencies in similar clinical doses for therapeutics with affinities differing by several orders of magnitude, and data from RMT3-23 in the TIM-3 driven mouse model. QSP model analysis predicted: (1) there would be diminishing returns on very tight binding biologics due to Target Mediated Drug Disposition (TMDD) that offsets potency, and (2) there is no advantage between FDC, 2-2 bispecific, and 2-1 bispecific formats, which are predicted to be roughly equivalent.

#5002

Anti-tumor activity of a novel anti-human PD-1 antibody BGB-A317 in mouse models.

Xiaomin Song, Zhiyu Tang, Tong Zhang, Mingming Guo, Wenfeng Gong, Yong Liu, Ningning Zhang, Yilu Zhang, Yanjuan Zhang, Jie Ma, Jing Song, Kang Li, Lai Wang. _BeiGene Inc., Beijing, China_.

Background: Programmed death receptor-1 (PD-1) is a co-inhibitory immune checkpoint molecule. The engagement of PD-1 by its ligands negatively regulates T cell function and induces immune tolerance in both normal and pathological conditions, especially in cancer. BGB-A317 is a novel humanized IgG4 anti-PD-1 antibody under clinical development. The anti-tumor activity of BGB-A317 was evaluated in mouse models and reported here.

Material and methods: NOD/SCID mice transplanted with human peripheral blood monocyte (PBL-SCID models) were employed to examine the anti-tumor activity and pharmacokinetics of BGB-A317. In these models, human PBMCs (4-5×106) from healthy donors were co-injected with human tumor cells (A431, 2.5×106) or primary tumor tissue fragments derived from cancer patients (BCCO-028 and BCLU-054, 3×3×3 mm3) subcutaneously in the right flank of the mice. BCCO-028 is human colon cancer and BCLU-054 is human non-small cell lung cancer (NSCLC). The mice were then treated with BGB-A317 or PBS intra-peritoneally once a week from the day of tumor implantation. The tumor infiltrated lymphocytes and PD-L1 expression were examined by immunohistochemistry staining and flow cytometry.

Results: In A431 model, 3 doses of BGB-A317, 1, 3, 10mg/kg, were examined. All three doses of BGB-A317 induced significant tumor growth inhibition (TGI) on day 29 (78%, 89%, and 89%, respectively) (p < 0.001 vs. vehicle treatment). Both AUC0-168h and Cmax of BGB-A317 after single dose (day 1, 1st dose) or at steady state (day 29, 5th dose) increased proportionally from 1 to 10 mg/kg. There was about 2-fold accumulation in drug levels (AUC0-168h and Cmax) at the steady state compared to those after single dose. Increased lymphocyte infiltration and PD-L1 expression were observed in BGB-A317 treated groups. In BCCO-028 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 83% TGI (p < 0.05 vs. vehicle treatment) on day 36. In BCLU-054 model, BGB-A317 at 10 mg/kg QW induced significant tumor growth inhibition, with 45% of TGI (p < 0.05 vs. vehicle treatment) on day 23. BGB-A317 treatment had no significant impact on animal body weight throughout all of the studies.

Conclusions: BGB-A317 demonstrated significant anti-tumor activity in 3 human cancer xenograft PBL-SCID mouse models, including A431 human epidermoid carcinoma, BCCO-028 colon cancer, and BCLU-054 NSCLC models.

#5004

**APX005M, a humanized anti-CD40 antibody with strong immune-modulatory activities capable of tumor eradication** in vivo **.**

Pia Björck, Erin Filbert, Xiaodong Yang, Ovidiu C. Trifan. _Apexigen Inc, San Carlos, CA_.

The success of immune checkpoint inhibitors validates the concept that immunotherapy is an effective approach for the treatment of solid tumors. In addition to reversing tumor-induced immune suppression, immune activating antibodies are being explored as the next generation of immuno-oncology therapeutics. CD40, which is expressed on antigen presenting cells such as dendritic and B cells initiates and regulates both innate and adaptive immunity and is essential for the activation of antigen-specific T cells. APX005M is a humanized IgG1 CD40 agonistic antibody developed using Apexigen's APXiMAB™ discovery platform. APX005M binds with high affinity to human (Kd=0.12nM) and monkey (Kd=0.37nM) CD40. It recognizes a unique epitope that overlaps with the CD40 ligand binding sites and thus blocks the binding of CD40 to CD40L. APX005M is a potent CD40 agonistic antibody capable of activating antigen presenting B cells (EC50=12pM) and dendritic cells (EC50=0.49nM). Its CD40 agonistic activity requires crosslinking by Fc-gamma receptors since F(ab)'2 fragment of APX005M loses the agonistic activity. Upon binding to CD40 expressing tumor cells APX005M induces antibody-dependent cellular phagocytosis (ADCP) and apoptosis. In vivo APX005M completely eradicates CD40+ lymphoma tumors and inhibits the growth of rituximab-resistant tumors. The data suggest that a CD40 agonistic mAb such as APX005M, that activates CD40 through binding to the CD40L binding site while being dependent on crosslinking via Fc-gamma receptors, may represent an ideal immune activating antibody drug candidate for stimulating effective immune responses against tumors. APX005M is currently being evaluated in clinical trials for the treatment of patients with solid tumors.

#5005

AGEN1884 and AGEN2041: Two functionally distinct anti-CTLA-4 antagonist antibodies.

Elise E. Drouin,1 Ana Gonzalez,1 Hao Tang,1 David Savitsky,1 Randi Gombos,1 Jeremy Waight,1 Benjamin Duckless,1 Andrea Schuster,2 Lili Wang,1 Shiwen Lin,1 Cornelia Mundt,3 Gerd Ritter,4 Taha Merghoub,5 Kyle Draleau,5 Jedd Wolchok,5 Daniel Levey,1 Jennifer Buell,1 Marc van Dijk,3 John M. Goldberg,1 Robert Stein,1 Nicholas S. Wilson1. 1 _Agenus, Inc., Lexington, MA;_ 2 _4-Antibody, a wholly-owned subsidiary of Agenus, Inc., Jena, Germany;_ 3 _4-Antibody, a wholly-owned subsidiary of Agenus, Inc., Basel, Switzerland;_ 4 _Ludwig Institute for Cancer Research, New York, NY;_ 5 _Memorial Sloan Kettering Cancer Center, New York, NY_.

CTLA-4 is an important negative regulator of T cell function. Together with CD28, these two co-receptors exemplify a co-inhibitory/stimulatory system that is critical in the regulation of T cell immune responses. Key to this regulatory system are the shared ligands, CD80 and CD86, whose engagement determines whether T cells receive stimulatory (CD28) or inhibitory (CTLA-4) signals. Pre-clinical and clinical studies have shown that anti-CTLA-4 antibodies can enhance tumor-specific immunity through a combination of mechanisms including: 1) blockade and or displacement of CD80/CD86 binding to CTLA-4, leading to CD28 activation; 2) prevention of the trans-endocytosis of CD80/CD86 from the surface of antigen presenting cells by CTLA-4 expressing regulatory T cells; and 3) the selective depletion of CTLA-4 expressing intratumoral regulatory T cells by an Fcγ receptor-mediated mechanism.

AGEN1884 and AGEN2041, two fully human anti-CTLA-4 antibodies identified using the Retrocyte Display™ platform, are being developed for the treatment of advanced malignancies. The antibodies share heavy and light chain complementarity determining regions (CDRs), but differ in their IgG Fc region (AGEN1884, an IgG1, and AGEN2041, an IgG2). AGEN1884 and AGEN2041 selectively bind to human and cynomolgus monkey CTLA-4 with low single digit nM affinity. Further, both antibodies bind CTLA-4 expressed on T cells, and potently block engagement of CD80 and CD86, leading to enhanced T cell responsiveness. In a T cell-dependent antibody response (TDAR) study in cynomolgus monkeys, administration of a vaccine in combination with either AGEN1884 or AGEN2041 augmented the antibody response to the vaccine antigen. This finding demonstrates that both antibodies are functional in non-human primates, and exemplifies their utility in promoting immunity to co-administered antigens in patients, such as therapeutic cancer vaccines. Consistent with this, an anti-mouse CTLA-4 antibody produced potent tumor regressions in combination with a heat-shock protein-based vaccine (a surrogate vaccine resembling Prophage™ heat shock protein-based autologous vaccine).

The distinct IgG backbones of AGEN1884 and AGEN2041 enable distinct optimal effector functions, such as the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). We anticipate that these differences in effector function may be exploited in certain tumors depending on immune cell composition. Taken together, the biochemical and functional attributes of AGEN1884 and AGEN2041 are ideally suited for clinical development, both as single agents and also in combination with other immune education approaches, such as cancer vaccines and immunomodulatory antibodies or small molecule therapies.

#5006

Docetaxel-platinum-fluorouracil (TPF) induction chemotherapy increases PD-L1 positivity and CD8+ / FOXP3+ immune infiltrates in advanced head and neck squamous cell carcinoma (HNSCC).

Charlotte Leduc, Julien Adam, Emilie Louvet, Marine Bernard, Elodie Maingot, Caroline Even, Ken A. Olaussen, Stéphane Temam, Jean-Charles Soria, Sophie Postel-Vinay. _Gustave Roussy, Villejuif, France_.

Introduction: Checkpoint inhibitors targeting programmed cell death-1/-ligand1 (PD-1/-L1) have demonstrated promising clinical activity in locally advanced head and neck squamous cell carcinoma (LA-HNSCC), with overall response rates (ORR) of ≈20% in unselected patients (pts). Moreover, significant improvements have been observed when selecting pts on PD-L1 positivity, with ORR reaching 50-78%. It is therefore crucial to optimize therapeutic sequence between induction chemotherapy (Docetaxel, Platinum, Fluorouracile,TPF) and anti-PD-1/-L1 in order to maximize the number of pts that benefit from these drugs. Methods: All patients with LA-HNSCC treated at Gustave Roussy between 2006 and 2013 by induction TPF followed by surgery were considered. Only patients for who paired samples (pre and post-TPF) were available were kept for further analysis. PD-L1 expression was quantified by immunochemistry (IHC) (clone E1L3N, Cell Signaling) on a Ventana Discovery Ultra autostainer by a senior pathologist according to a validated protocol. PD-L1 IHC staining on tumor cells (TC) and tumor-infiltrating immune cells (IC) was quantified with a positivity threshold of ≥5%. The density of CD8+ and FOXP3+ lymphocytes was also assessed. Results: Out of 313 pts receiving induction TPF, 86 underwent surgery; paired samples were available for 21. Baseline PD-L1 expression was positive in only 2 and 5 samples for TC and IC respectively. An increased PD-L1 expression was observed post-TPF in 3 (14%) and 15 (71%; p=0.0003, T-test) samples for TC and IC respectively. Tumor-infiltrating CD8+ and FOXP3+ populations also significantly increased post-TPF. Conclusion: TPF induction in LA-HNSCC increases PD-L1 positivity on tumor-infiltrating immune cells as well as CD8+ and FOXP3+ populations. This suggests that anti-PD-1/-L1 therapy might be more effective as a second-line than first-line strategy, but warrants clinical evaluation.

IHC staining results

---

|  | pre-TPF | |

post-TPF | |

|

paired samples | median | CI | median | CI | T-test

PD-L1 positive IC (%) | 21 | 1 | 0-10 | 10 | 0-30 | 0.0003***

PD-L1 positive IC (%) | 21 | 0 | 0-20 | 1 | 0-20 | 0.08 NS

CD8+ cell density (n/mm3) | 17 | 119 | 4-779 | 426 | 53-1190 | 0.016*

FoxP3 cell density (n/mm3) | 17 | 91 | 26-180 | 177 | 4-467 | 0.018*

### Predictive and Prognostic Biomarkers

#5007

Differential expression of neurogenes among breast cancer subtypes identifies high risk patients.

Mario Mancino. _IDIBAPS, Barcelona, Spain_.

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural

growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p<0.0001) and distant metastasis-free survival (p<0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p<0.0001) and metastasis-free survival (p<0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.

#5008

Elucidating HER2 molecular heterogeneity of circulating tumor cells among breast cancer patients.

Man Chun Leong,1 Tse Hui Lim,2 Chye Ling Tan,2 Yong Wei Chua,2 Elaine Hsuen Lim,3 Kiley Wei Jen Loh,3 Guek Eng Lee,3 Rebecca Dent,3 Raymond Chee Hui Ng,3 Andrew Wu,1 Wan-Teck Lim,3 Alvin Soon-Tiong Lim,2 Yoon-Sim Yap3. 1 _Clearbridge Biomedics, Singapore, Singapore;_ 2 _Department of Pathology, Singapore General Hospital, Singapore, Singapore;_ 3 _National Cancer Centre Singapore, Singapore, Singapore_.

Background: HER2-positive tumors are often associated with poor prognosis, chemo-resistances and some patients eventually develop refractory disease during HER2 targeted therapy. While different mechanisms of trastuzumab resistance are being identified in recent years, contribution of confounding factors such as inherent genomic heterogeneity, equivocal HER2 amplifications and presence of chromosome 17 polysomy has been less understood thus far. In this pilot study, we aim to examine HER2 heterogeneity in CTCs obtained from breast cancer patients in the Asian population setting.

Methods: CTCs were enriched from blood samples using a label-free spiral microfluidics-based ClearCell® FX system. A total of 26 samples were collected from patients diagnosed with HER2 positive (17/26) and HER2 negative (9/26) breast cancer. The enriched CTCs were analyzed using conventional diagnostic modalities (fluorescence in-situ hybridisation (FISH) and immunocytochemistry) to examine HER2 status. Concordance rate between CTCs and matched primary tumor was evaluated.

Results: HER2-positive CTCs were successfully identified in 14 out of 17 HER2+ patients (82.4%); HER2 gene amplification and chromosome 17 polysomy were observed in 10(58.8%) and 13(76.5%) patients respectively. HER2+ CTC counts ranged from 2 to 30 cells from 7.5ml blood (median: 4 HER2+ CTCs/7.5ml). HER2 amplification was not observed in any of the 9 patients with HER2-negative tumors, though 5 out of 9 patients (55.6%) were identified with CTCs harbouring gain in chromosome 17 (median: 2 HER2+ CTCs/7.5ml). A "false positive" cut-off of more than 2 cells/7.5ml blood were established using receiver operating characteristic (ROC) curve analysis and a concordance rate of approximately 70% between paired tumor tissue and CTC among the 26 patients. Immunofluorescence labelling of CTCs with cytokeratin, CD45 and HER2 antibodies further revealed heterogeneity in HER2 expression on CTCs.

Conclusion: CTCs capture the heterogeneity of breast cancer, and could potentially overcome limitations of tissue biopsy which are site specific. HER2- patients, as confirmed by tissue biopsy, with HER2+ CTCs pose interesting questions while determining treatment regime.

#5010

Prediction of therapy resistance by targeted massive-parallel sequencing in primary HER2-positive breast cancer.

Sibylle Loibl,1 Jan Budczies,2 Wilko Weichert,3 Jenny Furlanetto,1 Albrecht Stenzinger,3 Nicole Pfarr,3 Gunter von Minckwitz,1 Christian Jackisch,4 Andreas Schneeweiss,3 Peter A. Fasching,5 Sabine Schmatloch,6 Bahriye Aktas,7 Valentina Nekljudova,1 Karsten Weber,1 Michael Untch,8 Carsten Denkert carsten.denkert@charite.de9. 1 _German Breast Group, Neu-Isenburg, Germany;_ 2 _Charite Berlin, Germany;_ 3 _University Heidelberg, Heidelberg, Germany;_ 4 _Sana Klinikum, Offenbach, Germany;_ 5 _Universitätsklinikum Erlangen, Erlangen, Germany;_ 6 _Elisabeth Hospital, Kassel, Germany;_ 7 _Universitätsklinikum Essen, Essen, Germany;_ 8 _Helios Kliniken, Berlin, Germany;_ 9 _Charite Berlin, Berlin, Germany_.

Background: Massive parallel sequencing is a promising tool to investigate key molecular events in cancer. Genomic alterations, such as PIK3CA mutations, are important for response to therapy in HER2+ breast cancer (BC). We have investigated genomic alterations in 364 pretherapeutic core biopsies from two prospective clinical trials with or without anti-HER2 therapy.

Methods: A total of 417 formalin-fixed paraffin embedded samples from HER2+ tumors of the neoadjuvant GeparTrio (G3 without anti-HER2 treatment) and GeparSepto (G7 with dual blockade and randomization for paclitaxel vs nab-paclitaxel) study were analyzed by deep targeted massive parallel sequencing, which was successful in 364 tumors (87%). We interrogated hot spot regions of 22 genes (including TP53, PIK3CA, CDH1, FBXW7, PTEN, AKT1, ATM, BRAF, EGFR, ESR1, FGFR2, HRAS, KRAS, NRAS, SF3B1) with a minimum coverage of 500. Only non-synonymous mutations with allele frequencies ≥10% were taken into consideration.

Results: A total of 291 non-synonymously mutated genes were detected in the 364 tumor samples, the most commonly mutated genes were TP53 (47%), and PIK3CA (24%), respectively. EGFR, KRAS, NRAS, HRAS were combined to the XRAS group with 9 (2.5%) mutations. 151 tumors had no, 148 one, 57 two, and 8 tumors three or more mutated genes. There were no statistical significant differences between HR+ and HR- HER2+ tumors; overall 141/252 HR+ and 72/112 HR- tumors had a mutation. HR+ tumors had TP53 mutations in 112/252 (44.4%); PIK3CA mutations in 53/252 (21.0%) and XRAS mutations in 5/252 (2.0%). HR- tumors had 60/112 (53.6%) TP53 mutations, 34/112 (30.4%) PIK3CA mutations and 4/112 (3.6%) xRAS mutations.

Response rates (pCR, ypT0 ypN0) were evaluated separately for the two trials. In G7 the pCR rate was 64.7% in the group with a TP53 mutation vs 60.6% in the group without (p=0.545). The pCR rate was significantly lower in the PIK3CA mutant vs wild type (wt) group (47.7% vs 66.7%; p=0.009). This effect was seen in the HR+ (43.9% vs 61.3%; p=0.052) and the HR- cohort (54.2% vs 80.0%; p=0.029). In the nab-paclitaxel cohort, pCR rates were significantly lower in patients with PIK3CA mutations compared to those without (38.7% vs. 72.0%; p=0.001), whereas in the paclitaxel group, there was no significant difference between patients with and without PIK3CA mutations (55.9% vs. 60.9%; p=0.690; interaction p=0.039). Neither TP53 nor xRAS mutations showed a significant effect on response and treatment effect. In G3 the pCR rate was 16.3% in the PIK3CA wt cohort compared to 23.7% in the mutant cohort; p=0.339.

Conclusion: Targeted NGS on FFPE core biopsies reliably identified the most common genomic alterations in HER+ BC. PIK3CA mutation in HER2+ BC predicts resistance to anti-HER2 therapy. PIK3CA mutations were found to be predictive for response to nab-paclitaxel in G7. The results show that mutational alterations are relevant for response in HER2+ BC.

#5011

Strong FGFR3 staining is a marker for FGFR3 fusions and poor prognosis in diffuse gliomas.

Kirsi J. Granberg,1 Matti Annala,1 Birgitta Lehtinen,1 Juha Kesseli,1 Joonas Haapasalo,2 Olli Yli-Harja,3 Tapio Visakorpi,1 Hannu Haapasalo,4 Matti Nykter,1 Wei Zhang5. 1 _BioMediTech, University of Tampere, Tampere, Finland;_ 2 _Tampere University Hospital, Tampere, Finland;_ 3 _BioMediTech, Tampere University of Technology, Tampere, Finland;_ 4 _Fimlab Laboratories, Tampere University Hospital, Tampere, Finland;_ 5 _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled coil 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibitors. As high FGFR3 expression has been detected in fusion positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level and its potential to indicate FGFR3 fusions. We thus performed FGFR3 IHC on tissue microarrays containing 676 grade II-IV astrocytoma and 116 grade II-III oligodendroglial tumor specimens. Selected cases were further analyzed using targeted sequencing. Moderate-to-strong FGFR3 staining was detected in all tumor grades, was more common in females, and associated with poor survival. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 12 strongly stained cases (staining specificity 86%), whereas no fusions were found in 12 negatively-to-moderately stained cases (staining sensitivity 100%). Fusion-positive cases were predominantly female and negative for IDH, TP53, and EGFR/PDGFRA/MET alterations. Importantly, FGFR3 staining revealed intratumoral heterogeneity, with subclonal negative staining in a subpopulation of fusion-positive cases. Taken together, strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a cost-effective predictive marker for FGFR-inhibitor-based treatment regimens.

#5012

Nuclear expression of lysyl oxidase enzyme is an independent prognostic factor in rectal cancer patients.

Na Liu,1 Thomas R. Cox,2 Weiyingqi Cui,1 Gunnar Adell,1 Birgitta Holmlund,1 Janine T. Erler,2 Xiaofeng Sun1. 1 _Department of Oncology and Department of Clinical and Experimental Medicine, Linkoping University, Linkoping, Sweden;_ 2 _Biotech Research & Innovation Centre, University of Copenhagen, Copenhagen, Denmark_.

Emerging evidence implies that the complex and seemingly paradoxical role of lysyl oxidase (LOX) in cancer may depend on its subcellular localization. LOX expression has been reported extracellularly, as well as intracellularly in both the cytoplasmic and nuclear compartments. In this study we analyze previously unreported LOX expression patterns in the nucleus of rectal cancer patient samples and determine the clinical significance of this expression.

LOX expression was examined by immunohistochemistry in 137 primary rectal cancer patients who participated in the randomized Swedish rectal cancer trial of preoperative radiotherapy between 1987 and 1990. Location and expression of LOX protein were detected by confocal microscopy and Western blot in three colon cancer cell lines.

LOX nuclear expression was high in normal mucosal and showed a significant decrease in primary rectal cancer (P<0.001). Furthermore, a significant increase was observed from primary cancer to lymph node metastasis (P<0.01). High nuclear LOX expression was correlated with a high rate of distant metastasis and total recurrence (P=0.046, P=0.048, respectively). It was also confirmed that LOX (50-kDa and 32-kDa isoforms) are located in the nucleus of in vitro colon cancer cells. High nuclear LOX expression was found in the metastatic cell line SW620 compared to the parental primary cancer cell line SW480. Radiotherapy had no effect on LOX expression and localization in cancer tissues as well as in in vitro culture. Moreover, multivariable analysis showed that high nuclear LOX expression correlated with poor overall survival (P=0.003) and disease free survival (P=0.006), independent of age, gender, TNM stage, differentiation and radiotherapy treatment. Nuclear LOX also correlated with other biological factors (such as NF-κB) known to be associated with patient survival suggesting a possible mechanism. Cytoplasmic LOX is high in primary cancer and metastasis compared with normal tissue, but not prognostic.

So here we firstly proved the nuclear expression of LOX enzyme both in patient rectal cancer tissues and in in vitro colon cancer cells. The strong correlation of nuclear LOX expression to survival offers a promising prognostic biomarker in rectal cancer.

#5013

A meta-analysis of MSI frequency and race in colorectal cancer.

Hassan Ashktorab,1 Sadhna Ahuja,1 Lakshmi Kannan,1 Xavier Llor,2 Nathan Ellis,3 Rosa M. Xicola,2 Adeyinka O. Laiyemo,1 John M. Carethers,4 Hassan Brim,1 Mehdi Nouraie1. 1 _Howard University, Washington, DC;_ 2 _Yale University, New Haven, CT;_ 3 _University of Arizona, Arizona, AZ;_ 4 _University of Michigan, Ann Arbor, IL_.

BACKGROUND: African Americans(AA) are at a higher risk of colorectal cancer (CRC) and some studies report a higher frequency of microsatellite instability (MSI) in their tumors while others report lower frequency compared to Caucasians.

AIM: To determine and evaluate the association of race and clinical factors with MSI rate through meta- analysis.

METHODS: Twenty-two studies out of 15105 (1997-2015) were evaluated after a search in different literature databases, using keywords "colorectal cancer, microsatellite instability". We used random effect meta-analysis to calculate the MSI frequency in all studies as well as in African American and Caucasian samples. Meta-regression analysis was used to assess the univariate effect of race, gender, age, tumor location and stage on MSI rate.

RESULTS: The overall MSI frequency among CRCs was 17% (95%CI: 15%-19%, I²=91%). The MSI rate among racial groups were 12%, 12% and 14% in AAs, Hispanics and Caucasians respectively and was not significantly different. Sub-group analysis of studies with racial information indicates MSI OR (95% CI) of 0.78 (0.58-1.06) for AAs compared to Caucasians.

CONCLUSION: CRCs demonstrate an MSI frequency of 17%. MSI frequency differences between AAs and Caucasians were not pronounced, suggesting other factors contribute to the racial disparity. There is a large variation in MSI rate between different studies. Methodology approaches and biological sources of this variation should be investigated.

#5014

Predictive value of wild-type EGFR determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer.

Said Abdullah Khelwatty,1 Sharadah Essapen,2 Izhar Bagwan,3 Margaret Green,3 Alan Seddon,1 Helmout Modjtahedi1. 1 _Kingston Univ. London School of Life Science, Pharmacy and Chemistry, London, United Kingdom;_ 2 _Royal Surrey County Hospital, St. Luke's Cancer Centre, Guildford, United Kingdom;_ 3 _Royal County Surrey Hospital, Department of Histopathology, Guildford, United Kingdom_.

The aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in patients with colorectal cancer, and EGFR is an important therapeutic target in such patients. To date, of the anti-EGFR monoclonal antibodies (mAbs), only cetuximab and panitumumab have been approved for the treatment of patients with metastatic colorectal cancer (mCRC). While treatment with a combination of antibodies and cytotoxic drugs improves response rate and median time to progression in some colorectal cancer patients, the duration of response is often limited. In addition, no clear association has been found between the expression level of the EGFR protein in the tumours, determined by the FDA-approved EGFR PharmDx kit, or other standard anti-EGFR antibodies, and the response to the EGFR inhibitors. We have previously shown that kits such as the EGFR PharmDx™ kit, used to determine the expression of EGFR, does not discriminate between the wild-type EGFR (wtEGFR) and the type III deletion mutant EGFR (EGFRvIII), and as such could have contributed to the lack of association between the expression of EGFR and response to anti-EGFR antibodies. Therefore, the aim of this study was to determine the predictive value of wtEGFR for response to therapy with cetuximab. The expression levels of wtEGFR, mutated EGFRvIII, and phosphorylated EGFR (pEGFR Tyr1068 & Tyr1173) were determined using immunohistochemistry and specific antibodies in 61 FFPE tumour specimens from patients with mCRC treated with cetuximab. Sections were scored by the percentage of positive tumour cells, location and intensity of staining. Their associations with response and progression free survival were evaluated using Chi-squared and Kaplan-Meier survival curves and log-rank test respectively. Overall 11% and 46% of the cases were found to express membranous and cytoplasmic EGFR, using mAb specific for wtEGFR (DAK-H1-WT). On the other hand using anti EGFR.113 clone antibody, which does not discriminate between the wtEGFR and EGFRvIII, 11.5% and 18% of the cases were found to express the cytoplasmic and membranous EGFR respectively. While all cases were negative for the pEGFR, the expression of EGFRvIII was mainly cytoplasmic, occurring in 41% of the cases examined. Of these, only the cytoplasmic expression of wtEGFR was found to be significantly associated with poor response to treatment (P = 0.002) and progression free survival (95% CI, 0-3 months vs 4.5-17 months, P = 0.001). Taken together, our results suggest that cytoplasmic expression of wtEGFR is associated with a significantly shorter progression free survival, and is a predictive marker for poor response to treatment with the anti-EGFR mAb cetuximab. These findings underlie the need for further investigations, in a larger population of patients, to validate the predictive value of wtEGFR for response to therapy with anti-EGFR antibodies in patients with mCRC.

#5015

Pretreatment carcinoembryonic antigen levels predict survival in patients with rectal adenocarcinoma.

Oxana V. Makarova-Rusher, Julius Strauss, Susanna Ulahannan, Chul Kim, Jaydira Del Rivero, Austin Duffy, Tim F. Greten. _National Cancer Institute, National Institutes of Health, Bethesda, MD_.

Background: Population-based studies have reported elevated Carcinoembryonic Antigen (CEA) level as an independent prognostic factor in patients with colon cancer, thus supporting inclusion of CEA-based C stage in classical TNM staging for colon cancer. However, the effect of C-stage incorporation on outcomes for patients with rectal adenocarcinoma is unknown.

Methods: The Surveillance, Epidemiology and End Result (SEER) database was used to collect data from 2004 to 2007 for patients with rectal adenocarcinoma by topography code C20.9 and histology codes 8140-8144, 8210-8211, 8220-8221, 8260-8263, 8440, 8480-8481, and 8490. CEA stage C0 =normal CEA or C1=elevated CEA was assigned to patients with known pretreatment CEA levels. Observed survival (OS) by American Joint Committee on Cancer (AJCC) stages I-IV and CEA stage C0 or 1 was determined using Kaplan Meier method. Relative survival (RS) as a net measure of cancer survival adjusted for sex, race, age and date was calculated in addition to observed survival (OS). Log-rank was used to compare observed survival. Z-test with corresponding p values was used to compare 5-year relative survival.

Results: We identified 25,241 patients with a record of histologically confirmed invasive rectal adenocarcinoma. Approximately half (N=13,151) of these patients had records of pretreatment CEA levels: N=6,360 stage C1, N=6,690 stage C0 and a small number (101) with borderline CEA levels. Mean age at diagnosis was similar in both groups, 64.2 for C0 and 64.7 for C1. Among patients with C1 disease the leading AJCC stage was distant metastatic, stage IV (33.8%) followed by 25.8% stage III, 20.7% stage II, 13.8% stage I, and 5.9% unknown stage. In contrast to CI disease, the most common stage for C0 was stage I (35.2%), and only 6.3% of patients with C0 were diagnosed with stage IV disease. Observed survival by each of I-IV AJCC TNM stages was decreased for C1 stage relative to C0, p<0.001. The 5-year OS by AJCC TNM stage for C1 was as follows: 56.7% for IC1 (CI=53.3-59.9), 55.4% for IIC1 (CI=52.7-58.1), and 53.4 % for IIIC1 (CI=51.0-55.8). The 5-year OS by AJCC TNM stage for C0 was 75.8% for IC0 (CI=74.1-77.5), 68.8% for IIC0 (CI=66.5-71.0), 65.4% for IIIC0 (63.3-67.5). Stage shifting was observed with IIIC0 disease, which had superior OS as compared to stage IIC1 and IC1 (p<0.001). For stage IV disease, the 5-year OS for C0 was more than double the 5 year OS for C1, 20.8 (17.1-24.9) vs. 7.9 (6.8-9.1), p<0.001. In concordance with this OS data, the 5-year relative survival analysis also showed a significant difference between C1 and C0 stages of rectal adenocarcinoma in the respective AJCC TNM stages, p<0.001.

Conclusion: Our study suggests that pretreatment CEA levels predict survival in patients with rectal adenocarcinoma, in accordance with previous data in colon cancer. Therefore, our study supports C-stage inclusion in AJCC TNM staging for this neoplasm. Further prospective confirmatory studies are warranted.

#5016

Elevated serum monocyte chemotactic protein 4 (MCP4) as a novel noninvasive prognostic and predictive biomarker for detection of metastasis in colorectal cancer.

Yoshinaga Okugawa,1 Yuji Toiyama,2 Koji Tanaka,2 Mikio Kawamura,2 Takahito Kitajima,2 Tadanobu Shimura,2 Hiroki Imaoka,2 Hiroyuki Fujikawa,2 Susumu Saigusa,2 Yasuhiro Inoue,2 Motoyoshi Tanaka,1 Yasuhiko Mohri,2 Chikao Miki,1 Ajay Goel,3 Masato Kusunoki2. 1 _Department of Surgery and Medical Oncology, Iga Municipal Ueno General Citizen's Hospital, Iga, Mie, Japan;_ 2 _Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie, Japan;_ 3 _Center for Gastrointestinal Cancer Research; Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Research Institute and Charles A. Sammons Cancer Center, Baylor University Medical Center, Dallas, TX_.

Purpose. Despite the recent progress in diagnosis and treatment for colorectal cancer (CRC), prognosis of CRC patients still remains poor. Metastatic recurrence is the major causes of cancer-related death in CRC patients, and recent progress in treatment options, especially technical advances in invasive treatment for metastatic lesion, have improved the prognosis of CRC patients with metastasis. Furthermore, almost half of patients that receive chemotherapy derive no clinical benefit, though all are exposed to these severe toxic and expensive therapeutic regimens in patients with metastatic CRC patients. In view of these underlying issues, for improvement of patient's prognosis, there is a keen interest in developing robust biomarkers that can identify the subset of patients who can benefit from intensive post-treatment surveillance protocols for early detection of recurrence, and prompt decision of appropriate treatment protocol. Herein, we systemically and comprehensively evaluated differentially levels of serum cytokines using array-based techniques to identify novel and reliable serum biomarker to predict metastasis and poor outcome in CRC.

Methods. The study design included an initial dual screening phases, and followed by a subsequent clinical validation phase in this study.In discovery phase, we examined cytokine profiling using preoperative serum from two different CRC cohorts (n=30) to identify differentially expressed serum cytokines in CRC patients with metastasis. In validation phase, serum levels of MCP-4 were assessed in 194 CRC patients by enzyme-linked immunosorbent assay, and their relationships with clinicopathological findings, including recurrence and survival, were investigated.

Results. In discovery phase, three cytokines (elevated: MCP-4, ENA-78; decreased:Ckb8-1) were overlapped as a differentially expressed cytokines in serum from CRC patients with metastasis compared those without metastasis. High serum MCP-4 was significantly associated with older age (p = 0.033), advanced T stage (p=0.029), distant metastasis (p=0.011) and UICC stage classification (p = 0.006). Cox regression analysis showed that elevated MCP-4 was a significant and independent prognostic factor of disease free survival (HR:2.6, 95%CI:1.0-6.7) and overall survival (HR:2.7, 95%CI:1.3-5.9) in all CRC patients. Furthermore, logistic regression analysis revealed that high serum MCP-4 level was an independent marker in predicting distant metastasis in CRC (OR:3.8, 95%CI:1.3-10.9).

Conclusion. Our comprehensive study highlights the clinical feasibility of serum MCP-4 as a prognostic and predictive biomarker for distant metastasis and recurrence in CRC patients. Quantification of serum MCP-4 concentration might support the early detection/prediction of recurrence and may contribute to the prediction of clinical outcomes in CRC.

#5017

Tumor mismatch repair (MMR) status and prostate cancer: insights into the clinical and biological behaviour of MMR deficient versus MMR intact cases.

Brianan McGovern,1 Thomas U. Ahearn,2 Edward Giovannucci,2 David Gallagher,1 Christopher Sweeney,3 Lorelei Mucci,4 Stephen Finn5. 1 _St. James's Hospital, Dublin, Ireland;_ 2 _Harvard TH Chan School of Public Health, Boston, MA;_ 3 _Harvard Medical School and Dana Farber Cancer Institute, Boston, MA;_ 4 _Harvard TH Chan School of Public Health and Dana Farber Cancer Institute, Boston, MA;_ 5 _St. James's Hospital and Trinity College, Dublin, Ireland_.

Introduction:

Lynch syndrome, characterized by defective function of the DNA mismatch repair (MMR) system, results in a higher risk of developing certain cancers such as colorectal cancer. The role of the MMR system in prostate cancer is understudied. The MMR system is essential for repairing DNA damage and maintaining genetic stability. We evaluated the clinical and prognostic significance of MMR status in prostate cancer, as determined by immunohistochemical analysis, among a large cohort of prostate cancer patients with extensive clinical follow-up.

Methods:

In the Health Professionals Follow-up Study and Physicians' Health Study, we studied 985 incident prostate cancer cases diagnosed between 1982 and 2005. Immunohistochemistry was used on tissue microarrays to measure MMR status in prostate tumors by measuring expression of MLH1, MSH2, MSH6, and PMS2. Tumors were characterized as absent or present for staining, with endothelium or lymphocytes used as an internal positive quality control. We evaluated the association between MMR proteins and clinical and molecular features. Men were followed prospectively through 2012, and Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals to examine the association of MMR status with lethal disease adjusting for clinical factors.

Results:

On average, men were followed for 13.8 years, during which there were 90 lethal events. Loss of tumor MSH2 (p=0.03) and PMS2 (p=0.001) expression was significantly associated with lower Gleason grade. Tumors with loss of MLH1, MSH2, MSH6, and PMS2 expression were significantly less likely to be positive for the TMPRSS2:ERG gene fusion (all p-values <0.0005). Loss of expression of all 4 markers was significantly correlated with lower KI67 (p-values <0.0005) expression and positively correlated with TUNEL (p-values <0.005) expression. Concordant loss of MLH1/PMS2 expression (p=0.06) and loss of MSH2/MSH6 expression (p=0.002) were associated with having a positive family history of prostate cancer. After adjustment for clinical-pathologic variables, none of the MMR proteins were associated with lethal prostate cancer.

Conclusion:

We found that loss of MMR expression is associated with low frequency of TMPRSS2:ERG fusion, lower proliferation, and higher apoptosis. Collectively, our findings suggest the MMR system may play a key role in prostate cancer. The positive findings of MMR status and prostate cancer family history is intriguing and warrants future studies.

#5018

Immunohistochemical expression of lysyl oxidase in prostate cancer.

Virgilia Macias, Santiago Delgado, Andre A. Kajdacsy-Balla. _University of Illinois Medical Center, Chicago, IL_.

Background:

Lysyl oxydase (LOX) is a family of enzymes involved in catalyzing the cross-linking of collagen and elastin in the extracellular matrix, playing a crucial role in the building, strengthening and maintenance of tissues. Changes in its expression have been associated with many diseases. In oncology, these changes have been implicated in every step of tumor progression in different organs, with currently little existing data on prostate. The aim of this study is to analyze the nuclear expression of LOX in prostate tumor epithelial cells using a prostate cancer tissue array cohort.

Design:

PCa tumor samples were obtained from an outcome-based TMA set (5 tissue microarray blocks with 200 paired recurrence (R) and non-recurrence (NR) cases, 400 subjects total, each with quadruplicate 0.6 mm in diameter cores) from the Cooperative Prostate Cancer Tissue Resource (CPCTR). Each case was obtained from a PSA-recurrence subject matched to a non-recurrence subject based on age; race; interval since prostatectomy; Gleason sum-score and pTNM stage. All had at least 5 years follow up, and cases with known metastases were excluded. TMA sections were incubated with rabbit polyclonal anti-human LOX antibody (1:800, NB100-2527, Novus Biologicals) using the Bond™ polymer refine detection HRP (Leica Biosystems, DS9800). Nuclear LOX staining was scored semi-quantitatively as follows: 0, negative; 1+, weak; 2+, moderate; and 3+, strong. Percentage of positively stained cells at each intensity level was recorded to obtain a final h-score The Wilcoxon signed-rank test was used for the statistical analysis of the pair-matched dataset to assess the 2 groups (R vs. NR) for differences in LOX expression.

Results:

Data generated was available in 168 complete pairs. The Wilcoxon signed rank test showed no statistically significant differences for the h-score between the two groups (R vs. NR) (median=2.15625 [range 0-3] vs. median=2.2583333335 [range 0-3], p=0.24604).

Conclusions:

The role of LOX in prostate cancer has not been fully established, and inhibitory and stimulatory effects have been reported. This is the first study in a case-control cohort evaluating the expression of LOX in prostate cancer. Based on our results we are now completing the analysis comparing expression of LOX in normal epithelium and cancer associated stroma.

#5019

Impact of the ERG status on the prognostic relevance of prostate cancer biomarkers: a survey of 13,000 patients.

Ronald Simon, Martina Kluth, Claudia Hube-Magg, Maria Christina Tsourlakis, Sarah Minner, Christoph Burdelski, Katharina Grupp, Nathaniel Melling, Asmus Heumann, Christina Koop, Thomas Steuber, Markus Graefen, Hartwig Huland, Guido Sauter, Thorsten Schlomm. _Univ. Medical Ctr. Hamburg-Eppendorf, Hamburg, Germany_.

The TMPPRS:ERG fusion is the most frequent genomic alteration in prostate cancer leading to androgen dependent up-regulation of the ERG transcription factor. ERG activation deregulates more than 1,600 ERG-responsive genes, and some molecular alterations have been linked to either the subset of ERG negative or ERG positive cancers. While studies in large cohorts of prostate cancers treated by radical prostatectomy have demonstrated that ERG fusion alone has no impact on patient outcome, there is growing evidence that the prognostic relevance of many other biomarkers may be different in cancers with and without ERG fusion. In order to better understand the impact of the ERG status on the prognostic relevance of prostate cancer biomarkers, we took advantage of our molecular database obtained from immunohistochemistry and fluorescence in situ hybridization (FISH) analyses of more than 50 markers with respect to expression levels and gene copy number changes in 13,665 prostate cancers that had been assembled in a tissue microarray (TMA) format. Markers with strongest associations to presence of ERG fusion included CRISP3, HOOK3, RBM3, LPCAT1, BAZ2A, and deletions of 3p and PTEN (p<0.0001 each). Markers with strongest associations to absence of ERG fusion included SPINK1 and deletions of 6q and 5q (p<0.0001 each). Alterations of 47 markers were linked to reduced time to biochemical recurrence if all cancers were analyzed regardless if the ERG status. However, subset analyses revealed, that the prognostic relevance of 18 (38%) of these 47 markers was driven either by the subset of ERG positive (n=7) or ERG negative cancers only (n=11). Combined analysis of these markers identified ERG status dependent molecular classifiers that provided prognostic information exclusively in ERG negative or in ERG positive prostate cancers. Multivariate modeling indicated that the classifiers were independent of established prognostic factors, including pre-surgical (PSA, Gleason grade on biopsy, clinical stage) and post-surgical (pT stage, Gleason of the radical prostatectomy, nodal stage, resection margin stage) parameters. In conclusion, the results of our study demonstrate that the impact of many candidate prognostic markers in prostate cancer strongly depends on the ERG status. Our growing molecular database attached to more than 13,000 prostate cancers allows for the continuous validation of candidate prognostic markers and their impact in molecularly defined subsets of the disease.

#5020

Associations of clinical, molecular and immunohistochemical parameters in five different types of uterine smooth muscle tumors.

Margaux J. Kanis,1 Jennifer L. Bender,2 Anna E. Strohl,1 Denise Scholtens,1 Dachao Liu,1 John Lurain,1 Shohreh Shahabi,1 Jian-Jun Wei1. 1 _Northwestern University Feinberg School of Medicine, Chicago, IL;_ 2 _Weill Cornell Medical College, New York City, NY_.

Objective:

To evaluate clinical, molecular and pathologic factors associated with 5 types of uterine smooth muscle tumors.

Materials and Methods:

Seventy-nine patients with leiomyosarcoma [31 (39.2%) LMS], smooth muscle tumors of undetermined significance [10 (12.7%) STUMP], atypical leiomyoma [19 (24.1%) ALM], mitotically active leiomyoma [5 (6.3%) MALM], and cellular leiomyoma [14 (17.7%) CLM] treated from 1997-2015 were analyzed. Statistical analysis was performed using ANOVA, chi-square and logrank tests.

Results:

Mean age (years) differed across histologies: 54 (LMS), 36 (STUMP), 41 (ALM), and 47 (CLM/MALM) (p<.0001). Median follow up was 37 months (range 0-226). Those with LMS were more likely to have a family history of cancer (48%, p<.003). Tobacco use did not vary by histology but was significantly associated with recurrence (50 vs. 17%, p=.03). Fifty-four percent of LMS recurred and 48% died. There were no recurrences or deaths due to non-LMS histology. Patients who recurred were more likely to have a family history of malignancy (58 vs 41%, p =.0005). There was no association with recurrence or survival for: ethnicity, parity, hormone use, personal history of malignancy, tumor size or weight.

Of 6 gene markers examined by immunohistochemistry (IHC), ER and PR expression were significantly lower in LMS than in other types (p <.0001). High ER IHC intensity was protective against recurrence compared to negative ER IHC (3 vs 42%, p=.0014). High PR IHC intensity was also protective against recurrence compared to negative PR IHC (3% v 56%, p <.0001). Higher PFS was significantly associated with ER (p=.0019) and PR (p <.0001) positivity. PR was associated with a significant increase in OS (p<.0001). Ki67 index was higher in LMS than other types (p=.05). PTEN IHC intensity correlated with histology (p=.03) and a shorter PFS and OS (p=.01, p=.018, respectively). PTEN molecular analysis correlated with shorter survival (p<.0001). Using an H-score cut-off of 40 for ER and 25 for PR, LMS will be positive in 21-24% of cases compared to over 80% in other histologies. Ki67 was uniquely positive in 81% of LMS tumors. P53 mutations were detected in LMS, STUMP and ALM, and absent in CLM/MALM.

Conclusions:

ER/PR and Ki67 IHC are valuable biomarkers in differentiating histology and predicting clinical recurrence in uterine smooth muscle tumors. Smoking cessation is key to decreasing recurrence.

#5021

Glutathione-S-transferases polymorphisms are associated with increased risk of relapse in pediatric patients with acute lymphoblastic leukemia.

María M. Abbate,1 Daiana B. Leonardi,1 Javier N. Brandani,1 María C. Riccheri,2 Geraldine Gueron,1 Graciela Alfonso,2 Elba S. Vazquez,1 Javier Cotignola1. 1 _Iquibicen, Conicet/UBA, Caba, Argentina;_ 2 _Hospital Nacional Prof. A. Posadas, El Palomar, Buenos Aires, Argentina_.

Current treatment protocols of the International Berlin-Frankfurt-Münster Study Group (BFM) for childhood Acute Lymphoblastic Leukemia (ALL) include the stratification of patients into groups of risk for disease relapse. This stratification is based on biochemical, molecular and cytogenetic parameters at diagnosis, and the early response to treatment determined by the minimal residual disease. ALL treatment protocols are continuously revised and improved, but some patients still recur and die of disease. The inclusion of genotype at ALL diagnosis as a genetic predictor of disease outcome is under constant study. However, results are inconclusive and seem to be population specific. Hence, we aimed to analyze the predictive value of germline polymorphisms for childhood ALL relapse in order to support their inclusion as additional tools in disease diagnosis and therapeutic protocols.

We retrospectively recruited 140 Argentine patients diagnosed with de novo ALL (median age at diagnosis: 6.1 years old, range: 0-19). The protocol was approved by the Institutional Ethical Committee in compliance with the Ethical Principles enunciated by the Declaration of Helsinki. Genotypes were analyzed using PCR-RFLP (GSTP1 c.313A>G, MDR1 c.3435T>C, and MTHFR c.665C>T) and multiplex PCR (GSTT1 null, GSTM1 null). Relapse-free survival (RFS) was assessed using the Kaplan-Meier method and the risk for relapse was estimated using multivariate Cox proportional hazard models (median follow-up time: 52 months). Multivariate models included gender, risk group, treatment protocol, age at diagnosis and genotypes as covariates.

Patients with the GSTP1 c.313GG genotype had worse RFS (p=0.025) and a 2.6-fold increased risk for relapse (95%CI=1.1-6.1, p=0.030). In addition, since GSTs are enzymes that frequently participate in the same biological pathways with overlapping substrate specificity, we considered an additive score that captures information on the genotypes of GSTT1, GSTM1 and GSTP1. We found that patients with higher number of risk alleles genotype had statistically significant shorter RFS (p=0.021) and over a 2.5-fold increased risk for relapse (95%CI=1.1-5.6, p=0.027).

GST polymorphisms seemed to modify the risk of relapse and RFS of Argentine patients with childhood ALL. The inclusion of these genetic markers in ALL treatment protocols might improve risk stratification and reduce the number of relapses and deaths.

#5022

The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome.

Marina Martello,1 Daniel Remondini,2 Enrica Borsi,1 Barbara Santacroce,1 Mauro Procacci,1 Angela Flores Dico,1 Annalisa Pezzi,1 Elena Zamagni,1 Paola Tacchetti,1 Lucia Pantani,1 Giulia Marzocchi,1 Beatrice Anna Zannetti,1 Katia Mancuso,1 Serena Rocchi,1 Giovanni Martinelli,1 Michele Cavo,1 Carolina Terragna1. 1 _DIMES-Department of Experimental, Diagnostic and Specialty Medicine, Bologna, Italy;_ 2 _DIFA-Department of Physics and Astronomy, Bologna, Italy_.

Introduction In most human cancer p53 impairment is a driver event, which confers a survival advantage to affected cells. In Multiple Myeloma (MM), the role of p53 clonal aberrations (mainly 17p del) is well recognised, whereas the prognostic relevance of TP53 mutations is less clear, due to the very limited frequency of clonal lesions. Here we aim at characterizing by ultra-deep sequencing (UDS) the TP53 mutational state in both newly diagnosed and relapsed MM pts, to assess the prognostic role and evolution over time of small TP53 mutated sub-clones.

Pts and methods A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and ASCT was included in this study. In 29 cases, samples were collected also at relapse(s). DNA was obtained from CD138+ highly purified plasma cells and SNPs array profiled (Affymetrix). TP53 gene was analysed by amplicon-targeted UDS approach (GSJ, 454 Roche). A specific bioinformatics pipeline was set up to discriminate between low frequency TP53 variants and sequencing errors.

Results With a median coverage of 1386X, 129 correctly called TP53 variants were detected. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts). . According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with <2 variants (low load), respectively. The clinical impact of the TP53 sub-clonal mutational load was evaluated in 90/99 MM (median follow up=70 months). Pts carrying high TP53 sub-clonal mutational load had significantly shorter OS and OS after relapse (% at 5 years: 38 vs 74 and 30 vs 73, respectively), as compared to the others, while no difference between these two groups was seen regarding PFS and TTP. Multivariate analysis showed that high TP53 mutational load, resulted independent factors adversely affecting OS and OS after relapse (HR=2.38, CI: 1.31-5.98; HR=2.6, CI: 1.01-6.42). None of the detected genomic aberrations significantly influenced the response to front-line induction therapy. The distribution of both TP53 sub-clonal variants and genomic CNAs was overall modified in longitudinally collected samples: overall 90% of relapsed pts carried at least one sub-clonal variant (on average 1.63 variants per pts). Moreover, 5 different sub-clonal lesions proved a linear increment of both TP53 VAFs (from 29.4% to 54.6%; from 7.8% to 12.4%; from 0.5% to 4.3%) and TP53 CN loss smooth signal (from 7% to 89% and from 50% to 100%).

Conclusion The TP53 UDS analysis in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time.

Acknowledgements: Roche Diagnostics, FP7 NGS-PTL project, Fondazione Berlucchi.

#5023

Multiple myeloma with a clonal del17p aberration is characterized by somatic TP53 mutations, which negatively affect prognosis in this cytogenetic subgroup.

Davine Hofste op Bruinink,1 Remco Hoogenboezem,1 Eric Bindels,1 Mathijs Sanders,1 Claudia Erpelinck - Verschueren,1 Paulina van Strien,1 Jasper Koenders,1 Kristine Misund,2 Berna Beverloo,3 Bronno van der Holt,4 Ivo Touw,1 Anders Waage,2 Hervé Avet-Loiseau,5 Pieter Sonneveld1. 1 _Erasmus MC Cancer Institute, Rotterdam, Netherlands;_ 2 _Norwegian University of Science and Technology, St. Olavs Hospital, Trondheim, Norway;_ 3 _Erasmus MC, Rotterdam, Netherlands;_ 4 _Erasmus MC Cancer Institute - Clinical Trial Center, Rotterdam, Netherlands;_ 5 _University Cancer Center of Toulouse, Toulouse, France_.

Background

Multiple myeloma (MM) is a malignant plasma cell disorder, in which at diagnosis circa 10% of tumors are characterized by a deletion of chromosome 17p (del17p). Previously, others and we reported that this aberration is associated with a significantly inferior prognosis, classifying it as high-risk disease. As TP53 is located on chromosome 17p (Chr17p), most research has thus far been focused on this gene. However, only in a minority of del17p MM patients a mutation could be detected (Lodé et al. - Haematologica 2010, Kortüm et al. - Br J Haematol 2015). We therefore hypothesized that other relevant mutations might be present in TP53, its surrounding genes, or disease relevant pathways.

Aim

With this study, we sought to identify commonly mutated genes in del17p MM, which could explain its aggressive clinical behavior.

Material and Methods

We obtained high molecular DNA from CD138+ purified MM cells and matched peripheral blood from patients with a del17p in ≥50% of plasma cells, as detected with fluorescent in situ hybridization (FISH). Libraries were generated using a custom capture of 111 Mb (SeqCap EZ Exome Plus, Nimblegen), comprising the whole exome, Chr17p and the IgH, Igk, IgL and MYC regions. Paired-end sequencing was performed on a HiSeq2500 platform, followed by data-analysis using an in-house bioinformatics pipeline at Erasmus MC.

Results

Matched tumor and germline DNA was sequenced from 54 patients, with sequential tumor samples available from 12 patients. This resulted in an average coverage of 88x and overall coverage of 94%. Of 44 del17p patients, tumor DNA was obtained before treatment start. Clinical data were collected from 40/44 patients, with a median follow-up time of 18 months.

We found TP53 to be mutated most frequently. 31/54 (57%) patients had a somatic nonsynonymous mutation, insertion or deletion (71% missense, 19% stopgain/frameshift, 10% splicing). Although no hotspot mutations were detected, most TP53 mutations resided in the DNA binding domain. Of note, most TP53wt/- patients had no clonal somatic nonsynonymous mutations in other p53 pathway genes, or on Chr17p.

To assess the prognostic impact of TP53 mutations in del17p MM, we focused on the 40 chemotherapy-naive patients and stratified for age ≤65 years (n=25) and >65 years (n=15). TP53mut/- patients had a significantly worse overall survival (OS) than TP53wt/- patients (p=0.002). Strikingly, TP53 missense mutations showed the worst OS (p<0.001), as well as progression free survival (PFS) (p=0.006).

Conclusions

(1) In contrast with previous reports, we find TP53 to be somatically mutated in the majority of MM patients with a clonal del17p aberration.

(2) Particularly TP53 missense mutations have a significantly negative impact on both PFS and OS in del17p MM patients.

This study was supported by the Plaisier Foundation, Erasmus MC Grant and the Dutch CTMM Project.

#5024

Multiple biomarker combination increased both versatility and recurrence-predictive accuracy of molecular deep surgical margin in head and neck squamous cell carcinoma.

Hayashi Masamichi,1 Wayne M. Koch,2 Yasuhiro Kodera1. 1 _Nagoya University, Nagoya city, Aichi prefecture, Japan;_ 2 _Johns Hopkins University, Baltimore, MD_.

Oncologic evaluation of surgical margins has long depended on visible histologic diagnosis. Although intraoperative cytology or frozen section diagnosis directs surgery to be performed in a more adequate way, some histologic negative surgical margins still have genetic or epigenetic alterations associated with disease recurrence in head and neck squamous cell carcinoma (HNSCC). This phenomenon may be partly explained by "field cancerization" or "tumor budding". In order to further evaluate the locoregional recurrences from histologically negative deep surgical margins, we conducted molecular deep surgical margin analysis using prospectively collected HNSCC surgical samples.

Surgical specimens were collected from consecutive patients with head and neck cancer from whom written informed consent was obtained at Johns Hopkins Hospital (Baltimore, MD) between May 2009 and December 2013. Surgical margins of all cases were histologically negative. Deep surgical margin tissues (n=70) were collected, and DNA extraction, bisulfite treatment and quantitative MSP (QMSP) assay were performed for three candidate genes (PAX5, VGF and ZNF420).

First, methylation status of all 70 main tumors were examined. PAX5 methylation was detected in 50 cases (71%), VGF methylation in 34 cases (49%) and ZNF420 methylation in 25 cases (36%). Among the cases with PAX5 methylation positive tumors, PAX5 methylation of deep surgical margin tissue was positive in 10 cases and negative in 40 cases. The locoregional recurrence free survival (LRFS) of the cases with methylation-positive margin was worse than those with negative margin (P=0.059, log-rank test), but not significant. Similarly, LRFS difference between the cases with VGF methylated margin (14 cases) and those with unmethylated margin (20 cases), and the cases with ZNF420 methylated margin (5 cases) and those with unmethylated margin (20 cases) were not significant (P=0.606, P=0.059, log-rank test). Then, we combined these three gene markers and defined the case with at least one marker-positive surgical margin as margin positive. As a result, among total 62 cases (89%), margin positive 22 cases showed significantly worse LRFS than margin negative 40 cases (P=0.021, log-rank test).

Although the case coverage rate of each individual marker was relatively low, marker combination improved it up to 89% of all cases. Optimized gene marker combination would help us not only to detect histologically undetected cancerous cells but also to predict locoregional tumor recurrences.

#5025

PAX5 methylation as a novel biomarker of esophageal cancer for both potential tumor malignancy and cisplatin sensitivity.

Keisuke Kurimoto,1 Masamichi Hayashi,1 Masahiko Koike,1 Mitsuro Kanda,1 Yoko Nishikawa,1 Naoki Iwata,1 Yukiko Niwa,1 Hideki Takami,1 Daisuke Kobayashi,1 Chie Tanaka,1 Suguru Yamada,1 Goro Nakayama,1 Hiroyuki Sugimoto,1 Michitaka Fujiwara,1 Tsutomu Fujii,1 Guerrero-Preston Rafael,2 Yasuhiro Kodera1. 1 _Nagoya University Graduate School of Medicine, Aichi, Japan;_ 2 _Johns Hopkins University, Baltimore, MD_.

Therapeutic strategy of esophageal cancer largely depends on histopathological assessment just like tumor differentiation and TNM stage. In order to make appropriate choice for individual case, we have to pay attention to background molecular characteristics about tumor malignancy or chemotherapy sensitivity.

In order to find extremely tumor-specific marker of esophageal cancer, we picked several methylation markers from what we previously used in the research of head and neck cancer, which has the same background of smoking and alcohol and the same tissue-type. As a result, we identified PAX5 gene methylation as a very tumor-specific marker also in surgically resected 90 esophageal cancer cases. Then, we continued to examine PAX5 expression, function and other functions.

Quantitative MSP (QMSP) assay revealed significantly higher methylation score (100x[PAX5 QMSP]/[ACTB QMSP]) in tumor tissues (16.2±2.4) than in normal tissues (0.4±0.2) (P<0.001, Mann-Whitney U test). Also, 77/90 (85.6%) of these cases showed tumor-specific QMSP elevation. Similarly, all 9 esophageal cancer cell lines (TE1, TE2, TE3, NUEC1, NUEC2, NUEC3, T.T, T.Tn and WSSC) showed high QMSP scores over 10. The inverse correlation of PAX5 methylation and expression was confirmed by Quantitative RT-PCR of clinical samples and 5-aza-dC treated cell lines. PAX5 downregulated cases indicated significantly poor overall survival (P=0.037, log-rank test). For functional analysis, we examined WST1 assay and BrdU assay using ectopically PAX5 knock down NUEC1 cell line, which has secondly high PAX5 expression. The result showed significantly high cell proliferation and cell cycle acceleration. Since PAX5 is reported to bind p53 promoter and regulate its expression, we hypothesized epigenetically downregulated PAX5 might affect p53 expression and therefore cisplatin sensitivity of esophageal cancer. Actually, PAX5 downregulated NUEC1 cell line acquired significantly cisplatin-resistant character after siRNA transfection. Moreover, among postoperatively cisplatin-treated cases (25 cases), PAX5 hyper-methylated clinical cases (n=6) demonstrated significantly worse survival than PAX5 hypo-methylated cases (n=19) (P=0.002, log-rank test). We also surveyed another downstream mechanism of PAX5 dysregulation. Human Cancer Pathway Finder PCR Assay identified three key molecules including SLC2A1 (Fold change: 4.6), CCL2 (12.5) and IGFBP5 (-5.0).

Epigenetic inactivation of PAX5 gene may affects malignant potential of esophageal cancers and cisplatin-based chemotherapy through p53 dysregulation or other carcinogenic pathways. It could be utilized for planning individual multidisciplinary therapy.

#5026

EGFR copy number alterations in primary tumors, metastatic lymph nodes and recurrent tumors in areca-quid associated oral squamous cell carcinoma.

Shiang-Fu Huang,1 Huei-Tzu Chien,1 Sou-De Cheng,1 Chun-Ta Liao,2 Hung-Ming Wang,2 Ling-Ling Hsieh1. 1 _Chang Gung Univ., Taoyuan, Taiwan;_ 2 _Chang Gung Memorial Hospital, Taoyuan, Taiwan_.

EGFR and downstream signaling pathway plays important roles in the tumorigenesis in oral squamous cell carcinoma (OSCC). Gene copy number alteration is one the mechanism overexpressing the EGFR protein. We previously demonstrated that EGFR gene amplification was related with lymph node metastasis, tumor invasion and perineural invasion. We appraise the hypothesis that the EGFR gene copy number alteration in the primary tumor can predict that in recurrent tumors or in lymph node metastatic foci. Of 167 primary OSCCs, fluorescence in-situ hybridization (FISH) study showed EGFR polysomy in 19 (11.4%) patients and amplification in 33 (19.8%) patients. EGFR gene amplification was related with lymph node metastasis (χ2 trend test: P = 0.018) Of 57 metastatic lymph nodes, 9 (15.8%) had EGFR polysomy and 14 (24.6%) had EGFR gene amplification. The concordance rate of EGFR gene copy number in primary tumor and lymph node metastasis was 68.4% (McNemar test: P = 0.389). Of 41 recurrent tumors, 5 (12.2%) had EGFR polysomy and 5 (12.2%) had gene amplification. The concordance rate of EGFR gene copy number between primary tumor and recurrence tumor was 65.9% (McNemar test: P = 0.510). From our results, we can predict two-thirds of the EGFR gene copy number alteration in the lymph node metastasis or the recurrent tumor from the analysis of primary tumor. In clinical scenario, for OSCC patients that the lymph nodes or the recurrent tumors are unavailable for EGFR gene copy number analysis, studies from the primary tumors could provide part of the EGFR clonal information in the metastatic or recurrent lesions.

#5027

Detection of soluble protein biomarkers in NSCLC serum samples by meso scale discovery electrochemiluminescence platform.

Dana S. Gaffney, Katherine Bell, Gabriela Martinez, Jayaprakash Karkera, Suso Platero. _Janssen Research & Development, LLC, Springhouse, PA_.

Identification of soluble biomarkers has become a critical non-invasive approach for disease diagnosis and monitoring in the treatment of solid tumors. Non-Small Cell Lung Cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all lung cancers worldwide. The EGFR family of genes is known to be highly expressed in NSCLC tumors and play a critical role in the progression of the disease. Although patients initially respond well to EGFR TKI therapy, acquired resistance occurs through several mechanisms, including compensatory cMET pathway activation (Robinson et al. 2013). Previous reports have demonstrated that soluble forms of EGFR, Her2 and cMET are generated through either alternate splicing of mRNA or proteolytic cleavage of the full length receptors (Wilken et al. 2013). Serum concentrations of these receptors can be correlated with prognosis as well as treatment response (Gregorc, 2004, Kasahara, 2010 Heinmoller, 2003). Soluble receptor ligands, including Hepatocyte growth factor (HGF), have also been evaluated in NSCLC and a strong correlation has also been observed between the levels of serum HGF and disease outcome in patients treated with EGFR-TKIs (Kasahara et al. 2010).

In this study, we evaluated concentrations of sEGFR, sMET, sHER2, , and HGF in NSCLC versus healthy normal serum samples via meso scale discovery (MSD) platform. Inventoried and custom MSD assays were optimized to measure these proteins in a training set consisting of 19 NSCLC and 16 normal healthy serum samples. Significantly lower concentrations of sEGFR and sHER2 were observed in the NSCLC serum samples compared to normal (p value = 0.0092 for both receptors), whereas, in contrast, the levels of HGF were significantly higher in the NSCLC patient samples (p value <0.0001). No significant difference in sMET concentration was observed between NSCLC and normal serum. Based on this data, it was determined that a ratio of protein concentration of each soluble receptor (EGFR, Her2 and cMET) to HGF yielded a greater differentiation between NSCLC and normal samples (p value <0.0001, = 0.0041,and 0.0327 respectively). Cut-off values to define NSCLC versus normal patients were established for each ratio and validated in a blinded independent cohort , consisting of 50 NSCLC and 12 healthy normal serum samples. PPV and NPV were then calculated to determine sensitivity and specificity for each protein ratio. Results from the validation study show an almost complete separation between healthy and NSCLC patient samples (p value <0.0001, AUC =1 )for all three ratios tested (sEGFR/HGF , sHER2/HGF and sMET/HGF), with a PPV = 100%, 98%, 100% and NPV = 100%, 100%, 85.7%, respectively. Overall, these findings indicate potential clinical applications for these protein pairs as non-invasive biomarkers of NSCLC diagnosis, prognosis and treatment monitoring to support emerging TKI therapies

#5028

Reverse translational research for identifying the correlation between EGFR mutation status and DPD protein expression in lung adenocarcinoma.

Tomoshi Tsuchiya,1 Tetsuo Tominaga,1 Koji Mochinaga,2 Naoya Yamasaki,1 Keitaro Matsumoto,1 Takuro Miyazaki,1 Go Hatachi,1 Yuka Kitajima,1 Takeshi Nagayasu1. 1 _Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan;_ 2 _Oita Prefectural Hospital, Oita, Japan_.

[Background] Reverse translation research is often described as "From the Bedside to the Bench", which means taking an understanding gained by observations in a patient population to create hypotheses and confirmation of the hypothesis in the laboratory. According to current chemotherapy treatment for lung adenocarcinoma, empirically, clinicians know that epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) tend to be effective for radiological ground glass nodule- type lung adenocarcinomas. On the other hand, it has been shown that 5-FU sensitivity in patients with non-small cell lung cancer (NSCLC) is associated with EGFR mutation status. From such clinical hints, we identified a relationship between dihydropyrimidine dehydrogenase (DPD), a 5-FU degrading enzyme, and EGFR mutation status.

[Methods] We firstly focused on clinicopathologic factors and in vitro correlations between DPD expression and EGFR mutation status. Secondly, we analyzed crosstalk between the EGFR signal cascade and DPD protein expression using EGFR mutated and not mutated cell lines.

[Clinicopathological Study] EGFR mutations and messenger RNA (mRNA) levels of DPD were analyzed in 47 resected NSCLC tumors by laser-capture microdissection. EGFR mutation status and the immunohistochemical expression of DPD in 49 resected NSCLC tumors were also examined. As a result, adenocarcinoma in situ showed significantly higher DPD mRNA levels and more EGFR mutation frequency than other histological types (P < 0.05). DPD immunopositive cases were more frequently observed in adenocarcinoma, in females, and in nonsmokers; which were correlated with EGFR mutation status (P < 0.003).

[Basic Study] In vitro, EGFR-mutated cell lines (PC9, HCC827; exon19 E746-A750 and H1975; exon21 L858R, T790M, gefitinib resistant) showed higher DPD mRNA and protein expression than wild types (H1437, H1299). The DPD gene (DPYD), and DPD protein expression were known to be regulated by the transcription factor Sp1. In the PC9 study, EGF treatment induced up-regulation of both Sp1 and DPD. Gefitinib, an EGFR-TKI, and mithramycin A, specific Sp-1 inhibitors, suppressed them. However, the phenomena were not observed in EGFR-wild types and EGFR-TKI resistant cell lines. These results suggest DPD is regulated mainly in EGFR signal cascade enhanced lung cancers such as EGFR mutated adenocarcinomas. 5-FU and its derivatives such as uracil-tegafur (UFT) or S-1 might be effective for EGFR wild type adenocarcinomas because of the low DPD expression.

[Conclusion] Reverse translational research can provide useful information for clinical treatment. In the present study, we identified crosstalk between the EGFR signal cascade and DPD protein expression, which might affect therapeutic strategy for lung adenocarcinomas.

#5029

The clinical prognostic factors of 889 patients with non-small cell lung cancer.

Yiping Han. _Shanghai Changhai Hospital, Shanghai, China_.

Abstract Objective:To analyse the clinical characteristics and investigate the prognostic factors of the patients with non-small cell lung cancer. Method: We analysed the clinical characteristics of 889 cases with non-small cell lung cancer including age, gender, smoking history, TNM stage, Pathologic grade, the expression of Ki67 and p53 , chemotherapy, radiotherapy and targeted therapy. Then, we identified the prognosis through the follow-up. The prognostic factors of the non-small cell lung cancer patients were analysed through survival analysis.We chose the Kaplan-Meier method for the univariate analysis and the Cox proportional hazard regression model for multivariate analysis.Results:The overall median survival time of the patients were 12.87 months,and 1-year,2-year,3-year survival rates were 49%, 26% and 12% respectively. Conclusion: Smoking history,TNM stage,Performance Status score, Pathologic grade, treatment and the expression of Ki67 were independent prognostic factors.

#5030

Tumor necrosis serves as a prognostic predictor in operable and stage I lung adenocarcinoma.

Pei-Ying Lin,1 Tzu-Hung Hsiao,2 Pan-Chyr Yang1. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _Academia Sinica, Taipei, Taiwan_.

Background

A relatively high proportion of stage I lung adenocarcinoma patients suffer from disease relapse after surgical resection. This highlights an unmet need in clinical risk assessment for this patient group. Several previous studies suggested adopting the feature tumor necrosis as a risk stratification parameter, but consensus has not been reached. Here we evaluated whether gene signatures that represented three biological processes, cell proliferation, tissue hypoxia and nutrient supplement, underlying their pathological sum tumor necrosis, supported the assumption that necrosis is a prognostic predictor.

Methods and Results

The three selected gene signatures, cell-cycle, hypoxia and mTOR, were revealed to have higher signature activities in necrosis-positive lung adenocarcinoma tissues than in necrosis-negative ones. This phenomenon was observed in a Japanese dataset and was verified in The Cancer Genome Atlas lung adenocarcinoma dataset. In these two datasets, both the feature tumor necrosis and the signatures were shown to stratify operable and/or stage I lung adenocarcinoma patients into different risk groups. We further validated this observation in a 130 surgically resected lung adenocarcinoma patient cohort. The results showed that tumor necrosis predicted shorter overall and relapse-free survival in patients with operable (P<0.001 and P=0.007, respectively) and stage I (P=0.003 and P=0.03, respectively) lung adenocarcinomas.

Conclusions

Our study showed that tumor necrosis served as a prognostic predictor in operable and stage I lung adenocarcinomas. The findings supported the results from previous pure pathological studies.

#5031

Melanoma progression involves a profound nuclear to cytoplasmic shift of the transcription factor SUM-6 (specifically upregulated in melanoma gene six).

Laura J. Graziano,1 Xue Zhang,1 Yabin Cheng,1 Gang Wang,2 Mingwan Su,1 Magdalena Martinka,3 Youwen Zhou1. 1 _Department of Dermatology and Skin Science, University of British Columbia, Vancouver, British Columbia, Canada;_ 2 _Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada;_ 3 _Department of Pathology and Laboratory Medicine, Vancouver General Hospital, Vancouver, British Columbia, Canada_.

INTRODUCTION: SUM-6 is a human homeobox gene that encodes for a transcription factor, which plays a key role in normal embryogenesis. Overexpression of both the gene and nuclear protein has been shown to occur in a variety of cancers such as breast, colorectal, and pancreatic, and has been demonstrated to promote tumorigenesis. The purpose of the current study was to evaluate the expression of the SUM-6 protein, its clinical relevance, and biological function in melanoma.

EXPERIMENTAL PROCEDURES: Immunohistochemistry using a specific SUM-6 antibody was performed on a tissue microarray consisting of 438 patient biopsies, which included benign and malignant melanocytic tumors. Double-blinded scoring of distinct nuclear and cytoplasmic SUM-6 staining was performed. SPSS 16.0 statistical package and Microsoft Excel were used to carry out the analyses of data. Analyses included the χ2 test and univariate analysis by the log-rank test in conjunction with the Kaplan-Meier survival curve for visualization.

RESULTS: Nuclear staining of SUM-6 was detected in 100% of normal nevi (n=19), 81% of dysplastic nevi (n=32), 87% of melanoma in situ (n=23), 79% of primary melanoma (n=219), and 53% of metastatic melanoma (n=145), whereas cytoplasmic staining was detected in 63%, 94%, 56%, 94%, and 97%, respectively. Univariate analysis revealed that five year disease-specific survival decreased significantly when SUM-6 was excluded from the nucleus and when SUM-6 levels were elevated in the cytoplasm (p=0.001 and 0.019, respectively).

CONCLUSIONS: We concluded that melanoma progression and metastasis is associated with a profound shift of transcription factor SUM-6 from the nucleus to the cytoplasm. It is interesting to note that in most other cancers, the SUM-6 protein was instead overexpressed in the nucleus. The patients in whom SUM-6 was excluded from the nucleus demonstrated a significantly worse prognosis than the patients who retained nuclear expression. These findings suggest that either the absence of nuclear expression or elevated cytoplasmic presence (or both) of SUM-6 may play a role in the progression of malignant melanoma.

#5032

Glycoprotein NMB (gpNMB) overexpression is prevalent in human cancers: pancreatic cancer, non-small cell lung cancer, head and neck cancer, and osteosarcoma.

Abdel Halim,1 Rebecca G. Bagley,2 Tibor Keler1. 1 _Celldex Therapeutics, Inc., Hampton, NJ;_ 2 _Celldex Therapeutics, Inc., Needham, MA_.

Introduction: gpNMB is an internalizable transmembrane protein overexpressed in 20% of breast cancers, 40% of triple-negative breast cancer (TNBC) and 80% of melanomas. Glembatumumab vedotin (GV; CDX-011) is a novel antibody-drug conjugate (ADC) that delivers the potent cytotoxin monomethyl auristatin E to cancer cells expressing gpNMB. GV is in Phase II clinical trials for TNBC (the pivotal "METRIC" study; NCT01997333) and melanoma (NCT02302339). We investigated the prevalence of gpNMB overexpression in other human cancers to explore the potential for therapeutic benefit of GV beyond TNBC and melanoma.

Methods: Tumor and normal tissues were procured (Mosaic Laboratories, CA) under an IRB-reviewed protocol (MOS001) allowing in vitro analysis of remnant, anonymized human samples. The study included 20 pancreatic tumors, 21 normal pancreatic tissues, 20 head & neck (H&N) tumors, 21 normal H&N tissues, 20 lung squamous cell carcinoma (SCC), 20 lung adenocarcinoma, 20 normal lung and 21 osteosarcoma tissues but no normal bone tissues were available. A validated immunohistochemistry assay was performed centrally on formalin fixed, paraffin embedded tissues. A goat polyclonal anti-gpNMB antibody (R&D Systems, MN) was applied to slides after deparaffinization and antigen retrieval. Visualization was achieved with a rabbit anti-goat antibody (Vector Laboratories, CA) and EnVision+ anti-rabbit HRP detection system (Dako, CA). Hematoxylin-counterstained slides were scored by 2 pathologists for percent of positive cells and staining intensity (0, 1+, 2+, 3+). Considering only cells strongly stained (2+ and/or 3+), the highest percent of cells stained in normal corresponding tissues was used as a cut-off.

Results: Tumor epithelial cells staining exceeded the cut-off in 55%, 40%, 85% and 45% of pancreatic, H&N, lung SCC and lung adenocarcinoma respectively, and 62% of osteosarcoma samples stained in 1-90% of tumor epithelial cells. In pancreatic, lung, and H&N tumors, staining was both cytoplasmic and membranous but only cytoplasmic and 1+ intensity for normal tissue, normal tumor-adjacent tissue, and non-tumor stromal cells. The pattern of gpNMB staining in osteosarcomas was obviously cytoplasmic. However, both cytoplasmic and membranous staining resulted in a broader panel of 67 osteosarcoma tissues and glembatumumab vedotin had strong activity in 3/6 osteosarcoma xenografts (Roth, PBC, 2015; Kolb, PBC, 2014).

Conclusions: As compared to normal tissue, differential expression of gpNMB in human osteosarcoma, pancreatic, lung, and H&N tumor samples suggests that patients with these cancers may derive a benefit from treatment with the investigational agent glembatumumab vedotin that targets cell surface gpNMB. Clinical studies are being initiated in squamous cell lung cancer, osteosarcoma and uveal melanoma with ongoing studies in TNBC and melanoma.

#5033

Clonal origin of therapy-related myeloid neoplasms.

Koichi Takahashi, Feng Wang, Hagop Kantarjian, Doss Denaha, Erika Thompson, Curtis Gumbs, Keyur Patel, Sattva Neelapu, Yan Wang, Zachary Bohannan, Courtney DiNardo, Farhad Ravandi, Zeev Estrov, Simona Colla, Jianhua Zhang, Xifeng Wu, Guillermo Garcia-Manero, Andrew Futreal. _The University of Texas MD Anderson Cancer Center, Houston, TX_.

Background: Therapy-related myeloid neoplasms (t-MNs) are secondary malignancies that develop in patients (pts) treated with chemo-radiation therapy (CRT). Patient-related risk factors for t-MN susceptibility is unknown. Recent studies reported that canonical hematological driver mutations (DMs) could be detected in peripheral blood (PB) from healthy individuals or pts with solid tumors, a phenomenon referred to as clonal hematopoiesis of indeterminate potential (CHIP). We hypothesized that there are canonical DMs likely important in t-MNs that pre-exist as detectable CHIP in pts at time of diagnosis of their original cancer.

Methods: We sequenced 300 leukemia-relevant genes on diagnostic bone marrow (BM) samples from 13 t-MN pts to detect DMs. We then assessed the presence of the same DMs in the pts' matched PB samples, which were obtained after primary cancer diagnosis but before CRT.

Results: Four pts (31%) had t-AML and 9 (69%) had t-MDS. The median age at primary cancer diagnosis and t-MN diagnosis was 62 years (y) (range: 25-74 y) and 66 y (range: 28-77 y), respectively. The median latency time to t-MN development was 3 y (range: 1-8 y).

Targeted gene sequencing revealed 17 canonical DMs in 8 genes. At least one clonal DM was detected in each pt. The most frequently detected mutation was TP53 mutations in 4 patients (31%), followed by DNMT3A (N = 3, 23%), IDH2 (N = 2, 15%), TET2 (N =2, 15%), and RUNX1 mutations in 2 (15%).

Analysis of deep sequencing PB sample revealed that DMs identical to those detected in t-MN BM were detected in 7 of 13 pts (54%). For example, Case UID6982 had small cell lung cancer and received concurrent CRT with carboplatin and etoposide. He developed t-AML 3 y later and was found to have an IDH2 p. R140Q mutation in the diagnostic BM with a VAF of 40%. His PB obtained before CRT showed the same IDH2 p.R140Q mutation with a VAF of 14%.

The most common DM detected in paired PB samples was TP53 mutation in 3 cases, followed by DNMT3A, TET2, IDH2, and RUNX1 mutation in 1 case each. Of note, DMs that were subclonal in t-MN BM were not detected in the PB at the time of primary cancer diagnosis. The median VAF of the detected variants in the paired PB samples was 6% (range: 0.6-27%). We found no significant differences between pts with and pts without evidence of CHIP in terms of median age at primary cancer diagnosis or median latency time to t-MN development.

Conclusion: In this study we have demonstrated evidence of detectable mutations in multiple canonical leukemia driver genes at time of diagnosis and before therapy of primary cancer in pts whose subsequent t-MN also harbored identical DMs. This work suggests that a substantial fraction of t-MN pts likely exhibit CHIP with detectable canonical mutations in a number of leukemia driver genes at time of diagnosis for their primary cancer. Further, these data suggest the potential to develop a risk stratification model based on presence of CHIP with canonical DMs at the time of primary cancer diagnosis.

#5034

Overexpression of MYST4 induces tumor progression in hepatocellular carcinoma.

Tsui Lien Mao,1 Jinn-Chyuan Sheu2. 1 _National Taiwan University Hospital, Taipei, Taiwan;_ 2 _National Sun Yat-Sen University, Taiwan_.

MYST4 is a member of histone acetyltransferases which are involved in transcriptional activation of genes important for cellular growth and development. Our previous study revealed that overexpression of MYST4 in ovarian high-grade serous carcinoma correlated with worse survival. To further explore the role of MYST4 in human cancers, we selected hepatoma cell line Huh 7 due to its high level of MYST4 expression among various cancer cell lines tested. Immunohistochemical study showed that overexpression of MYST4 correlated with worse overall survival in hepatoceullular carcinoma (P=0.02). Furthermore, tumor cells at invasion front and in lymphovascular spaces are frequently overexpressed. Knockdown of MYST4 in Huh 7 resulted in significantly reduced cellular growth, migration and perturbed cell cycle progression. Mouse xenograft model revealed that MYST4-knockdown Huh 7 cells had lower growth rate, consistent with the in vitro findings. Together, our studies suggest that MYST4 promotes cell growth, migration and lymphovascular spread in hepatocellular carcinoma. Thus, MYST4 may serve as a druggable target in treating hepatocellular carcinoma.

#5035

MDB-seq screening of muscle-invasive urothelial carcinoma revealed unpredicted novel tumor suppressor genes.

Elisa Guida, Luigi Marchionni, Rachel Goldberg, Mark Schoenberg, Trinity Bivalaqua, George J. Netto, Mohammad O. Moque, David Sidransky. _Johns Hopkins University- SOM, Baltimore, MD_.

Bladder cancer (BC) is among the most frequent cancers in the United States. More than 70% patients suffer from superficial disease called non-muscle invasive bladder cancer (NMIBC) which, after the first successful surgery, has favorable survival but high risk of recurrence; on the other hand, muscle-invasive urothelial carcinoma (MIBC) is less prevalent but typically associated with a relatively poor prognosis. Since BC has been largely described as a multifactorial disease, with some DNA mutational component and strong evidence supporting an important role performed by the exposure to environmental factors, mainly derived from tobacco smoking and occupational hazards, consequent epigenetic DNA alterations could play important pathological role and also be used as markers for detection and stratification of the disease. In order to discover new methylated genes we performed novel sequential high throughput Methyl-DNA Binding (MDB) proteins Pull-down followed by large genome sequencing of 5 primary MIBC (not subject to previous therapy nor BCG) versus their normal counterparts.

After Bioinformatic cross-analysis between the enriched sequences in our MDBseq experiment (corresponfing to 85 genes) and publicly available database (BC TCGA), we have come up to a list containing 16 hypermethylated genes in BC (our «consensus genes»). From the first unbiased analysis of this list, we have found some genes already found as important and/or hypermethylated in BC (as for example, CDKN2A, Sall3, Nkx6-2, CDO1), confirming the validity of our approach. Interestingly, we have observed high presence of transcription factors (homeobox, Paired, Zn fingers) and proteins involved in fundamental developmental processes. Among the newly hypermethylated genes in BC, we have chosen VIPR2 for further characterization. VIPR2 encodes the vasoactive intestinal peptide receptor VPAC2, a G-protein-coupled receptor that is expressed in a variety of tissues (expressed throughout the central nervous system and the periphery); it binds VIP14, activates cyclic AMP (cAMP)-signaling and PKA. The VIP-VPAC2 signaling pathway has been implicated in several neural and behavioral processes and is known to influence circadian rhythms and cognition. We first confirmed inverse correlation between diminished expression and promoter methylation in 11 BC cell lines (5637, HT-1376, J82, SW780, UM-UC-3, BFTC-905, BFTC-909, Scaber, RT4, T24) and a normal human urinary tract epithelium immortalized cell line SV-HUC-1 (HUC-1). Next, we determined that pharmacological unmasking with DNA methylation transferases (DNMTs) inhibitors (5-Aza-deoxycytidine) re-expressed the transcript in the cells analyzed. We are in the process of analyzing VIPR2 expression and promoter methylation in primary BC samples. After confirming in primary BC samples, we will further characterize the role of VIPR2 in bladder cancer pathogenesis.

#5035A

Intratumoral HPV16-specific T-cells determine clinical outcome of HPV16+ oropharyngeal carcinomas.

Marij J. Welters, Lilly-Ann van der Velden, Vanessa J. van Ham, Ilina Ehsan, Renske Goedemans, Saskia J. Santegoets, Sjoerd H. van der Burg. _Leiden University Medical Center, Leiden, Netherlands_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer. While the number of HNSCC cases is decreasing, the number of oropharyngeal SCC (OSCC) is rising, especially in young adults. OSCC are increasingly (45-90%) caused by human papillomavirus type 16 (HPV16). Intriguingly, patients with HPV-induced OSCC respond better to therapy than HPV-negative OSCC. This is independent of nodal status, age, stage, tumor differentiation or gender. We hypothesized that the expression of the viral antigens E6 and E7 in HPV16+ OSCC equips the immune system with two strong tumor-specific antigens resulting in the development of a type 1 T-cell immune contexture supporting the better response of HPV+ OSCC to standard therapy.

To test our hypothesis we studied 87 patients with OSCC. The tumors of these patients were tested for the presence of HPV by GP5+/6+ PCR and the expression of p16 by immunohistochemistry. Moreover, fresh tumor tissue was dispersed, cryopreserved and subsequently used for in-depth analyses regarding the composition of the leukocyte infiltrates using a large set of antibodies either by CyTOF or BD Fortessa. In addition, T-cell reactivity was tested directly ex vivo and after a short-term culture period.

57% of OSCC patients were HPV16+ and they showed the best overall survival (p<0.0001, HR 6.9). Whereas none of 37 HPV-negative patients displayed intratumoral HPV16 E6/E7-specific T-cell reactivity this was detected in 25 of 40 (63%) HPV16+ OSCC. Notably, for most patients these T-cells were already detectable directly ex vivo, indicative for their activity in vivo. The presence of intratumoral HPV16-specific T-cell reactivity for the response of patients to therapy was extremely important for a better clinical outcome (p=0.0003, HR 23.9) as estimated by Kaplan-Meier curves and log-rank analysis. Division of HPV16+ patients on the basis of p16 expression revealed that p16+ patients had a much better clinical outcome (p<0.0001, HR 11.8). In line with the notion that overexpression of p16 is directly associated with expression of the HPV16 E6 and E7 oncogenes, a strong association between p16 expression and HPV16-specific TIL reactivity was found (p<0.0001).

Mechanistically, we found that OSCC cell lines promoted the differentiation of monocytes to M2 macrophages. In vitro experiments showed that the presence of HPV-specific T-cell secreted cytokines prevented this and was even able to redifferentiate macrophages into better antigen-presenting cells, both phenotypically (including co-stimulatory molecules) and functionally (more IL-12 and less IL-10 production). In addition, these cytokines mediated a direct effect on the tumor cell proliferation and metabolism, thereby slowing down tumor growth as well as changing their impact on myeloid cells in the tumor microenvironment.

In conclusion, HPV-specific T cells elicited by HPV+ OSCC are highly predictive for response to therapy.

## TUMOR BIOLOGY:

### Cell Adhesion and Tumor Treatment

#5036

Elucidating an oncogenic role of an extracellular matrix protein, Agrin, in hepatocellular carcinoma.

Sayan Chakraborty, Wanjin Hong. _Institute of Molecular and Cell Biology, Singapore, Singapore_.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers worldwide. Despite offering poor survival advantage, broad spectrum receptor tyrosine kinase inhibitors are the sole chemotherapeutic treatments for advanced stages of HCC. Hence, there is a pressing clinical need to broaden the scope of HCC treatments through the systematic identification of suitable candidate(s) overexpressed in liver cancer. Therefore, we performed a SILAC based quantitative proteomics of cell surface proteins in HCC lines and identified proteoglycan Agrin to be overexpressed and secreted in HCC. Our systematic cell biological analysis indicated a role of Agrin in maintaining focal adhesion integrity through stimulation of focal adhesion kinase (FAK). We showed that Agrin functions in cellular migration, invasion and tumor growth. However, the exact mechanisms by which Agrin's mediates liver tumorigenesis remain unknown. The present study dissects how Agrin mediated signals from the extracellular matrix are sensed by tumor cells. Our evidences reveal that Agrin binds to its co-receptor(s) Lrp4 and subsequently activates MuSK to mediate oncogenic effects through activating focal adhesions. Additionally, integrins and focal adhesions collaborate closely with Agrin in communicating ECM signals to intracellular components in multiple HCC cell lines, thereby stimulating their invasive, migratory and growth capabilities. More importantly, Lrp4-MuSK as well as integrin-FAK pathways are crucial downstream signaling axes of Agrin's functions in liver tumorigenesis. The signal transduction network initiated by Agrin activates the oncogenic program in liver by negatively regulating tumor suppressor pathways. These novel insights will have profound impact on the possibility of targeting Agrin mediated oncogenesis in HCC.

#5037

Upregulated superoxide dismutase 2 by specificity protein 1 mediates temozolomide resistance in glioma stem cells.

Jian-Ying Chuang,1 Jr-Jiun Liu,2 Shao-Wen Chou,2 Wen-Chang Chang,1 Kwang-Yu Chang2. 1 _Taipei Medical University, Taiwan;_ 2 _National Health Research Institutes, Taiwan_.

The resisitant mechanism of glioblastoma mutliforme (GBM) to the standard chemotherapy temozolomide (TMZ) can be attributed to methylguanine methyltransferase (MGMT) expression in only half of the clinical cases. For the other half, it is not fully understood. To elucidate the mechanism, MGMT-negative GBM cell lines, U87MG and A172, were used to develop the TMZ-resistant variants. The variants showed reduced reactive oxygen species (ROS) accumulation by TMZ treatment comparing to the parental one. Further analysis of the cells revealed enhanced expression of superoxide dismutases 2 (SOD2), an antioxidative enzyme, as well as the stem cell-like properties. The protein was shown to associate with glioma stem cells (GSC). Notably, inhibition of SOD2 by diethyldithiocarbamate attenuated the stemness properties and the viability of the resistant variants. Further investigation of the promotor binding study suggested specificity protein 1 (Sp1) as the key factor of the SOD2 response. Moreover, Sp1 expression was even enhanced in the spheroid cells and the TMZ-resistant U87MG cell line. Conversely, treatment with Sp1 inhibitor mithramycin A attenuates SOD2 expression and the stemness properties of the resistant variants. In summary, our results suggested a novel role of SOD2 in TMZ resistant mechanism of GBM. Further study of Sp1-SOD2 pathway as a therapeutic target to restore susceptibility of chemotherapy is therefore warranted.

#5038

The repression of the extracellular matrix protein TGFBI induces chemoresistance in nasopharyngeal carcinoma.

Pierre-Antoine Bissey,1 Jeff W. Bruce,1 Jacqueline Law,2 Kenneth W. Yip,1 Fei-Fei Liu3. 1 _Ontario Cancer Institute, University Health Network, Toronto, Ontario, Canada;_ 2 _Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada;_ 3 _Department of Radiation Oncology, Princess Margaret Cancer Centre and University of Toronto, Toronto, Ontario, Canada_.

Introduction: Nasopharyngeal carcinoma (NPC) is a malignancy that is strongly associated with Epstein-Barr virus (EBV). The five-year overall survival (OS) rate of locally-advanced NPC patients is ~65%. Therapeutic options for such patients are limited; ionizing radiation is the primary mode of therapy for early-stage NPC, while chemoradiotherapy is used for more advanced stages. The use of chemotherapy (combined with radiation) provides a modest benefit in OS, but causes significant toxicities. The major cause of death in NPC is distant metastasis (DM), which remains a clinical challenge. In order to identify novel mechanisms underlying this process, our laboratory has recently completed global RNA sequencing of NPC samples which were fully clinically annotated. The extracellular matrix protein TGFBI (Transforming Growth Factor, Beta-Induced) was identified as a potential marker for chemoresistance. The objective of this study was to elucidate the role of TGFBI, and acquire greater insight into the biological pathways leading to chemoresistance and contributing to DM in NPC.

Methods: Nasopharyngeal cell lines including NP69 (normal nasopharyngeal, SV-40 immortalized), and C666-1 (NPC, EBV positive) were used to assess the differential expression of TGFBI between normal and malignant cells. TGFBI under-expressing NP69 cells were engineered using shRNA vectors, and the impact of the loss of TGFBI expression was assessed by cell death, clonogenic, and migration assays. Downstream target gene expression was evaluated by quantitative real-time PCR (qRT-PCR) and Western blot. C666-1 cells, which had no expression of TGFBI (RNA or protein), were used as controls.

Results: RNA-seq data identified poorer distant relapse-free survival in chemoradiotherapy-treated patients with low TGFBI expression vs. those with high TGFBI expression. In concordance with this observation, in vitro down-regulation of TGFBI induced cisplatin resistance via the phosphorylation of Akt and PTEN. TGFBI downregulation also increased cellular migration in spheroid cultures. Furthermore, TGFBI was identified to be a target of miR-449b, one of the 4 microRNAs in a DM prognostic signature that was previously identified by our group (Oncotarget 6:4537, 2015). In turn, over-expression of miR-449b in nasopharyngeal cells increased phospho-Akt expression, phenocopying the results observed in the TGFBI experiments.

Conclusion: Together, these data suggest that the miR-449~TGFBI-Akt axis plays a significant role in both treatment resistance and metastasis in NPC. Further studies will allow us to elucidate additional components of this pathway, and identify novel therapeutic targets which can benefit future patients with NPC.

#5039

Co-targeting polo-like kinase 1(Plk1) and the Wnt/β-catenin signaling pathway in castration-resistant prostate cancer.

Jie Li, Xiaoqi Liu. _PURDUE UNIVERSITY, West Lafayette, IN_.

The Wnt/β-catenin signaling pathway has been identified as one of the predominantly upregulated pathways in castration-resistant prostate cancer (CRPC). However, whether targeting the β-catenin pathway will prove effective as a CRPC treatment remains unknown. Polo-like kinase 1 (Plk1) is a critical regulator in many cell cycle events and is significantly elevated upon castration of mice carrying xenograft prostate tumors. Indeed, inhibition of Plk1 has been shown to inhibit tumor growth in several in vivo studies. Here, we show that Plk1 is a negative regulator of Wnt/β-catenin signaling. Plk1 inhibition or depletion enhances the level of cytosolic and nuclear β-catenin in human prostate cancer cells. Furthermore, inhibition of Wnt/β-catenin signaling significantly potentiates the anti-neoplastic activity of the Plk1 inhibitor BI2536 in both cultured prostate cancer cells and CRPC xenograft tumors. Mechanistically, Axin2, a negative regulator of the β-catenin pathway, serves as a substrate of Plk1 and Plk1 phosphorylation of Axin2 facilitates the degradation of β-catenin by enhancing the binding between GSK3β and β-catenin. Plk1 phosphorylated Axin2 also exhibits resistance to Cdc20-mediated degradation. Overall, this study identifies a novel Plk1-Wnt signaling axis in prostate cancer, offering a promising new therapeutic option to treat CRPC.

#5040

Genomic and proteomic characterization of anti-androgen resistant cell lines.

Jennifer Hertzog, Mike Russell, Michael Quigley, Marco Gottardis, Theresa McDevitt. _Janssen R &D, Spring House, PA_.

Background: Prostate cancer is the most common cancer and second most frequent cause of cancer-related deaths for men in the United States. AR-axis inhibitors have been shown to improve both progression-free survival (PFS) and overall survival (OS) in castration resistant prostate cancer (CRPC) patients however an estimated 50% of patients will go on to develop resistance to these therapies. Some mechanisms of resistance observed in the clinic include Androgen Receptor (AR) dependent events such as increased AR copy number, activating point mutations in AR, or splice variants (ARV7) both of the latter leading to constitutively active versions of AR. Alternatively, AR-independent resistance mechanisms include up-regulation of other oncogenic signaling pathways such as FGFR and PI3/Akt and induction of the nuclear hormone Glucocorticoid Receptor (GR). Finally, an increase in the emergence of a neuroendocrine phenotype is observed in the clinic with CRPC patients and is thought to be associated with chronic AR-antagonist treatment. To better understand the specific proteomic and genomic changes that occur during chronic AR inhibitor treatment we developed two enzalutamide (ENZA) resistant cell lines and performed targeted proteomic and global gene expression analysis.

Methods: We generated LnCaP and VCAP ENZA-resistant (ENZA-Res) prostate cancer cell lines. We performed gene expression analysis by using both cDNA microarray and RNA-SEQ techniques to identify novel or differential gene expression patterns between ENZA-Res and sensitive phenotypes. We also employed a targeted proteomic approach to identify changes in the relative expression levels of proteins that have historically been associated with anti-androgen resistance including, ARV7, GR and SGK-1.

Results: We have shown that ENZA-Res cell lines can undergo a significant change in morphology that is associated with an elongated neuronal-like appearance. This phenotype is associated with an induction in the mRNA expression of neuroendocrine markers, CHGA and REST along with a strong induction of GR mRNA and protein expression. We have shown that the neuroendocrine derived prostate cancer cell line, H660, also demonstrates high GR mRNA and protein content suggesting that GR may have a role in the maintenance of neuroendocrine and/ or ENZA-Res phenotypes when AR is absent. In VCaP ENZA-Res cells a significant increase in ARV7 mRNA and protein content was observed compared to parental ENZA sensitive VCaP cells. Lastly, we employed a combination of cDNA microarray and RNA-SEQ techniques to identify a molecular signature associated with anti-androgen resistance. We have identified a unique subset of genes that are differentially up-regulated in ENZA-Res cell lines compared to ENZA-sensitive cell lines. This novel molecular signature identifies several candidate genes as potential therapeutic targets that may be important in mediating anti-androgen resistance.

#5042

A CRISPR/Cas9 knockout screen identify novel drug resistance genes in AML.

Jian Huang. _Temple University, School of Medicine, Philadelphia, PA_.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. Despite remarkable progress made in the therapy of AML, the mainstays of treatment have not significantly changed over the last 20 years. Technological advances have greatly accelerated genomic discovery and precision medicine holds promise to better tailor medications to AML with specific mutations. Importantly, one major obstacle to greater success with target therapy of leukemia is drug resistance. But the mechanisms underlying drug resistance in AML are poorly understood.

The activating mutations of FLT3 are now recognized as the most common molecular abnormality in AML and FLT3ITD mutations are found in nearly 30% of AML patients. The poor prognosis of patients harboring FLT3 mutations renders FLT3 an obvious target of therapy. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3ITD positive and negative AML patients and as a maintenance therapy. So far, those clinical trials have showed very promising result. However, drug resistance to AC220 has also been reported through the early clinical studies. To understand the underlying mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confer resistance to AC220. In our screen, we identified SPRY3 and GSK3 as positive hits and the related Wnt and FGF-Ras-ERK signaling activation as the major mechanisms to cause resistance to the FLT3 inhibitor AC220. In the next step, we will explore the detailed underlying mechanisms by which SPRY3 and GSK3 mutations lead to drug resistance to AC220. We will also confirm our findings in primary AML patient samples. Taken together, our study will provide new insight into signaling pathways that contribute to the acquired resistance in AML. The knowledge learned from this study may lead to the development of more efficient therapeutic avenues for AML.

#5043

Molecular mechanism of resveratrol induced cell death in multidrug resistance prostate cancer.

Santosh K. Singh, Saswati Banerjee, James W. Lillard, Rajesh Singh. _Morehouse School of Medicine, Atlanta, GA_.

In order to understanding the molecular mechanisms of drug resistance as well as enhancing the efficacy of docetaxel we established docetaxel-resistant prostate cancer (PCa) cells. The IC50 for docetaxel in PCa resistant cells was about 50-fold higher than parental PCa cells. In the current study we used resveratrol (RES), a natural compound with chemo preventive potential to test its ability to enhance the effectiveness of docetaxel on PCa as well as to explore the mechanism of acquired docetaxel resistance. PCa (PC3, C4-2B and DU145) cell lines treated with different dose of RES or DTX alone or in combination followed by analyzed cell proliferation (MTT assay), apoptosis (flow-cytometric analysis of annexin V/propidium iodide-stained cells and western blot methods).The expression of ABC transporter markers in the development of docetaxel resistance was examined using RT-PCR. Our result shows that the expression of 18 ABC transporter genes were down regulated in PCa cells at 48 and 72 hrs in RES treatment group. The up regulations of pro-apoptotic proteins (Bax, Bad, Bim) and down regulation of anti-apoptotic protein (Bcl2, Mcl1) further confirmed the effectiveness of RES or DTX alone or in combination with DTX. In conclusion these studies suggest that RES inhibits ABC gene expression and resensitizes docetaxel resistant PCa cells to DTX treatment with enhancing apoptotic cell death.

#5044

CtBP1 regulates olfactory and adhesion pathways in prostate cancer and metabolic syndrome.

Guillermo N. Dalton,1 Juliana Porretti,1 Cintia Massillo,1 Georgina Scalise,1 Paula Lucía Farré,1 Cristian P. Moiola,1 Alejandra Paez,2 Geraldine Gueron,2 Paola De Luca,1 Adriana De Siervi1. 1 _Instituto de Biología y Medicina Experimental (IByME) - CONICET, C.A.B.A, Argentina;_ 2 _Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET, C.A.B.A, Argentina_.

Metabolic syndrome (MeS) is a cluster of pathophysiological disorders that comprise at least three of the following factors: abdominal obesity, elevated triglycerides, dyslipidemia, high blood pressure and impaired glucose tolerance. Several studies associated MeS with increased risk for several cancer types, including prostate cancer (PCa). C-terminal binding protein 1 (CtBP1) is a potent transcriptional co-repressor of tumor suppressor genes. This protein is activated by low NAD+/NADH ratio produced by highly energetic environment such as high fat diet (HFD) intake. Previously, we identified CtBP1 as a novel molecular link between MeS and prostate tumor growth. The aim of this work was to assess the CtBP1 related pathways in PCa and MeS. We developed a MeS in vivo model by chronically feeding male nude mice with HFD. Control diet (CD) fed animals were maintained at the same conditions. These mice were inoculated with PC3 stable CtBP1 depleted or control cells. RNA obtained from xenografts was used for Genome-wide expression profiles (Affymetrix) and Gene Set Enrichment Analysis (GSEA). These analyses yielded several important pathways regulated by CtBP1, such as "Cell Adhesion" (COL17A1, PRRS2, CDH3, ITGB4, LCN2, CDH1, GJB5, TGM2 and SPARC) and "Olfactory" (OR4C45, OR5P2, GUCA1C and CLCA2). CtBP1 significantly diminished the capability of PCa cell lines to adhere to a collagen matrix, repressing the epithelial marker CDH1 and inducing the mesenchymal marker VIM expressions. Interestingly, CtBP1 associated to ITGB4 promoter gene, strongly repressing its expression. CtBP1 depletion increased the plasma membrane of the cell attached to the substrate and the number of filopodia.

From olfactory pathway, we particularly focus on Chloride Channel Accessory 2 (CLCA2), a reported breast cancer tumor suppressor gene that function inhibiting epithelial to mesenchymal transition (EMT) and invasion processes. We found that CtBP1 regulates the transcription of CLCA2 in xenografts generated on HFD-fed mice. Using a panel of luciferase reporter plasmids with variable length of the CLCA2 promoter region, we determined that CtBP1 represses CLCA2 promoter activity in PC3 cells. Moreover, we established that CtBP1 associates to the CLCA2 proximal promoter region by chromatin immunoprecipitation (ChIP). Finally, several proteins regulate CLCA2 promoter activity independently (p53, ET2 and BRCA1) or dependently of CtBP1 (p300 and HDAC2). Altogether, these results demonstrated a new role for CtBP1 in the regulation of cellular adhesion, EMT and invasion reinforcing a potential function for this protein in cancer progression. Hence, CtBP1 pathway might help to identify new molecular candidates for better prediction of PCa progression in a subset of patients with MeS

#5045

Overexpression of ABC transporters through HGF/c-MET activation results in anti-cancer drug resistance in SCLC.

Yoshitaka Yagi,1 Hiroaki Ozasa,2 Takahiro Tsuji,1 Yuichi Sakamori,2 Takeshi Nomizo,1 Hiroki Nagai,2 Young Hak Kim,2 Ken Maeno,3 Tetsuya Oguri,3 Michiaki Mishima2. 1 _Graduate School Student of Medicine, Kyoto University, Kyoto, Japan;_ 2 _Kyoto University Hospital Respiratory Medicine, Kyoto, Japan;_ 3 _Dept. of Med. Oncol. and Immun., Graduate Sch. of Med., Nagoya City Univ., Nagoya, Japan_.

Upregulation of hepatocyte growth factor (HGF)/c-MET pathway causes drug resistance to anti-cancer agents such as epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in non-small lung cancer (NSCLC) with EGFR mutation and cytotoxic agents in small cell lung cancer (SCLC), but the roles and mechanisms of c-MET in drug-resistant cancer cells are not clarified. We studied to reveal the role of c-MET overexpression in the resistant lung cancer cell lines and to demonstrate whether MET-inhibition could restore drug sensitivity in anti-cancer drug resistant cell lines via ABC transporters down-regulation. In this study we used the 7-ethyl-10-hydroxycamptothesin (SN-38)-resistant cell line (PC-6/SN-38) that was derived from the human SCLC cell line PC-6. We compared the expression levels of the purposed genes by quantitative real-time PCR (qRT-PCR) and western blotting (WB). MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay was used to measure cell viability of the resistant cells compared with the parental cells. Inhibition of c-MET activation was performed by c-MET inhibitors, PHA665752 and Crizotinib or small interfering RNA against c-MET. We found MET inhibitors (4 or 8μM) reduced cell growth and restored drug sensitivity (2μM) of resistant cells, PC-6/SN38, compared to parental cells. To reveal the mechanisms of c-MET in drug resistance we examined ATP-binding cassette (ABC) transporters expression in PC-6/SN-38 compared to the parental cells by qRT-PCR, and found the mRNA level of ABCG2, which effluxes SN-38 as substrate, in PC-6/SN-38 cells was extremely increased and another ABC transporters that do not efflux SN-38 were not. Recently we reported PC-6/SN-38 cells had c-MET overexpression, and c-MET inhibition in resistant cells such PC-6/SN-38 resulted in restoration drug resistance. Based on this report we examined c-MET inhibition by c-MET inhibitor and small interfering RNA against c-MET in PC-6/SN38, and qRT-PCR and WB were performed to investigate alteration of ABCG2 expression, and found both two c-MET inhibition reduced ABCG2 expression. In addition, to demonstrate c-MET upregulation in resistant cells is addicted to the ligand HGF, PC-6 cells were incubated with HGF treated for 3 weeks and made PC-6/HGF, then continued to incubate with HGF and SN-38 was added two times in a week. Although c-MET expression is equal in PC-6 and PC-6/HGF, after SN-38 treatment c-MET was overexpressed. Furthermore, serum HGF level in SCLC patients correlated with clinical course. We demonstrated upregulation of ABC transporters via HGF/c-MET signaling activation might be one of the mechanisms of acquired resistance to cytotoxic anticancer agents in lung cancer cells. Decreased ABC transporters expression through inhibition of c-MET activation might overcome drug resistance to cytotoxic agents.

#5046

High-throughput chemical screening for sensitization of bladder cancer to gemcitabine and cisplatin chemotherapy.

Yuki Kita, Takashi Kobayashi, Norihiko Masuda, Atsuro Sawada, Yoshiyuki Matsui, Takahiro Inoue, Tomomi Kamba, Osamu Ogawa. _Kyoto university, Kyoto, Japan_.

Background

Gemcitabine and cisplatin chemotherapy (GC) is the current standard regimen for locally advanced and metastatic bladder cancer (BC). Despite a relatively high initial response rate, some cases do not regress (intrinsic resistance) and the remaining cases often show regrowth after initial shrinkage (acquired resistance). To identify novel therapeutic agents for overcoming these resistances, we applied a high-throughput screening of chemicals administered in combination with GC.

Methods and Findings

As a high-throughput screening, 2100 compounds were administered alone or in combination with GC to human BC cell lines (J82, UMUC-3). Cell viability was determined after 3-day incubation and chemicals that enhanced inhibitory effect of GC were screened. The initial screening identified 26 compounds and further validation narrowed them into the most synergistic agent disulfiram (DSF), an FDA-approved drug for alcoholism. Combination index assay showed synergistic effects of DSF with cisplatin but not with gemcitabine in J82, UMUC-3, T24, HT1197 and HT1376 cells. Co-administration of DSF significantly enhanced apoptosis by GC treatment in UMUC-3 and T24 cells, with significant increase of reactive oxygen species (ROS) production. DSF-induced apoptosis was attenuated in the presence of N-acetyl L-cysteine and Trolox, ROS scavengers. Use of DSF in combination with GC (GCD) significantly inhibited tumor growth of UMUC-3 subcutaneous xenograft on athymic mice (by 39% compared with GC alone, p = 0.02). GCD regimen was as tolerable as GC and no significant differences were observed in body weight of treated mice between the two regimen.

Conclusion

Repositioning of DSF to a chemotherapy sensitizer is a promising treatment strategy, which can be translated rapidly in the future.

#5047

The MUC1 membrane-bound mucin increases tumor cell properties and chemoresistance in renal clear cell carcinoma.

Kelly Gaudelot, Viviane Gnemmi, Audrey Bouillez, Jean-Baptiste Gibier, Mélanie Fanchon, Justine Woszczyk, Brigitte Hémon, Isabelle Van Seuningen, Sébastien Aubert, Michael Perrais. _INSERM UMR-S1172, Lille Cedex, France_.

Introduction: MUC1, an O-glycoprotein membrane-bound mucin, is overexpressed in renal clear-cell carcinomas (CRCC) with correlation to two major prognostic factors, Tumor-Node-Metastasis stage and nuclear Fürhman grade. Previously, we have shown that (i) MUC1 was significantly overexpressed in metastatic CRCC vs non-metastatic CRCC and (ii) MUC1 is a target gene of HIF-1 transcription factor which is a part of the hypoxia pathway, the main renal carcinogenetic pathway. Furthermore, CRCC is highly resistant to common systemic chemotherapies.

Material and method: To better understand the roles of MUC1 in CRCC, we used two renal cell lines expressing MUC1 (786-O cells) or not (ACHN cells). 786-O cells were stably transfected with shRNA targeting MUC1 while ACHN cells with full-length MUC1. Proliferation, drug resistance, migration and invasion properties were studied in vitro in the different cellular clones using MTS cell proliferation assay, wound healing assay and Boyden chambers coated with Matrigel, respectively. Signaling pathways were screened by proteome profiler and western blot.

Results and discussion: We showed that MUC1 expression was associated with increased invasion and migration properties of renal carcinomatous cells and a decrease of cell-cell interactions. MUC1 overexpressing cells (i) expressed higher levels of anti-apoptotic factors and MDR genes involved in chemoresistance processes and (ii) were more resistant to chemotherapeutic drugs.

Conclusion: Our results show that MUC1 plays a role in biological properties of renal cancer cells suggesting important function for this mucin in tumour progression and chemo-resistance. Our data confirm its potential as a therapeutic target in this type of cancer.

#5048

Platelet FAK is a critical regulator of tumor growth after withdrawal of anti-angiogenic therapy.

Monika Haemmerle,1 Justin Bottsford-Miller,1 Sunila Pradeep,1 Morgan L. Taylor,1 Hyun-Jin Choi,1 Rebecca L. Stone,2 Min Soon Cho,1 Alpa M. Nick,1 Gabriel Lopez-Berestein,1 Vahid Afshar-Khargan,1 Anil K. Sood1. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _The John Hopkins University, Baltimore, MD_.

Objective: In most clinical trials, anti-angiogenic therapies have offered only modest improvements in progression-free survival without impacting overall survival. Following cessation of anti-angiogenic therapy, concerns have been raised about a possible rebound in tumor growth, but the underlying mechanisms are poorly understood. Therefore, in this study we aimed at comparing tumor growth and the effects on tumor microenvironment after therapy withdrawal compared to continuous treatment with anti-angiogenic agents.

Methods: Mice were intraperitoneally injected with human or mouse ovarian cancer cells and were treated with the anti-angiogenic drugs pazopanib, bevacizumab or B20 for either a short-term with subsequent withdrawal or continuous therapy until necropsy. Immunohistochemical staining was used to evaluate platelet infiltration into the tumor microenvironment, tumor angiogenesis and vascular leakage. To assess the significance of focal adhesion kinase (FAK) in the process of platelet infiltration and tumor rebound after withdrawal of therapy, we either used the FAK inhibitor GSK2256098 or a mouse model with platelet-specific FAK deletion.

Results: Cessation of therapy with pazopanib, bevacizumab and the cross-human and murine anti-VEGF antibody B20 was associated with up to 4-fold increased tumor growth in mouse models of ovarian cancer when compared to continuous treatment. Tumor outgrowth was associated with significant tumor hypoxia, increased tumor angiogenesis and vascular leakage. More importantly, we found 380% increased hypoxia-induced ADP production and 3-fold increased platelet infiltration into tumors where anti-angiogenic therapy was withdrawn. Lowering platelet levels significantly inhibited tumor rebound after withdrawal of anti-angiogenic therapy. Interestingly, FAK in platelets regulated their migration into tumor microenvironment and FAK knock-out specifically in platelets completely prevented the rebound tumor growth. Additionally, combined therapy with the FAK inhibitor GSK2256098 and the anti-angiogenic agents pazopanib and bevacizumab led to up to 5-fold reduced orthotopic tumors and likewise inhibited negative effects of withdrawal of anti-angiogenic therapy.

Conclusions: Collectively, our results characterize a previously unknown role for platelets in the tumor microenvironment and provide a potential therapeutic benefit for FAK inhibitors in preventing rebound in tumor growth following discontinuation of anti-angiogenic agents. Additionally, dual targeting of FAK and VEGF could have important therapeutic implications for ovarian cancer management.

#5049

SIAH2 E3 ligase targets LIM-domain proteins for degradation to modulate focal adhesion and cell motility.

Minglei Bian,1 Yang Liao,1 Monicah Njogu,1 Rebecca Schmidt,2 Rie Takahashi,2 Zandra Walton,2 Amy H. Tang1. 1 _Eastern Virginia Medical School, Norfolk, VA;_ 2 _Mayo Clinic, Rochester, MN_.

Introduction: The RAS signaling pathway is an important growth-promoting pathway that controls cell proliferation and survival in multicellular organisms. During oncogenesis, activation of EGFR/HER2/K-RAS signaling pathway reduces focal adhesion, increases cell motility, promote tumorigenesis and accelerate tumor invasion and metastasis. SIAH2, which is the human homologue of a highly conserved family of RING-domain E3 ubiquitin ligase, is a key downstream signaling component which is essential for RAS signal transduction. We noticed that SIAH2 can regulate cell migration, tumorigenesis and metastasis by unknown mechanisms.

Methods: Using HIV-based lentiviral system, we introduced an anti-SIAH molecule, the proteolysis-deficient mutant form of SIAH2 (SIAH2PD), into multiple types of human cancer cell lines. The changes of cell morphology, focal adhesion and adherens junction were examined by immunofluorescence (IF) staining. Images were captured on day 3 post infection experiments to show the changes in cell focal adhesion/junction. Using lentiviral system, we introduced exogenous LIM-domain proteins into human cancer cell lines which already expressed SIAH2PD, and then check the cell focal adhesion rescue by immunofluorescence staining.

Results: We identified and validated three proteins that interact with SIAH2, contain LIM domains, and regulate focal adhesion and cell attachment. Through in vitro and in vivo experiments, we show that SIAH2 binds, ubiquitinates and degrades thyroid receptor-interacting protein 6 (TRIP6), Four-and-a-half LIM domain protein 2 (FHL2) and Leupaxin (LPXN), indicating that these evolutionarily conserved LIM-domain proteins are bona fide SIAH2 substrates and their double zinc finger motif might serve as a degron signal for SIAH2 E3 ligase. Our staining results showed that SIAH-deficiency would disrupt Trip6/FHL2/LPXN localization at the focal adhesion site, and inhibit cell motility. By introducing exogenous Trip6/FHL2/LPXN proteins into cancer cells, the focal adhesion deficiency and cell death caused by SIAHPD can be rescued, and the ability of cell migration and tumor initiation can be restored.

Conclusions: We show that in cancer cells, SIAH2 antagonizes TRIP6, FHL2 and LPXN in modulating focal adhesions and cell motility. This interaction could be important in cell dissemination, invasion and metastasis. The suppression of cell motility and tumorigenesis in response to SIAH-deficiency could provide a new molecular mechanism to reveal the importance of RAS/SIAH signaling pathway in cancer cells. By circumventing this essential cell growth signaling pathway in cancer, we hope to develop novel anti-SIAH-based therapeutic drugs, which may help to inhibit cancer cell dissemination, invasion and metastasis.

#5050

The significance of the expression of E-cadherin and CD44 in patients with unresectable metastatic colorectal cancer.

Yasuhito Iseki, Masatsune Shibutani, Kiyoshi Maeda, Hisashi Nagahara, Tatsuro Tamura, Go Ohira, Sadaaki Yamazoe, Katsunobu Sakurai, Kenjiro Kimura, Takahiro Toyokawa, Ryosuke Amano, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. _Osaka City University Graduate School of Medicine, Osaka, Japan_.

The loss of the adhesion molecules is reported to correlate with tumor invasion and metastasis in patients with various cancers. E-cadherin is an important molecule for cell-to-cell adhesion. While, CD44 is important for cell-to-extra cellular matrix adhesion. The aim of the present study is to evaluate the significance of the expression of E-cadherin and CD44 in patients with the unresectable metastatic colorectal cancer who are undergoing palliative chemotherapy. Formalin-fixed, paraffin-embedded samples were obtained from 49 patients who underwent primary tumor resection and who were receiving palliative chemotherapy for unresectable metastatic colorectal cancer. We evaluated the expression of E-cadherin and CD44 by immunohistochemistry. The expression of E-cadherin was not significantly associated with progression-free survival (PFS) or overall survival (OS). Although, the expression of CD44 did not correlate with PFS, it tended to correlate with the OS (p=0.0699). According to the combination of CD44 and E-cadherin, both low expression group showed a significant poor PFS (p=0.0101) and OS (p=0.0009). Objective response rate and disease control rate were significantly decreased in both low expression group (p=0.0076 and p=0.0294, respectively). A univariate analysis to determine the variables that were associated with PFS indicated that the both low expression of E-cadherin and CD44 (p=0.0474) and gender (p=0.0330) were significantly associated with decreased survival. A multivariate analysis showed that the both low expression of E-cadherin and CD44 was an independent risk factor for decreased PFS (hazard ratio [HR]: 8.276, 95% confidence interval [CI]: 1.383-43.311; p=0.0227). According to OS, the both low expression of E-cadherin and CD44 expression was revealed a prognostic factor by univariate and multivariate analysis(HR:15.118, 95%CI: 2.645-77.490; p=0.0039). This study suggested that the combined low expression of both E-cadherin and CD44 was a strong independent predictor of decreased chemotherapeutic outcome and survival in patients with unresectable metastatic colorectal cancer.

#5051

Docosahexaenoic acid inhibits cancer cell invasion and migration by modifying focal adhesion complex structure and signaling.

Michael Mouradian, Palvinder K. Bains, Ronald S. Pardini. _University of Nevada, Reno, Reno, NV_.

The extracellular matrix (ECM) is responsible for the regulation of many cellular processes, including survival, proliferation, migration and invasion. Cellular-ECM contact induces a coordinated process recruiting functional protein complexes, consisting of scaffold, adapter, linking and docking proteins, to form focal adhesion (FA) structures. Once formed, FA complexes then recruit signaling proteins to activate downstream cellular pathways. Aberrations in FA structure and signaling have shown to contribute to cancer initiation, progression and cellular motility. Studies investigating the molecular mechanisms accounting for FA structure and function in cancer cells have demonstrated unique growth behavior and migratory properties in vitro. While docosahexaenoic acid (DHA), an omega-3 PUFA, has been shown to inhibit growth of a number of cancer cell lines, the effects of DHA on FA dynamics and signaling have not been investigated. In the present study, we show that DHA induced changes in the morphological growth phenotype in cancer cell lines grown in Matrigel™. DHA treatment also inhibited planar cell movement (cell migration) and amoeboid motility (invasion) in breast and lung cancer cell lines. Associated with the observed morphological changes and inhibited growth, were changes in the expression of a number of proteins involved in the FA complex. Upon further characterization, cell line specific changes in protein expression levels and protein localization of FAK, e-cadherin, paxillin and vinculin were observed with DHA treatment, which suggests that DHA is modifying FA complex structure. Additionally, changes in the phosphorylation patterns of major signaling pathways associated with the FA complex, like EGFR, ERK and c-Myc, were also observed with DHA treatment. Overall, these results provide new insight into FA activation and signaling and demonstrate a new mechanism for DHA treatment in cancer.

#5052

Pro-oncogenic role of desmosomal plaque-related proteins in non-small cell lung cancer (NSCLC).

Pedro P. Medina, Laura Boyero, Joel Martin-Padrón, Esther Farez. _University of Granada / GenyO, Granada, Spain_.

Squamous cell carcinomas and adenocarcinomas are the most frequent forms of lung carcinoma, and have different prognoses and therapeutic approaches.

During recent years, accumulating evidence has supported an important role of several junctional proteins in carcinogenesis, tumor invasion and metastasis. Contact between epithelial cells is mediated by several types of cell-cell junction, which are composed by complex arrays of transmembrane and plaque proteins and are connected typically to cytoskeletal components.

Desmosomes are cell-cell complexes found primarily in epithelial tissues. In addition to the well-known structural role of desmosome genes, evidences such as their dual subcellular location, the interaction with single-stranded DNA and translation initiation factors, etc. suggest there is a poorly understood role of these proteins in signalling pathways.

Desmosomal components have been traditionally considered as tumoral suppressors although current evidences suggest potential pro-oncogenic functions of desmosomal-plaque proteins, probably due to an unknown regulatory role in intracellular signaling -such as in Wnt and Akt pathways.

Previously, our group showed differentially expressed gene sequences corresponding to several desmosomal plaque-related proteins as a function of tumor type, stage and differentiation grade in NSCLC.

The staining pattern of some junctional proteins also differed between squamous-cell carcinomas and adenocarcinomas. Expression of these proteins marked intercellular junctions that are characteristic of the squamous stratum of stratified flattened epithelium and of neoplasias with this type of differentiation. We observed more intense staining of these proteins in better-differentiated areas.

In order to gain greater insight into the specific function of some desmosomal components in NSCLC tumors, we have performed several functional assays, as cell cycle and proliferation among others, in selected SCC lung cancer cell lines. Furthermore, we are developing stable cell lines with over-expression and knockdown of some desmosomal proteins by lentiviral vectors and inducible clonal cell lines; and also carrying out knockout cell lines using CRISPR-Cas9 system. Presumably, they will allow us to perform long-term functional assays and expression arrays in order to identify subcellular location and potential pro-oncogenic roles of desmosomal proteins in NSCLC carcinogenesis which may lead to improvements in the treatment and prevention of this disease.

Our preliminary results indicate some desmosomal proteins as potential targets for NSCLC diagnosis and treatment.

In a future perspective, xenografts will lead to understand the molecular origins and progression of lung cancer in regard to structural components of the desmosome.

Corresponding authors: pedromedina@ugr.es, efarez@ugr.es

#5053

Tumor cell clustering - Identification of new regulators.

Valérie LOBJOIS, Bernard Ducommun. _Univ. of Toulouse, Toulouse, France_.

Cancer cell aggregation is a key process involved at different stages of tumor evolution, during primary tumor growth, metastatic cells dissemination and secondary tumor evolution. Thus, understanding principles that govern and modulate the ability of cancer cells to aggregate might pave the way for new therapeutic opportunities.

We investigated cancer cell aggregation independently of cell migration or matrix adhesion and demonstrated that the cell-cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are both involved this process (Saias, 2015). Strikingly, elevated expression of these proteins in primary tumors has also been reported to be associated with blood detection of clusters of highly metastatic circulating tumor cells and poor prognosis (Aceto, 2014). We also reported that Myosin IIa applies a counter force that slows down aggregation indicating that acto-myosin cytoskeleton tension favors aggregation.

To further decipher the regulation of this process, we developed arrays of hydrogel microwells and robust microscopy-based quantification assay to monitor the kinetics of aggregation of tumor cells in the absence of cell - substrate adhesion. We performed a candidate approach and found that several other members of the cadherin superfamily are involved in the aggregation process. We also conducted a small molecule screen to identify other pathways involved in regulating aggregation of tumor cells. We will report unpublished results on the characterization of the molecular bases of aggregation properties in various tumor cell types and on the identification of new regulatory mechanisms that modulate this process.

\- Saias L, Gomes A, Cazales M, Ducommun B, Lobjois V. Cell-cell adhesion and cytoskeleton tension oppose each other in regulating tumor cell aggregation. Cancer Res. (2015) 75(12); 1-8

\- Aceto N, Bardia A, Miyamoto DT, Donaldson MC, Wittner BS, Spencer JA, et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 2014;158:1110-22.

#5054

Role and signaling of desmosomal DSP and PKP1 in ovarian cancer.

Yan Xu, Qingchun Cai. _Indiana University School of Medicine, Indianapolis, IN_.

Identification of driver genes/proteins in EOC tumorigenic processes is critical to increase the cure rate for late stage epithelial ovarian cancer (EOC), which has not been in improved over several decades. We have developed a highly aggressive EOC cell line (ID8-P1 or P1) from ID8-P0 (without in vivo passage, or P0) through in vivo passage in

C57BL6 mice. Two genes encode important desmosome components, desmoplakin (Dsp) and plakophilin 1 (Pkp1) were found to be significantly over-expressed in P1

vs. P0 cells, as well as human EOC cells. DSP is an obligate component of functional desmosomes that anchors intermediate filaments to desmosomal plaques. Pkp1 encodes a member of the arm-repeat (armadillo) and plakophilin (PKP) gene families. Our understanding of the functions of desmosomes in cancer is just emerging and the role of desmosomes in EOC is totally unknown. Our data showed the novel functions of DSP in anoikis-resistance and SRC activation using shRNA-mediated DSP-knocked down (KD) stable cell lines. In addition, we found that ID8-P1 cells formed spheroids and P0 cells tended to be single cells when they were cultured in suspension. KD-Dsp dissociated spheroids, suggesting that desmosomes play an important role in spheroid formation, which may be directly related to anoikis-, drug-resistance, and/or the cancer stem cell (CSC) concepts. Moreover, we found that enhanced glycolysis was involved in the increased anoikis-resistance of ID8-P1 cells and an inhibitor of glycolysis, shikonin (SHK) was a potent anoikis-resistance inhibitor and P1 cells gained more drug-resistance, when compared to P0 cells, with the IC50s were 1.7 and 7.4 µM for P0 and

P1 cells, respectively. Similarly, the IC50s were 2.4 and 8.1 µM for human EOC PE01 and PE04 cells, respectively. PE01 and PE04 cell lines are identified to represent high-grade serous ovarian cancer (HGSC), which were derived from the same patient before (PE01) and after (PE04) the onset of drug resistance to cis-platinum (CDDP), chlorambucil, and 5-fluorouracil. PP2 (a SRC nhibitor) and SHK were able to dissociate the cell-cells aggregates/spheroids in EOC cells. The previously unknown role of DSP and/or PKP1 in EOC may be related to their signaling activities outside desmosomes. Cells staining of PKP1 protein showed its cytosolic and perinuclear location, providing supporting and proof-of-concept data. Together, our data have suggested the novel links among DSP, PKP1, anoikis-resistance, SRC activation, cell-cell-aggregation, and

drug-resistance. These data provide a basis for developing new modalities for EOC treatment.

### Cellular and Molecular Dynamics of Cancer Migration and Invasion

#5055

SPEN stimulates primary cilia formation, cell migration and is associated with metastasis in breast cancer.

Stéphanie Légaré, Catherine Chabot, Mark Basik. _McGill University, Montreal, Quebec, Canada_.

The primary cilium (PC) is a microtubule-based and non-motile organelle that functions as animal cells' antennae, regulating a number of signalling pathways, including the mTOR, PDGFR and Wnt pathways, in addition to being essential for activation of the canonical Hedgehog (Hh) pathway. In breast cancer, the PC is a structure that is frequently lost due to transcription- and mutation-driven alterations in cilium-related genes. In a study aiming to delineate the hormone-independent transcriptional program regulated by SPEN, a gene that we have identified as a tumour suppressor gene in estrogen receptor (ER)-positive breast cancers, we found that SPEN was co-expressed (R>0.95) with a number of genes involved in ciliary biology (P=1.64E-19), including the ciliogenic transcription factors RFX2, RFX3 and FOXJ1. Interestingly, the overexpression of SPEN resulted in the restoration of the PC in T47D cells, which have previously been shown to lack this structure. Consistent with the re-establishment of a PC in T47D cells, SPEN-overexpressing T47D cells had decreased mTOR signalling, hyperactivation of PC-dependent pathways, including the Hh and PDGFR pathways, and increased migration. Silencing SPEN expression in the non-tumorigenic MCF10A cells, which express high endogenous levels of SPEN and show a high prevalence of PC (36%), considerably decreased PC levels in MCF10A cells and was accompanied with increased mTORC1 activity as well as attenuated cell migration. We also found that SPEN silencing in MCF10A cells decreased the expression of the ciliogenic transcription factor RFX3 and that SPEN interacts with DNA upstream of RFX3 gene transcription start site in chromatin immunoprecipitation experiments. Collectively, these observations indicate that SPEN may mediate its effects, at least in part, through the transcriptional regulation of RFX3. Accordingly, RFX3 expression levels were strongly positively correlated (R=0.55, P<0.0001) with SPEN levels in a cohort of 82 clinical samples from triple negative breast cancer patients. Notably, high SPEN RNA levels were also predictive of early metastasis in two independent cohorts of 77 (HR=2.25, P=0.03) and 170 (HR=2.23, P=0.004) ER-negative breast cancer patients. Together, our findings highlight a role for SPEN in the regulation of PC formation and support important functions in the inhibition of mTORC1 and activation of Hh and PDGFR pathways as well as cell migration, a process that may contribute to early metastasis in ER-negative breast cancers.

#5056

RET-mediated invasion in three-dimensional microenvironment models.

Sarah M. Maritan, Eric Y. Lian, Lois M. Mulligan. _Queen's University, Kingston, Ontario, Canada_.

RET is a receptor tyrosine kinase expressed in cells derived from the neural crest. RET activation and signalling is mediated by soluble ligands of the Glial Cell-Derived Neurotrophic Factor (GDNF) family. RET signalling plays critical roles during embryogenesis, mediating directional cell migration, proliferation, and survival. In addition to its normal developmental roles, oncogenic mutations or aberrant expression of RET are also linked to tumor spread and metastasis in multiple human tumor types. Specifically, activating mutations of RET have been identified in multiple-endocrine neoplasia type 2, while expression of wildtype RET is linked to increased local invasion in breast and pancreatic tumors. Previous studies of RET-mediated invasion and metastasis have used standard 2D culture methodologies, which may not be reflective of the in vivo microenvironment. Here, we have developed an in vitro 3D model of tumor growth, invasion, and metastasis to characterize the signals critical to these processes.

Using SH-SY5Y cells, a neuroblastoma cell line that endogenously expresses RET, we assessed anchorage-independent growth and invasion into a surrounding collagen matrix in response to RET stimulation with GDNF. We have quantified the contributions of several RET downstream signalling pathways by individually blocking SRC, PI3K, FAK, STAT3, MEK, and integrin β-1 signals, for their effects on RET-mediated cell growth and invasion in 3D culture. Our data demonstrate that inhibiting either PI3K or MEK decreases the invasive potential of SH-SY5Y cells in response to RET activation, while inhibition of STAT3 had little effect. We have also shown that integrin β-1, FAK or SRC inhibition completely blocked invasion, consistent with an integrin dependent mode of invasion in SH-SY5Y cells. Additionally, we showed that FAK and SRC signalling were critical for SH-SY5Y survival in 3D microenvironments. While several RET-mediated signalling pathways have been implicated in cell motility and invasion in two-dimensional culture models, our findings suggest that specific signals may differ in their importance in a three-dimensional microenvironment. We are currently using our 3D models to further characterize the contributions of RET signalling to tumor cell invasiveness. These data may provide further insight into the role of RET expression in in vivo tumor spread.

#5057

Mena at the nexus of chemotaxis and haptotaxis during tumor progression.

Madeleine J. Oudin,1 Miles A. Miller,2 Tatsiana Kosciuk,1 Joelle Klazen,1 Alisha Lussiez,1 Douglas Lauffenburger,1 James E. Bear,3 Frank B. Gertler1. 1 _MIT Koch Institute for Integrated Cancer Research, Cambridge, MA;_ 2 _MIT Department of Biological Engineering, Cambridge, MA;_ 3 _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Directed-cell migration is key step in the metastatic cascade, and multiple cues can promote local invasion. The most well studied mode of cell motility for metastasizing tumor cells is chemotaxis, the directional movement of cells attracted to a source of soluble cues. In contrast, haptotaxis, migration of cells on gradients of substrate-bound factors, remains poorly understood. Mena, an actin regulatory protein, plays an important role in cell motility and is upregulated in various cancers, driving migration in response to chemotactic and haptotactic cues. We have recently shown expression of the pro-metastatic isoform of Mena, MenaINV, increases sensitivity to growth factors (EGF, HGF and IGF) via dysregulation of the phosphatase PTP1B, as well as high concentrations of fibronectin (FN) via its interaction with the integrin α5β1. We hypothesized that MenaINV may also promote crosstalk between these two pathways to drive metastasis. First, we show that MenaINV-driven haptotaxis on FN gradients and FN-driven invasion requires crosstalk between α5β1 and EGFR. Indeed, we show evidence that MenaINV expression is associated high phosphorylation of EGFR in high FN environments in vitro and in human breast cancer databases. Second, we find that MenaINV promotes synergy between EGF and FN in vitro and in vivo, leading to hyper-invasive phenotypes that drive more invasion than which each cue alone. Finally, we show that MenaINV-driven haptotaxis, ECM reorganization and 3D invasion requires RCP-dependent recycling of α5β1 and EGFR. Together, our data demonstrate that MenaINV is a shared component of the machinery that mediates both haptotactic responses to FN and chemotactic responses to EGF, two pathways that promote tumor progression.

#5058

Hitting the brakes on the migratory capacity of tumoral cells: Targeting key regulators of actin dynamics in prostate cancer.

Alejandra V. Paez,1 Carla Pallavicini,2 Federico Schuster,1 Jimena Giudice,3 Pia Valacco,1 Estefania Labanca,4 Nicolas Anselmino,1 Emiliano Ortiz,1 Maria Binaghi,1 Javier Cotignola,1 Marcelo Marti,1 Luciana Bruno,2 Valeria Levi,1 Nora Navone,4 Elba Vazquez,1 Geraldine Gueron1. 1 _IQUIBICEN-UBA/CONICET, Buenos Aires, Argentina;_ 2 _Department of Physics, FCEN, University of Buenos Aires, Buenos Aires, Argentina;_ 3 _Department of Cell Biology and Physiology. School of Medicine. University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 4 _Department of Genitourinary Medical Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX_.

Prostate Cancer (PCa) is the second leading cause of cancer in men. PCa cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize other organs. We have previously shown that heme-oxygenase 1 (HO-1) has a strong anti-tumoral effect in prostate cancer (PCa) and regulates the adhesive properties of PCa cells. Innovative high-throughput proteomic platforms are now identifying and quantifying new specific and sensitive biomarkers for PCa detection, stratification and treatment. Towards this end, we undertook an in-depth mass spectrometry-based proteomics study to build the Heme-oxygenase 1 (HO-1) interactome in PCa, in an effort to identify HO-1 molecular partners associated to the integrity of the cellular architecture and assess actin dynamics of PCa cells under HO-1 modulation.

The proteomics analysis of HO-1 interacting proteins yielded several cytoskeletal-associated proteins regulating actin filament dynamics, such as gelsolin, lasp1, SIPA1L1, testin, moesin, tropomodulin and vinculin. The bioinformatics screening across the Oncomine platform revealed that the RNA expression profiles of the cytoskeletal HO-1 interacting proteins, lie within the 10 percent of the most consistently low-expressed genes in prostate adenocarcinoma compared to normal tissue.

Motility changes were assessed on fiber-like 2D migration scenarios displaying a reduced frequency in migration events and in migration speed under hemin exposure, a specific pharmacological inducer of HO-1. A significant higher proportion of filopodia-like protrusions among neighboring cells and cellular contacts were observed when HO-1 was induced; effects reversed under HO-1 silencing. Altogether, our experimental findings demonstrate that HO-1 modulation in PCa induces the remodeling of the actin filament architecture at filopodia, alters the migratory patterns and cellular morphology, showcasing the relevance of the cytoskeletal proteins as potential therapeutic targets against the aggressive and metastatic disease.

#5059

Cancer cell invasion dynamics in microchannels during stromal cell coculture.

Andrew W. Holle,1 Verena Kast,2 Ralf Kemkemer,2 Joachim Spatz1. 1 _Max Planck Institute for Intelligent Systems, Stuttgart, Germany;_ 2 _University of Reutlingen, Reutlingen, Germany_.

Metastasizing cancer cells must escape their tumor microenvironment and invade into surrounding healthy stromal tissue. During this process, they must balance intercellular crosstalk with ECM degradation, physical force generation, and cytoskeletal rearrangement. To mimic this process in vitro, PDMS microchannels fabricated with photolithography and replica molding were used to examine the interaction of cancer cells with healthy stromal cells during physical confinement in three dimensional environments. A focus was placed on understanding the variations in invasion behavior between healthy cells and cancer cells, then attempting to explain these differences by cell mechanical properties and gene expression. Distinct differences in cancer cell invasion characteristics were observed between wide 10 μm channels and narrow 3 μm channels, with the latter exhibiting smooth velocity profiles and increased channel permeation. Live cell actin staining, ECM protein alteration, and chemical inhibition of both Rac1 and RhoA mechanical pathways were all used to better understand these differences, leading to the conclusion that the actin cytoskeleton is greatly altered during invasion into very confined channels. To better understand the role of stromal cells in this process, both indirect and direct coculture of MDA MB-231 basal breast cancer cells and stromal cells were performed in microchannel chips with the aid of live cell permeable tracking dyes. To reflect a diverse range of stromal cell types, cocultures were performed with MCF 10A non-tumorigenic breast epithelial cells, macrophage-differentiated THP-1 monocytes, and HN-CAFs (head and neck carcinoma-associated fibroblasts). Both the cancer cells and the stromal cells exhibited increases in invasion speed upon direct co-culture of the two cancer cell lines, both in wide and narrow channels. The use of stromal cell-conditioned media as well as observed differences in migration speed and persistence when unconfined provided further understanding of the complex processes underlying cancer cell and stromal cell invasion.

#5060

Myosin-IIA heavy chain phosphorylation on S1943 regulates tumor cell invasion.

Laura E. Norwood Toro, Jonathan M. Backer, Anne R. Bresnick. _Albert Einstein College of Medicine, Bronx, NY_.

Nonmuscle myosin IIA (NMHC-IIA) heavy chain phosphorylation has been shown to regulate motility and chemotaxis in invasive breast cancer cells; phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances two-dimensional cell migration. To test the role of NMHC-IIA S1943 phosphorylation in regulating the invasive properties of tumor cells, we produced MDA-MB-231 breast cancer cells in which the endogenous NMHC-IIA was stably knocked down via shRNA and replaced with exogenously expressed murine GFP-tagged wild type, phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA. Since previous studies have shown that the matrix-degrading activity of invadopodia is regulated via a myosin II-FAK-Cas pathway (Alexander et al, Curr Biol 18:1295, 2009), we examined the ability of MDA-MB-231 cells expressing wild type, S1943E or S1943A NMHC-IIA to form invadopodia and degrade matrix. Only 39.4% of S1943A NMHC-IIA expressing cells exhibited matrix degradation as compared to wild type NMHC-IIA (90.5%) and S1943E NMHC-IIA (90.3%). In addition, the average percent degradation area for cells expressing S1943A NMHC-IIA was 15-fold lower than for cells expressing wild type NMHC-IIA, whereas S1943E NMHC-IIA cells exhibited a 2-fold increase in degradation area. There was no significant difference in the numbers of immature invadopodia between the three cell lines. However, S1943A NMHC-IIA cells had significantly fewer mature invadopodia, while S1943E NMHC-IIA cells had increased numbers of mature invadopodia compared to wild type NMHC-IIA cells. To investigate the consequences of altered invadopodia maturation and activity, we examined the secretion of MMP-9 from cells expressing wild type, S1943E or S1943A NMHC-IIA. Gelatin zymography and MMP-9 activity assays of conditioned medium showed that S1943E NMHC-IIA expression enhanced MMP-9 secretion by 2-fold, whereas S1943A NMHC-IIA expression reduced MMP-9 secretion by 4-fold. In experimental metastasis assays in SCID mice, S1943A NMHC-IIA cells rarely formed lung metastases as compared to wild type NMHC-IIA cells, which formed poorly differentiated metastatic foci with some pleural localization. The S1943E NMHC-IIA cells also produced poorly differentiated carcinomas, which primarily localized along the pleural lining. Overall, S1943A NMHC-IIA cells produced approximately 2-fold fewer and S1943E NMHC-IIA cells produced 2-fold greater metastases than wild type NMHC-IIA cells. Together, these observations indicate that NMHC-IIA S1943 phosphorylation contributes to tumor cell invasion and metastasis via the regulation of extracellular matrix degradation.

#5061

Evaluating the role of durotactic migration in the tumor microenvironment.

Brian J. DuChez, Kenneth M. Yamada. _NIDCR, Bethesda, MD_.

Durotaxis is a mechanism of directional migration in which cells respond to a stiffness gradient in their local microenvironment. While durotaxis has been characterized primarily in cells of mesenchymal origin, its role in cancer biology has not been clearly defined. At a cellular and molecular level, evidence suggests cancer cells sense and respond to the stiffening tumor microenvironment in a manner that promotes malignant and invasive characteristics. Given the gradual stiffening of tumors, it is interesting to speculate how durotaxis might contribute to cell flux to and from primary tumors and metastatic sites. We hypothesize that a durotactic mechanism may, in part, contribute to the dissemination of cancer cells into the stiffened microenvironment associated with primary tumors and also possibly with pre-metastatic sites.

To directly characterize the impact of local stiffness on directional migration, cancer cells were cultured on a hydrogel possessing a stiffness gradient, then imaged by time-lapse microscopy and tracked using custom automated tracking software. Automated cell tracking allows for unbiased, accurate, and high-speed generation of large migratory datasets not feasible with conventional manual tracking and enables more robust analysis of cell populations. Preliminary data reveal that multiple cancer cell lines respond to a stiffness gradient with directed migration towards the stiffer part of the gel. The strength of the directional migratory response to stiffness gradients was found to depend on the magnitude of stiffness a cell encounters. By manipulating stiffness gradients to accurately reflect stiffness encountered by cancer subtypes, we will better understand if a durotactic mechanism has a global or cancer-specific impact on disease progression.

#5062

Hypoxia-induced amoeboid cancer cell migration.

Veronika te Boekhorst,1 Liying Jiang,1 Steffi Lehmann,2 Alba Zuidema,1 Peter Friedl1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _ETH, Switzerland_.

Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of epithelial-to-mesenchymal transition (EMT) and subsequent cancer cell invasion and metastasis. However, how hypoxia impacts on tumor cell invasion programs and translates into modes of dissemination of moving tumor cells remains unknown. Using breast cancer (BC) and head and neck cancer (HNC) cells which efficiently invade from multicellular spheroids into 3D fibrillar collagen, we addressed which signaling programs that control cell-cell adhesions, cell-matrix interactions and cytoskeletal dynamics and which modes of tumor cell migration are induced by hypoxia.

While both cancer models primarily invaded collectively under normoxic conditions, severe hypoxia (0.2% oxygen) or pharmacological stabilization of HIF-1 using the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced EMT-like detachment and migration of single cells into collagen, exhibiting a 2- and 4-fold increase of single BC and HNC cells, respectively. Besides infrequent spindle-shaped movement, hypoxia or DMOG predominantly induced the transition from collective to amoeboid single-cell migration, with blebby amoeboid migration as the predominant migration mode (4- and 3-fold increase for BC and HNC, respectively) next to filopodial amoeboid migration.

Hypoxia-induced amoeboid migration is characterized by variably fast and persistent migration, reaching up to several 100 μm/day, mediated by actin-rich filopodial or blebby protrusions at the leading edge. Criteria for amoeboid movement included (i) actin-containing blebs that interacted with collagen structures in a polarized fashion, (ii) weak dependence on matrix-metalloproteinase-mediated collagen remodeling, (iii) low dependence on focalized integrin-mediated matrix adhesions, and (iv) high dependence on RhoA-mediated actomyosin contraction for efficient bleb formation and migration speed. Interference with ROCK signaling using Y-27632 and myosin using blebbistatin significantly inhibited hypoxia-induced amoeboid dissemination and bleb formation. Thus, hypoxia induces plasticity of cancer invasion modes, with efficient blebby amoeboid dissemination as primary outcome. While blebby amoeboid motion is known as a rudimentary migration mode in embryonic stem cells, here we observe blebby amoeboid migration as a simple (low MMP- and integrin dependent) but efficient salvage migration mode of cancer cells under severe hypoxic stress.

#5063

The receptor for urokinase-plasminogen activator controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style.

Anastasia Chillà, Francesca Margheri, Anna Laurenzana, Cristina Luciani, Alessio Biagioni, Gabriella Fibbi, Mario Del Rosso. _University of Florence, Firenze, Italy_.

Introduction. The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility.

Materials and methods. To evaluate the mesenchymal ameboid transition we performed RhoA and Rac1 activation assay, immunofluorescence analysis of protein involved in cytoskeleton organization, and collagen degradation assay. For a clear visualization of collagen fiber breakdown in the process of proteolytic migration we used reconstruction by time-lapse video microscopy. Cell invasion was studied in Boyden chambers using filters coated with Matrigel. uPAR gene expression was inhibited using a 18-mer phosphorothioate aODN, and as a negative control we used a degenerated oligodeoxynucleotide, which is a mixture of all possible combinations of bases that compose the aODN.

Results and discussion. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the "path generating" mesenchymal to the "path finding" amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one.

Conclusion. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly.

#5064

CORO1B amplification and expression status identifies an aggressive subset of chromosome 11q13 amplified HNSCC.

Jessica L. Allen,1 Elyse L. Walk,1 Kristen L. Rhodes,1 Colleen J. Beatty,1 Steven M. Markwell,1 Brenen W. Papenberg,1 Erik T. Interval,1 Hong Wu,2 Marileila Varella Garcia,3 James E. Bear,4 Scott A. Weed1. 1 _West Virginia University Randolph Cancer Center, Morgantown, WV;_ 2 _Fox Chase Cancer Center, Philadelphia, PA;_ 3 _University of Colorado Cancer Center, Aurora, CO;_ 4 _University of North Carolina Lineberger Comprehensive Cancer Center, Chapel Hill, NC_.

Head and neck squamous cell carcinoma (HNSCC) is highly invasive cancer type that occurs with greater incidence in West Virginia and the rest of Appalachia. The chromosome 11q13 region is amplified in approximately 30% of late stage HNSCC cases and is associated with a poor patient prognosis. The core 11q13 region encodes the well-studied tumor genes CCND1 (cyclin D1) and CTTN (cortactin). The CORO1B (coronin 1B) gene flanks the 11q13 core and is amplified in 9-14% of all HNSCC, but how CORO1B amplification contributes to HNSCC is unknown. Coronin 1B governs cell motility by negatively regulating F-actin stability through displacement of cortactin at actin related protein 2/3 (Arp 2/3) branch points, generating dynamic turnover of Arp2/3-F-actin networks required for productive cell movement. Kaplan-Meier analysis of two independent patient cohorts indicates that HNSCC cases with CORO1B amplification have significantly reduced overall survival. Cox hazard ratios also indicate the CORO1B amplification is strongly associated with increased mortality in both cohorts. Automated quantitative analysis (AQUA) of coronin 1B expression in HNSCC tissue microarrays indicates that coronin 1B overexpression decreases median survival of HNSCC patients from 81 to 29 months. Reduced expression of coronin 1B by RNAi-mediated knockdown in established HNSCC cell lines decreases several actin-mediated invasive processes, including invadopodia formation and extracelluar matrix degradation. Collectively these results suggest that elevated coronin 1B expression levels in HNSCC function to enhance tumor invasion, potentially through accelerated downregulation of cortactin stabilizing activity at Arp2/3-F-actin junctions. Importantly, CORO1B amplification and coronin 1B expression status may serve a role as a precision biomarker for identification of a subset of late-stage HNSCC patients that would benefit from more aggressive forms of initial clinical treatment.

#5065

Role of EpCAM in EGFR signaling and tumorigenesis.

Lincoln Muhoro, Timothy Fleming, Narendra Sankpal, William Gillanders. _Washington University in St. Louis, St. Louis, MO_.

EpCAM is a transmembrane glycoprotein that functions as a homophilic cell adhesion molecule. EpCAM is expressed in normal epithelium and at even higher levels in epithelial cancers, including breast, pancreatic and lung cancers. Since its discovery in the 1980s, EpCAM has been targeted in several different epithelial cancers. Even though numerous novel therapeutic strategies targeting EpCAM have been investigated, the biological role and functional significance of EpCAM expression in cancer remains unclear. EpCAM expression appears to be associated with prognosis in several epithelial cancers -in some cancers it is associated with a favorable prognosis (renal cell, rectal) and in others it is associated with a poor prognosis (lung, pancreatic, breast). Thus, the function of EpCAM appears to be context-dependent in epithelial cancers. To understand the impact of EpCAM in human cancer, we specifically ablated EpCAM expression in over 40 cancer cell lines, and assessed the impact on invasion. We identified the A431 epidermoid cancer cell line as a cell line that overexpresses EpCAM, but is exquisitely sensitive to manipulation of EpCAM expression. A431 is known to overexpress EGFR and serves as a model cell line for EGFR signaling. Specific ablation of EpCAM significantly enhanced the migration and invasion of the A431 cell line. Specific ablation of EpCAM was also associated with increased EGFR activity, as demonstrated by phospho-immunoblot. Immunoprecipitation studies demonstrate that EpCAM binds directly to EGFR. Using specific EpCAM mutant constructs, we showed that the N-terminal domain of EpCAM is required for binding and suppression of EGFR activity. Using a soft agar colony growth assay, we were able to confirm that N-terminal EpCAM domain mutants (NTD288) inhibited colony growth in 3T3-transfected cells relative to the wildtype. In addition, a transformation assay showed that the same N-terminal mutant was able to inhibit transformation of 3T3 cells. To assess the effects of EpCAM ablation on in vivo tumor growth, we specifically ablated EpCAM and then performed tumor challenge experiments using immunocompromised mice. Specific ablation of EpCAM increased tumor growth relative to control. These data suggest that EpCAM may modulate EGFR signaling. Further studies are ongoing to define the mechanisms by which EpCAM inhibits EGFR activity.

#5066

Impact of Brk-inhibitors on nondirectional and directed migration of breast cancer cells.

Markus Oelze,1 Franziska I. Kluwe,1 Tom Wersig,2 Wolfgang Sippl,2 Andreas Hilgeroth,2 Christoph A. Ritter1. 1 _Ernst-Moritz-Arndt-Universität Greifswald, Greifswald, Germany;_ 2 _Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany_.

The non-receptor tyrosine kinase Brk (breast tumor kinase, PTK-6) has been linked to various effects in tumor cells including cell proliferation, differentiation and migration. Modulation of cellular signaling occurs due to protein scaffolding and substrate phosphorylation, while effects depend on cell type and molecular context. Recent findings suggest, that some hallmark effects of cancer may be linked to the active kinase whereas inactive Brk seems to be involved in different physiological processes of epithelial cells. Novel α-carboline derivatives were described previously and identified as Brk inhibitors in in-vitro testing. We examined the effect of four candidates MK5, MK136, MK138 and MK150 in breast cancer cell lines regarding Brk as a modulator of cell migration.

Cell lines MDA-MB-231 and MDA-MB-468 were cultivated as indicated by the distributor. Inhibition of non-directional migration over a given period of time was determined by scratch assays barring proliferation by addition of 50 mM hydroxyurea. The gap was created using silicon inserts and TScratch software was used to evaluate cell free surface area. Directional migration was investigated by trans-well assay utilizing FCS as attractant and an 8 µm pore membrane in combination with xCellingence real time measurement. Statistical evaluation was conducted using 1-way ANOVA followed by Dunnett's test.

In scratch assays samples incubated with 1 µM of each substance showed a larger cell free area after 24 hours for MDA-MB-231 cells and after 30 hours for MDA-MB-46 cells. The increase was significant in MDA-MB-231 cells for substance MK136 (32.07±17.21 %, p<0.1) and highly significant for MK138 (52.37±7.00 %, p<0.001) compared to DMSO treated control (7.73±3.22 %). Furthermore, the effect of MK138 increased in a concentration-dependent manner yielding consistently highly significant differences at a concentration of 1 µM (36.77±14.53 %, N=8 vs. 9.870±1.12 %, N=4). MK138 displayed a significant difference in MDA-MB-468 cells as well (37.27±12.56 % vs. 10.07±3.04 %, p<0.01). In transwell experiments MDA-MB-231 cells showed significantly impaired migration when incubated with MK138 and MK150. For MK138 a concentration-dependent effect was found similar to the scratch assay. In cell line MDA-MB-468 the inhibitor MK138 accounted for a significant decrease in directional migration as well.

Our findings indicate an influence of pharmacological Brk-inhibition on the migratory ability of breast cancer cells. These effects were evident for all tested substances and constantly significant and concentration-dependent for MK138 making unspecific effects unlikely. The mechanism of Brk activity in modulation of migratory processes needs to be further addressed to better understand and exploit its influence on tumor cell migration, dissemination and metastasis.

This work was supported by the German Research Foundation (DFG, Grant No. RI1196/4-1).

#5067

Casein kinase 2 alpha phosphorylation of cortactin governs actin cytoskeletal regulation of invadopodia function.

Steven M. Markwell,1 Amanda Gatesman Ammer,1 Erik T. Interval,1 Dorothy A. Schafer,2 River A. Hames,1 Scott A. Weed1. 1 _West Virginia University Randolph Cancer Center, Morgantown, WV;_ 2 _University of Virginia, Charlottesville, VA_.

Malregulation of signal transduction events controlling cortical actin cytoskeleton dynamics is responsible for to enhancing tumor cell motility and invasion. The actin‐binding protein cortactin facilitates branched actin network formation through activation of the actin related protein (Arp) 2/3 complex. Increased cortactin expression due to gene amplification is observed in many cancers, corresponding with increased tumor progression and poor patient outcome. In many cancer types, elevated Arp2/3 complex activation is responsible for driving increased migration and extracellular matrix (ECM) degradation by governing invadopodia formation and activity. While cortactin‐mediated activation of Arp2/3 complex has been well established, the objective of this study is to identify upstream signaling pathways responsible for modulating the interaction between cortactin and Arp2/3 complex in invasive carcinoma cells. We have determined that casein kinase (CK) 2α is responsible for disrupting the ability of cortactin to bind and/or activate Arp2/3 complex. CK2α directly phosphorylates cortactin at a conserved threonine 24 (T24) adjacent to the canonical Arp2/3 binding motif in the cortactin amino‐terminal acidic (NTA) domain. CK2α phosphorylation of cortactin T24 impairs the ability of cortactin to activate Arp2/3 actin nucleation as determined by spectrofluorometric analysis. Decreased invadopodia formation and ECM degradation activity is observed in head and neck squamous cell carcinoma (HNSCC) cells with shRNA‐mediated CK2α knockdown, expression of a CK2α phosphorylation‐null cortactin mutant (T24A), and with treatment of the Phase I CK2α inhibitor CX‐4945 (Silmitasertib). Collectively, these data suggest that CK2α‐mediated cortactin phosphorylation at T24 is a critical event in regulating the interaction with Arp2/3 complex, identifying a key anti-invasive mechanism impacted by targeted anti-CK2α therapeutics.

#5068

Carcinoma-associated fibroblast-derived CXCL12 enhances MDA-MB-231 tumor cell migration though mDia2 formin suppression.

Kaitlyn Dvorak, Kathryn M. Eisenmann. _University of Toledo Health Science Campus, Toledo, OH_.

The tumor microenvironment (TME) is a heterogeneous region comprised of tumor cells, stromal cells, and secreted factors that make the environment favorable for cancer formation and progression. An important step in the shift to a pro-cancerous environment is the transformation of normal stromal fibroblasts to carcinoma-associated fibroblasts (CAFs). CAFs are a critical cell type to understand. They are present in a majority of solid tumors and can have a direct effect to enhance adjacent tumor cell motility via their innate ability to secret cytokines, chemokine and growth factors into the TME. We sought to understand how CAF-derived chemokines impact breast tumor cell motility through modification of the F-actin cytoskeleton. We collected cultured media (CM) from WS19T fibroblasts, a patient-derived breast tumor CAF cell line. CAF-CM dramatically enhanced wound-closure in MDA-MB-231 monolayers, relative to normal fibroblast cultured media. The chemokine CXCL12 (SDF1 alpha) has been identified as a potential secreted CAF-directed signal driving tumor cell migration. To determine if CXCL12 was the soluble factor in CAF-CM promoting tumor cell migration, MDA-MB-231 cells were preincubated with AMD3100, an inhibitor of the CXCL12 receptor, CXCR4 prior to CAF-CM application and wounding. AMD3100 effectively blocked CAF-CM-induced MDA-MB-231 migration. CXCL12 incubation with MDA-MB-231 cells equally promoted MDA-MB-231 wound closure, supporting the notion that CXCL12 is secreted by CAFs to promote MDA-MB-231 motility. We previously showed a link between CXCL12 directed CXCR4 signaling and a critical regulator of the dynamic F-actin cytoskeleton, mammalian Diaphanous-related formin (mDia2). This formin regulates the assembly and bundling of unbranched actin filaments and plays a role in tumor cell migration and invasion programs. Western blotting CAF-CM-treated MDA-MB-231 cells revealed a near complete loss of mDia2 protein. mDia1 and ROCK expression were unaffected with CAF-CM treatment. These data were consistent with treatment with a functional mDia FH2-domain inhibitor SMIFH2, which likewise suppresses mDia2 protein levels in MDA-MB-231 cells. It remains unclear if CAF-derived CXCL12 first suppresses mDia2 FH2 activity, leading to loss of mDia2 protein, thus promoting tumor cell motility. Current experiments are designed to address this gap in the knowledge, potentially indicating a role for CAF-derived soluble factors in modulating mDia2 protein function and expression in migrating tumor cells.

#5069

p66Shc regulates the migration of castration-resistant prostate cancer.

Matthew A. Ingersoll,1 Sakthivel Muniyan,1 Fen-Fen Lin,1 Ta-Chun Yuan,2 Yu-Wei Chou,3 Surinder K. Batra,1 Ming-Fong Lin1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _National Dong Hwa University, Shou-Feng, Taiwan;_ 3 _Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan_.

Purpose and Background: Prostate cancer (PCa) remains the most commonly diagnosed solid tumor and is the second leading cause of cancer-related death in United States men. While androgen deprivation therapy is the current standard-of-care treatment for metastatic PCa, most patients eventually relapse and develop castration-resistant (CR) tumors for which there is currently no effective treatment. p66Shc, a 66 kDa Src and collagen homologue oxidase, is elevated in clinical PCa as well as multiple PCa cell lines which correspond with advanced CR PCa. Additionally, p66Shc has been demonstrated to promote proliferation in PCa cell lines via generation of reactive oxygen species (ROS). This study is the first to demonstrate p66Shc also regulates PCa cell migration. Understanding of the mechanism through which p66Shc promotes migration may lead to the development of therapeutic strategies to treat metastatic CR PCa.

Experimental Methods: Human prostate adenocarcinoma cell lines LNCaP C-33 and C-81, androgen-sensitive (AS) and androgen independent (AI) MDA PCa2b, PC-3, and DU145 were used to determine the role of p66Shc in PCa migration. Stable p66Shc cDNA transfected subclones S-31, S-32, and S-36 were established from LNCaP C-33 cells. p66Shc's impact on migration was determined using transwell migration assays. Immunoblotting was performed to profile potential proteins involved in the mechanism through which p66Shc influences migration. Small-molecule inhibitors were used to confirm these proteins' involvement in migration.

Results: Levels of p66Shc protein positively correlate with metastatic potential across multiple PCa cell lines as well as LNCaP and MDA call progressive models. p66Shc is an authentic oxidase and promotes ROS production. In this study, we further demonstrate peroxide treatment is able to induce C-33 cell migration in a dose-dependent manner, while antioxidants inhibit migration. Similarly, p66Shc subclones possess increased migratory ability which is abolished upon antioxidant treatment and demonstrates the involvement of ROS in p66Shc-mediated migration. Molecular profiling reveals the correlation of p66Shc protein level with the activation of ErbB-2, PYK2, AKT, ERK, p38, RAC1, STAT3, and STAT5 as well as suppression of cPAcP.

Conclusions: Altogether, our results indicate p66Shc is involved in PCa cell migration, in part, by inducing ROS generation. Understanding the role p66Shc in advanced PCa progression will help to determine its potential as a therapeutic target, and elucidating its mechanism of intracellular signaling will enable us to design new treatments for metastatic CR PCa.

#5070

CD44 alternative splice variants are associated with prostate cancer cell identity and migration.

David J. Bond, John D. Lewis. _University of Alberta, Edmonton, Alberta, Canada_.

Patient tumors can demonstrate tremendous cell-to-cell genetic heterogeneity while cultured cancer cell lines are often more homogeneous. This fundamental difference can undermine in vitro findings and their application to the more complex in vivo tumor environment. However, it should not be assumed that cultured cells are homogeneous even in well established and widely used cell lines. In my study of CD44 alternative splicing in the prostate cancer cell line PC3, I encountered varying CD44 alternative splicing expression profiles dependent on the origin of the PC3 cell line. CD44 is a cell surface glycoprotein that binds to components of the extracellular matrix, including hyaluronan, and is primarily involved in cell-cell and cell-matrix interactions. CD44 contains 10 variable exons that when combined in particular combinations significantly alter/guide CD44 downstream activities. In the context of cancer biology, alternative splicing of CD44 can control the epithelial to mesenchymal transition (EMT) of cancer cells and is associated with metastasis in many types cancers such as breast and prostate. To determine if cellular heterogeneity was the source of variable CD44 alternative splicing profiles, I isolated PC3 single cell clones. From these clones I identified two cell populations based on colony morphology: a compact colony subset with cells of a epithelial phenotype (PC3-EL), and a diffuse colony subset with cells of a mesenchymal phenotype (PC3-ML). I then performed reverse transcription-PCR (rt-PCR) on cDNA generated from these two populations with primers that specifically amplify the CD44 variable region to generate a fingerprint of CD44 alternative splicing. I found that PC3 cells with an epithelial phenotype express multiple CD44 variant exons (CD44v), including the epithelium-associated CD44v1,v8-10 variant (CD44E). However, phenotypically mesenchymal PC3 cells show a complete loss of CD44 splice variants and primarily express standard CD44v1 (CD44S). These differences in CD44 mRNA alternative spicing are manifested at the protein level as phenotypically epithelial PC3 cells express predominantly high molecular weight CD44 variants. I then monitored the association of CD44v expression on cell migration and determined that PC3 cells that express CD44E are non-motile, while PC3 cells that predominantly express CD44S are highly motile. Acquisition of CD44S over CD44E can signal EMT, an early and critical step in cancer metastasis. These data indicate that the CD44 alternative splice fingerprint may provide a predictive biomarker for EMT and the acquisition of other early pro-metastatic features in prostate cancer cells.

#5071

**Expression and cellular distribution of Profilin1/VASP** p157 **and cofilin1/VASP** p239 **is altered by docosahexaenoic acid and suppresses cancer cell migration and survival.**

Mehboob Ali, Kathryn M. Heyob, Lynette K. Rogers. _Nationwide Children's Hospital, Columbus, OH_.

The major risk factor for cancer associated death is uncontrolled metastasis and longer cancer cell survival. Mechanisms associated with actin binding protein-mediated actin dynamics and subcellular distributions are not well defined. Secondary messengers (cAMP and cGMP)differentially phosphorylate actin binding proteins like VASP and are associated with actin remodeling in cancer cells. We tested the hypothesis that differential phosphorylation of VASP precipitates association with two additional actin binding proteins, profilin1 and cofilin1. Furthermore, we speculate that the associations of VASP with profilin1 or cofilin1 are causing changes in subcellular location of the protein complexes. Briefly, non-invasive lung epithelial (MLE12) and cancer (A549) cells were treated with 8-Br-cAMP and/or DHA for 6h and 24h. Actin binding proteins (profilin1, cofilin1, VASP, VASPp157and VASPp239) expressions were analyzed by western blot in cell lysate. Wound assay and transwell apparatus were used to study cell migration. Confocal analysis was performed to analyze F-actin content and actin binding proteins at the wound leading edges and in human lung cancer biopsy samples. Flow cytometry, using ki-67, and cleaved-caspase-3 antibodies were performed to study proliferation and cell viability. Our data indicate an association between profilin1/VASPp157 and cofilin1/VASPp239 and changes in the relative cytoplasmic/nuclear distribution of these protein complexes that coincide with the invasive potential of the cell line or tissue. Additional studies showed that docosahexaenoic acid, inhibited cancer cell migration and viability and altered the expression and cellular distribution of profilin1/VASPp157 and cofilin1/VASPp239. In summary, profilin1/VASPp157 and cofilin1/VASPp239 protein complex expression and subcellular distribution could serve as novel, new areas of research, as biomarkers of disease progression, and/or as innovative therapeutic targets against metastasis.

#5072

Paracrine interactions between Schwann cells and cancer cells promotes perineural invasion via L1cam secretion.

Shorook Na'ara, Ziv Gil, Moran Amit. _Rambam Medical Center, Rappaport School of Medicine, the Technion Israel Insitute of Technology, Ha, HAIFA, Israel_.

Cancerous neural invasion (CNI) is a well-known route of cancer spread in head and neck cancer. Patients with CNI suffer from severe morbidity due to neuropathic pain and most succumb to cancer within months. The exact mechanism that drives cancer cells to disseminate along nerves remained to be defined. The overall objective of our research was to identify candidate proteins and host cells that contribute to CNI. L1 cell adhesion molecule (L1cam) is a 200kDa transmembrane glycoprotein of the immunoglobulin superfamily. In many human cancers, L1cam is constitutively over-expressed, and its expression is generally associated with poor prognosis and metastases formation. Immunohistochemical analysis of the neural niche in human cancer specimens showed overexpression of L1cam in both cancer cells and Schwann cells. To address the role of L1cam in vitro, we used the Dorsal Root Ganglia. Treatment of Cancer Cells with Anti L1cam mAb resulted in a significant reduction in CNI of cancer cells. Next, we used Schwann cells conditioned media (SCCM) to study the role of soluble Schwann cells derived L1cam. Immunopercipitation of L1cam in SCCM resulted in a significant reduction in L1cam concentration and led to reduced cancer cell migration, invasion, and motility. To study the role of SCCM as a chemo attractant to cancer cells we utilized a modified in-vitro nerve invasion assay that consists of 3 layers Fibroblasts-ECM-Schwann. In this model blocking the activity of L1cam in Schwann cells inhibited the cancer cells invasion through these layers. Finally, in-vivo studies in transgenic mice treated with L1cam mAb triweekly, showed a significant reduction in CNI in the tumors compared to the control group. Taken together, our results identify a paracrine interaction between Schwann cells in the neural niche and the cancer cells promotes perineural invasion via L1cam secretion.

#5073

C5a receptor promotes invasive ability of gastric cancer.

Takayoshi Kaida, Hidetoshi Nitta, Yuki Kitano, Kensuke Yamamura, Risa Inoue, Kota Arima, Takaaki Higashi, Katunobu Taki, Hiromitsu Hayashi, Katsunori Imai, Daisuke Hashimoto, Akira Chikamoto, Toru Beppu, Hide Baba. _Graduate school of life Sciences Kumamoto University, Kumamoto, Japan_.

Background: C5a is one of the important factors produced via the complement pathway and a strong chemokine. The C5a receptor (C5aR) is expressed in various cancer cells and is linked to C5a-induced promotion of cancer cell invasion. However, the role of C5aR in gastric cancer (GC) is still mostly unknown.

Aim: To investigate the clinical role of C5aR-expression in human GC.

Method: 1) We investigated the C5aR expression for 237 GC patients who underwent gastrectomy from Jan. 2004 to Dec. 2011 by immunostaining using monoclonal human C5aR-antibody and analyzed the association between C5aR expression and clinicopathological factors. 2) We investigated the association between C5aR expression and invasiveness of GC cells, in vitro.

Result: 1) From immunostaining analysis, High-expression of C5aR was observed in 86 patients (36.8%) and was significantly related to depth of invasion, stage and vascular invasion. The 5-years RFS and OS rates of the patients with high-expression of C5aR were significantly worse than those of the patients with low-expression. 2) We found that several GC cell lines had high-expression of C5aR with western blotting. By using AGS and NUGC3 cell, the cell line which had low-expression of C5aR, We produced AGS (AGS/C5aR) and NUGC3 (NUGC3/C5aR) which had the overexpression of C5aR. Matrigel chamber assay revealed that C5a recombinant enhanced the cell invasion of AGS/C5aR and NUGC3/C5aR cell. Migration assay by using the timelapse imaging revealed that C5a recombinant enhanced the cell motility of AGS/C5aR and NUGC/C5aR cell. In addition, C5aR-antagont decreased the cell invasion and motility of those cell lines. The immunofluorescence microscopy revealed that C5a recombinant enhanced the cytoskeletal rearrangement, such as stress fiber, fillipdia and lamellipodia, in NUGC/C5aR cell. Then, C5a recombinant enhanced RhoA activity of NUGC3/C5aR.

Conclusions: The stimulation of C5a receptor may enhance the cell motility via RhoA activity, then it may enhance the cell invasion ability. In addition, High-expression of C5aR was associated with poor prognosis for GC patients. Therefore, C5a receptor might be a target of medical treatment.

#5074

The impact of RHOA mutation related with invasion in diffuse-type gastric cancer.

Tsugio Eto, Takatsugu Ishimoto, Daisuke Izumi, Yuka Tamaoki, Daisuke Kuroda, Kota Arima, Takayoshi kaida, Mayuko Ohuchi, Kenichi Nakamura, Ryuma Tokunaga, Hironobu Shigaki, Junji Kurashige, Masaaki Iwatsuki, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. _Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan_.

Background: Gastric cancer (GC) is one of the most general causes of cancer related death, especially in East Asia and is divided into intestinal type and diffuse type by Lauren classification. Five-year survival rate of diffuse-type gastric cancer (DGC) is poor compared with the Intestinal-type gastric cancer (IGC). To improve the prognosis of advanced GC patients, molecularly targeted drugs against gastric cancer have been developed including trastuzumab, a recombinant monoclonal antibody against HER2. But, HER2 gene amplification rate is about 10% in DGC, though 30% in IGC. DGC-specific molecular carcinogenic mechanisms have still remained unclear. Recently, some studies revealed RHOA mutations occur specifically in DGCs. They also demonstrated that proliferation and organoid formation are related with RHOA mutations in DGC. However, there is no report in terms of the functional difference among RHOA mutational hotspots in DGCs.

Aim: To investigate the functional difference among RHOA mutations and the impact on prognosis in DGCs.

Method: We prepared RHOA wild type vector and three RHOA mutation vectors (G17E, Y42C, and L57V). We introduced these RHOA vectors into DGC cell lines and examined invasion assay. In invasion assay of RHOA introduced DGC cell lines, we conducted experiments with or without TGFβ1 (RHOA activator).

Result: RHOA overexpression in DGC cells did not exhibit significant upregulation of invasiveness compared with empty vector introduced DGC cells. However, all RHOA mutation introduced DGC cells acquired more significant elevation of invasiveness as well as wild type after treatment with TGF-β1. (wild:p=0.0001, G17E:p=0.0001, Y42C:p=0.0001, L57V: p<0.0001). Among these RHOA mutations, G17E and Y42C RHOA mutation introduced DGC cells exhibited remarkable upregulation of invasiveness compared with wild type RHOA introduced DGCs (G17E:p<0.0001 Y42C:p=0.009).These results indicate particular RHOA mutations cause higher response to TGF-β1 compared with wild type.

Conclusion: We evaluated the functional difference among RHOA mutations in DGC. These results suggest particular RHOA mutation might have possibility to cause invasion in DGCs. A large cohort study across RHOA mutations in DGCs will be required to identify the clinical significance between hotspot RHOA mutations.

#5075

Hedgehog Pathway Transcription Factor Gli1 Promotes Glioma Invasiveness through Up-regulating Aquaporin 1.

Zhenzhen Yao,1 Chaoxu Yang,2 Lie Ma,1 Yexiong Tan3. 1 _Department of Biochemistry and Molecular Biology, Shanghai, China;_ 2 _Department of Basic Medicine, Shanghai, China;_ 3 _Department of Signal Transduction, Shanghai, China_.

Gliomas, especially Glioblastoma multiformes (GBMs) express increased aquaporin1(AQP1) compared to normal brain. AQPs may contribute to brain edema, cell motility, and tumor angiogenesis. Although the physiology of AQP1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP1 gene expression and the relationship between the regulation of the AQP1 expression and the major intracellular signaling pathways is also poorly concerned. Hedgehog signaling pathway activation is required for sustained glioma growth and survival. playing an important role in the genesis of glioma and In the present study, we report that Gli1, the transcriptional activator of the Hedgehog pathway, is co-expressed with AQP1 in human glioma tissues and can enhance AQP1 gene transcription by binding to conserved core Gli-binding site in the 5'-flanking promoter region of the AQP1. Transfection of 293T and U251 (human glioblastoma cell line) with Gli1 can markedly increase the AQP1 promoter reporter activity and the AQP1 expression level. Moreover, suppressing the Gli1 expression with the specific RNAi can abort this effect. The ChIP and reporter mutation analysis showed that the -406~-398 region of the AQP1 promoter is essential for regulation activity of Gli1. To elucidate the possible pathophysiological importance of this molecular interaction, we investigated the migration and invasion ability of cell lines expressing different levels of Gli1 and AQP1. The results demonstrated that restoring the AQP1 expression in the Gli1-knockdown cell line can abort the decrease in the migration ability, which suggested that Gli1 may contribute to the invasion ability of glioma cells through AQP1. The up-regulation of Gli1 and its consequences of increased AQP1 in gliomas may provide a new therapeutic target, either as a cell surface marker or as a functional intervention.

#5076

Ras association (RalGDS/AF-6) domain family member integrates with notch signaling to regulate tumor cell migration.

Lakshmi R. Perumalsamy, Mahalingam S. _Indian Institute of Technology Madras, Chennai, India_.

Tumor metastasis, the spread of tumor cells to distant tissues from its site of initiation is a multi-step and multi-factorial process. Hyperactivation of RAS pathway is reported in >50% of different cancers and is associated with metastasis and poor prognosis. Since Ras signaling pathway is essential for normal development, its been difficult to develop drugs against it in tumors. This led to the identification of several Ras effectors. Ras association (RalGDS/AF-6) domain family (RASSF) proteins are non-enzymatic effectors of Ras. RASSF7 is a member of the less characterized N-terminal RASSFs. Similar to other RASSFs, RASSF7 also associates with Ras. Several mutations within RASSF7 open reading frame as well as differential expression pattern were observed in various tumors. The role of RASSF7 and its downstream signaling is not yet clearly understood.

Microarray analysis for RASSF7 identified several differentially regulated genes with enrichment for pathways including cell adhesion, inflammation, actin cytoskeleton and cell-cell communication suggesting that RASSF7 might regulate cell migration. Ectopic expression of RASSF7 inhibited cell migration of multiple cancer cell lines in vitro. RT-qPCR and western blotting analysis suggest that RASSF7 can transcriptionally upregulate Notch1 expression. Knockdown of Notch1 abrogated RASSF7 mediated inhibition of cell migration. Our experiments suggest that RASSF7 alters the localization of Notch1. Cross-talk Notch signaling is a key signaling pathway regulating tumor progression across several different cancers. Collectively, our data suggests that RASSF7 impinges on Notch1 to regulate cell migration.

#5077

The insulin-like growth factor-2 (IGF-2): A potential therapeutic target for triple-negative breast cancer metastasis.

Nalo Hamilton, Diana Marquez-Garban, Richard Pietras. _University of California Los Angeles, Los Angeles, CA_.

Introduction: Triple-negative breast cancers (TNBC) occur in 15-17% of patients, yet this subtype accounts for almost half of all breast cancer (BC) deaths. This breast cancer sub-type often occur in younger and African American (AA) women. TNBCs cannot be treated with current chemotherapies. Although initially responsive to chemotherapy, TNBCs tend to relapse and metastasize early. As there are no specific treatments for TNBCs, we aim to identify the therapeutic potential of insulin-like growth factor- 2 (IGF-2) in TNBC management.

Methods: Using immunohistochemistry archival breast tissue from women with metastatic TNBC was evaluated and scored for IGF-2 expression. To assess the ability of IGF-2 to stimulate cell proliferation and migration we used commercially available TNBC cell lines validated to be ER-alpha-negative (ER-), progesterone receptor-negative (PR-) and Her2 overexperssion-negative (HER2).

Results: We have found IGF-2 to be highly expressed archival breast tissue of women with metastatic TNBC. Using TNBC cell lines MDA-MB-231, HCC 38, HCC 1806 and HCC 1937, our in vitro studies demonstrate the ability of IGF-2 to stimulate TNBC proliferation and migration (P = 0.05). Further, the combination of NVP-AEW541, an insulin-like growth factor-1 receptor (IGF-1R) inhibitor, and BMS-754807, an inhibitor of IGF-1R and the insulin receptor (IR), cause a significant (P < 0.05) reduction in TNBC proliferation and migration. Investigation into the cellular characteristics of the TNBC cell lines used, suggest that BRCA1 mutations increase cell susceptibility to IGF-1R inhibitors.

Conclusion: Our data indicate that IGF-2 plays a role in TNBC metastasis, an action that can be inhibited by targeting multiple IGF-2 mediated receptors.

#5078

**Tumor treating fields (TTFields) reduce migration and invasion properties of human glioma cancer cells** in vitro **.**

Rosa S. Schneiderman,1 Anna Shteingauz,1 Moshe Giladi,1 Tali Voloshin,1 Yaara Porat,1 Mijal Munster,1 Roni Blat,1 Eilon D. Kirson,1 Uri Weinberg,2 Yoram Palti1. 1 _Novocure, Haifa, Israel;_ 2 _Novocure, Luzern, Switzerland_.

The ability of glioblastoma cells to invade adjacent brain tissue remains one of the major obstacles in obtaining therapeutic success. The development of novel treatment modalities that hinder glioma cancer cell motility could therefore facilitate disease control. TTFields are an effective treatment modality delivered via continuous, noninvasive application of low intensity, intermediate frequency, alternating electric fields. This therapy is approved for the treatment of patients with glioblastoma. Previous investigations have shown that TTFields disrupt microtubules and septin filaments, both of which govern key roles in mitosis. The goal of this study was to evaluate the possible effect of TTFields on human glioma cell migration and invasion properties. Four human glioma cell lines: U-87 MG, A-172, LN-229, and LN-18 (ATCC, USA) were treated with TTFields (1.75 V/cm RMS, 200 kHz) for 24 hours using the inovitro system. Cell migration was measured using in vitro wound healing assays. These were analyzed with Image Pro Premier (Media Cybernetics, USA) to determine migration rates. Invasion assays were performed using modified Matrigel coated Boyden chamber. Cells were stained, photographed and counted using image J (NIH, USA).

Application of TTFields in-vitro led to a significant reduction in both migratory and invasive phenotype in all tested cell lines. Specifically, cell migration velocity, as assessed by the wound healing assay, was significantly reduced in U-87 MG (60%, P<0.001), and in A-172 (33%, P<0.001) compared with untreated control cells. The number of invading cells, as assessed by the modified Boyden chamber assay, was reduced in U-87 MG (54%, P<0.01), A-172 (51%, P<0.001), LN-229 (52%, P<0.001) and in LN-18 (30%, P<0.001) compared with untreated control cells.

Our results suggest that human glioma cell motility is impaired by exposure to TTFields. Further studies are needed to elucidate the mechanism by which TTFields disrupts cellular motility in glioma cancer cells.

#5079

Sunitinib suppresses ovarian cancer metastasis through the negative modulation of TGF-β-mediated epithelial-mesenchymal transition.

Zibo Chen, Lin-lin Chang, Tian-yi Zhou, Dan-dan Wang, Ying Chen, Hong Zhu, Meidan Ying, Bo Yang, Qiao-jun He. _Zhejiang Univ. College of Pharmaceutical Sci., Hangzhou, China_.

The mechanism of the negative regulation TGF-β mediated of epithelial-mesenchymal transition by sunitinib to inhibit ovarian cancer metastasis. We evaluated the inhibitory ability possessed by sunitinib against SKOV3 ovarian cancer cells motility by wound-healing and transwell assays. The results showed that sunitinib inhibited the migration of SKOV3 cells. The results obtained from wound-healing and transwell assays showed that sunitinib suppressed the motility of ovarian cancer SKOV3 cells in a concentration-dependent manner. In another,the effects of sunitinib on TGF-β-induced E-cadherin protein levels was assessed by western-blot, qRT-PCR and immunofluorescence assay. As a result, sunitinib attenuated the downregulation of E-cadherin protein level induced by TGF-β. Western-blot, qRT-PCR and immunofluorescence analyses displayed that the TGF-β stimulation reduced E-cadherin protein level, which could be attenuated by sunitinib. Our recent work reveals that the protein level of snail and the transcriptional activity of smad in sunitinib-treated cells were examined by western-blot and SBE-luciferase assay. Our results imply that sunitinib abolished TGF-β-induced upregulation of snail protein and decreased the transcriptional activity of smad complexes. The western-blot analyses revealed that sunitinib inhibited the upregulation of snail protein level caused by TGF-β treatment. The SBE reporter was constructed by linking the Smad-binding elements promoter upstream of luciferase reporter gene. A remarkable increment of transcriptional activity of Smads complexes was observed in SKOV3 cells exposed to TGF-β, which was significantly prohibited by sunitinib. The current study explores the prohibition of EMT, illustrates the anti-metastatic activities of sunitinib and the underlying mechanisms.

[Keywords] Sunitinib; EMT; TGF-β; E-cadherin; Snail; Smad signal pathway

#5080

Amylase overexpression promotes ovarian cancer invasion.

Mai Mohamed, Patricia Kruk. _University of South Florida, Tampa, FL_.

Introduction: Hyperamylasemia is an overexpression of amylase. Hyperamylasemia is associated with poor clinical outcome in ovarian cancer and reportedly serves as a prognostic biomarker for ovarian cancer. However, the function of hyperamylasemia in ovarian cancer is thus far unknown. Since amylase cleaves α1, 4-glycosidic bonds in carbohydrates, hyperamylasemia may play an active role in the etiology of ovarian cancer.

Purpose: The purpose of this study was to characterize amylase expression in ovarian cancer cell lines and establish an active role for hyperamylasemia in ovarian cancer.

Hypothesis: We hypothesized that secreted amylase enzymes cleave α1, 4-glycosidic bonds found in the carbohydrate shell which may result in a modified glycocalyx structure facilitating ovarian cancer cell invasion. Consequently, inhibiting amylase expression could abrogate ovarian cancer cell invasion/metastasis.

Methods: To characterize amylase isozyme expression, we assayed amylase expression in normal and cancerous ovarian epithelial cell lines by western immunoblot, qPCR, amylase assays and computational analyses. We measured the ability of amylase to promote cellular proliferation by MTS assays. We assayed invasive capacity of cultured cells using collagen coated Boyden chambers and abrogated amylase expression with either amylase siRNA or treatment with the blue-green algae, Spriulina.

Results: It was found that the amylase genes have identical or similar biochemical and bioinformatics properties: similar molecular weights, theoretical isoelectric points, disorder and hydropathy profiles, secondary structure and posttranslational modifications. In contrast to normal cell lines, ovarian cancer cell lines overexpress amylase at the messenger and protein levels and secrete high levels of metabolically active amylase. Amylase isozymes 1 and 2B were preferentially overexpressed in ovarian cancer cell lines. Inhibition of amylase abrogated ovarian cancer cell migration/invasion through collagen coated Boyden chambers which was associated with amylase-dependent alteration of extracellular carbohydrates. Further, the blue-green algae, Spirulina, was found to be a novel inhibitor of amylase.

Conclusion: Herein, we describe for the first time an active function of amylase to promote ovarian cancer cell invasion potentially mediated by amylase-dependent alteration of extracellular carbohydrates. Further, transcriptional inhibition of amylase by Spirulina demonstrates its clinical potential as an adjuvant nutraceutical therapy.

#5081

hnRNP A18: an emerging novel target for cancer therapy.

Palak R. Parekh, Elizabeth Chang, Qingyuan Yang, France Carrier. _University of Maryland, Baltimore, Baltimore, MD_.

Heterogenous ribonucleoprotein A18 (hnRNP A18) increases de novo protein translation during cellular stress. Although hnRNP A18 has recently been associated with several cancers, the exact role of it during the multifactorial process of cancer growth and progression is still unknown. Accordingly, we examined the dependability of various malignant cancer cell lines on hnRNP A18. Down-regulation of hnRNP A18 impaired the proliferative, invasive and migratory properties of melanoma and triple negative breast cancer cell lines in vitro, while overexpression of hnRNP 18 caused an increase in invasiveness. Consistent with these observations, we detected remarkable reduction of tumor growth in two mouse xenograft models, melanoma and breast cancer. Moreover, down-regulation of hnRNP A18 increased the sensitivity of melanoma cells and breast cancer cells to paclitaxel and 5-fluoro uracil, respectively. In conclusion, hnRNP A18 plays a key role in tumor growth and progression. It may serve as a novel target for the treatment of cancer.

#5082

A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype.

Mirko Corselli,1 Xiao Wang,1 Lissette Wilensky,1 Aaron Middlebrook,2 Smita Ghanekar,2 Jacob Rabenstein,1 Nil Emre1. 1 _BD Biosciences, San Diego, CA;_ 2 _BD Biosciences, San Jose, CA_.

Cell migration occurs during physiological and pathological processes, which include wound healing and tumor metastasis. In vitro migration assays are necessary to understand the mechanism underlying cell migration and also to identify inhibitory or stimulatory molecules. The Boyden chamber assay is commonly used to measure cell motility in vitro where the number of cells that migrate through the transwell membrane is quantitated manually using an inverted microscope. Alternatively, cells can be detached from the membrane and stained with a fluorescent probe, prior to counting cells using a plate reader. These conventional assays are limited by an inability to simultaneously characterize individual cells while monitoring migration. To address this, we have developed a multi-parameter flow cytometry-based migration assay for the quantitative measurement of cell migration and simultaneous immunophenotypic analysis of the migrated cells. Invasive HT-1080 and non-invasive MCF-7 cancer cell lines were plated in the upper layer of a cell permeable membrane, as per the standard Boyden chamber assay. BD Horizon™ Fixable Viability Stain (FVS) was used to determine the optimal detachment conditions providing maximum yield with minimal impact on cell viability. Migrated cells were also co-stained with multiple conjugated antibodies to assess cell surface marker expression. Cells were analyzed on BD Accuri™ flow cytometer for rapid quantitation of migrated cells and analysis of up to 4 parameters. Alternatively, BD FACSCelesta™ flow cytometer was used for higher parameter analysis. Migrated cells were identified based on light scatter properties and viability staining with FVS, while immunophenotype was assessed in live cells. Our results demonstrate that this flow cytometric assay can be used to rapidly quantify changes in the migratory ability of different cell lines in the presence of inhibitors or stimulators. Additionally, we were able to assess the expression of hallmark cancer cell markers (CD44, CD24, CD326 (EpCAM)) before and after migration. The ability to quantify the number of migrating cells in response to specific stimuli and to simultaneously define cell immunophenotype represents a significant advancement, as compared to conventional cell motility assays. Our results demonstrate that this novel approach allows for a deeper analysis of cell migration that could enable complex drug discovery studies and the discovery of new cell signatures associated with metastatic progression.

#5083

A comprehensive workflow for screening and real-time analysis of cell invasion.

Amedeo J. Cappione, Timothy Nadler. _EMD Millipore Corp., Danvers, MA_.

Cell migration and invasion are crucial steps in many normal, as well as, aberrant physiological processes, in particular, the highly lethal metastatic progression of cancer. Once the normally restrictive basement membrane boundaries of the tumor have been breached, invasive cells can gain access to the vasculature and lymphatic system permitting systemic spread of the disease. Invasion is a complex multistep process involving cell adhesion, directed migration, and proteolytic degradation of surrounding extracellular matrix (ECM) barriers. Multiple factors present in the local microenvironment, including soluble growth factors or chemokines and ECM-anchored integrins and proteases, act coordinately to regulate the invasive potential.

Understanding the cellular and molecular mechanisms underlying invasion is essential to the development of therapeutics that target tumor metastasis. Therefore, techniques for the quantification and dynamic visualization of penetrating cells have become central to oncology research. The most widely employed method for analyzing cell invasion in vitro is a modified Boyden chamber assay using Matrigel, a basement membrane matrix preparation. While amenable to semi-quantitative "screening" assays, these systems suffer from the inability to establish continuous gradients. As a result, this method, such gradients cannot be stably maintained under static culturing conditions, precluding longer-term real-time cell analysis. Moreover, these platforms are not strictly designed for microscope-based visualization and thus cannot be employed for dynamic mechanistic studies through imaging analysis at the inter- and intra-cellular levels.

Here we present a two-tiered platform for the analysis of cell invasion. Cell culture insert plates (24 and 96-well capacity), provide the ideal tool for initial compound screening for functional response (inhibit invasion) and toxicity. For selected compounds, mechanistic studies were then performed using a microfluidic-based system. The platform consists of a microfluidic culture plate and environmental control system; the latter regulates matrix loading, media perfusion, the establishment of stable continuous concentration gradients within the plate, as well as temperature and gas control. Most significantly, the plate can be paired with an inverted microscope enabling dynamic analysis of cell movement in real-time. Fluorescent visualization further permits selective discrimination of unique cell types in heterogeneous samples as well as changes in expression patterns of labeled proteins. In summary, the combined workflow platform presented here provides a framework for studying the effects of signaling molecules and growth factors on the migratory and invasive propensities of tumors cells.

#5084

Real-time, quantitative cellular analysis of migration and invasion.

Meagan Roddy, John Rauch, Lindy O'Clair, Daniel M. Appledorn. _Essen BioScience, Ann Arbor, MI_.

Chemotactic-driven cell migration is an important biological process impacting many areas of life science research including immunology, cancer and developmental biology. The dominant high-throughput solution for measuring cellular chemotaxis in vitro is the Boyden-chamber. However, traditional Boyden chamber products have inherent liabilities including: a) the inability to directly visualize the cell migration process, b) a requirement of tens of thousands of cells per well, c) the need to label cells for quantitation, d) a relative insensitivity to surface integrin signaling, and e) high random migration artifacts. Microfluidic solutions have sought to eliminate some of these deficiencies, specifically by providing the ability to visualize the cell migration process, but at the expense of relatively low throughput and cumbersome quantitation methods. Using a live-cell imaging approach and real-time image analysis quantitation, we have developed a new technology which eliminates most of the deficiencies of existing Boyden chambers and most importantly, provides direct visualization of both migration and invasion as it occurs. Integrated metrics precisely quantify the chemotactic response using significantly fewer cells (500-5000 cells per well) in a 96-well format. We exemplify this novel image based technique by studying the migration and invasion of both non-tumor and tumor-derived cell lines. Using Fetal bovine serum (FBS) as a chemoattractant, HT-1080 and NIH-3T3 cells were evaluated for their ability to directionally migrate across a coated surface or invade through a basement membrane extract by embedding the cells in an extracellular matrix prior to seeding. Qualitatively, we observed significant morphological differences between migrating and invading cells, the latter adopting a mesenchymal morphology reminiscent of cells that have undergone epithelial-mesenchymal transition (EMT) and exhibiting thinner, spindle-like morphology as they invade through the matrix. Using both cell count and cell area metrics, we observed a significant difference between migration and invasion kinetics of the relatively non-invasive cell line, NIH-3T3, and the highly invasive human fibrosarcoma derived cell line, HT-1080. Moreover, we observed significant and selective inhibition of HT-1080 cell invasion in the presence of GM6001, a broad inhibitor of Matrix Metalloproteinases (MMPs) with an IC50 of 25.7 nM. This new, real-time, quantitative cellular analysis technique illustrates a novel method to simultaneously study migration and invasion of tumor cells. 

### Extracellular Microenvironment

#5085

Cancer-associated fibroblasts transcriptional pattern reveals common changes in extracellular matrix-related genes across different cancer types.

Francesca Andriani,1 Marta Giussani,1 Federica Facchinetti,1 Maurizio Callari,2 Elda Tagliabue,1 Ugo Pastorino,1 Luca Roz,1 Gabriella Sozzi,1 Gabriella Sozzi1. 1 _Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy;_ 2 _Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom_.

Interactions between cancer cells and the surrounding microenvironment are crucial determinants of cancer progression. Fibroblasts isolated from carcinomas (cancer associated fibroblasts, CAF) have been shown to fuel the neoplastic process. While normal fibroblasts from different organs generally present very specific transcriptional programs, CAF from different tumors display a similar phenotype reminiscent of myofibroblasts found at sites of wound healing, indicating that common transcriptional programs could be induced by cancer cells on the surrounding microenvironment.

To investigate the existence of common mechanisms in fibroblast activation across different cancer types, gene expression profiles of tumor stroma or cancer associated fibroblasts (CAF) derived from breast, prostate and lung cancer were previously evaluated performing an inter-pathology bio-informatics analysis. For each comparison, genes were ranked and subjected to Gene Set Enrichment Analysis to identify Canonical Pathways significantly enriched. Most of commonly enriched gene sets were related to extracellular matrix (ECM) structure and remodeling, cytoskeletal organization and cell-cell adhesion. We specifically evaluated a previously identified ECM-related signature (ECM3) defining a breast cancer subtype likely to progress. We found that ECM3 signature was significantly enriched (p-value < 0.05) in cancer associated fibroblasts compared to the normal counterpart across different pathologies supporting the implication of ECM-related genes in fibroblasts with activated status. Furthermore increased expression of some of the most ECM3 genes (COL10A1, COMP and COL11A1) was detected in fibroblasts co-cultured with breast cancer cells, both in cellular extracts and in the conditioned medium (CM), suggesting their potential importance as circulating markers for early detection and risk assessment. To explore the significance of ECM-related molecules also in lung, we finally selected some of enriched ECM3-related molecules in lung fibroblasts (SPARC, COL11A1, MMP11, FN1, COMP, COL10A1) and we evaluated their presence in the conditioned medium. The analysis showed that the ECM molecules could be detected in medium conditioned by lung fibroblasts and generally enriched in CAF_CM compared to NF_CM (SPARC p=0.004, COMP p= 0.003, MMP11 p=0.012). Interestingly, analysis of de-cellularized extracellular matrix showed that Fibronectin 1 (FN1) was preferentially deposited by CAFs compared to NFs (p=0.003) denoting a possible role of this molecule in the modulation of extracellular matrix during tumor development and/or progression.

These findings support the hypothesis that identification of common factors responsible for proficient tumor-stroma cross-talk could be instrumental in novel strategies for risk assessment in different diseases.

#5086

Legumain-contained exosomes mediate remodeling of tumor extracellular matrix and protumor effects of TAMs.

Long Shen,1 Yuzhi Shi,2 Jing Xun,3 Chuanai Chen,4 Lingfang Du,4 Xiaoyue Tan,1 Rong Xiang1. 1 _Key Lab of Tumor Microenvironment and Neurovascular Regulation, Nankai University, Tianjin, China;_ 2 _The 2011 Project Collaborative Innovation Center for Biological, School of Medicine, Nankai University, Tianjin, China;_ 3 _The International Collaborative Laboratory for Biological Medicine of the Ministry of Education, School of Medicine, Nankai University, Tianjin, China;_ 4 _Department of Immunology, Nankai University, Tianjin, China_.

It has been recognized that tumor associated macrophages (TAMs) in tumor microenvironment play a variety of pro-tumor effects during the tumor progression, while the role of extracellular matrix in mediating these effects is still unclear.

In this study, we focus on elucidating the way as well as the underlying mechanism about the involvement of an asparaginyl endopeptidase, legumain in the crosstalk between TAMs and ECM. Our data showed that co-culture of breast cancer cells 4T1 with macrophages Raw 264.7 promoted asparaginyl endopeptidase, legumain, expression with the introduction of markers of M2 - polarized macrophage. Legumain was detected in the condition medium (CM) collected from RAW 264.7 co-cultured with 4T1 cells by western blotting, with the existence of asparagine endopeptide enzyme activity assessed by enzyme activity assay. Result of western blotting showed that adding the CM of RAW 264.7 co-cultured with 4T1 cells depleted extracellular matrix (ECM) contents, fibronectin and collagen I. Transwell assay showed increased infiltration of 4T1 cells through the ECM contents after treatment with the CM of RAW 264.7 co-cultured with 4T1 cells. Furthermore, treatment with CM of RAW 264.7 co-cultured with 4T1 suppressed the adhesion of endothelial cells with ECM, promoting tube formation of endothelial cells compared with control. All the above results could be abolished by synthesized legumain inhibitor, RR-11a. Interestingly, legumain was also detected in the exosomes isolated from CM of RAW 264.7 co-cultured with 4T1. Applying legumain contained exosomes could replicate the effect of CM of RAW 264.7 co-cultured with 4T1 on the depletion of ECM contents, promotion of 4T1 infiltration and suppression of endothelial cell adhesion with ECM.

Altogether, our results suggest that legumain is overexpressed and secreted out by TAMs; TAMs exosomes contain legumain and help delivering legumain to the ECM thus exert its effect on the depletion of ECM, pro-infiltration and angiogenesis.

#5087

EMT in cancer cells promotes the extracellular matrix remodeling by cancer-associated fibroblasts.

Shinya Neri,1 Genichiro Ishii,2 Atsushi Ochiai,2 Toshi Menju,1 Makoto Sonobe,1 Hiroshi Date1. 1 _Kyoto University Graduate School of Medicine, Kyoto, Japan;_ 2 _National Cancer Center Hospital East, Kashiwa, Japan_.

Microenvironmental factors in tumor tissue induce epithelial-mesenchymal transition (EMT) in cancer cells. However, it remains unclear how EMT-induced cancer cells affect mechanisms of invasion by cancer-associated fibroblasts (CAFs). In this study, we investigated how EMT-induced cancer cells develop supportive functions of CAFs in the process of local invasion. Using an in vitro collagen invasion assay model, EMT-induced cancer cells invaded the collagen matrix to a greater extent compared to control cells when they were cultured alone. Interestingly, CAFs co-cultured with EMT-induced cancer cells invaded to a greater extent compared to those with control cancer cells. We revealed that platelet-derived growth factor (PDGF)-B was upregulated in EMT-induced cancer cells, and the invasion ability of CAFs decreased by shRNA knockdown of PDGF-B in EMT-induced cancer cells. Treatment of a PDGF receptor inhibitor cancelled the increased invasion abilities of CAFs and EMT-induced cancer cells. Our results indicated that EMT-induced cancer cells enhanced the CAF invasion ability through the secretion of PDGF-BB as well as increased the invasion ability themselves, suggesting that EMT-induced cancer cells in the tumor microenvironment could not only exhibit their increased invasion but also promote CAF invasion which might lead to local invasion of cancer cells.

#5088

ACVRIB-dependent regulation of extracellular matrix protein in squamous cell carcinoma progression.

Holli A. Loomans,1 Laura L. Quast,1 Claudia A. Andl2. 1 _Vanderbilt University, Nashville, TN;_ 2 _University of Central Florida, Orlando, FL_.

Squamous cell carcinomas (SCC) of the head and neck and esophagus pose a substantial public health risk globally. As SCC is often diagnosed in late stage, therapeutic options are limited. A more thorough understanding of the mechanisms involved their progression is needed. One potential mode by which SCCs progress is via Activin A signaling. The downstream effects of Activin A signaling have been primarily characterized as growth inhibiting and anti-angiogenic. However, Activin A is commonly overexpressed in SCC. Therefore, we hypothesized that SCC become insensitive to Activin A stimulation through the dysregulation of the signaling pathway. To model this dysregulation we utilized CRISPR-mediated regulation of ACVRIB (ACVRIB-kd), the necessary type IB receptor to transduce Activin A signaling in three SCC cell lines. Downregulation of ACVRIB enhanced invasiveness in the OSC19 SCC three-dimensional organotypic cultures. Additionally, ACVRIB-kd cells showed significant changes in protein expression, when compared to control. In particular, the correlative markers of SCC aggressiveness and putative downstream Activin A targets, podoplanin and fibronectin, were substantially altered in ACVRIB-kd cells. Integrin expression, particularly integrins β3, α5, and αv, was markedly changed in ACVRIB-kd cells, indicating additional ACVRIB-mediated effects to ECM proteins. We are currently analyzing ACVRIB-kd SCC tumor growth and metastasis in vivo using tongue orthotopic xenografts and subcutaneous flank injections. In conclusion, SCC-specific disruption of Activin A signaling promotes an aggressive phenotype. Re-activation of the canonical Activin A pathway may provide a novel therapeutic strategy to combat SCC.

#5089

Extracellular Survivin uptake by cancer cells via receptor-mediated endocytosis.

Amber Gonda, Salma Khan, Heather R. Ferguson Bennit, Ron B. Moyron, Nathan R. Wall. _Loma Linda University, Loma Linda, CA_.

The tumor microenvironment is a crucial player in tumorigenesis, proliferation, and survival. Communication between its various components through microvesicular trafficking is central to the interaction between tumor cells and the surrounding milieu. Exosomes, nanovesicles released by all cell types, carry various proteins and nucleic acids. Once released into the extracellular environment, exosomes are taken up by surrounding cells or the exosomes travel in various bodily fluids to reach distant parts of the body. Those exosomes derived from tumor cells contain proteins and nucleic acids essential to the development and maintenance of tumors. One such protein, Survivin, a member of the Inhibitor of Apoptosis (IAP) protein family, has been identified in virtually all cancer cells. Survivin function is closely linked with subcellular location. Nuclear Survivin plays a key role in cell division and cell cycle regulation as a member of the chromosome passenger complex. Cytosolic and mitochondrial Survivin primarily facilitate the inhibition of apoptosis. Recent research has identified a unique pool of Survivin in the extracellular environment. Functions tied to this location are as yet undefined, however, evidence suggests that this population may contribute to increased proliferation, invasiveness and therapeutic resistance when taken into recipient cancer cells.

Using tandem affinity purification we have identified a relationship between Survivin and TNF receptor 1 (TNFR1), as well as transferrin receptor 2 (TfR2). Both of these receptors play a role in endocytosis of extracellular proteins. Transferrin receptors are upregulated on cancer cells to facilitate the increased iron demand of proliferating cells. We therefore hypothesize that exosomal Survivin is taken up by adjacent cancer cells via receptor-mediated endocytosis. We have shown that in the presence of antibodies to these receptors, there is a decreased uptake of extracellular Survivin (from conditioned media). Results were similar when conditioned media was introduced to HeLa cells with siRNA knockdowns of each receptor, as well as to CHO cells deficient in these receptors. We conclude that uptake of extracellular Survivin into cancer cells is dependent upon these two endocytosis receptors. The relationship of Survivin and exosomes in the extracellular environment is indicative that exosomes transport Survivin out of tumor cells and into the surroundings. These results suggest that exosomal Survivin enters cancer cells by way of receptor-mediated endocytosis. Understanding this mechanism of signaling between cancer cells shows the potential intercellular trafficking of oncoproteins may play in tumor maintenance and progression. It also provides us with a novel means to combat the resulting enhanced aggressive nature of the disease.

#5090

α1β1 is pro-proliferative and promigratory integrin and its expression is upregulated by the MYC oncogenic factor in colorectal cancer.

Salah Boudjadi, Gérald Bernatchez, Blanche Sénicourt, Marco Beausejour, Pierre-Henri Vachon, Julie Carrier, Jean-Francois Beaulieu. _Université de Sherbrooke, Sherbrooke, Quebec, Canada_.

Background

Colorectal cancer (CRC) is a multi-step process that involves successive mutation, epigenetic alteration and gene dysregulation. Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling and participate in the regulation of cell shape, adhesion, migration, survival and proliferation. The integrin α1 subunit is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in mammary carcinoma cell proliferation and migration. In the small intestine, α1 is present in the crypt proliferative compartment and absent in the villus. In mouse models, the α1β1 integrin, together with the Kras oncogenic factor, potentiates tumor growth. Very little is known about α1β1 function in CRC.

Aims: As we have recently shown that α1 is present in 65% of CRC (Boudjadi et al, 2013), that its expression is controlled by the MYC oncogenic factor and that the expressions of α1 and MYC correlate in 72.3% of colon adenocarcinomas (Boudjadi et al, Oncogene 2015) we postulated that integrin α1β1 has a pro-tumoral contribution to CRC related to α1 function.

METHODS: α1β1 function was studied in HT29, T84 and SW480 CRC cell lines using shRNA silencing targeting α1 (shα1) compared to an shRNA control (shCtrl). Cell proliferation was assessed by cell count and BrdU incorporation. Migration was tested by the scratch test assay. For the anoikis test, cells were kept in suspension without serum for 24 hours on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated dishes and were then lysed and subjected to caspase3 activity measurement and cleaved PARP expression. To test tumorigenic capacity, shα1 and shCtrl HT29 cells were injected into the dorsal subcutaneous tissue of female CD1 nu/nu mice. The tumor volume was assessed by external measurement. After resection, α1 knockdown was confirmed at the mRNA and protein levels.

RESULTS: In HT29, T84 and SW480 cells, α1 mRNA silencing resulted in reduced cell growth and proliferation compared to the control. Caspase3 activity measurement and cleaved PARP expression in HT29 and T84 cells showed that resistance to anoikis was altered in shα1 cells compared to shCtrl. Wound healing was delayed in shα1 HT29 and T84 cells compared to shCtrl. Moreover, tumor development in xenografts was reduced in HT29 shα1 cells. Histopathological analysis showed extensive necrosis areas and low mitotic index in shα1 tumors compared to the shCtrl tumors.

CONCLUSION: Our results show that α1β1 is involved in tumor cell proliferation, survival and migration. This finding suggests that α1β1 is involved in colorectal cancer progression. (Supported by the CIHR)

#5091

Synergistic apoptosis induced by combined PI3 kinase and bcl-xl inhibition in genotypic subsets of prostate cancer is attenuated by a feedback signaling loop via the alpha-5 integrin.

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

Background: Integrin-mediated cellular adhesion to extracellular matrix components is a crucial regulator of tumor cell survival and chemoresistance. The integrin alpha 5 (ITGA5) plays a vital role in the migration and adhesion of prostate cancer (PCa) cells to fibronectin secreted by bone-derived mesenchymal stromal cells (Joshi, 2015), architects of a metastatic niche (Shiozawa, 2011). Hypothesis: The mechanism(s) whereby ITGA5 regulates the survival and chemoresistance in PCa may define novel therapeutic approaches. Methods and Results: To assess the pro-survival function of ITGA5 in PCa, we found that ITGA5 shRNA reduced bcl-2 and bcl-xl expression, and induced apoptosis in PC-3 cells. In these PTEN-mutant (mt) AR-negative(-) cells, pharmacological inhibition of the PI3K signaling pathway (BKM120) in combination with ITGA5 knockdown synergistically induced apoptosis as assessed by induction of cleaved caspase-3 and cleaved PARP. Similarly, BKM120 in combination with pharmacological inhibition of bcl-2/bcl-xL (ABT263) also induced apoptosis in synergistic fashion in PC-3 cells. Synergistic apoptosis with PI3-kinase inhibitors (BKM120, pictilisib) and ABT-263 was restricted to PTEN-mt PC-3 and LNCaP cells and was not observed in PTEN-wt, AR- DU145 cells. The pan-Akt-inhibitor GDC-0068 in combination with ABT263 induced apoptosis specifically in PTEN-mt AR+ LNCaP and C4-2B cells. By contrast, there was diminished evidence of synergy of PI3-Kinase inhibitors and bcl-2 specific inhibition with ABT199 pointing toward the requirement of bcl-xl inhibition. However, resistance to longer-term therapy with PI3K and bcl-xl/bcl-2 inhibition was observed in PC-3 but not LNCaP cells. Although single-agent PI3K inhibitor therapy downregulates ITGA5 expression in PC-3 cells, by contrast bcl-2/bcl-xl inhibitor therapy, alone or in combination with PI3K inhibitor therapy, strongly upregulates ITGA5 expression together with Akt activation suggesting that ITGA5 represents a pathway of resistance to combinatorial therapy through feedback loop signals generated with bcl-xl inhibition. In support of this hypothesis, ITGA5 knockdown together with BKM120 and ABT263 combinatorial therapy blocked the upregulation of ITGA5 and associated Akt activation, further reduced bcl-xl expression, and enhanced apoptosis in PC-3 cells. In PTEN-wild type AR-positive VCAP cells, evidence for synergistic apoptosis with PI3K and bcl-2/bcl-xl inhibition was also obtained suggesting that the TMPRSS2-ERG fusion may phenocopy the pro-survival pathways of PTEN-deficient cells. Conclusions: These data point toward a personalized pharmacological strategy combining PI3-kinase or Akt inhibition with bcl-2/bcl-xL inhibition in genotypic subsets of prostate cancer and identify a potential resistance pathway mediated by ITGA5-mediated signaling.

#5092

CD151-α3β1 integrin complexes are prognostic markers of glioblastoma and cooperate with EGFR to drive tumor cell motility and invasion.

Pengcheng Zhou,1 Sonia Erfani,2 Rosalind Segal,3 Craig Horbinski,4 Xiuwei H. Yang2. 1 _Department of Cancer Biology and Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 2 _Dept of Pharmacology & Nutritional Sciences, University of Kentucky, Lexington, KY; _3 _Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA;_ 4 _Dept. of Pathology, University of Kentucky, Lexington, KY_.

Glioblastoma, one of the most aggressive forms of brain cancer, is featured by high tumor cell motility and invasiveness, which not only fuel tumor infiltration, but also enable escape from surgical or other clinical interventions. Thus, better understanding of how these malignant traits are controlled will be key to the discovery of novel biomarkers and therapies against this deadly disease. Tetraspanin CD151 and its associated α3β1 integrin have been implicated in facilitating tumor progression across multiple cancer types. How these adhesion molecules are involved in the progression of glioblastoma, however, remains largely unclear. Here, we examined an in-house tissue microarray-based cohort of 96 patient biopsies and TCGA dataset to evaluate the clinical significance of CD151 and α3β1 integrin. Functional, biochemical and signaling analyses were also conducted to understand how these molecules promote the aggressiveness of glioblastoma at molecular and cellular levels. Results from our analyses showed that CD151 and α3 integrin were significantly elevated in glioblastomas at both protein and mRNA levels, and exhibited strong inverse correlation with patient survival (p <0.006). These adhesion molecules also formed tight protein complexes and synergized with EGF/EGFR to accelerate tumor cell motility and invasion. Furthermore, disruption of such complexes enhanced the survival of tumor-bearing mice in a xenograft model, and impaired activation of FAK and small GTPases. Also, knockdown- or pharmacological agent-based attenuation of EGFR, FAK or Graf (ARHGAP26)/small GTPase-mediated pathways markedly mitigated the aggressiveness of glioblastoma cells. Collectively, our findings provide clinical, molecular and cellular evidence of CD151-α3β1 integrin complexes as promising prognostic biomarkers and therapeutic targets for glioblastoma.

#5093

Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS.

Mary Topalovski, Miao Wang, Zachary Moore, David Boothman, Rolf Brekken. _UT Southwestern Medical Center, Dallas, TX_.

Pancreatic ductal adenocarcinoma (PDA) is a highly recalcitrant disease. One factor contributing to chemoresistance is thought to be the desmoplastic response seen in these tumors. Fibronectin (FN) is a major constituent of the desmoplastic reaction of PDA. It has been shown that integrin activation by fibronectin can induce reactive oxygen species (ROS) production by stromal cells. Increased ROS has been observed in many cancers, including PDA. Therefore, we propose that oxidation therapy is an attractive approach to increase chemoresponse in PDA as this strategy takes advantage of the sensitivity of these tumors to further ROS insults. We discovered that the matricellular protein Fibulin5 (Fbln5) is upregulated in PDA and reduces FN-mediated integrin-induced ROS production by competing with FN for binding to α5β1 integrin and blocking integrin-induced ROS production. Mutation of the RGD-integrin binding domain in Fbln5 to RGE abolishes the binding of Fbln5 to the integrin and increases integrin-induced ROS production. Fbln5RGE/RGE (RGE) mice, display reduced tumor growth in implant and genetic models of PDA (KIC and KPC) as a direct consequence of elevated ROS levels in the tumor microenvironment. Furthermore, ROS induction correlates with reduced angiogenesis in tumors from RGE animals and had an additive therapeutic effect when combined with standard therapy. We have identified multiple oxidative stress-responsive genes that are elevated in stromal fibroblasts isolated from tumors from RGE animals. One of the most promising targets is NAD(P)H dehydrogenase (quinone 1) (Nqo1). In support of this observation we found that the level of Nqo1 is significantly higher in RGE fibroblasts compared with WT fibroblasts when plated on FN but not collagen or plastic. Furthermore we have demonstrated that FN-mediated induction of ROS and Nqo1 is dependent on β1 integrin and 5-lipooxygenase (5-LOX). Our studies reveal a unique integrin-based mechanism to enhance ROS production to a level that is specifically toxic to the tumor by exploiting the tumor-specific expression of Fbln5. Current studies are focused on understanding the mechanism by which Fbln5 is induced in PDA.

#5094

Acid-induced collagen remodeling promotes cancer progress as a result of niche engineering competition between cancer and stroma cells.

Mehdi Damaghi, Tinjan Chen, Josef Johnson, Robert J. Gillies. _Moffitt Cancer Center, Tampa, FL_.

Early carcinogenesis is an avascular and hypoxic disease that makes tumor cells export metabolically derived acid through glycolysis. Even in presence of oxygen cancer cells keep the glycolytic phenotype (Warburg effect) that acidifies the environment more and constantly. Hence the physical microenvironment of solid tumors is profoundly acidic. Pre-malignant cells within this niche must adapt to acidosis in order to survive this hostile microenvironment. We think pre-malignant cells adapt to acidosis to not only survive in this hostile microenvironment but also to acquire an advantage that helps them to outcompete the others and eventually help them to invade. Recently many evidences have been provided showing the collaboration of tumor associated stroma cells such as Fibroblasts (TAF) with tumor cells in cancer progress. Here we also show how these cells use acidification and acid adaptation as a strategy to construct and engineer their niche in their own favor for growth and proliferation. In this study we focused on collagen as major proteins of extra cellular matrix (ECM) and even arguably the most dominant one. The unique mechanical properties of fibrillar collagen are mainly controlled by its structure and any change in it can lead to ECM induced tumor growth. ECM unique compositions and topographies are generated through a dynamic biochemical and biophysical interplay between the various cells and the evolving microenvironment. In this study we developed the window chamber and intra vital microscopy to study the collagen remodeling lively. Later we used 3D cell culture and co-culture to validate our findings. Finally we used proteomics to understand molecular mechanism behind the changes in acid adapted versus non-adapted cancer cells and TAfs. We found cancer cells take different strategy than TAFs, although both changes promote cancer progression.

In conclusion, there are evolutionary advantages behind acid production and adapting to it for cancer and cancer associated cells. We discuss how acquiring these advantages can helps us to design new therapeutics against these unique phenotypes.

#5095

Hic-5 modulation of the stromal matrix is required for mammary tumor progression.

Gregory J. Goreczny, Jessica Ouderkirk, Eric Olson, Mira Krendel, Christopher Turner. _SUNY Upstate Medical University, Syracuse, NY_.

Primary tumors grow and invade into surrounding tissues/stroma, frequently resulting in metastasis to distant organs. Tumor cell-stroma crosstalk plays a crucial, but incompletely understood role during tumor progression and metastasis. For example, increased stromal matrix density and/or rigidity correlates with poor prognosis in human breast cancer patients. Previous studies have implicated Hic-5, a focal adhesion scaffold protein, in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in mammary tumor progression, Hic-5 -/- mice were generated and crossed with mice expressing Polyoma Middle T Antigen (PyMT) driven by the MMTV promoter. The PyMT mouse is a well-established model for human breast cancer progression. Tumors from the Hic-5 -/- PyMT mice demonstrated an increased latency and had reduced growth as compared to Hic-5 +/- PyMT controls. The absence of Hic-5 did not affect the progression of the tumor to a carcinoma stage, but there was a decrease in metastasis to the lungs. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs) and the Hic-5 -/- PyMT tumor stroma exhibits reduced extracellular matrix (ECM) deposition. Furthermore, 3D cell-derived matrices (CDM) generated in vitro using isolated CAFs, confirmed that Hic-5 -/- PyMT CAFs do not efficiently deposit and organize ECM. The organization of the stromal matrix has been shown previously to increase the rigidity of the tumor, which in turn promotes FAK Y397 phosphorylation in the tumor cells to promote cell invasion and proliferation. Accordingly, the Hic-5 -/- PyMT tumors exhibited a reduction in FAK phospho-Y397 staining and attenuation of downstream ERK activation, suggesting that their stromal matrix is not optimally organized for tumor cell signaling. Taken together, these data demonstrate that Hic-5 expression in CAFs is important for stromal matrix organization/remodeling during tumor progression and that the loss of the organization of the ECM in the Hic-5 -/- tumor stroma may contribute to the reduced tumor growth and metastasis observed in these animals.

Supported by NIH CA163296 to CET, NS066071 to EO and Carol M. Baldwin Breast Cancer Fund to CET.

#5096

Development of a decellularized tumor model for the evaluation of breast carcinomas.

Elizabeth Martin, Nicholas Pashos, Jeffrey Gimble, Bruce Bunnell, Matthew Burow, Bridgette Collins-Burow. _Tulane University, New Orleans, LA_.

Breast cancer recurrence is a clinical manifestation of tumor progression in patients which has been shown to significantly increase mortality rates. While up to one-fifth of breast cancer patients will experience recurrence, the mechanisms underlying this process remain poorly understood. Recently, gene expression profiling of human tumor metastasis and recurrence demonstrated that the extracellular matrix (ECM)-receptor interactions pathway was enhanced. In breast carcinomas, the ECM is known to vary with cancer progression. The ECM acts as a chemical reservoir, structural component, and mediator of tumorigenesis to tumors. To date, the ECM of breast carcinomas has been evaluated within the context of a seeded cellular population, i.e. intact tumor. Here we demonstrate for the first time a mechanism to evaluate the ECM devoid of breast cancer cells. We have employed a model of tissue decellularization to breast cancer cell line derived tumors and demonstrate that tumor ECM architecture remains intact when devoid of cells. Validation of loss of cell content is evident through DNA quantification, nuclear DAPI stain, and H&E stain. Furthermore we demonstrate that structural proteins, fibronectin, collagen, and elastin are still retained within the decellularized scaffold. Applications of the tumor decellularization model will aid in the evaluation of ECM structural and molecular characteristics independent of cancer cell populations. The data presented here will enhance methods used to evaluate the cancer microenvironment and further our understanding of breast cancer recurrence, altering existing treatment strategies to extend survival.

#5097

Tissue stiffness regulates multinucleation in mammary epithelial cells.

Allison K. Simi,1 Derek C. Radisky,2 Celeste M. Nelson1. 1 _Princeton University, Princeton, NJ;_ 2 _Mayo Clinic, Jacksonville, FL_.

This study investigates how increased stiffness of the tumor microenvironment (TME) can facilitate cellular multinucleation.

Multinucleation is an easily observable marker for polyploidy, facilitating the quantification of this phenotype. Polyploidy is commonly an intermediate to aneuploidy, defined as an abnormal number of chromosomes, a phenotype found in 85% of solid cancers that can drive tumorigenesis. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promoted increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture.

Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells.

Cells cultured on "stiff" substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on "soft" substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of MMP3, a protease commonly upregulated in cancer. This signaling depended on expression of the Rac1 splice variant, Rac1b, production of reactive oxygen species (ROS), and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation.

We also investigated biochemical effectors downstream of Snail. Aurora A kinase (Aurora A) is a mitotic regulator commonly elevated in cancer that has been shown to induce multinucleation in mammary epithelial cells and increase tumor incidence in mice. We found using microarray analysis that MMP3 increases the levels of Aurora A in mammary epithelial cells. We further discovered that upregulation of Aurora A in response to MMP3 signaling was limited to one transcript variant.

These data suggest that an increase in multinucleation is driven by the stiffening of the tissue during tumorigenesis, in part by MMP3-induced signaling and regulation of Aurora A splicing. More broadly, these data suggest a key role for the mechanical properties of the tumor microenvironment in cancer progression.

#5098

In vitro designer microenvironments for modeling the endothelin axis in brain cancer.

Simone C. Rizzi, Charlotte Mermier, Sophie Crettaz, Valeria Blumer-Nesca, Céline Gandar. _QGel SA, Lausanne, Switzerland_.

Preclinical studies in vivo have demonstrated that the endothelin (ET) axis is implicated in drug resistance of several tumors (e.g. brain and ovarian). Indeed, antagonists, blocking the binding of endothelin-1 to endothelin receptors (ETRs) overexpressed by cancer cells, have shown to make tumors more sensitive to conventional chemotherapies. In vitro, however, mechanistic studies have been hampered by the lack of adequate models, in which cell lines sufficiently express all key elements of the ET axis (e.g. ETRs), as they do in xenografts.

We argue that limitations of conventional in vitro systems are mainly due to their inability to take into account the role of the extracellular microenvironment (ME). To overcome these limitations we have exploited the design flexibility of the QGel Matrix technology to identify 3D cell microenvironments that induced cancer cells to naturally express ETRs in vitro. Briefly, brain cancer cells LN229 were grown clonogenically in differently functionalized poly(ethylene glycol) (PEG)-based gels whose biological and biophysical characteristics were combinatorially varied. AlamarBlue™ assays and intracellular calcium release measurements (ICRM) were employed to measure cell growth and functionality of ETRs in the different gel ME over time. All assays were performed in 384-well plate format with liquid handling devices.

ETR expression and functionality was confirmed in control cell lines when grown as clonogenic spheroids in MEs or as monolayer on tissue culture plastic (2D). Likewise, LN229 growth was observed in all tested MEs and in 2D. However, ETR functionality of LN229 differed considerably depending on the ME. For instance, in 2D, ICRM did not show ETR functionality. In contrast, clonogenically grown cell spheroids, in gel MEs containing cell integrin binding sites, showed significantly higher ETR functionality compared to LN229 in control gel MEs. Ongoing studies are further investigating the influence of other matrix characteristics (stiffness and degradability) to optimise expression and functionality of ETRs by LN229 for drug testing applications.

These data suggest that engineered MEs are powerful tools for the development of cell-based assays with enhanced physiological relevance. With their proven compatibility with standard laboratory automation systems and assay miniaturization, these in vitro disease models can be translated to industry for making more relevant decisions on compound efficacy in preclinical tests.

#5099

Weaning-induced tissue remodeling, metastasis, and young women's breast cancer.

Virginia Borges,1 Pepper J. Schedin2. 1 _University of Colorado, Denver, CO;_ 2 _Oregon Health & Science University, Portland, OR_.

Women diagnosed within five years of a completed pregnancy have a ~3 fold increased risk of metastatic disease independent of tumor stage and biologic subtype, and account for 30-50% of all young women's breast cancer cases in the US. We propose that weaning-induced breast involution supports breast cancer metastasis due to activation of a COX-2 dependent program that coordinately regulates immune suppression, fibroblast activation, collagen deposition, and lymphangiogenesis. In preclinical models, we find the microenvironment of the involuting gland supports breast cancer growth, dissemination, and escape to secondary organs. A rate-limiting step to metastasis is successful tumor cell seeding and expansion at secondary sites. A potential mechanism by which secondary sites may facilitate tumor cell seeding after lactation is by undergoing an involution process such as occurs in the post-weaning breast. Here, we show weaning-induced liver involution in rodents, including a 50% reduction in liver volume, hepatocyte cell death, deposition of collagen and tenascin-c, increased matrix metalloproteinase activity, and influxes in CD11b+F4/80+ macrophages, CD11b+F4/80-Ly6C+Ly6G- monocytes, and CD4+FoxP3+ T regulatory cells; these observations are also consistent with the establishment of a pro-metastatic niche. In an intracardiac metastasis model, the post-weaning liver supports increased seeding of 4T1 murine mammary tumor cells when compared to livers of nulliparous control mice. Relevance to women is suggested by data obtained from a cohort of young women's breast cancer patients, where liver metastatic tropism is observed specifically in the postpartum patients. These findings shed light on how normal reproductive physiology may alter site-specific metastasis and lay the groundwork necessary to investigate postpartum involution as a target for the prevention of breast cancer metastasis in young women.

Funded by grants from the DoD and NIH

#5100

Interplay between ECM stiffness, miR-203 and mammographic density.

Ivory Dean,1 Irene Acerbi,1 Alfred Au,1 Yunn-Yi Chen,1 Shelley Hwang,2 Valerie Weaver1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _Duke University, Durham, NC_.

High mammographic density (MD) is associated with a greater risk of breast cancer and is characterized by high extracellular matrix (ECM) collagen. Elevated collagen increases ECM stiffness and can promote the malignant transformation of oncogenically-primed mammary epithelial cells in culture and in vivo. Whether or not high MD reflects a breast stroma that is stiff and if ECM stiffness accounts for the elevated breast cancer risk in high MD tissue has yet to be resolved. To address this question we are conducting a comprehensive biophysical and molecular analysis of BIRADS status in prophylactic mastectomies in women with low (BIRADS 1) versus high MD (BIRADS 4). Data revealed that the intra-lobular ECM associated with the terminal end-buds contains anisotropic compliant and relaxed collagen fibrils. By contrast, the inter-lobular ECM contains stiff, oriented collagen fibrils. Preliminary data suggest that the ECM associated with the terminal end-buds in the upper outer quadrant may be stiffer in women with high MD as compared to low MD. These same tissues also expressed lower levels of the microRNA miR-203, which has been implicated in epithelial-to-mesenchymal transition (EMT) inhibition. Consistently, in vitro studies revealed that miR-203 was repressed in MECs cultured on a stiff ECM. We also found that a stiffened mammary ECM decreased miR-203 expression and promoted an EMT. Additionally, inhibiting ECM stiffening restored miR-203 levels and reduced an EMT. Moreover and importantly, we showed that triple negative breast tumors that often express EMT markers have reduced miR-203 levels. Studies are underway to elaborate these preliminary findings and to assess their clinical relevance. (This work is supported by the: NIH NSRA T32 CA 108462-11 to Ivory Dean, Susan G Komen PDF12230246 to Irene Acerbi, W81XWH-13-1-0216, USMRAA DOD-BCRP BC122990, NIH R01 CA138818-01A1 and NIH R01 CA192914-01 to Valerie Weaver.)

#5101

Tumor-secreted acids alter macrophage phenotype.

Asmaa El-Kenawi, Arig Ibrahim-Hashim, Kimberly Luddy, Brian Ruffell, Shari Pilon-Thomas, Robert Gatenby, Robert Gillies. _Moffitt Cancer Center, Tampa, FL_.

Within tumors, tumor-associated macrophages (TAMs) usually adopt an M2 phenotype with tumor-promoting characteristics, as opposed to the M1 phenotype responsible for host protection. As acidic pH can affect the characteristics of immune cells, we aimed to test whether low pH as a common stress factor in the tumor microenvironment could be partially responsible for the phenotypic characteristics and/or functional activity of macrophages in prostate cancer. To test this hypothesis, we allowed TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice to develop neoplastic lesions of variable histologic severity. Thereafter, we isolated tumor-associated macrophages from control tumors and analyzed their expression of the M1 and M2 markers; iNos, Il-6, Cd206 and Arg1. Gene expression analysis confirms previous studies which reported higher expression of both M1 and M2 markers in TAMs compared with peritoneal macrophages. Treatment of TRAMP mice with sodium bicarbonate to continuously buffer acids secreted by neoplastic cells, decreased TAMs infiltration into the stromal compartment of prostate. In addition, sodium bicarbonate decreased iNos, Arg1, and Cd206 but not Il-6 expression in TAMs isolated from subcutaneously growing TRAMP-C2 tumors, a TRAMP-derived prostate tumor cell line injected into syngeneic male hosts. Since decreased expression of Arg1 and iNos has been shown to mediate the suppressive activity of myeloid cells, tumor acidity might promote the suppressive microenvironment by upregulating both enzymes. In conclusion, extracellular acidosis increases the density of macrophages and their L-arginine metabolic potential; coincident with delayed tumor progression in prostate tumors.

#5102

Heparan sulfate modifying enzymes deliver cancer, stromal and macrophage-produced heparin-binding growth factors in the prostate cancer microenvironment.

Fabio H. Brasil,1 Valeria Ferrer,1 Nora M. Navone,2 Mary C. Farach-Carson,1 Daniel D. Carson1. 1 _Rice University, Houston, TX;_ 2 _University of Texas MD Anderson Cancer Center, Houston, TX_.

We previously showed that the heparan sulfate proteoglycan, perlecan (PLN)/HSPG2, is deposited into the matrix of reactive stroma surrounding tumors, where it concentrates heparin binding growth factors (HBGFs) and cytokines at the tumor-stromal border both in primary prostatic tumors and bone metastatic lesions. Heparan sulfate (HS) modifying enzymes produced in tumor-associated stroma play key roles in the delivery of these perlecan-sequestered cancer, stromal and tumor macrophage-produced HBGFs and cytokines, serving to enhance tumorigenesis and recruit other bystander cells to sites of metastasis. Studies presented here focused on the production of secreted HS-modifying enzyme heparanase and the two extracellular sulfatases, SULF1 and SULF2, in the prostatic tumor microenvironment. Our studies showed that SULF1 and heparanase mRNA levels are elevated in prostate cancer (PCa) cells of the LNCaP lineage in the presence of stroma, indicating that both stroma and PCa cells can be sources of these enzymes. We are presently examining a number of PCa patient derived xenografts to determine the prevalence of SULF-1 and heparanase elevation in castrate resistant disease. To study function, we developed a modified 3D hyaluronate gel system that contains HS-bearing recombinant perlecan domain I (PlnDI) to retain and deliver HBGFs and cytokines to various PCa cells cultured with bone marrow stromal cells, osteoblasts, or tumor macrophages. We are using this system to study the expression and individual functions of heparanase and the two SULFs in promoting HBGF-dependent PCa progression in a physiologically relevant system. Overexpression and gene deletion techniques allow us to determine the role that heparanase or SULFs play in each relevant cell type present in the tumor microenvironment, and thus determine the need for co-targeting of both tumor and reactive stroma. (Supported by NIH P01 and CAPES).

#5103

Crosstalk between osteoprotegerin (OPG), fatty acid synthase (FASN), and cyclooxygenase-2 (COX-2) in breast cancer: implications in carcinogenesis.

Sudeshna Goswami, Neelam Sharma-Walia. _Rosalind Franklin University of Med. & Science, North Chicago, IL_.

The cross-talk between malignant and nonmalignant cells in the tumor microenvironment, as maneuvered by cytokines/chemokines, drives breast cancer progression. We demonstrated that OPG is expressed and secreted at very high levels from the highly invasive breast cancer cell lines SUM149PT and SUM1315MO2 as compared to human normal mammary epithelial HMEC cells (1). OPG was involved in modulating aneuploidy, cell proliferation, and angiogenesis in breast cancer (1). Mass spectrometry analysis performed in this study revealed OPG interacts with fatty acid synthase (FASN), which is a key enzyme of the fatty acid biosynthetic pathway in breast cancer cells. Further, electron microscopy, immunofluorescence, and fluorescence quantitation assay highlighted the presence of a large number of lipid bodies (lipid droplets) in SUM149PT and SUM1315MO2 cells in comparison to HMEC. We recently showed upregulation of COX-2 inflammatory pathway and its metabolite PGE2 secretion in SUM149PT and SUM1315MO2 breast cancer cells (2). Interestingly, human breast cancer tissue samples displayed high expression of OPG, PGE2 and FASN. Immunofluorescence staining exposed the co-existence of COX-2 and FASN in the lipid bodies of breast cancer cells. We reasoned that there might be cross-talk between OPG, FASN, and COX-2 that sustains the inflammatory pathways in breast cancer. Here, we identified cis-acting elements involved in the transcriptional regulation of COX-2 and FASN by recombinant human OPG (rhOPG). Treatment with FASN inhibitor C75 decreased the number of lipid body/cell, downregulated phosphorylation of ERK, GSK3β, and induced apoptosis by caspase-3 and caspapse-9 activation. Collectively, the novel biological cross-talk between OPG, FASN, and COX-2 advocates for combinatorial drug treatment to block these players of carcinogenesis as a promising therapeutic to target highly invasive breast cancer.

References:

1. Goswami, S., & Sharma-Walia, N. (2015). Osteoprotegerin secreted by inflammatory and invasive breast cancer cells induces aneuploidy, cell proliferation and angiogenesis. BMC. http://www.biomedcentral.com/1471-2407/15/935

2. Chandran, K., Goswami, S., & Sharma-Walia, N. (2015). Implications of a peroxisome proliferator-activated receptor alpha (PPARα) ligand clofibrate in breast cancer. Oncotarget, 5. http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=6402

#5104

The RNA binding protein HuR enhances exosome secretion in colorectal cancer.

Ranjan Preet,1 Shufei Zhuang,1 Wei-ting Hung,2 Lane K. Christenson,2 Dan A. Dixon1. 1 _University of Kansas Cancer Center, Kansas City, KS;_ 2 _University of Kansas Medical Center, Kansas City, KS_.

Enhanced secretion of extracellular vesicles such as microvesicles and exosomes by cancer cells has been recently recognized as a new means of transferring oncogenic information within the tumor microenvironment. Through their ability to carry specific RNA and protein cargo, tumor-derived exosomes can directly impact neighboring cells to support tumor progression. However, it remains unclear what regulates exosome production in tumor cells and the factors influencing loading of tumor-promoting RNA cargo. Our prior work has established that colorectal cancer (CRC) cells and tumors overexpress the key RNA-binding protein HuR early in GI tumor development. When overexpressed and present in the cytoplasm, HuR can promote mRNA stabilization of tumor-promoting genes through binding of 3'UTR AU-rich elements (ARE). While the ability of HuR overexpression to promote gene expression is well recognized, our understanding of how HuR communicates this information within the tumor microenvironment is limited. To test if HuR overexpression could impact secreted exosomes, Tet-regulated HuR-inducible HeLa cells were used to demonstrate that cytoplasmic HuR overexpression promoted a 4-fold increase in exosomes produced. Furthermore, HuR was detected in exosomes produced only from HuR-overexpressing cells. The presence of HuR in exosomes directly impacted mRNA cargo, as selective uptake of ARE-containing mRNAs was observed in exosomes derived from HuR overexpressing cells. To test if these effects were seen in CRC cells that endogenously overexpress HuR, exosome levels from CRC cells were compared to normal primary human intestinal epithelial and myofibroblast cells. When normalized to total cell numbers, CRC cells secrete ∼3-fold greater exosome levels than normal cells and enhanced exosome production was dependent upon HuR. Knockdown of HuR in CRC cells directly impacted exosome secretion to levels observed in normal cells. HuR was also only detected in exosomes produced from CRC cells. Using an inducible model of RasV12-mediated transformation of intestinal epithelial cells, endogenous HuR overexpression was observed associated with 4-fold greater levels of exosomes containing HuR as cargo. These findings were reflected in vivo where GI-tumor bearing APCMin/+ mice produced ~3-fold more serum exosomes, with HuR as exosomal cargo in APCMin/+ mice where as no expression of exosomal HuR was detected in wild-type mice. This work has identified a novel connection between HuR-mediated post-transcriptional regulation and tumor-derived exosome production, along with providing the first evidence indicating the presence of exosomal HuR as a serum-based CRC biomarker.

#5105

Exosomes secreted by proinflammatory macrophages modulate breast cancer.

Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. _New York Medical College, Valhalla, NY_.

Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression.

#5106

Unexpected roles for stromal MMP3 during breast cancer.

Emilia Hartland,1 Charley Jang,1 Ricardo Romero Moreno,1 Megan McGarel,1 Sunil Badve,2 Laurie Littlepage3. 1 _University of Notre Dame, Notre Dame, IN;_ 2 _Indiana University School of Medicine, Notre Dame, IN;_ 3 _Harper Cancer Research Institute, Notre Dame, IN_.

Matrix metalloproteinases (MMPs) are a family of proteases that have been shown to contribute to the degradation of components of the extracellular matrix and other substrates to promote cancer progression and metastasis. Stromelysin / matrix metalloproteinase-3 (MMP3) is one member of the matrix metalloproteinase family that is overexpressed in human breast cancer. During breast cancer, MMP3 is expressed primarily by stromal cells but also by cancer cells. Overexpression of MMP3 induces an epithelial-to-mesenchymal transition (EMT), promotes hyperplastic growth, and expands a mammary gland stem cell population. In addition, MMP3 is required for the mammary gland stem cell population. In this study, we determined if stromal MMP-3 is required for breast cancer progression for primary and metastatic lung tumor growth. To study this, we transplanted mammary epithelial cancer cells (V0PyMT) into the mammary glands of MMP3 knockout and heterozygous syngeneic mice. We quantified the tumor burden of both primary and metastastic tumors over time and discovered that elimination of stromal MMP3 increased the tumor burden. The tumor pathologies, proliferation rate, and apoptosis were similar between cancer and not cancer tissue. In contrast, the vasculature density decreased. These data suggest that stromal MMP3 inhibits, but is not required for, the development of the primary breast tumor. However, in contrast to our primary tumor data, we interestingly discovered that stromal MMP3 was required for visible lung metastases. These experiments begin to distinguish between the contrasting roles of stromal and epithelial MMP3 during breast cancer. Our data reveal that MMP3 contributes to the metastatic tumor formation within the lungs. This study will help to elucidate new mechanisms through which MMP3 functions within particular microenvironmental contexts during breast cancer progression.

#5107

The involvement of connective tissue growth factor in nonalcoholic steatohepatitis and liver cancer development under diabetic condition.

Liya Pi, Marda Jorgensen, Seh-Hoon Oh, Altin Gjymishka, Bryon E. Petersen. _Univ. of Florida, Gainesville, FL_.

Introduction: Obesity and type 2 diabetes are risk factors for liver cancer due to a transition from steatosis to nonalcoholic steatohepatitis (NASH), resulting in liver fibrosis. If left untreated, liver fibrosis can progress into cirrhosis leading to hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF) is a pro-fibrotic matricellular protein that is highly expressed during hepatocarcinogenesis. This study aims to investigate the involvement of CTGF in nonalcoholic steatohepatitis (NASH) and liver cancer development during diabetic conditions induced by streptozotocin (STZ) treatment and the feeding of a high fat diet (HFD) in mice.

Methods: Transgenic male mice expressing green fluorescent protein (GFP) under the control of Ctgf promoter (Ctgfp-GFP), liver specific CTGF knockouts (CtgfΔhep/Δhep), and control mice that contained two alleles of floxed Ctgf (Ctgffloxed/floxed) were given STZ (200 ng/pup) at postnatal day 2 (P2) through subcutaneous injection and were fed HFD at postnatal week four to induce NASH and HCC. The treated animals were harvested at postnatal week 5, 12, and 20 for early steatosis, fibrosis and liver cancer development respectively. CTGF expression was analyzed by immunofluorescent staining, Western analysis, and qRT-PCR. In addition, the mouse liver cancer RT2 profiler PCR array was screened to identify cancer-related genes that were differentially expressed during NASH and HCC development comparing CtgfΔhep/Δhep and Ctgffloxed/floxed mice.

Results: All three types of animals developed NASH and HCC in response to STZ and HFD feeding. Liver pathologies ranging from steatosis, NASH, liver fibrosis, and nodule formation, to intratumoral angiogenesis were observed as discrete molecular and histological stages in the STZ/HFD induced NASH-HCC model. Immunofluorescent analysis of Ctgfp-GFP reporter mice showed induction of the Ctgf gene in vascular endothelial cells, biliary epithelial cells, hepatocytes, and inflammatory cells in STZ/HFD livers. We utilized the mouse liver cancer RT2 profiler PCR array and compared the expression of 90 liver cancer related genes between CtgfΔhep/Δhep and Ctgff/f tumors that developed after 12-week HFD feeding. 12 upregulated genes and 10 downregulated genes were identified in Ctgfk/k mice compared to Ctgff/f animals.

Conclusion: We successfully established a NASH-HCC model combining STZ and HFD treatment. CTGF expression was found in multiple cell types including endothelial cells, cholangiocytes, inflammatory cells, and hepatocytes. Liver specific deletion of CTGF was associated with differential expression of groups of genes involved in cell proliferation and apoptosis. The functional relationship between these genes and CTGF in liver cancer development in the setting of metabolic syndrome needs to be characterized in future studies.

#5108

Glucagon like peptide 1 (GLP1) up-regulation in hypoxic and nutrient deprived microenvironment promotes neuroblastoma cell survival.

Umadevi V. Wesley, Nausheen Singh, Robert J. Dempsey. _University of Wisconsin, Madison, Madison, WI_.

Introduction: Glucagon-like peptide 1 (GLP1) an incretin has demonstrated to increase insulin secretion in a glucose-dependent manner and is shown to be neuroprotective. GLP1 also promotes angiogenesis. Hypoxia, a common feature in solid tumors including neuroblastoma, promotes tumor progression by upregulation of hypoxia responsive factors. Little is known about alterations of GLP1 expression in neuroblastoma under hypoxic condition. GLP1 is rapidly inactivated by dipeptidyl peptidase IV (DPPIV) mediated enzymatic cleavage. Previously we have shown that DPPIV suppresses neuroblastoma survival and angiogenesis. In this study, we examined whether GLP1, its receptor GLP1R, and DPPIV expressions are altered after exposing neuroblastoma cells to hypoxic conditions of oxygen and nutrient deprivation (OND). We then examined the direct effects of GLP1 on neuroblastoma proliferation and survival, and evaluated if DPPIV modulates GLP1 mediated functions.

Methods: We used Neuro-2a, a mouse neuroblastoma cells as a model. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation, followed by re-oxygenation. Immunofluorescence staining was used to analyze the protein expression of GLP1, GLP-1R, and DPPIV. RT-PCR was used to measure mRNA levels. Effects of DPPIV on GLP1 mediated Neuro-2a cell proliferation and survival was evaluated by MTT and live/dead cell assay. Levels of PI3K/AKT were determined by western blot analysis.

Results: Neuro-2a cells subjected to OND showed about 2 fold increase in the GLP1 protein and mRNA levels at four hours of

re-oxygenation after 4 hours of OND. However, GLP1 protein levels decreased drastically after one day of re-oxygenation, while DPPIV levels were highly upregulated. GLP1R was also downregulated at one day of re-oxygenation. At this time point, cell viability was decreased by 30-40%. Pre-treatment with DPPIV inhibitor rescued the protective effects of GLP1. Furthermore, exogenous GLP1 stimulated cell proliferation and increased Neuro-2a cell viability by 2-3 fold. GLP1dependent action was mediated via increased levels of phosphorylated AKT, a pro-survival signaling molecule. The effects of GLP1 were suppressed in presence of DPPIV.

Conclusion: The results indicate an inverse correlation between DPPIV and GLP1 levels in Neuro-2a cells. Together, these studies indicate that GLP1-DPPIV interaction may play an important role in promoting neuroblastoma cell proliferation and survival.

#5109

MDM2 regulates intracellular pathways in TNBC and osteosarcoma that affect osteoclastogenesis.

David J. Olivos, Lindsey D. Mayo. _Indiana University School of Medicine, Indianapolis, IN_.

Advanced cancer cells infiltrate and colonize new organ microenvironments through transformation of the dynamic metastatic niche. In bone, tumor cells secrete soluble factors that distort and manipulate normal bone remodeling leading to osteolytic lesions and inflammation. Clinically, primary high-grade bone sarcomas and secondary metastatic cancers exhibit elevated levels of murine double minute 2 (Mdm2), the principal cellular antagonist and E3 ubiquitin ligase of tumor suppressor p53. We demonstrate a novel function of mdm2, independent of p53, to drive osteolytic disease influenced by the signaling cascade of RANKL, ICAM1, and RANTES. To elucidate the direct influence of Mdm2 in cancer cells on osteoclast differentiation, several cell lines were created to overexpress or silence Mdm2. We found that osteoblast-like osteosarcoma (MG63, Saos2) and metastatic breast cancer cell lines (MDA468, MDA231, TMD-231, T47D, and BT474) lead to significant increased osteoclast differentiation, proliferation, and bone resorption. Co-cultures of Mdm2 over-expressing cancers and normal monocytes resulted in increased numbers of osteoclasts. In cancer cell lines where Mdm2 is silenced, we observed lower number of osteoclasts. The presence of Mdm2 in cancer cells also increased osteoclast resorption in an in vitro organ model. To determine the effects of ICAM1, we used an ICAM antibody to block ICAM1 in the media and showed a decrease in osteoclastogenesis. Understanding the role of Mdm2 in osteoclastogenesis may be amendable to translational benefit in disease models of advanced bone resorption and bone metastases.

#5110

The role of autophagy in the tumor microenvironment.

Christopher M. Dower, Hong-Gang Wang. _Penn State Hershey Medical Center, Hershey, PA_.

The tumor microenvironment (TME) is a complex and dynamic breeding ground for the selection of aggressive tumor cells with an advantage of survival and metastatic potential. Autophagy has emerged as a powerful cellular process that can greatly influence how cancer cells respond to nutrient deprivation and hypoxic stress, the hallmarks of the TME. However, the physiological importance of hypoxia-regulated autophagy remains unknown. Here, we used a novel hypoxia-dependent reversible dominant-negative strategy, which allowed for physiological regulation of autophagy at the cellular level during tumor growth and metastasis and creates a heterogeneous population comprised of autophagy-competent and autophagy-deficient tumor cells. We demonstrate that the suppression of autophagy by HIF1-α dependent ULK1 inhibition within the hypoxic tumor microenvironment enhances the metastatic potential of breast cancer cells both in vitro and in vivo. Functional proteomic and transcriptome analysis indicated that the loss of hypoxia-regulated autophagy promotes metastasis through extracellular remodeling pathways, primarily through induction of the fibronectin-integrin signaling axis, along with LOX-mediated extracellular remodeling and pre-metastatic niche formation. In this way, the role of physiological hypoxia regulated autophagy was assessed for the first time in a triple negative breast cancer model. Currently, we are exploring the interplay between autophagy-competent and autophagy-deficient tumor cells within the TME, and how it relates to metastasis. Although the importance of autophagy in tumor-stromal interactions has recently become appreciated, the role autophagy plays in tumor cell-tumor cell interactions remains unknown. Through investigation of these intra-tumor cell networks, we can elucidate the precise mechanism of how physiologically-regulated autophagy in tumor cells influences the surrounding TME and tumor progression.

#5111

A novel β 2 adrenergic-nerve growth factor feed forward loop promotes pancreatic cancer.

Bernhard W. Renz,1 Marina Macchini,2 Yoku Hayakawa,2 Xiaowei Chen,2 Zhengyu Jiang,2 Takayuki Tanaka,2 C. Benedikt Westphalen,1 Yagnesh Tailor,2 Jens Werner,1 Axel Kleespies,1 Alina C. Iuga,2 Daniel L. Worthley,2 Kenneth P. Olive,2 Timothy C. Wang2. 1 _Ludwig Maximilian University of Munich, Munich, Germany;_ 2 _Columbia University, New York, NY_.

Introduction and objectives: There is increasing evidence that chronic activation of sympathetic neurons can lead to increased noradrenaline levels in the tumor microenvironment (TME) in PDAC. However, the mechanisms by which adrenergic neurotransmitters are delivered to the TME are not well understood. In this study we aimed to investigate the effect of locally and systemically delivered catecholamines into the TME in two well established genetically engineered mouse models of PDAC (Pdx1-Cre/KRasG12D (KC) and Pdx1-Cre/KRasG12D/Trp53R172H (KPC)).

Methods: Adrenergic signaling was induced or inhibited in KC or KPC mice and in pancreatic organoids using various pharmacological compounds. Adrenergic receptor expression was assessed by RT-PCR, WB and IHC. Downstream pathways were identified by phosphorylation assays and WB. Adrenergic signaling was modeled in vivo applying restraint stress. To elucidate the crosstalk between nerves and cancer cells, pancreatic spheres and PDAC cells were co-cultured with DRGs and neuronal plasticity was quantified. NGF secretion was measured by RT-PCR and ELISA. Sympathetic denervation of the pancreas and blockage of the 2-receptor was employed as a treatment strategy in KPC mice. Overexpression of NGF was targeted to the mouse pancreas using a novel NGF knockin mouse, which was crossed to KC and KPC mice.

Results: ADRB2 was upregulated during pancreatic cancer development. Furthermore, in vitro stimulation of cells harboring an oncogenic KRas mutation with catecholamines resulted in significantly increased sphere forming efficiency. The non-specific -blocker propranolol and the selective 2-blocker ICI 118,551 inhibited this effect. Selective blockade of 2-adrenergic signaling suppressed pancreatic sphere formation caused by co-cultures with DRGs.

NGF secretion was stimulated through beta2-adrenergic signaling. Furthermore, restraint stress accelerated PDAC development in KC mice. The effects of stress were prevented by treatment with the selective ADRB2 antagonist ICI 118,551. Specific β2-blocker treatment as well as sympathetic denervation in addition to GEM significantly increased the survival of mice with 3-5 mm tumors in comparison to controls treated by GEM alone. Targeted overexpression of NGF in the mouse pancreas using a novel NGF knockin mouse resulted in marked acceleration of PanIN lesions in the KC mouse and shortened overall survival in KPC mice significantly. Finally, retrospective analysis of a PDAC patient cohort revealed a extension of overall survival in PDAC patients on non-selective -blockers.

Conclusions: Taken together, these studies suggest that increased catecholamines can engender a feed forward loop, whereby upregulation of NGF can lead to increased innervation and local NE accumulation, thereby enhancing tumor growth. Therapies targeting these adrenergic and NGF pathways may prove useful in the treatment and prevention of pancreatic cancer.

#5112

Galactose probes hit neuroblastoma cells through a specific galactose-galectin association.

Giuseppina Simone,1 Mirco Ponzoni,2 Fabio Pastorino2. 1 _University of Naples, Naples, Italy;_ 2 _IRCCS, Istituto G. Gaslini, Genoa, Italy_.

Galectins are a family of proteins that decorate the cell membrane and form extracellular molecular associations with β-galactoside sugars.

In order to mimic multivalent molecular recognitions, galactoside moieties have been immobilized on microbeads and used in cytofluorimetric analysis and in microfluidic assay to study the molecular association between carbohydrates and proteins in neuroblastoma (NB) cells. The hypothesis behind this investigation was that the molecular mechanisms by which glycans modulate neural metastatic cells involve protein-carbohydrate association.

To evaluate the binding between microbeads and tumor cells, two human NB cell lines were used in cellular association experiments. GI-LI-N cells, derived from the peripheral blood of a bone marrow-infiltrated, high risk, stage IV NB patient, were chosen for metastatic characteristics; IMR-32 cells were chosen because derived from the primary tumor in the abdominal site of an unknown-stage NB patient.

In vitro experiments have revealed molecular binding preferences of the metastatic NB cells. This result underscores that the efficiency of the cellular association might be related to the differences in the expression of the galectins between the two cell lines (GI-LI-N cells >> IMR-32 cells) and that the formation of the molecular association galectin-galactose could discriminate the tumor stages.

Ex vivo investigations have supported in vitro results; indeed, galactose moieties mostly recognized GI-LI-N cells derived from orthotopic tumor-bearing mice respect to explanted IMR-32 cells. This molecular association was tumor cells specific because it discriminated healthy tissues.

The understanding of this mechanism of recognition, and of possible cellular discrimination, can be relevant for the diagnostic monitoring of metastatic NB cells, and for producing probes tailored to interfere with galectin activities associated with the malignant phenotype.

#5113

CXCL12/CXCR4 and integrin signal activation by cancer-associated fibroblasts enhances the chemoresistance in gastric cancer.

Yuka Tamaoki, Takatsugu Ishimoto, Keisuke Miyake, Daisuke Izumi, Daisuke Kuroda, Tsugio Eto, Kota Arima, Hironobu Shigaki, Junji Kurashige, Masaaki Iwatsuki, Yoshifumi Baba, Yasuo Sakamoto, Naoya Yoshida, Hideo Baba. _Graduate school of Medical science Kumamoto University, Kumamoto, Japan_.

Purpose: Gastric cancer (GC) is an aggressive cancer metastasizing with a high propensity for peritoneal dissemination and liver metastasis. Some studies have been shown that not only cancer cells but also their surrounding stroma, especially cancer-associated fibroblasts (CAFs) play an important role in tumor development. Previously, we reported that CXCL12-CXCR4 and integrin β1 signal activation by CAFs co-operates for promoting FAK phosphorylation and enhances the invasiveness of GC cells in extracellular matrix (ECM).On the other hand, a previous study presented that CXCR4 and integrin signaling co-operates in mediating adhesion and chemoresistance in small cell lung cancer cells. Therefore, we hypothesized that CXCL12-CXCR4 and integrin signal activation by CAFs enhances the chemoresistance in GC as well. The aim of current study is to elucidate the significance of CAFs mediating signal activation for chemoresistance in GCs.

Experimental Design: We first performed chemo-sensitive assays using GC cells cultured with fresh medium or with CAF-conditioned-medium (CM), and these experiments were conducted on non-coated and Matrigel-coated plates. Next, we examined the CXCL12-CXCR4 and integrin related signaling by western blotting analysis. Furthermore, we investigated the critical molecule for chemoresistance using silencing CXCR4 or integrin β1 in GC cells.

Results: Chemo-sensitive assays revealed that GC cells cultured with CAF-CM showed more resistant to cisplatin and reactive oxygen spices (ROS) than those cultured with fresh medium. Moreover, GC cells with CAF-CM on Matrigel-coated plates exhibited remarkable resistance in these assays. On the other hand, western blotting analysis showed that Akt activity in GC cells cultured with CAF-CM was up-regulated in concurrence with FAK phosphorylation. Finally, the chemoresitance induced by CAFs was significantly suppressed by CXCR4 or integrin β1 silencing.

Conclusions: These results suggest that CXCL12/CXCR4 and integrinβ signal activation by CAFs promoted the chemoresistance through enhancing their interaction with ECM in GC. This mechanism underlying the chemoresistance may provide a novel therapeutic target in advanced GCs.

#5114

The role of microvesicles on immune function in response to cancer.

Ritu Jaiswal, Deep Pokharel, Mary Bebawy. _University of Technology Sydney, Ultimo, NSW, Australia_.

Cell to cell communication is vital for the co-ordination of physiological process and the regulation of an organism's phenotype. More recently communication via extracellular membrane vesicles has gained recognition. We first described a novel mechanism for the spread and dominance of multidrug resistance (MDR) and enhanced metastatic capacity in cancer via submicron microparticles (MPs). MPs are plasma membrane vesicles released spontaneously from various cell types, carrying bioactive material and are implicated in different physiological and pathophysiological processes. Through this communication apparatus, cancer cells can acquire and secure a survival advantage by various mechanisms. This study aims to examine a role of MPs in altering immune cell function in cancer.

The effects of MPs isolated from human breast cancer cells were examined on antigen presenting cells (APC) in vitro. MP-mediated effects on cell phenotype and functionality was assessed by cytokine profiling and migration assay. We observed a cancer cell induced change in immune cell phenotype and functionality which have the potential to support a reduced global immune response in cancer. The elucidation of this pathway provides novel therapeutic strategies which can be exploited for the treatment of cancer.

### Immunomodulation and Immunotherapy

#5115

Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies.

Alan Wise, Barry E. McGuinness, Sarah C. Trewick, Thomas J. Brown, Phillip M. Cowley. _IOmet Pharma Ltd, Edinburgh, United Kingdom_.

The enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1) catalyse oxidation of the essential amino acid tryptophan (Trp) leading to the formation of immunosuppressive kynurenine (Kyn) pathway metabolites that dampen the immune response in the tumor microenvironment. Both IDO1 and TDO have been shown to be up-regulated in a variety of cancers and blockade of their activity has been shown to stimulate the anti-tumour immune response in pre-clinical animal models. We have discovered and optimized multiple novel chemical series of both highly selective and dual-acting inhibitors of IDO1 and TDO. We describe the characterization of molecules that we are progressing into the clinic.

We have characterized the IDO1 and TDO-selective inhibitors and dual-acting inhibitors which demonstrate nM potencies. In IFNγ-stimulated A172 glioblastoma cells expressing both enzymes, the dual-acting molecules fully inhibit Kyn production, whilst selective inhibitors yield only a partial response, demonstrating the utility of dual enzyme blockade in cancer cells. The compounds also relieve inhibition of T-cell proliferation in cancer cell conditioned media/T-cell co-culture assays and demonstrate highly favourable physico-chemical and pharmacokinetic properties. These properties translate to superior PK/PD effects following oral dosing in rodents with profound and sustained modulation of plasma Kyn levels, combined with an exceptional safety profile. In a mouse model of LPS-induced IDO1 activation, our IDO1 inhibitors fully ablate elevation of Kyn in both plasma and lung in a dose-dependent manner. Following oral administration, the IOmet IDO1 inhibitors provide excellent single-agent tumor growth control in PAN02 pancreatic adenocarcinoma syngeneic models. In addition, combining IOmet IDO1 inhibitors with standard-of-care chemotherapy (gemcitabine/abraxane) in the PAN02 model completely inhibits tumor growth.

In summary, we describe the characterization of novel potent, selective and dual-acting IDO1 and TDO inhibitors. These candidate molecules demonstrate excellent rodent in vivo PK/PD, safety and anti-tumor profiles, supporting their progression into the clinic.

#5116

Phosphatidylserine-targeting antibodies augment anti-tumor activity of PD-1 antibodies and alter immuno-profiles in murine triple negative breast cancers.

Michael J. Gray,1 Jian Gong,1 Ryan N. Parks,1 Michaela M.S. Hatch,2 Van Nguyen,1 Christopher C.W. Hughes,2 Jeff T. Hutchins,1 Bruce D. Freimark1. 1 _Peregrine Pharmaceuticals, Inc., Tustin, CA;_ 2 _University of California, Irvine, CA_.

Current treatments for triple negative breast cancers rely primarily upon surgical intervention and chemotherapy. Unfortunately, chemotherapies have the propensity to help establish an immunosuppressive condition in part though exposure of phosphatidylserine (PS) in the tumor microenvironment. We demonstrate that treatment of the murine triple negative breast cancer E0771 using a PS-targeting antibody inhibits in vivo growth, and significantly enhances the anti-tumor activity of antibodies directed to the checkpoint inhibitor PD-1. Combinational treatment by antibodies targeting PS and PD-1 increased the number of tumor infiltrating lymphocytes (TILs) to a greater extent than observed with single-arm therapies. Furthermore, combination of PS and PD-1 targeting antibodies provides a distinct survival advantage over treatment by either agent alone, which correlates with an increase in complete tumor regression and the onset of a durable immune mediated response capable of rejecting a secondary challenge by the same tumor. Finally, immune-profiling analysis of tumor RNA using Nanostring revealed that the combination antibody therapy enhanced immune regulatory pathways including MHC class I/II expression, antigen processing and presentation, and leukocyte and macrophage functions. Combined, our data suggest that in triple negative breast cancers antibody therapy blocking PS-associated immunosuppression may further enhance immunotherapies directed at immune checkpoint inhibitors, including those targeting the PDL-1/PD-1 axis.

#5117

An automated 5-plex fluorescent immunohistochemistry enabled characterization of PD-L1 expression and tumor infiltrating immune cells in lung and bladder cancer specimens.

Wenjun Zhang,1 Antony Hubbard,1 Adriana Racolta,1 Nick Cummins,1 Mehrnoush Khojasteh,1 Liping Zhang,1 Karl Garsha,1 Joerg Bredno,1 Dustin Harshman,1 Srabani Bhaumik,1 Tobin Jones,1 Marcin Kowanetz,2 Sanjeev Mariathasan,2 Ian McCaffery,2 Dustin Smith,2 J. Andrew Williams,2 Lidija Pestic-Dragovich,1 Larry Morrison,1 Lei Tang1. 1 _Ventana Medical Systems, Inc., Tucson, AZ;_ 2 _Genentech, San Francisco, CA_.

Cancers may escape immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1) on tumor cells and in the tumor microenvironment. PD-L1 expression has been reported in various cell populations within the tumor, and its expression associated with prognosis for various tumors. Further, clinical studies have shown that this pathway is an important target for immunotherapy and PD-L1 expression on tumor cells and in the tumor microenvironment has been associated with enhanced response. Understanding PD-L1's complex biological function not only on tumor cells but also within the tumor microenvironment requires simultaneous interrogation of multiple biomarkers, ranging from cancer immunology checkpoint markers, tumor infiltrating immune cell markers, and tumor specific markers, etc. Multiplex immunohistochemistry (IHC) allows simultaneous detection of multiple markers to explore the potential cellular composition of immune/stromal/cancer cells in tumor microenvironment. Development of a multiplex IHC assay remains challenging due to antibody species similarity and cross reactivity, stability of fluorophores through multiple rounds of processing, balancing high and low signals and measurement of weakly expressed markers. We present here the development of a fully automated multiplex IHC assay (PD-L1, CD3, CD8, CD68 and FoxP3) using rabbit primary antibodies with a heat deactivation process between each antigen staining cycles on the BenchMark ULTRA automated slide stainer. As part of the technology validation, we compared the 5-plex IHC to the respective single-plex chromogenic IHC assays. Using the automated 5-plex fluorescent IHC assay, we tested a cohort of non-small cell lung (NSCLC) and bladder cancer tissue specimens and characterized PD-L1 and immune marker expression in both tumor and infiltrate immune cells. To provide an objective and reliable readout of the assay, image analysis tools are being developed for automated identification and quantification of the labelled biomarkers and their co-expression on a cell-by-cell basis. This automated multiplex PD-L1 5-Plex IHC assay could be utilized as a tool for further characterizing tumors and its microenvironment and gain a better understanding of which patients may benefit from immune-therapies.

#5118

5'-deoxy-5'-methylthioadenosine (MTA) impairs human T-cell functions and constitutes a novel immuno-suppressing tumor metabolite.

Carolin Strobl,1 Frederik Henrich,1 Marina Kreutz,2 Anja-Kathrin Bosserhoff,3 Andreas Mackensen,1 Michael Aigner1. 1 _University Hospital Erlangen, Erlangen, Germany;_ 2 _Universityhospital Regensburg, Regensburg, Germany;_ 3 _University Erlangen-Nuremberg, Erlangen, Germany_.

Establishment of an immunosuppressive microenvironment is one mechanism of tumor immune escape. Elevated levels of 5'-deoxy-5'-methylthioadenosine (MTA) were detected in various tumor entities and spurred interest as putative immuno-inhibitory metabolic dysregulation. Loss of MTA phosphorylase (MTAP) activity in tumor cells was identified as major cause of MTA accumulation. Lack of MTAP expression is associated with an inferior response towards adjuvant interferon-α therapy and a higher risk for metastatic disease in malignant melanoma. Here we examined the role of tumor-secreted MTA in suppression of anti-tumor T cell responses.

The effect of MTA was investigated in human polyclonal or antigen-specific T cells. We evaluated proliferation, viability, activation, differentiation, clonal expansion and effector function of the T cells as well as the phosphorylation of major signaling pathways. In addition, co-culture experiments of T cells with an MTA-secreting tumor cell line were performed. Finally, we used retroviral transduction to generate MTAP-overexpressing T cells as a strategy to overcome MTA-mediated inhibitory effects.

MTA strongly reduced proliferation, expression of activation markers, viability, induction and expansion as well as differentiation and effector functions of antigen-specific T cells in a dose dependent manner. Moreover, MTA-mediated suppression could be confirmed using an MTA-secreting tumor cell line, which impaired T cell proliferation in mixed lymphocyte-tumor cell cultures. Mechanistically, we found MTA to interfere with different T cell signaling pathways, with the most noteworthy influence on AKT phosphorylation, thereby explaining the reduction in proliferation and viability. Moreover, we found that MTA also influences intracellular protein methylation, also a critical factor for proper T cell function. To address these broad inhibitory functions of MTA as tumor metabolite we aimed to enhance T cell resistance to MTA by equipping T cells with higher levels of the MTA catabolizing enzyme MTAP. We thus successfully generated stable MTAP-overexpressing T cells, which revealed less sensitivity against MTA mediated effects such as inhibition of proliferation, cytotoxicity, or viability upon co-culture with MTA when compared to mock controls. These results suggest a promising approach to reconstitute T cell function in presence of a normally inhibitory tumor microenvironment.

Our data emphasizes the importance of tumor metabolites such as MTA in the tumor microenvironment for tumor immune escape. Furthermore, we were able to identify potential molecular mechanisms for MTA induced T cell inhibition, which offer the opportunity to study pharmacological approaches to overcome tumor induced immune inhibition in T cells and will help to develop more effective immune-based therapies against MTAP-deficient tumors.

#5119

Complex, 3D tissues for modeling the immune response in cancer and predicting the activity of immunotherapies.

Tessa M. DesRochers,1 Lillia Holmes,1 Qi Guo,1 Lauren O'Donnell,1 Stephen Shuford,1 Larry Puls,2 Jeff Elder,2 Jeff Edenfield,2 Hal E. Crosswell1. 1 _KIYATEC, Inc., Greenville, SC;_ 2 _Greenville Health System, Greenville, SC_.

The immune system plays an active role in both the prevention and the promotion of cancer dependent upon its interaction with the tumor cells. The roles of both macrophages and T-cells in cancer progression have been heavily studied over the past few years. Macrophages have been found to be either tumor promoting or tumor preventing depending upon their differentiation status and the tumor microenvironment while the homing and cell destroying capabilities of T-cells have been manipulated to effect better, more specific tumor cell cytotoxicity through the development of therapies such as chimeric antigen receptor T-cells (CAR-T cells). Unfortunately the majority of research in the area of immune-oncology has relied upon either 2D cell culture or animal models. While a large amount of information has been learned from these models, it has been well established that 2D cell culture does not mimic in vivo biology and the immune system of mouse models differs from that of humans in numerous ways including T-cell subsets, cytokine receptors, and costimulatory molecule expression. To overcome these limitations, we have developed a number of 3D in vitro tissue models including multi-cell type models of glioblastoma (GBM), breast, and ovarian cancer. Our GBM model combines tumor cells, endothelial cells, and macrophages; the ovarian cancer model includes patient-derived tumor cells and macrophages; our breast cancer model incorporates 5 cell types including tumor cells, fibroblasts, adipocytes, endothelial cells, and macrophages. We have also begun preliminary work to incorporate patient-specific T-cells into these models. Preliminary work in GBM and breast has utilized a combination of cell lines and primary cells while ovarian modeling has relied solely upon primary patient cells from either solid tumors or ascitic fluid. These models have been characterized for tumor cell viability, biomarker expression, and drug response. We have noted changes in both tumor cell viability and biomarker expression dependent upon macrophage differentiation and have also observed the reciprocal effect of tumor cells upon the macrophages. Additionally, employing multiphoton microscopy we have identified the incorporation of macrophages into the 3D microtumors. Utilizing primary patient tumor cells and immune components, we can test these models for patient-specific responses to not only traditional chemotherapeutics, but also immunotherapies such as CAR-Ts and antibody drug conjugates.

#5120

The novel claudin-low molecular subtype of high-grade urothelial bladder cancer is highly immunogenic yet immunosuppressed.

Benjamin G. Vincent, William Kim, Jordan Kardos, Shengjie Chai, Joel Parker, Lisle Mose, Sara Selitsky, Michael Iglesia, Matthew Milowsky. _University of North Carolina at Chapel Hill, Chapel Hill, NC_.

Rationale: High-grade urothelial carcinoma of the bladder is a heterogeneous disease, with molecular subtypes characterized by distinct tumor biologies and prognoses. Our group and others have described the basal and luminal subtypes, and we now report on the discovery of the claudin-low subtype, all of which resemble analogous subtypes in breast cancer.

Methods: We analyzed mRNA sequencing and whole exome sequencing data for The Cancer Genome Atlas (TCGA) bladder tumors and tumors collected at UNC. We performed unsupervised hierarchical clustering on relative gene expression values with significance testing using SigClust to identify the claudin-low subtype. Basal, luminal, and claudin-low bladder tumors were compared for enrichment of genetic features (single nucleotide and copy number variation), gene set expression, immune gene signature expression, T cell receptor (TCR) and B cell receptor (BCR) gene segment expression, and number of predicted MHC Class I and Class II neoantigens. We further report on the first use of our VDJician software to reconstruct full-length rearranged BCR sequences from short-read RNA sequencing data in bladder cancer.

Results: Claudin-low bladder tumors were defined by low expression of tight junction claudin-proteins, high expression of epithelial-to-mesenchymal transition genes and immune gene signatures, and were associated with reduced overall survival compared to luminal tumors. A minimal gene set classifier to identify claudin-low tumors showed some but not complete overlap with an optimal classifier derived in breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features: increased rates of RB1, EP300, and NCOR1 mutations, increased frequency of EGFR amplification, decreased rates of FGFR3, ELF3, and KDM6A mutations, and decreased frequency of PPARG amplification. Claudin-low tumors showed the highest expression of immune gene signatures (including an immunosuppression signature), however in contrast to basal tumors, increased immune gene signature expression was not associated with prolonged survival. Claudin-low tumors also showed the highest overall expression of rearranged BCR sequences but lowest BCR repertoire diversity. Predicted neoantigen burden did not vary significantly by subtype, however broad cytokine and chemokine expression levels were elevated in claudin-low tumors, potentially related to low PPARG activity driving increased NFKB activity.

Conclusions: Claudin-low bladder cancer is a novel molecular subtype with distinct molecular and immunologic features and prognostic significance. Given the presence of dense immune infiltrates, BCR repertoire characteristics consistent with an antigen-driven response, and high expression of immunosuppression genes, claudin-low bladder tumors may be primed to respond to immune checkpoint inhibitor therapy.

#5121

Immunomodulating cytokine profiling of docetaxel-treated human prostate cancer specimens.

Debapriya G. Mehrotra,1 Nilesh Brijwani,1 Biplab Tewary,1 Vasathkumar Sekar,1 Abhishek Krishnan,1 Dency D. Pinto,1 Muthusamy Oliyarasi,1 Biswanath Majumder,1 Saravanan Thiyagarajan,1 Padhma Radhakrishnan,2 Pradip K. Majumder1. 1 _Mitra Biotech, Bangalore, India;_ 2 _Mitra Biotech, Boston, MA_.

Prostate cancer is one of the leading malignancies in adult males worldwide and existing therapies often result in treatment failure and emergence of castration resistant prostate cancer (CRPC). Outcome of some recent clinical trials highlighted the benefit of Docetaxel as a part of initial therapy in CRPC. However, a significant number of patients still remain chemoresistant. Context dependent response profiling using a clinically oriented functional platform would further improve this scenario. Here, we elucidated the role of different immune cells and type 1 and type 2 cytokines in improving Docetaxel sensitivity to prostatic carcinoma. We tested Docetaxel sensitivity in prostate tumors (n=10) using CANScriptTM platform, a personalized next generation functional testing tool (Majumder B et al. Nat. Commun 2015, Goldman A et al. Nat. Commun. 2015). Of these 10 tumors, 4 were found to be sensitive to Docetaxel and 6 were insensitive. The immune profiling of these tumors suggested that despite preferential CD45, CD8 and CD4 levels in majority of the responders both responders and non responders display modest numbers at baseline. We also observed similar heterogeneity in CD69 (T cell activation marker) in these two groups. Altered expression of cytokines was observed in Docetaxel sensitive and resistant tumors upon their co-culture with peripheral blood mononuclear cells (PBMC), suggesting a defined role of these cytokines in Docetaxel response. We extended the study further by testing the efficacy of PD-1/ PD-L1 inhibitors in Docetaxel resistant tumors. Our results provide the basis for validation of immune phenotypes and cytokines as therapeutic response biomarkers in Docetaxel treated prostatic tumors.

1. Majumder B et al. Nat. Commun.6:6169 (2015).

2. Goldman A et al. Nat. Commun. 6:6139 (2015).

#5122

Characterization of the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets on cell lines used for translational research.

Bruce A. Adams, James Lim, Luke Cassereau, Tianna Chow, Susan Bernstein. _Xcell Biosciences Inc., San Francisco, CA_.

INTRODUCTION

The characterization of the tumor microenvironment includes mechanotransduction, hypoxia, acidosis, and tissue remodeling. These factors can influence the gene and protein expression of various cell types that make up the tumor as well as influence the selection of cells that can thrive in a given microenvironment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native extracellular matrix (ECM). We designed a system to study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating culturing environments relevant for translational studies involving key immunotherapeutic targets.

METHODS

First, we studied how hypoxia with or without pressure influences cell biology and gene expression, using transcriptome profiling across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body. To do this, we utilized Xcell's primary cell culture platform that allows for the precise control of oxygen concentration (0.1%-20% O2) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig). Additionally, we studied the influence of biomimetic substrates by ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml). Cell lines studied include models for brain (U-87, A172), pancreatic (PANC10.05), and prostate cancer (DU-145, PC-3, 22Rv1, LNCaP). We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of key targets, including immunotherapeutic targets CTLA-4, PD-1, and PD-L1. Finally, we applied these culturing conditions to characterize primary tissues obtained from cancer patients for studies focused on biomarker discovery, patient monitoring and treatment decision-making.

CONCLUSIONS

We identified both common and unique gene expression signatures across different cells lines, with hypoxic conditions activating HIF1 signaling, whereas hydrostatic pressure resulted in restricted signatures of high clinical value. Cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key drug targets. Analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and immune cells. Therefore, these results support the presence of physiologically-relevant drug targets in tumors and immune cells characterized by low oxygen and high interstitial fluid pressure.

#5123

Multiplex in situ immunoprofiling and survival outcomes in non-small cell lung cancer.

Steven H. Lin,1 James R. Mansfield,2 Xiayu Rao,1 Wen Jiang,1 Penny Fang,1 Kristin Roman,2 Chichung Wang,2 Ritsuko Komaki,1 Stephen Hahn,1 Stephen G. Swisher,1 Junya Fujimoto,1 Ignacio Wistuba,1 Jing Wang,1 Clifford C. Hoyt2. 1 _UT MD Anderson Cancer Center, Houston, TX;_ 2 _PerkinElmer, Inc., Hopkinton, MA_.

The clinical activity of immune checkpoint blockade ("immunotherapies") in non-small cell lung cancer (NSCLC) has highlighted the importance of the immune system in controlling cancer growth. Immune cells play a central role in determining response to immunotherapies, yet obtaining phenotypic information about the various immune cells in intact tissue sections and measuring cell-to-cell interactions is challenging. For this study, we applied multiplex immunofluorescence imaging to simultaneously resolve both phenotypic and morphologic information in order to determine the immune subset in the tumor -microenvironment that dictated the prognosis of NSCLC. One-hundred-twenty nine non-metastatic NSCLC tumors were studied using Tissue Microarray (TMA), with each tumor represented in triplicate. Majority of the patients were females (56%), had stage 2 (11%) or stage 3 (84%) disease, and all patients had primary resection and postoperative radiotherapy. TMA sections were stained for CD4, CD8, CD68, FOXP3, CD274 (PD-L1) and cytokeratin, with a DAPI counterstain, using a sequential multiplex IHC approach involving antibody stripping and tyramide signal amplification. Slides were imaged using an automated multispectral microscope. We determined cellular phenotype counts and per-cell marker expression in tumor and stromal areas in each core of the TMA, using machine learning-based pattern recognition and cell classification approaches. On average, 4,770 cells were analyzed in each TMA core. The densities of phenotyped cells (counts/mm2) for tumor and stroma were calculated. We analyzed the tumor to stroma ratios in each core for each of the immune subsets (CD8, CD4, FoxP3+ Treg, and CD68+ macrophages). In multivariate analysis, after controlling for patient and tumor characteristics, high CD4 tumor:stroma ratio predicted significantly for poorer overall survival (OS) (5 year OS of CD4 high: 27% vs. CD4 medium/low: 50%, p=0.001). High CD4 content also correlated significantly to high FoxP3+ Treg levels (p<0.0001, Fisher's Exact test) and lower PD-L1 expression in the tumor (p=0.038). This initial data suggests the immunosuppressive tumor microenvironment, as reflected by high tumor CD4 content, portends to poor prognosis in NSCLC patients. This is an example of assessments enabled by data sets such as this, that include per-cell expression of checkpoint proteins that retain the exact cellular locations of the various cell phenotypes within the microenvironment context. We will present analyses that explore more in depth on the immuno-biology cell-to-cell interactions, including associations between PD-L1 expression and outcomes. This study highlights the power of multiplex immunofluorescence characterization to unravel the complex immunologic network within the tumor microenvironment that may not only be prognostic but also predictive of immunotherapy responsiveness.

#5124

Tumor and immune cell infiltration are enhanced by irradiation of normal tissues in immunocompromised mice.

Marjan Rafat, Marta Vilalta, Todd A. Aguilera, Amato J. Giaccia, Edward E. Graves. _Stanford University, Stanford, CA_.

Breast cancer recurrence remains high in triple-negative cases despite aggressive surgical, radiological, and chemotherapeutic intervention. Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence. However, the tumor microenvironment's role in recurrence is not well understood. We hypothesize that the irradiated tumor stroma and microenvironment may influence tumor cell migration. In this study, we characterize the effects of normal tissue irradiation on tumor and immune cell migration to evaluate how tumor-stromal interactions modulate recurrence after therapy. This work represents the first step toward elucidating how stromal tissue radiation response contributes to tumor and immune cell recruitment.

Mouse embryonic fibroblasts (MEF) were irradiated to 20 Gy with a cesium source. Supernatant was collected after 2 or 7 d incubation to be used as a chemoattractant in a transwell assay to investigate the induction of 4T1 murine or MDA-MB-231 human mammary carcinoma cell invasion. An orthotopic breast cancer model was used to evaluate the effect of radiation on tumor cell migration to normal tissues. Nude mice were inoculated with luciferase labeled 4T1 or MDA-MB-231 cells in the mammary fat pad (MFP), and BALB/c mice with depleted CD4 and CD8+ T cells were inoculated with 4T1 MFP tumors. The contralateral normal MFP was irradiated to a dose of 20 Gy with a 250 kVp cabinet x-ray machine when tumors were palpable. Cell migration was monitored with bioluminescence imaging (BLI) 10 d after irradiation. Irradiated and control tissues were evaluated using immunohistochemistry (IHC). Tissue sections were stained with F4/80 to determine the extent of macrophage infiltration. Flow cytometry was also performed on dissociated irradiated and control tissues to characterize immune cell populations. Of particular interest were CD11b+F4/80+ macrophages and CD11b+GR1+ myeloid-derived suppressor cells (MDSCs).

Radiation enhanced tumor cell migration to normal tissues both in vitro and in vivo. 4T1 and MDA-MB-231 cells exhibited an increase in invasion (p<0.05) in vitro when exposed to irradiated MEF media. Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the MFP and surrounding tissues, including the peritoneum and muscle (p<0.001), in nude and BALB/c mice with depleted CD4 and CD8+ T cells. An increase in macrophage infiltration was found in irradiated tissue sections. Flow cytometry confirmed increases in macrophage and MDSC infiltration in irradiated areas.

This study establishes that normal tissue radiation response may play a role in modulating tumor and immune cell migration after radiation. The increase in macrophage and MDSC infiltration after irradiation indicates the immune contribution in tumor cell migration. These results suggest that the tumor stroma may facilitate tumor cell invasion and tumor regrowth following radiotherapy.

#5125

Immunomodulatory activity of bitter melon extract in head and neck squamous cell carcinoma.

Sourav Bhattacharya, Robert Steele, Naoshad Muhammad, Guangyong Peng, Ratna B. Ray. _Saint Louis university, Saint Louis, MO_.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and leading cause of cancer related mortality worldwide. Despite the advancement in treatment procedures the overall survival rate of patients has not considerably enhanced in the past few decades. Therefore, new strategies to achieve a favorable response for the improvement in the prognosis of HNSCC are urgently needed. In this study we examined the immune modulatory activity of bitter melon extract (BME) in HNSCC tumor microenvironment. For this, mouse head and neck cancer (SCCVII) cells were subcutaneously injected into the flanks of C3H mice. BME was fed orally, and tumor growth was evaluated. We observed that BME treatment significantly inhibits the tumor growth in BME fed mice as compared to control group. Flow cytometry data showed that BME treatment not only decreases the infiltrating T regulatory (Treg) cells in the tumor but also in splenocytes. Additionally, BME treatment reduces Th17 cell population in the tumor and augments in the spleen. However, BME treatment did not alter Th1 and Th2 populations. Further study suggested that BME inhibits cell proliferation as evident from low expression of PCNA and c-Myc in the tumors of BME fed mice as compared to that of control group. To our knowledge, this is the first report showing BME exerts immunomodulatory effect in regressing HNSCC tumor growth in preclinical model.

#5126

The link between therapy-induced senescence and anti-tumor immune microenvironment in melanoma.

Anna E. Vilgelm,1 C Andrew Johnson,1 Nripesh Prasad,2 Jinming Yang,1 Sheau-Chiann Chen,3 Gregory D. Ayers,3 Jeffrey A. Sosman,3 Jeffrey A. Ecsedy,4 Shyr Yu,3 Shawn E. Levy,2 Ann Richmond1. 1 _Vanderbilt University and TVHS, Nashville, TN;_ 2 _HudsonAlpha Institute for Biotechnology, Huntsville, AL;_ 3 _Vanderbilt University, Nashville, TN;_ 4 _Takeda Pharmaceuticals International Co, Cambridge, MA_.

Tumor cell senescence is often induced by cancer therapeutics. Senescent cells secrete many proteins that may affect both malignant and non-malignant components of the tumor. The goal of this study was to determine how therapy-induced senescence (TIS) affects the immune microenvironment in melanoma. We used inhibitors of cell cycle kinases AURKA and CDK4/6 (AURKAi, CDK4/6i) to induce senescence in melanoma tumors. RNA sequencing analysis of AURKAi-treated syngeneic mouse tumors demonstrated that response to AURKA inhibition was strongly associated with induction of an immune transcriptome (p=3.5E-29). Immunofluorescent staining and flow cytometric analysis of digested tumors showed correlation between the numbers of tumor-infiltrating leukocytes (TILs) and AURKAi response (Spearman r=-0.87, p<0.001). T cells were among the TILs recruited into AURKAi-sensitive tumors. Therapeutic benefit of AURKAi was limited in immunodeficient host (67% of tumors regressed in immunocompetent mice vs 0% in immunodeficient mice, p=0.01) suggesting that recruitment of TILs augments response to senescence-inducing therapy. We found that TILs were driven into tumors in response to chemokine CCL5 that was induced in AURKAi and CDK4/6i-treated melanoma cells (up to 20 fold, p≤0.005) on mRNA and secreted protein levels. Knockdown of CCL5 (KD) inhibited leukocyte recruitment (6.8% in KD vs 29.5% in control, p=0.002) and response to AURKAi in vivo (p=0.61 in KD vs p=0.02 in control). In contrast, therapeutic benefit was greatly enhanced when senescence-inducing therapy was combined with T cell-activating immunotherapy using CD137 (4-1BB) agonist (p<0.001). We also evaluated CCL5 expression in human melanoma tumors in relation with TIS and TIL markers. Expression of CCL5 was induced by AURKAi and/or CDK4/6i in patient derived xenografts (3 patients, 3 mice each, p≤0.02), in tumors from patients who responded on AURKAi clinical trial (3 patients, before/after therapy) and was associated with expression of "Immune response" genes in The Cancer Genome Atlas (TCGA) dataset of 278 melanoma tumors (p=1.40E-93). In conclusion, our data demonstrates that TIS promotes recruitment of TILs via the induction of CCL5 which, in turn, augment therapeutic response in melanoma. Further improvement of melanoma outcome is achieved when senescence-inducing drugs are combined with immunotherapy that promotes tumor cells killing by TILs. This study presents a novel promising approach to improve melanoma therapy by utilizing TIS to reprogram tumor immune microenvironment.

#5127

Clinical verification of antitumor autoimmune response in eribulin chemotherapy for breast cancer.

Wataru Goto,1 Shinichiro Kashiwagi,1 Yuka Asano,1 Kento Kurata,1 Tamami Morisaki,1 Satoru Noda,1 Tsutomu Takashima,1 Naoyoshi Onoda,1 Sayaka Tanaka,2 Masahiko Ohsawa,2 Masaichi Ohira,1 Kosei Hirakawa1. 1 _Department of Surgical Oncology, Osaka City University, Abeno-ku, Osaka, Japan;_ 2 _Department of Diagnostic, Osaka City University, Abeno-ku, Osaka, Japan_.

Background: The immune environment of the cancer tissue not only influences the response to immunotherapy, but also the response to and outcomes after other anti-cancer therapies. Thus, the importance of regulating and improving the cancer immune microenvironment is increasingly being recognized. Tumor-infiltrating lymphocytes (TILs) can be used to monitor the immune response and are important in predicting treatment responses and outcomes in various types of carcinoma. On the other hand, it was reported that eribulin suppresses epithelial-mesenchymal transition (EMT) in basic studies, and we investigated it with clinical

samples. In addition, it is thought that antitumor autoimmune response relates to suppression of EMT. In the present study, we investigated clinically antitumor autoimmune response in breast cancer with eribulin chemotherapy.

Materials and Methods: Eribulin chemotherapy was administered to 52 patients with locally advanced or metastatic breast cancer. Samples of cancer tissue obtained before and after treatment were available from 10 patients. We evaluated response rate (RR) and analyzed protein expression by immunostaining of specimens before and after treatment. We evaluated antitumor autoimmune response by PD-1, CD8, FOXP3 expression in stromal TILs and PD-L1, PD-L2 expression in cancer cells. And, we examined the relation between the expression of these immune related molecules and EMT markers (evaluated by E-cadherin, N-cadherin, Vimentin

expression).

Results: In 11 patients (21.2 %), it was possible to excise the lesion after treatment. In 10 evaluable cases without one pathological complete response (pCR) case, PD-1, PD-L2, and FOXP3 expression turned to negative in 5 cases, PD-L1 expression turned to negative in 6 cases, and CD8 expression turned to positive in 3 cases before and after treatment with eribulin chemotherapy. Looking at the relation between the transition in this protein expression and therapeutic effect, cases observed with negative conversion in PD-L1, FOXP3 expression had significantly high RR (p=0.024) (p=0.004). Among high RR cases, CD-8 expression was increased, the PD-1, PD-L1 and FOXP3 expression were remarkably reduced. Furthermore, we found a inverse correlation between PD-L1, FOXP3 expression turning to negative and E-cadherin expression turning to positive (p=0.024) (p=0.004).

Conclusions: In the chemotherapy with eribulin, PD-L1 and FOXP3 expression turning to negative before and after treatment suggested improvement of the tumor immune microenvironment, and this mechanism related to suppression of EMT.

#5128

Characterization of tumor immune microenvironment in response to PD-L1 therapy initiated at different time points post inoculation in syngeneic mouse models.

Geeta Sharma, Susan Wu, Robert Mihalek, Ann Fiore, Matthew Gale, Christa Dias, Wendy Grant, Nidia Hernandez. _Agilux Labs, Worcester, MA_.

The tumor microenvironment is an important aspect of cancer biology that contributes to tumor initiation, progression and responses to therapy. Cells and molecules of the immune system are fundamental components of the tumor microenvironment which have been known to vary widely and are important in determining the anti-tumor immune response. The approval of immune checkpoint inhibitors like PD-1 and CTLA-4 antibodies has resulted in a lot of research activity in immune-oncology. Syngeneic mouse models have been utilized to understand the preclinical immune response as a surrogate to the clinical response. The timing of immune checkpoint inhibitor dosing is supposed to play an important role on the tumor growth in mouse models. In order to understand the correlation of tumor regression, survival and immune response in a preclinical mouse model, we evaluated the effect of different times of treatment initiation with Program Death Ligand-1 (PD-L1) on tumor volume, survival and tumor infiltrating leukocytes (TIL's). Various dosing regimens evaluated included initiation of PD-L1 therapy on the day of inoculation, 6 and 12 days post inoculation. Tumors were collected from three different syngeneic mouse models (CT26, RENCA and B16F10) and profiled for the presence of tumor infiltrating leukocytes (TIL's) using flow cytometry. Major sub-population of TIL's examined included T-regulatory cells (Treg's), Tumor Associated Macrophages (TAM's) and Myeloid Derived Suppressor Cells (MDSC's). Finally, an attempt was made to identify any correlation or lack thereof between tumor growth and tumor immune microenvironment.

#5129

Delineating immune networks in colorectal cancers to predict effects of immune checkpoint inhibitors using CANScript platform technology preserving tumor immune microenvironment.

Pradip K. Majumder,1 Nilesh Brijwani,2 Nikita N. Karandikar,2 Vinod D. Radhakrishna,3 Muthusamy Oliyarasi,3 Dency D. Pinto,3 Babu Balakrishnan,3 Vasathakumar Sekar,3 Manoj Rajappa,3 Debapriya G. Mehrotra,3 Priyanka Chevur,4 Priyanka Chevur,4 Arkasubhra Ghosh,4 Saravanan Thyiagarajan,3 Padhma Radhakrishnan,1 Biswanath Majumder2. 1 _Mitra Biotech, Boston, MA;_ 2 _Mitra Biotech Pvt Ltd, Bangalore, India;_ 3 _Mitra Biotech, Bangalore, India;_ 4 _Grow Research Lab, Bangalore, India_.

Colorectal cancer (CRC) is one of the leading causes of cancer related mortality. Recent findings from clinical studies showed that immune checkpoint inhibitors are making good progress towards clinical practise. However, existing biomarkers and 3D platforms are not equipped to offer reliable prediction of response of these drugs. We recently engineered a personalized ex vivo systems biology based platform using patient tumors called CANScriptTM (Majumder B et al Nat. Commun. 2015, Goldman A et al Nat. Commun. 2015) and a companion in vivo patient derived immune reconstituted xenograft model (Mi-HTX) preserving multiple critical phenotypic profiles orchestrating tumor-immune cross talk and dysfunction. We further performed baseline characterization of immune markers (CD4, CD8, CD68, CXCR4 and CD45-RO). Intratumor heterogeneity of both M1 and M2 phenotypes were observed irrespective of clinico-pathological features. Baseline CD68 enrichment status was found to be correlated with therapy failure for some patients where clinical outcome (PERCIST) was available. Flow cytometry and microarray analysis showed preservation of diverse sets of functional CD4, CD8 and NK phenotypes along with degranulation markers (CD107a), IFN-γ and FoxP3 in both in vivo and ex vivo tumors reconstituted with ex vivo primed PBMC. The comparative response profiling of these models following treatment with immune checkpoint inhibitors suggested enhanced antitumor effects (as measured by multiple functional parameters like viability, proliferation, morphology and apoptosis). Together, these findings highlight the strengths of immune-competent functional testing tools to stratify patients for precision immunotherapy where direct response prediction biomarkers are still elusive.

1. Majumder B et al. Nat. Commun.6:6169 (2015).

2. Goldman A et al. Nat. Commun. 6:6139 (2015).

#5130

Immune profiling of malignant effusion from metastatic gastric cancer patients as a predictive biomarker for immune checkpoint inhibitors.

Woo Sun Kwon,1 So Jung Lim,1 Won Suk Lee,1 Eunji Jo,1 Hyun-jeong Kim,1 Kyung Hee Lee,2 Hyun Cheol Chung,3 Sun Young Rha3. 1 _Song-Dang Institute for Cancer Research, Yonsei University College of Medicine, Seoul, Republic of Korea;_ 2 _Department of Hematology-Oncology, Yeungnam University Medical Center, Daegu, Republic of Korea;_ 3 _Song-Dang Institute for Cancer Research, Brain Korea 21 PLUS Project for Medical Science, Division of Medical Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei University Health System, Seoul, Republic of Korea_.

Many types of cancers are able to circumvent immune surveillance by the host's immune system. It has been well established that many tumor infiltrating T cells are in a state of non-responsiveness due to the immune suppressive tumor microenvironment. The main mechanisms of T cells that confer suppressive anti-tumor immune response in tumor microenvironment could be regulatory T cells (Treg) and exhausted T cells. In advanced stage cancer patients, detailed evaluation of Treg cells in tumor microenvironment is hampered by limitation for enough tumor tissue, because surgical resection for sample is not standard treatment in these patients. In order to overcome this limitation of research, we performed the studies using lymphocytes and tumor cells isolated from malignant effusion including pleural effusion or ascites from advanced gastric cancer patients.

We collected 25 malignant effusion specimens from advanced gastric cancer patients, including 20 paired peripheral blood samples. Lymphocytes from malignant effusions and peripheral bloods were analyzed for subtype of Memory, Activated T cell and Treg cells, and expressions of inhibitory molecule using by flow cytometry. To evaluate the expression of PD-L1, we used the tumor cells of malignant effusions.

Firstly, the percentage of CD8+ T cells in malignant effusion seems to increase compared to those in peripheral blood, but the percentage of CD4+ T in malignant effusion were significantly lower than those in peripheral blood. Interestingly, the percentage of CD45RO+ (Memory) on CD4+ and CD8+ T cells was significantly higher in malignant effusion than peripheral blood. And Early activated molecule (CD69 and CD25) on CD4+ and CD8+ T cells was significantly higher in malignant effusion than peripheral blood. The percentage of PD-1 expression on CD8+ T cells was significantly higher in malignant effusion than peripheral blood (p=0.017). And the percentage and MFI of Treg cells (Foxp3+/CD4 T cell) tend to increase in malignant effusion was comparable with those of peripheral blood but did not show the statistical significance. Collectively, we found that T cells derived from malignant effusion demonstrated the exhausted phenotype showing highly expression of PD-1, compared to those from peripheral blood. Finally, the PD-L1 expression on tumor cells from malignant effusions was 0.04~12.78%. The upregulation of PD-1 on Treg cells and PD-L1 on tumor cell of malignant effusions could be a potential predictive marker and therapeutic target for immune checkpoint blockade.

#5131

The XL313 cryptic collagen epitope regulates immune checkpoint molecules by a αVβ3-integrin-associated mechanism.

Jennifer M. Caron, Liangru Contois, Jacquelyn Ames, Peter C. Brooks. _MMCRI, Scarborough, ME_.

Proteolytic remodeling of extracellular matrix (ECM) results in structural changes that facilitate the generation of cryptic regulatory sites that promotes angiogenesis, tumor growth and metastasis. While alterations in the biophysical characteristic of the ECM can help create a tumor permissive microenvironment, little is known concerning whether structural changes in the ECM contributes to the ability of tumors to escape immune control. Molecular insight into the signaling pathways operating in stromal cells has contributed to the development of new cancer therapies. While clear progress has been made, as indicated by the recent approvals of new therapies such as immune checkpoint inhibitors, the overall survival of patients with metastatic disease remains alarmingly low. Accumulating evidence suggests that stromal components of the tumor microenvironment may contribute to the development of multiple resistance mechanisms including adaptive immune resistance. Thus, there is an urgent need for a more detailed understanding of how immune and inflammatory mechanisms govern tumor progression in order to enhance long-term durable responses with current therapies in a larger percentage of patients.

Tumor-associated macrophages (TAMs) have been suggested to play roles in tumor growth and metastasis by multiple mechanisms including structural remodeling of the ECM. TAMs may also contribute to the development of resistance to current anti-cancer therapies. Our studies indicate that distinct subsets of macrophage may facilitate the generation of the RGDKGE containing XL313 cryptic collagen epitope that promotes angiogenesis and inflammation in vivo. Here we provide the first evidence that cellular interactions with the XL313 collagen epitope may regulate immune checkpoint molecules by a αVβ3 integrin-associated mechanism. Cellular interactions with the XL313 epitope and denatured forms of collagen, which are present within the tumor microenvironment, enhanced the levels of the immune checkpoint molecules PD-L1 and LAG-3. Selective targeting of the XL313 collagen epitope with a monoclonal antibody inhibited tumor growth and metastasis and tumors from these mice exhibited reduced levels of immune checkpoint molecules. Importantly, anti-XL313 epitope antibody significantly enhanced the anti-tumor activity of anti-PD-L1 therapy in vivo. These data suggest that the XL313 epitope may play a functional role in promoting immune suppression in tumors and that selective targeting of this cryptic collagen epitope may reduce immune suppression and significantly enhance the efficacy of immune checkpoint inhibitors. Taken together, our studies are consistent with the possibility that the endogenously generated XL313 epitope may regulate tumor growth in part by facilitating the escape of tumors from immune control.

#5132

IDH mutation-induced suppression of type-1 anti-glioma immune responses.

Gary Kohanbash,1 Brian Ahn,2 Shruti Shrivastav,1 Diego Carrera,1 Joseph Costello,1 Hideho Okada1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _University of Pittsburgh Medical Center, Pittsburgh, PA_.

Isocitrate dehydrogenase (IDH) mutations are the first mutations that occur during the oncogenic process of lower-grade glioma (LGG) and confers a novel gain-of-function activity by converting α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), promoting DNA hyper-methylation. Our analysis of LGG cases from The Cancer Genome Atlas (TCGA) database revealed that IDH-mutant (IDH-Mut) cases exhibit decreased expression of type-1 effector T-cell response-related genes, which are critical for anti-glioma immunity, including: CD8A, IFNG, OAS2, GZMA, EOMES, CXCL9 and CXCL10, compared with IDH-wild type (IDH-WT) cases. On the other hand, type-2 and regulatory T-cell response-related genes, such as IL5 and TGFB1, are not significantly different between IDH-Mut vs. WT cases, indicating that the observed down-regulation of type-1 response-related genes does not merely represent a possible global gene suppression. Furthermore, IDH-Mut cases exhibit increased CXCL10 promoter methylation compared with WT cases. We thus hypothesized that IDH mutation-mediated tumor intrinsic mechanisms occurring within glioma cells may inhibit anti-tumor immunity to promote tumor growth. In vitro, a normal human astrocyte (NHA) cell line transfected with IDH1-Mut cDNA expressed lower levels of CXCL10 compared to NHA cells transfected with WT IDH1. Consistently, C57Bl/6 mouse-syngeneic astrocyte and glioma cell lines transfected with IDH1-Mut expressed lower levels of CXCL10 gene and protein, compared to control cells transfected with IDH-WT, which was restored following 30 day treatment of the cells with the IDH1 inhibitor, IDH-C35. Furthermore, in vivo orthotopic IDH1-Mut gliomas at 21 days post-intracranial injection in syngeneic mice expressed lower levels of T-cell chemokines CXCL9 and CXCL10 as determined by RT-PCR and ELISA and reduced infiltration of CD3+CD8+ T-cells as determined by flow cytometry and quantitative immunohistochemistry compared with control IDH1-WT gliomas. Our analyses of the TCGA 450K gene methylation data suggest that the suppressed expression of OAS2 and CXCL10 in IDH1-Mut cases is associated with hypermethylation of the promoter for these genes. Indeed, treatment of IDH-Mut cell lines with demethylating agent 5-Aza-CdR restored CXCL10 expression levels. Further, an in vitro migration assay demonstrated reduced migration of T-cells towards culture supernatants from IDH1-Mut cell lines compared with control IDH1-WT supernatants. Finally, despite loss of vaccine efficacy in IDH-mutant syngeneic gliomas, compared to controls, the use of an IDH-inhibitor, AGI-5198, robustly improved vaccine efficacy in these mice. Overall, our data demonstrate that IDH mutations in tumor cells lead to reduced T-cell attracting chemokines and reduced T-cell accumulation in gliomas. Our data suggest that IDH inhibitors and demethylation agents may be used to enhance T-cell recruitment to LGG in combination with T-cell based immunotherapies.

#5133

Targeting the eicosanoid 20-HETE suppresses pancreatic cancer growth and metastasis through regulation of inflammation.

Yael Gus-Brautbar,1 Julia Piwowarski,1 Jessica A. Casper,1 Diane R. Bielenberg,2 Jennifer Cheng,3 Birgitta Schmidt,4 Bruce D. Hammock,5 Darryl C. Zeldin,3 Mark W. Kieran,6 Dipak Panigrahy1. 1 _Center for Vascular Biology Research and Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA;_ 2 _Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 3 _Laboratory of Respiratory Biology, NIH-NIEHS, Research Triangle Park, NC;_ 4 _Department of Pathology, Boston Children's Hospital, Harvard Medical School, Boston, MA;_ 5 _Department of Entomology and Cancer Research Center, UC-Davis, Davis, CA;_ 6 _Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA_.

Pancreatic cancer incidence and death rates are on the rise. Most available therapies fail partly due to the uniquely inflammatory pancreatic tumor stromal compartment. Prolonged and unresolved inflammation is implicated in the pathogenesis of pancreatic cancer, yet the critical inflammatory mediators remain largely unknown. The role of lipid autacoids in pancreatic cancer, which are metabolites of arachidonic acid and other related fatty acids, has not been thoroughly studied. Among these, metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been mostly neglected in cancer. We previously showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. Here we show that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of ω-hydroxylases of the CYP4A/F subfamilies, stimulated pancreatic primary tumor growth and metastasis. We measured 20-HETE and other lipid autacoids via LC/MS/MS in multiple cell lines and found that 20-HETE was produced primarily by pancreatic cancer cells, which express the 20-HETE generating enzyme, CYP4F2. Overexpression of CYP4F2 and exogenous 20-HETE induced Ras activity in HEK293 cells or pancreatic cancer and macrophage cell lines, respectively. 20-HETE attracted human macrophages in vitro, while macrophage migration was reduced towards pancreatic cancer cells treated with an inhibitor of 20-HETE synthesis, HET0016. Inhibition of 20-HETE synthesis in human macrophages and pancreatic cancer cells decreased HB-EGF and M-CSF production. Systemic administration of 20-HETE promoted primary human and murine pancreatic tumor growth in subcutaneous and orthotopic murine models. Transgenic mice overexpressing 20-HETE (Tie2-CYP4F2 Tr) exhibited spontaneous liver and mediastinal lymph node metastasis following resection of subcutaneous primary pancreatic tumors, in contrast to wild type control mice. Targeting 20-HETE by HET0016 or siRNA towards CYP4F2 suppressed primary pancreatic tumor growth. Systemic treatment of orthotopic pancreatic tumors with HET0016, 14,15-EEZE, and gemcitabine regressed established pancreatic tumors and prolonged survival without overt toxicity. Thus, 20-HETE critically regulates Ras activation, inflammation, and pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for pharmacologically targeting 20-HETE as a novel approach to treat pancreatic cancer.

#5134

Evaluation of immune tolerance mechanisms in pulmonary squamous cell and adenocarcinomas - how to interfere beyond PDL1 blockade.

Helmut H. Popper,1 Luka Brcic,1 Gudrun Absenger,1 Barbara Prietl,2 Joerg Lindenmann,1 Marija Balic1. 1 _Medical Univ. of Graz, Graz, Austria;_ 2 _Competence Center for Biomarker Research, Medical University Graz, Graz, Austria_.

Introduction: Immunotherapy has emerged as a new tool to treat patients with NSCLC. Programmed death ligand 1 (PDL1) expressed on carcinoma cells serve as a marker predicting the response of anti-PDL antibody therapy. However, the results of clinical studies are conflicting as patients might profit from therapy despite not expressing PDL1 on their tumors, and also some tumors expressing PDL1 do not respond to therapy. It is evident that NSCLC cells use other mechanisms of immunotolerance.

Methods: Surgically removed specimen of lung cancer are evaluated for infiltrating innate as well as specific immune cell in tumor centers and invasive front. In addition bronchoalveolar lavage is taken simultaneously and evaluated by flow cytometry for the same immune cells as the tissues. These cell types are evaluated: dendritic cells (classical, plasmocytoid, monocytoid), macrophages (M1 and M2), lymphocytes of Bcell lineage, lymphocytes of Tcell lineage, regulatory Tcells, natural killer cells. In addition tumor cells and lymphocytes are studied for their expression of PD1, PDL1, and PDL2, respectively

Results and discussion: Within lung cancer several different mechanisms of inducing immune tolerance emerged: PD1-PDL1 interaction of tumor cells and CD8 lymphocytes, plasmocytoid as well as monocytoid dendritic cell induced downregulation of cytotoxic Tcell action, influx of myeloid derived cells inducing immune tolerance against tumor cells, M2 macrophage polarization with enhancement of stroma remodeling for tumor cell invasion and reduced Tcell reaction, increase of Treg cells inducing immune tolerance. Some of these mechanisms are seen in combination with PD1-PDL1 induced immune tolerance. This might explain, why some patients do not benefit from anti-PDL1 therapy. BAL emerged as a powerful tool in the evaluation of the immune reaction in NSCLC patients and might be a tool for evaluation of immune responses as well as a diagnostic tool to evaluate resistance mechanisms.

#5135

**Detection of IFNγ induced PDL1 expression by combined** in situ **RNA analysis and protein profiling from a single FFPE slide.**

Qingyan Au, Kathy Nguyen, Michael S. Lazare, Edward J. Moler, Nicholas Hoe. _Clarient GE Health Care, Aliso Viejo, CA_.

PD1 ligands (PDL1) are often upregulated on the cell surface of many different tumors. The primary role of PDL1 in cancer is to inhibit T-cell mediated immune response. Two general mechanisms for PDL1 expression on tumor cells have been proposed. Innate immune resistance, in which PDL1 expression is induced by the constitutive oncogenic signaling, and adaptive immune resistance, in which PDL1 expression is induced by T-cells releasing interferon-γ (IFNγ) and activating the STAT signaling pathway. In order to differentiate between these two mechanisms, IFNγ mRNA expression is measured as an effective alternative to detecting IFNγ protein. Detection of cytokines by IHC is challenging as secreted proteins are widely diffused and the associated staining pattern appears to lack cellular specificity. RNAscope RNA in situ hybridization (ISH) assay is utilized to measure Interferon-γ (IFNγ) mRNA expression, and MultiOmyxTM multiplexed assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD56, CD68, PD1, and PDL1 protein expression. In this study, combined MO and RNAscope ISH assays, enabled identification of individual cells with characteristic mRNA and protein expression profile.

The MultiOmyx assay utilizes a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each Cy-dye conjugated antibody recognizes different target proteins. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining. RNAscope is a novel RNA ISH assay capable of single-molecule detection in individual cells, utilizing hybridization mediated signal amplification. The assay utilizes a pair of RNA target specific oligonucleotide probes, which sequentially hybridize to preamplifier, amplifier, and fluorophore label probes.

Utilizing MultiOmyx and RNAscope assays, this study proposed to profile both RNA and protein expression in lung, breast, melanoma, colorectal, esophageal, and prostate cancer samples.

Differentiating PDL1 expression induced in response to inflammatory signals produced by an activated T-cell, from PDL1 expression induced by constitutive oncogenic signaling, has potential implications in effectiveness of PD1 blockade therapy. According to the proposed mechanisms, PD1 blockade as a mono therapy may only benefit individuals with strong endogenous immune response. In individuals with weak endogenous immune response, combinational therapies consisting of both immune activation and PD1-pathway blockade may be more effective than either mono therapy alone.

#5136

HER2 or EGFR inhibition interacts with tumor microenvironment by downregulation of PD-L1 and chemokines.

Jin Won Kim, Ji Hea Sung, Mi Hyun Kang, Ji-Won Kim, Se-Hyun Kim, Keun-Wook Lee, Hye Seung Lee, Jee Hyun Kim. _Seoul National University Bundang Hospital, Seongnam-si, Gyeonggi-do, Republic of Korea_.

Background:

Agents targeting HER2 or EGFR have been applied in several cancer types. Tumor microenvironment including tumor-infiltrating lymphocytes could be a predictive marker in HER2- or EGFR- targeted therapy, although there are some controversies. However, the effects of HER2 and EGFR inhibition on tumor microenvironment are unclear.

Method:

We screened HER2, EGFR, and PD-L1 expression in gastric cancer cell lines by western blot (SNU216, SNU668, SNU719, AGS, N87, YCC3, and YCC10). HER2 and EGFR was inhibited by using dual inhibitor (afatinib, lapatinib). After HER2 and EGFR inhibition, the change of PD-L1 expression was evaluated in HER2 overexpressed cell lines (SNU216, N87, SKBR3) at western blot, FACS, PCR, and qPCR. Selective blockade for down-steam molecules of HER2 and EGFR was conducted by using pictilisib (PI3K inhibitor) and trametinib (MEK inhibitor). The change of chemokines such as CXCL1, CCL2, and CCL21 was evaluated after HER2 and EGFR inhibition by PCR. In clinical data, PD-L1 expression and HER2 expression in resected gastric cancer were also evaluated by immunohistochemistry analysis.

Results:

EGFR-overexpressed or HER2-overexpressed gastric cancer cell lines (SNU216, SNU668, N87) showed higher protein expression of PD-L1. PD-L1 expression decreased with dose-dependent manner in afatinib- or lapatinib- treated cell lines (SNU216, N87, SKBR3). In pictilisib-treated cell lines, PD-L1 was also down-regulated. However, trametinib-treated cell lines did not show down-regulation of PD-L1. After lapatinib treatment in HER2 overexpressed cell lines, CXCL1, CCL21, and CCL2 decreased with dose-dependent manner. In 289 patients with resected gastric cancer, there was a significant association between HER2 and PD-L1 expression (p=0.03).

Conclusions:

PD-L1 is associated with HER2 and EGFR expression. PD-L1 is regulated by inhibition of PI3K pathway. Therefore, HER2 or EGFR inhibition could interact with tumor microenvironment by down-regulation of expression of PD-L1 and chemokines.

#5137

Humoral factors of breast tumor environment may boost PD-1 and CTLA4 gene expression of T cells, which has already been upregulated by CD28 stimulation.

Shun Kawaguchi, Eiji Suzuki, Kosuke Kawaguchi, Fengling Pu, Ayane Yamaguchi, Moe Tsuda, Masakazu Toi. _Kyoto University, Kyoto City, Japan_.

Background:

It has been reported that PD-1 and CTLA4 expression of T cells is up-regulated by CD28 stimulation in vitro, however the influence of humoral factors on their up-regulation is still incompletely understood. Therefore, in the current study, we evaluated if humoral factors of breast tumor environment induce further enhancement of their expression in vitro.

Materials and Methods:

Conditioned medium from MDA-MB 231 (triple negative human breast cancer cell line)(M), THP-1 (human monocytic cell line) (T) and co-culture of MDA-MB 231 and THP-1 (MT) were prepared. Pan T cells were isolated from PBMC of a healthy volunteer by MACS Pan T cell isolation kit (Miltenyi Biotec) and activated with Dynabeads Human T-activator CD3/CD28 (Thermo Fisher scientific). Activated and non-activated T cells were cultured with each conditioned medium M, T, MT and original RPMI-1640 medium. After cultivation for 24 hours, we analyzed mRNA expression level of PD-1, CTLA4 and FOXP3 of activated and non-activated T cells stimulated with each conditioned medium by quantitive real time-PCR using TaqMan gene expression probes. We also measured cytokines released into each conditioned culture supernatants by using human Bio-Plex multiplex assay system (Bio-Rad).

Results:

As it has been reported, mRNA expression of PD-1 and CTLA4 was up-regulated by CD28 stimulation in normal medium. Interestingly, PD-1 and CTLA4 mRNA of activated T cells incubated with MT was further up-regulated by 2 times and 1.5 times as compared to that of activated T cells with normal medium (PD-1 and CTLA4, respectively) while there was no up-regulation of PD-1 and CTLA4 in non-activated T cells incubated with MT. We further found that abundance of MIP-1β, VEGF, IL-7 and IP-10 was significantly higher in supernatant of MT-treated T cells than that of activated T cells in normal medium (98135.37 +/- 22.50 vs 4957.47 +/- 281.00 pg/ml (MIP-1β), 670.18 +/- 5.00 vs 311.77 +/- 26.75 pg/ml (VEGF), 4.65 +/- 0.50 vs 2.69 +/- 0 pg/ml (IL-7), 1654.53 +/- 53.00 vs 1013.91+/- 16.50 pg/ml (IP-10)).

Conclusions:

These findings suggest that humoral factors may contribute to immune escape by further up-regulation of PD-1 and CTLA4 on T cells which has already been activated by CD28 stimulation (in addition to antigen-TCR signal activation). It is assumed that a certain humoral factor, which might not be detected in this experiment although the candidate factors may be IL-7 and VEGF according to our cytokine study, induces inhibition of T cells via immune checkpoints.

#5138

The PD-1/PD-L1 axis and clinical outcomes in head and neck squamous cell carcinomas.

Sumita Trivedi, Raja R. Seethala, Robert L. Ferris. _University of Pittsburgh, Pittsburgh, PA_.

Although novel therapeutic agents targeting PD-L1 and PD-1 have emerged, our understanding of the complex interaction between the tumor microenvironment and this checkpoint receptor-ligand axis remains incomplete. PD-L1 protein expression has been demonstrated by immunohistochemistry in several solid tumors including head and neck squamous cell carcinomas (HNSCC). However, the prognostic significance of PD-L1 overexpression in HNSCC remains inconclusive. Furthermore, the interaction between human papillomavirus (HPV) positive tumors, PD-L1 on tumor cells and PD-1 on immune cells has yet to be demonstrated in an architecturally intact tumor environment.

Here we demonstrate PD-L1 expression in a large cohort of mixed HPV+ and HPV-, surgically treated HNSCC using immunohistochemistry. Our findings confirm that more HPV+ tumors express PD-L1 compared with HPV- tumors (72% compared with 44%). Clinically, this correlates with improved overall and disease-free survival in in patients with HPV+ PD-L1+ tumors compared with HPV- PD-L1- tumors. By examining the intact tumor, we visualized distinct patterns of PD-L1 staining, either peripherally, at the tumor front or non-peripherally. Tumors with a peripheral staining pattern showed a trend towards decreased overall and disease-free survival, compared to those with a non-peripheral pattern of staining. By double staining PD-L1 and PD-1, we analyzed the co-expression of the ligand on tumor cells and its receptor on neighboring immune cells. Interestingly, patients whose tumors displayed co-localization of PD-1 and PD-L1 showed a trend towards improved disease-free survival compared to tumors where there was no co-localization of PD-1 and PD-L1.

In conclusion, out data suggest that the presence of PD-L1 and PD-1 alone may not be sufficient to guide clinical treatment rather; careful examination of the intact tumor microenvironment may inform more targeted therapy.

#5139

Adjuvant immunotherapy targeting CSF1R to limit metastatic progression.

Justin Evans, Amber J. Giles, Meera Murgai, Miki Kasai, Caitlin Reid, Rosandra Natasha Kaplan. _National Cancer Institute, Bethesda, MD_.

Metastasis is often considered a late stage event, although the first changes in the metastatic site occur very early during localized primary tumor development. Although understanding of these initiating events is limited, immune suppression plays an essential role in allowing for the outgrowth of disseminating tumor cells. Targeting this immune suppressive milieu can hold promise to effectively inhibit metastatic progression. Colony stimulating factor-one (CSF-1) is overexpressed by many diverse tumor types and can induce expansion and recruitment of CSF-1 receptor (CSF-1R) expressing cells. CSF-1R is expressed on a multitude of myeloid cells including inflammatory monocytes, myeloid derived suppressor cells and macrophages, which are key immune suppressive cells in a primary tumor site. Using a metastatic rhabdomyosarcoma model, we have identified expansion of immune suppressive myeloid cells in the early metastatic microenvironment. PLX3397, which is small molecular inhibitor that selectively targets CSF-1R, Kit and Oncogenic FLT3, was used to determine the impact of targeting this immune suppressive microenvironment in limiting metastatic progression. PLX3397 and GW2580, a selective CSF1R inhibitor, reduce the immune suppressive capacity of bone marrow-derived monocytes on activated T cells. Adjuvant therapy with PLX3397 in resected rhabdomyosarcoma reduces metastatic spread. Targeting CSF-1R is associated with an expansion of CD11C expressing antigen presenting cells in the metastatic tissue and in combination with PD-1 blockade improves anti-tumor immunity. Continued investigation of combination immunotherapy with metastatic microenvironment targeting in the adjuvant setting holds promise to limit metastatic spread.

#5140

PD-L1 expression and its relationship with other driver genes in non-small cell lung cancer (NSCLC).

Liyan Jiang,1 Xinying Su,2 Tianwei Zhang,2 Xiaolu Yin,2 Meizhuo Zhang,3 Haihua Fu,2 Hulin Han,2 Yun Sun,2 Lili Dong,1 Jialin Qian,1 Yanhua Xu,4 Xuan Fu,4 Paul Gavine,2 Yanbin Zhou,5 Tian Kun,6 Jiaqi Huang,7 Haiyi Jiang,4 Yihong Yao,7 Baohui Han,1 Yi Gu2. 1 _Shanghai Chest Hospital, Shanghai, China;_ 2 _Asia & Emerging Markets, iMed, AstraZeneca, Shanghai, China; _3 _R &D Information, AstraZeneca, Shanghai, China; _4 _Global Medicines Development, AstraZeneca, Shanghai, China;_ 5 _The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China;_ 6 _General Hospital of Chengdu Military Region of PLA, Chengdu, China;_ 7 _R &D, MedImmune, AstraZeneca, MD_.

Aims: In order to understand the potential patient population who could benefit from anti PD-1/PD-L1 mono or combinational therapies, this study aimed to profile a panel of immune-mediated therapy for cancer (IMT-C) related biomarkers (CD8, PD-1, PD-L1 and CTLA-4) and molecular targeted therapy biomarkers (EGFRmut, KRASmut, ALK, ROS1 and MET) in NSCLC patients.

Methods: Tumor samples from 356 Chinese NSCLC patients including 191 adenocarcinomas (AD) and 142 squamous carcinomas (SCC) were analyzed in this study. There were 221 early stage (I to IIIa) and 113 late stage (IIIb and IV) patients.

PD-L1, PD-1, CTLA-4 and MET expression were detected using immunohistochemical (IHC) staining on formalin fixed paraffin embedded (FFPE) tissues. PD-L1 and CD8 expression were also simultaneously observed using a dual-color immunofluorescence (IF) assay. EGFR and KRAS mutations were detected by sequencing analysis. ALK and ROS1 gene rearrangements were defined by break-apart FISH assays, and MET gene amplification was also detected using a FISH assay.

Results: Among 356 NSCLC patient samples, 145 (40.7%) had PD-L1 positive staining on ≥5% tumor cells (TC). The PD-L1 positive rate on TC was significantly higher in SCC than in AD (47.9% vs 34.0%, p=0.011). In AD patients, PD-L1 expression was significantly elevated in males (p=0.0059), smokers (p=0.0003), higher tumor grade (p<0.00001) and late stage (p=0.0009) patients. In addition, we found that 53.1% (113/213) patient samples had PD-L1 expression on tumor infiltrating immune cells (IC). Again, the PD-L1 positive rate on IC was higher in SCC than in AD (66.0% vs 36.6%, p<0.0001). Comparing the two cell types, 70% of the total cases had consistent PD-L1 staining status (positive or negative) on TC and tumor infiltrating IC (p<0.0001). Among 117 cases detected using dual-color IF, all had CD8 positive lymphocytes in the tumor samples while 54 cases were PD-L1 TC positive. The most interesting finding was CD8 and PD-L1 double stained lymphocytes were mainly located in the tumor center in 37 out of 42 cases. Furthermore, combined analysis of the eight biomarkers studied in this cohort of NSCLC samples showed that tumor PD-L1 positive staining (on ≥5% tumor cells) had overlaps with the other NSCLC driver genes' alterations in 28% of SCC and 69% of AD samples.

Conclusion: This study showed almost half of NSCLC patients have PD-L1 positive expression on TC, which to some extent overlaps with EGFR/KRAS mut, ALK/ROS1 rearrangement and MET alteration. As a potential patient selection biomarker, the PD-L1 positive prevalence in NSCLC TC and/or tumor infiltrating IC provides a strong evidence for PD-1/PD-L1 immune therapy in single agent or in combination with the molecular targeted agents. Furthermore, CD8+ lymphocyte' infiltration in the tumor center and its PD-L1 expression status might also be considered as part of patient selection biomarker strategy.

#5141

Determinants of metastatic competence in breast carcinoma: a role for immune cells.

Robiya Joseph,1 Rama Soundararajan,1 Anurag Paranjape,2 Sendurai Mani1. 1 _MD Anderson Cancer Center, Houston, TX;_ 2 _National Cancer Institute, Bethesda, MD_.

Dynamic interactions between cancer cells and their surrounding microenvironment influence tumor survival, growth and metastasis. The tumor microenvironment harbors a complex variety of cells including T lymphocytes and macrophages that produce a host of cytokines, chemokines, growth factors or interleukins, all of which presumably work in coordination to determine net tumor biology. Immune cells can offer a critical check-point in tumor progression. Interestingly however, infiltration of the tumor by immune cells appears to have dual functionality - they can reportedly either be pro-metastatic or anti-metastatic in their contribution. These clearly conflicting roles of immune cells limit our comprehension of their actual input to tumor advancement. Tumor infiltrating lymphocytes, in particular, the infiltration by T cells, has emerged as a good prognostic marker for a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and T lymphocytes, and the underlying signaling pathways are still ill-defined. Moreover, the impact of T lymphocytes of the inflammatory tumor microenvironment, on the epithelial-mesenchymal plasticity and associated metastatic traits in breast cancer cells, is incompletely understood. We previously demonstrated that two key EMT/CSC factors - FOXC2 and Twist - are critical requirements for breast carcinoma cells to metastasize. The objective of the current study is to systematically investigate the role of regulatory T cells in EMT/CSC-dictated breast tumor progression, using two isogenic breast cancer cell lines (67NR and 4T1) that form primary mammary tumors similarly, but differ drastically in their ability to metastasize.

#5142

Correlation of peripheral blood S100A9+ myeloid-derived suppressor cells and tumor-associated macrophages in lung adenocarcinoma patient.

Kang-Yun Lee, Po-Hao Feng. _Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan_.

Background: Peripheral blood S100A9+ monocytic myeloid derived suppressor cells (MDSCs) is a predictive factor for treatment response of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) of lung adenocarcinoma patients. Tumor associated macrophages (TAMs), which might derived from peripheral blood monocyte, is also a predictive factor to treatment response of EGFR-TKI. However, the correlation of S100A9+ monocytic MDSCs and TAMs is not well understood in lung adenocarcinoma patients.

Methods: Stage IV lung adenocarcinoma patients with paired formalin fixed paraffin embedded (FFPE) tumor tissue and peripheral blood mononuclear cell (PBMCs) were enrolled. CD68+ cells were calculated in 3 different 200X microscopic field of FFPE as TAMs, and S100A9+ cells also calculated with same method as MDSCs in tumor microenvironment. S100A9+ MDSCs percentage was calculated by flow cytometry from CD14+S100A9+ in PBMC.

Results: Twenty-four lung adenocarcinoma patients with paired FFPE tissue and PBMC data were enrolled. PBMC S100A9+ MDSCs percentage is 17.9± 13.6; average number of S100A9+ cells and CD68+ TAMs in tumor tissue is 96.8±45.7 and 79.3±13.3, respectively. The correlation between S100A9+ MDSCs percentage and tissue S100A9+ cells and CD68+ TAMs is significant (Spearman r =0.54, p=0.003 for S100A9+; Spearman r= 0.03, p=0.03 for CD68; n=24) CD68+ and S100A9+ cells were also correlated (Spearman r=0.41, p=0.04, n=24). Clinically, high or low ( cutoff value as median number) tissue S100A9+ cells and CD68+ TAMs were both predictive factors for treatment PFS for EGFR-TKI. (For CD68, median: 56, high vs low TAM PFS= 7.4 vs 15.4 months, p=0.04; For S100A9+ cells, median: 76, high vs low S100A9+ cells PFS=7.2 vs 13.6 months, p=0.05)

Conclusion: Peripheral blood S100A9+ MDSCs is well correlated to tumor tissue S100A9+ cells and CD68+ TAMs. Both tissue S100A9+ cells and CD68+ TAMs were predictive factors for EGFR-TKI PFS. Our study suggests that peripheral blood S100A9+ monocytic MDSC might be one of the origin of tumor tissue CD68+ TAMs.

#5143

MHC class I polypeptide-related sequence A (MICA) as a factor of aggressive prostate cancer.

Marcelo J. Sakiyama,1 Angel Garcia,2 Ingrid Espinoza,3 Jack Lewin,3 Xu Zhang,3 Elizabeth Tarsi,3 Tara Craft,3 Logan Fair,3 Jesus Monico,3 Lisa Sullivan,3 Charles R. Pound,3 Srinivasan Vijayakumar,3 Christian R. Gomez3. 1 _University of Mississippi Medical Center. Coordination for the Improvement of Higher Education Personnel (CAPES), Jackson, MS;_ 2 _Tougaloo College, Tougaloo, MS;_ 3 _University of Mississippi Medical Center, Jackson, MS_.

Prostate cancer (PCa) remains the second most common cancer in American men, with higher incidence and death rates in African American men (AAM) relative to Caucasian American men (CAM). MHC class I polypeptide-related sequence A (MICA) is a transmembrane protein responsible for activation of immunocytes, by binding the NKG2D receptor. The expression of MICA under normal conditions is low. However under stress conditions such as those found in neoplasms, MICA expression is increased. The soluble form of MICA (sMICA) is released by tumor cells and promotes immune evasion by binding and downregulating the NKG2D receptor in immune surveillance cells. Increased levels of sMICA have been found in patient sera of several types of cancer, including PCa. We hypothesized that low oxygen levels, a condition of the microenvironment present in aggressive tumors, would modify the levels of sMICA in PCa cell lines. Likewise we speculated that when compared to CAM, MICA would be differentially expressed in tumors from AAM, known to suffer a more aggressive disease. To address those questions, LNCaP cells were cultured under 20% O2 (normoxia) and 2% O2 (hypoxia) at various time points, supernatants were collected and the presence of sMICA in culture supernatant and patient sera was detected by ELISA. After 48 hours, the cells grown in hypoxia expressed about 5-fold more sMICA than cells grown in normoxia. When assessed in patient plasma, PCa patients had 2-fold more sMICA compared to healthy controls. In additional experiments cells growing under different oxygen levels were lysed and used in Western blot analysis to detect cellular MICA. Normal oxygen levels increased MICA protein by 3.5-fold. Hypoxia reduced MICA expression by 11%. A tissue microarray (TMA) was stained with MICA polyclonal antibody. The TMA was composed of 34 tumor samples and 30 controls. The samples came from 32 CAM and 32 AAM. The staining intensity was scored as 0, 1, 2 or 3 and scores 2 and 3 were defined as high intensity. Association of the staining result to presence of tumor was evaluated using the Fisher's exact test. A higher proportion of high intensity in tumor site (50%) was found when compared to non-tumor site (20%, p=0.019). Interestingly, stronger association was expressed in CAM (p=0.036) than in AAM (p=0.265). The results found in the cell line suggest that different oxygen levels affect the expression of sMICA. Higher expression of MICA in prostate tumor tissue and sMICA in patient sera are consistent with data previously reported in other cancers. Lower intensity in MICA staining in AAM patients may be related to better capacity of the tumor to evade the immune system. Overall our findings suggest the involvement of MICA on aggressive PCa. More studies are needed to decipher the biological mechanisms of MICA's immune evasion in prostate tumors and to establish its contributing role to aggressive disease in minorities.

Funding sources: PCRP W81XWH-14-1-0151, UMMC Office of Research

#5144

Role of Neuropilin-2 in the maintenance of tumor associated macrophages.

Sohini Roy,1 Samikshan Dutta,1 Samuel Schellenburg,2 James E. Talmadge,1 Michael Muders,2 Kaustubh Datta1. 1 _University of Nebraska Medical Center, Omaha, NE;_ 2 _Institute of Pathology, University Hospital of Dresden, Dresden, Germany_.

Purpose: Macrophages are key regulators of the immune system and act as a bridge between the innate and adaptive arms of the host immune responses. However, they also comprise an important component of immune cell infiltrates in various solid tumor microenvironments (TME) and impede therapy. Tumor Associated Macrophages (TAMs) are inflammatory as well as pro-tumorigenic and perform key functions which aid in tumor growth, evasion of host immune surveillance, and correlate with poor prognosis in many cancers. The objective of our current study is to elucidate the role of Neuropilin-2 (NRP2), a non-tyrosine kinase receptor in TAMs and illustrate the impact of targeting the molecular pathways regulated by NRP2 in the reversal of TAM polarization and facilitation of tumor specific host immune response.

Methods: Human U937 monocyte cell line, human peripheral blood monocytes as well as mouse bone marrow derived progenitors were differentiated to macrophages to investigate whether the expression of NRP2 could be induced under conditions mimicking normal tissue homeostasis, acute inflammation and TAM polarization. NRP2 expression in TAMs was also evaluated in certain human malignancies using Immunohistochemistry. Using cellular approaches and transgenic mouse model, phagocytosis assay was performed under above-mentioned conditions. Also, macrophages were stained for endocytic markers in the presence and absence of NRP2 and viewed using Confocal Microscopy.

Results: We observed that NRP2 expression was induced in human and murine macrophages under conditions mimicking normal tissue homeostasis, acute inflammation and TAM polarization. Immunohistochemical analysis revealed that macrophages isolated from human malignancies also exhibited a significant expression of NRP2. Phagocytosis assay in combination with Immunofluorescence data indicated a possible defect in endocytic compartments in NRP2 depleted conditions.

Conclusions: Our data demonstrate the expression of NRP2 in macrophages is induced under normal tissue homeostasis, acute inflammation as well as in macrophages invading tumor stroma. Also, we report a novel function of NRP2 in the regulation of the endosomal maturation and thereby phagocytosis in macrophages. This will elicit a favorable immune response in acute infections, but promote cancer growth by efficient processing of efferocytosed tumor cell debris. Previous reports have established that blockade of tumor cell efferocytosis suppresses TAM polarization in the TME, breaks immune tolerance and reduces metastasis. Our ongoing and future studies will establish the therapeutic impact of targeting NRP2 regulated pathways in the tumor infiltrating macrophages in combating therapy resistance. Hence, our study carries the potential to open up new strategies to selectively target and facilitate anti-tumor host immune response in multiple human malignancies.

### Molecular Carcinogenesis

#5145

To explore the tumorigenic regulation mechanism of SPZ1 in hepatocellular carcinoma.

LI-TING WANG, SHIH-HSIEN HSU. _Kaohsiung Medical University, Kaohsiung, Taiwan_.

Cancer directly affects at least one-third of the human population. Despite this extensively, the genetic determinants of cancer risk remain unknown. Spermatogenic leucine zipper 1 (SPZ1), a basic helix loop helix leucine zipper (bHLH-Zip) transcription factor, acted as a proto-oncogene in mouse and human. It has been demonstrated that SPZ1 was involved in early embryonic development, cell growth and differentiation. In this study, we compared the expression of SPZ1 in 168 paired specimens of HCCs and adjacent normal tissues and also in paired specimens from 140 patients with non-HCCs. In pathologic analysis, SPZ1 exhibited a tumor-specific expression pattern and a high correlation to patient survival time, tumor size, tumor number, and progression stage. Enforced expression of SPZ1 accelerated cell proliferation and tumorigenic activity in hepatoma cells. In contrast, short hairpin RNA-mediated attenuation of SPZ1 in hepatoma cells decreased cell proliferation and malignant transformation in vitro and in vivo. Together, we speculated that SPZ1 as a potential therapeutic target of HCCs

#5146

GM2 mediates tumor cell migration and induces EMT through involvement of the integrin signaling machinery and targeting YAP, a major component of the HIPPO signaling pathway.

Manjari Kundu, Barun Mahata, Avisek Banerjee, Shibjyoti Debnath, Kaushik Biswas. _Bose Institute, Kolkata, India_.

Background: In the present study we report a novel role of tumor derived ganglioside, GM2 in mediating tumor cell migration and anchorage independent growth suggestive of EMT and uncovered its mechanism.

Approach : Specific role of GM2 in cancer cell migration was evaluated by in vitro transwell migration assay using either GM2-synthase silenced or GM2-overexpressed cancer cells or by adding exogenous GM2. Mechanistic details were revealed by studying gene chip profiling and validated by real time PCR, western blot analysis and immunofluorescence. Interaction of GM2 with upstream specific molecules were determined by confocal microscopy, co-IP and surface plasmon resonance (SPR). TALEN mediated targeted genome editing was used to generate stable GM2-synthase knockout mouse tumor cells, to study the role of GM2 in EMT.

Results : siRNA mediated knockdown of GM2-synthase in cancer cells resulted in significant inhibition of tumor cell migration in vitro. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in dramatic increase in migration. TALEN mediated GM2-synthase knockout in Renca-v cells resulted in significant reduction in AIG and increased sensitivity to anoikis, suggesting a plausible role of GM2 in EMT. Gene expression profiling and DNA microarray analysis of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin signaling involving the Erk-MAP kinase pathway, as well as genes targeted by YAP, a major component of the HIPPO signaling pathway which is involved in EMT. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk while, exogenous GM2 treatment caused increased Erk phosphorylation. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Renca-v GM2-synthase knockout cells exhibited significant downregulation of several YAP target genes namely, Ctgf, pdgf C, Cyr61 and LOX while induction of these genes in both Renca as well as 4T1 in response to GM2 treatment suggests that GM2 mediates EMT in cancer cells possibly by targeting YAP. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor.

Conclusion: Our findings confirm a novel role of GM2 in tumor cell migration involving the integrin signaling pathway and in triggering EMT by targeting YAP, a major component of the HIPPO signaling pathway ultimately leading to enhanced migration and metastasis in tumor cells.

#5147

Characterization of the tissue-specific properties of cancer driver mutations suggests spontaneous mutation in normal tissues drives tumor susceptibility.

Barbara L. Parsons, Meagan B. Myers, Karen L. McKim, Malathi Banda. _US FDA-NCTR, Jefferson, AR_.

Cancer driver mutations (CDMs) are being developed as theranostic biomarkers. This study used high-sensitivity quantification by ACB-PCR, with a sensitivity of 10-5, to elucidate the tissue-specific properties of CDMs. We employed the paradigm of comparing levels of specific hotspot CDMs in normal human tissues and tumors. Specifically, we measured KRAS G12D, KRAS G12V, PIK3CA E545K, and PIK3CA H1047R mutant fractions (MFs) in normal breast, colon, lung, and thyroid and quantified the same mutations in mammary ductal carcinomas (DCs), colonic adenocarcinomas, lung adenocarcinomas, and papillary thyroid carcinomas. Three major findings were: 1) these mutations occur in normal human tissues at relatively high frequencies, 2) there is considerable variability in CDM MFs in normal tissues across individuals, and 3) tumors frequently carry CDMs at levels greater than that present in normal tissue, but below that detected by standard DNA sequencing. Understanding the prevalence of mutant subpopulations is important because they can drive resistance to molecularly-targeted therapies. The PIK3CA H1047R profile for normal breast and DC were similar, with large mutant subpopulations present in both tissue types. Because the PIK3CA H1047R mutation is the most prevalent point mutation reported in breast cancer, we conclude that it can drive breast carcinogenesis as a subpopulation, potentially through a paracrine mechanism, and may be a useful biomarker of breast cancer susceptibility. Other properties of CDMs elucidated were gender differences, the accumulation of mutation with donor age, and the correlation between MF and maximum tumor dimension (MTD). Male lung had significantly greater PIK3CA H1047R MFs than female lung. The PIK3CA H1047R MF in normal breast was positively correlated with donor age, and the mutation in DCs was positively correlated with MTD. By contrast, KRAS MFs showed non-significant decreases in normal lung with increasing donor age. For colorectal adenocarcinomas, KRAS G12V MF was negatively correlated with MTD, consistent with previous findings that KRAS G12V MF decreases during adenoma to adenocarcinoma progression. This suggests that the selective advantage provided by KRAS G12V mutation is context-dependent and can be either positive or negative. Across the four mutational targets and four tissue types, a significant positive correlation was observed between the variability (Log10 standard deviation) of MF measured in normal tissues and the prevalence with which the mutation reportedly occurs in tumors, as per The Cancer Genome Atlas database. This suggests that level and inter-individual variation in CDMs can be used to identify promising tissue-specific, mutational biomarkers of cancer susceptibility. This is not a formal dissemination of information by FDA and does not represent agency position or policy.

#5148

Angiotensin II type I receptor drives cancer progression through the FLJ10540/S100A9 pathway in oral cancer.

Chang-Han Chen. _Institute for Translational Research in Biomedicine, Kaohsiung, Taiwan_.

Oral cancer is the eighth most common cancers and a growing problem worldwide. Despite the easy approach for visual examination, oral cancer is often detected at advanced stages leading to severely decreased patient survival. Identifying reliable molecular markers for oral cancer provided in developing new therapeutic interventions is urgently needed. In this study, we explored that angiotensin II type I receptor (AT1R) was overexpression in tumor tissues of oral cancer patients, and its expression also significantly correlated with advanced TNM-stage, lymph node stage, and the poor 5-year survival rate. Functionally, gain-of function of AT1R in oral cancer cells promoted tumor cell growth in vitro and in xenograft model by MTT/colony formation/BrdU incorporation/time-lapse/IVIS, and 3D-Sonographic imaging appraoches. Furthermore, using the same cell panels, the migratory and invasive abilities were increased by wound healing and Transwell assays and the epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, fibronectin and vimentin were also elevated. Conversely, loss-of function of AT1R in oral cancer cells by using siRNA and CRISPR/Cas9 systems demonstrated that the abilities of tumor proliferation, and motility were decreased in vitro and in vivo. By using syn-expression concept combinging with bioinformatic appraoch, we found that FLJ10540 was a novel dwonstream target of AT1R. FLJ10540 expression was not only regulated by AT1R, but also participated in AT1R-elicited cancer progression in vitro and in vivo. We established FLJ10540 knockout oral cancer cell lines by CRISPR/Casp9 to perform DNA microarray and found that S100A9 was a top ranking target among the differrentially expression in down-regulated candidate sets. By using biochemical approaches and xenograft model, the post-transcriptional level of S100A9 was modulated by FLJ10540. In addition, the AT1R-elicited cancer progression was required for FLJ10540/S100A9 expression. Finally, immunohistochemical analysis of human aggressive oral cancer specimens showed a significant positively correlation between AT1R/FLJ10540 and S100A9 expression. Taken together, these findings suggest that AT1R is not only an important prognostic factor but also a new therapeutic target in the FLJ10540/S100A9 pathway for human cancer.

#5149

A novel regulatory circuit involving STIM1 and HIF-1 mediates hypoxia-driven hepatocarcinogenesis.

Yongsheng Li. _Xinqiao Hospital, Third Military Medical University, Chongqing, China_.

Hypoxia and intracellular Ca2+ transient are fundamental traits of cancer. Signaling cascades initiated or regulated by HIF-1 is essential for hypoxic responses. Stromalinteraction molecule 1 (STIM1) is well known as a Ca2+ sensor on endoplasmic reticulum (ER) that mediates SOCE activation and promotes tumor invasion and migration. However, there was no general consensus about the relationship between HIF-1 and STIM1. Also, the significance of STIM1, as well as the Ca2+ mobilization route in cancer cells during hypoxia remains unknown. Our results show that STIM1 correlates with elevated hypoxia-inducible factor-1 alpha (HIF-1α) in hypoxic hepatocarcinoma cells (HCC) and is upregulated during hepatocarcinoma growth. HIF-1 directly transcripts STIM1 and contributes to store-operated Ca2+ entry (SOCE), while STIM1-mediated SOCE is also required for HIF-1 accumulation in hypoxic HCC, via activating Ca2+/calmodulin-dependent protein kinase II and p300. Administration of YC-1, a HIF-1 inhibitor, or knockdown of HIF1A significantly diminishes hypoxia-enhanced STIM1 and suppresses tumorigenesis. Moreover, ectopic expression of STIM1 or HIF-1α partially reverses the impaired growth of the tumor treated with YC-1. These results suggest a mutual dependency and regulation of STIM1 and HIF-1 in controlling Ca2+ mobilization and hypoxic tumor growth, and highlight a potential target for early hypoxia-related intervention.

#5150

Identification of higher mRNA expression of both Kitenin and ErbB4 CYT-2 isoform as a molecular marker for the prediction of transition from colon adenoma to carcinoma.

Jeong A Bae,1 Eun Gene Sun,1 Yoo-Seung Ko,1 Hui Jeong Choi,1 Woo-Kyun Bae,1 Sang-Hee Cho,1 Ik Joo Chung,1 Kyung-Sub Moon,1 Young-Hyun Yu,2 Hyung-Ho Ha,2 Hangun Kim,2 Kyung Keun Kim1. 1 _Chonnam National Univ. Med. School, Kwangju, Republic of Korea;_ 2 _Sunchon Natl Univ. Coll Pharmacy, Sunchon, Republic of Korea_.

Purpose: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer (CRC) cells and we recently identified a novel EGFR-independent oncogenic signal of EGF that works under co-expressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contributes to further progression of intestinal adenoma following APC loss.

Results & Discussion: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven elevated KITENIN induced increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes, compared with those of non-transgenic mice. Among the 4 ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells co-expressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of ErbB4 CYT-2 isoform mRNA as well as increased EGFR expression were observed in intestinal adenoma of APCmin/+ mice making the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APCmin/+ mice, intestinal tumor tissues in the crossed mice showed the characteristics of early stage invading adenocarcinoma. In CRC patients, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no differences in tumoral expression between the stages, and a positive correlation exists between KITENIN and ErbB4-CYT-2 mRNA expression. Thus, elevated expression of KITENIN and ErbB4-CYT-2 is newly identified as a factor that promotes the transition of colon adenoma to adenocarcinoma within an APC-loss-associated tumor microenvironment.

Conclusion: Higher mRNA expression of both KITENIN and ErbB4-CYT-2 in colon adenoma tissues can be used as a molecular marker of the possible development of malignant transformation.

#5152

Inhibition of TLR/IRAK pathway in hepatocellular carcinoma augmented therapeutic responses.

Yik Ling Bowie Cheng, Oi Lin Irene Ng, Kin Wah Terence Lee. _The University of Hong Kong, Hong Kong, Hong Kong_.

Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Frequent relapse and drug resistance accounts for the poor prognosis, which could be attributed by the presence of cancer stem cells (CSCs), a subpopulation that harbor the properties of self-renewal, clonal tumor initiation and long-term repopulation capacity. Toll like receptors (TLRs) are widely expressed on immune cells and TLR/Interleukin-1 receptor associated kinase (IRAK) pathway was originally best known for their role in the induction of innate immune responses, but it was recently found to be frequently up-regulated in various cancers, although its role in cancer development remains largely unexplored.

By RNA sequencing analysis of 16 pairs of HCC clinical samples, we found IRAK1 to be significantly overexpressed in HCC among all members of IRAK family. By quantitative PCR and western blot analyses, overexpression of IRAK1 at both mRNA and protein level was confirmed, which correlated with advanced tumor stage (p=0.04). By lentiviral-based over-expression and knockdown approaches, we demonstrated that IRAK1 promoted cell proliferation and invasiveness. Interestingly, IRAK1 regulated CSC properties including tumorigenicity, self-renewal, drug resistance and expression of liver CSC markers including CD24 and CD47. To further examine the therapeutic potentials of targeting IRAK1 in HCC, we treated HCC cells with IRAK1/4 inhibitor. Suppression of IRAK1 at 10μM not only consistently inhibited CSC properties, but also augmented the effect of sorafenib, a molecularly targeted drug for treatment of advanced HCC. The cancer-promoting and drug desensitization effect of IRAK1 could be partly explained by activation of NF-κB signaling pathway as well as down-regulation of apoptotic cascade.

Conclusion:

Overexpression of IRAK1 in HCC promotes cancer progression by enhancing proliferation and invasiveness; Importantly, IRAK1 may promote cancer stemness and confers resistance to sorafenib. All in all, targeting IRAK1 with specific small molecule inhibitor alone or in combination with other drugs may be a novel therapeutic regimen for treatment of HCC.

#5153

Dual inhibition of STAT3 and IL-8 pathways as a novel therapeutic approach in osteosarcoma.

Yang Cao, Hui Xiao, Xiaojuan Wu, Jiayuh Lin. _The Research Institute at Nationwide Children's Hospital, Columbus, OH_.

Abstract

Objective: Targeting STAT3 signaling represents a potential therapeutic approach to treatment of osteosarcoma. However, the use of STAT3 inhibitor as a single agent may not be effective for cancer therapy. Identify the pathway(s) that may generate synergy in tumor suppression may be more effective in osteosarcoma therapy.

Methods: Inflammatory cytokines IL-6, IL-11, and IL-8 secretion of osteosarcoma after STAT3 inhibition were determined by ELISA assay. The sensitivity to STAT3 inhibitor was examined by cell viability assay. Cell migration and colony forming ability were evaluated when combination treatment.

Results: In this study, we observed that the levels of IL-11 were significantly decreased and IL-6 was slightly decreased by STAT3 inhibitor. Interestingly, IL-8 levels were increased upon STAT3 inhibition. IL-8 was originally discovered as one of the major chemokines associated with the promotion of neutrophil chemotaxis and inflammatory responses. Previous studies have demonstrated that IL-8 contributes to cancer progression through its induction of tumor cell proliferation, survival, migration, and angiogenesis. Knocking down IL-8 expression using RNA interference resulted in enhanced sensitivity to STAT3 inhibitor. ELISA assay showed that MEK inhibitor (AZD6244 or Trametinib) and P38α inhibitor (LY2228820) had the ability to reduce the levels of IL-8 induced by STAT3 inhibition. Combination treatment of STAT3 and with a MEK inhibitor or with a P38α inhibitor showed more effective cytotoxic effect on osteosarcoma cell lines compared to single agent alone. Furthermore, the combination treatment exhibited stronger inhibition of cell migration and colony forming ability than single agent alone.

Conclusion: These results suggest the IL-8 induction as a possible feedback mechanism for resistance to STAT3 inhibitor and suggest that simultaneous inhibition of STAT3 and IL-8 may be a more effective therapeutic approach in osteosarcoma.

#5154

Modeling cancer driver-like events in barrier bypass-clonal expansion in vitro assays.

Michael Korenjak, Hana Huskova, Maude Ardin, Maria Zhivagui, Kathryn Guyton, Dinesh K. Barupal, Kurt Straif, Zdenko Herceg, Magali Olivier, Monica Hollstein, Jiri Zavadil. _Int. Agency for Research on Cancer, Lyon, France_.

BACKGROUND Cancer genomes harbor mutational spectra that document exposures to external factors and endogenous events underlying tumor development. Information on candidate cancer driver alterations is accessible from public compendia of somatic mutations, yet much of this knowledge remains descriptive and of limited mechanistic insight. Simple, robust and rapid systems are thus needed for well-controlled experimental investigations of functional impact of carcinogenic exposures on the genome and on cancer cell growth.

METHODS We use barrier bypass-clonal expansion (BBCE) assays based on primary human and murine cell cultures, in which mutations are introduced by mutagenic carcinogens and examined by deep sequencing, after the exposed cells have bypassed a selective pressure barrier and have clonally immortalized. A customized deep sequencing data analysis pipeline is used to decipher both the mutational signatures and the putative functional driver events selected and enriched for during the clonal outgrowth phase.

RESULTS Using the BBCE assays, we tested the global mutagenic effects of a number of known human carcinogens. We obtained 25 independently arising clones, altogether harboring 15,200 acquired mutations, with varying numbers per clone, of which ~7,600 were non-synonymous. These affected 250 genes currently listed in the COSMIC Cancer Gene Census. Eighty-four genes were recurrently mutated across the BBCE clone panel, including well-established oncogenes (HRAS, ABL1, EGFR, MYC, PIK3CG) and tumor suppressors (APC, ATM, BRCA2, PTCH1, TP53). A number of epigenetic and chromatin regulators also acquired recurrent mutations, among them ASH1L, BAZ1A, BAZ1B, EP400, HDAC6, and members of histone lysine demethylase and methyltransferase families. Collectively, the recurrent alterations affected pathways regulating DNA damage response, DNA repair, cell cycle, cell death, transcription and chromatin structure, and developmental pathways of TGF-beta, Notch, WNT and ERBB signaling. Thus, as in human cancers, mutations driving critical steps of cellular stress bypass and clonal immortalization arise and become selected for when these processes are modeled in vitro.

CONCLUSIONS The BBCE assays constitute a unique resource amenable to follow-up functional studies of particular mutations in cancer genes. Data will be presented describing systematic genome editing and pharmacological manipulation of select mutated genes, followed by assessment of resulting phenotypic and molecular traits. In summary, our BBCE approach may yield new mechanistic insights into driver-like events underlying cancer development.

ACKNOWLEDGMENTS Funding from International Agency for Research on Cancer; ITMO CANCER-INSERM Plan Cancer 2015 grant to J.Z.

#5155

Integrated genomic analysis of colorectal cancer progression reveals activation of EGFR through demethylation of the EREG promoter.

Xueping Qu,1 Thomas Sandmann,1 Henry Frierson,2 Ling Fu,1 Eloisa Fuentes,1 Kimberly Walter,1 Kwame Okrah,1 Craig Rumpel,2 Christopher Moskaluk,2 Shan Lu,1 Yulei Wang,1 Richard Bourgon,1 Elicia Penuel,1 Andrea Pirzkall,1 Lukas Amler,1 Mark Lackner,1 Josep Tabenero,3 Garret Hampton,1 Omar Kabbarah1. 1 _Genentech/Roche, South San Francisco, CA;_ 2 _University of Virginia, Charlottesville, VA;_ 3 _Vall d'Hebron University Hospital, Barcelona, Spain_.

The molecular drivers that underlie transformation of colonic epithelium into colorectal adenocarcinoma (CRC) have been well described. However, the mechanisms through which some of the clinically targeted pathways are activated during CRC progression have yet to be elucidated. Here, we employed an integrative genomics approach to examine CRC progression. Transcriptional profiling of laser capture microdissected colonic crypt cells, differentiated surface epithelium, adenomas, carcinomas, and metastases, showed distinctive patterns in the activation of developmental and oncogenic pathways, including the clinically important EGFR axis. We observed a dramatic up-regulation of the EGFR ligand EREG in primary and metastatic cancer cells as compared to normal and adenomatous tissues that was indicative of autocrine tumor production, and confirmed this pattern of gene expression by in situ hybridization. Global methylation analysis indicated that up-regulation of EREG during the adenoma-carcinoma transition was associated with de-methylation of two key sites within the EREG promoter and this was accompanied by an increase in the levels of EGFR phosphorylation, as assessed by reverse phase protein analysis. In a clinical trial setting, we observed that low levels of EREG methylation in patients who received cetuximab as part of a Phase II study were associated with high expression of the ligand and a favorable response to therapy. Conversely, high levels of promoter methylation and low levels of EREG expression were observed in tumors that progressed after treatment. We also noted an inverse correlation between EREG methylation and expression levels in several other cancers from the TCGA datasets, including those of the head and neck, lung, and bladder. We propose that up-regulation of EREG expression through promoter de-methylation might be an important means of activating the EGFR pathway during the genesis of CRC and, potentially, other types of cancer.

#5156

The roles of x-linked Midline 1 interacting protein 1 (MID1IP1) in gender disparity and development of hepatocellular carcinoma.

Elley Yung Tuen Chiu, Irene Oi Lin Ng. _Univ. of Hong Kong, Hong Kong, Hong Kong_.

HBV-associated hepatocellular carcinoma (HCC) has a male predominance, with a male-to-female ratio of about 5:1, suggesting gender difference is one of the major risk factors for HCC development. However, up till now, no concrete evidence is obtained to elucidate the male predominance of HCC. It is believed that the gender-specific signaling pathway is critically involved and targeting this pathway may provide a significant treatment outcome.

To target candidates that are implicated in the male predominance in HCC, aberrant expression of sex-linked genes between HCC tumors and non-tumorous livers (NT-L) draws particular attention. Previously, we performed transcriptome sequencing on sixteen pairs of human HCC tumors and NT-Ls. Our preliminary data show that a potential target, Midline 1 interacting protein 1 (MID1IP1) which is involved in hepatic lipogenesis and microtubule stabilization, was remarkably overexpressed in HCC tumors as compared to the NT-Ls (fold change=2.73; p=0.017). Moreover, the expression of MID1IP1 was significantly higher in male non-tumorous liver tissue than that of female (fold change=2.63; p=0.001). In addition, the upregulation of MID1IP1 correlated with less tumor encapsulation, more tumor microsatellite formation, more venous and direct liver invasion, and poorer tumor stage, features of a more aggressive biological behavior in our clinicopathological correlation study. Moreover, upregulation of MID1IP1 in the tumors was associated with a poorer overall survival. In its functional study, upon knockdown of MID1IP1, HCC cells (BEL7402, SMMC7721 and HepG2) had reduced proliferation rates in the cell proliferation assay and colony formation assay. Here, we also showed that the migratory abilities of the HCC cell lines with MID1IP1 knockdown were remarkably reduced in the transwell assay. Furthermore, the STRING interaction network analysis suggests that MID1IP1 interacts with Suppressor of Fused (Sufu), which is a regulator of Gli in the hedgehog (HH) pathway. To examine its regulatory pathway, we showed that the mRNA expression of the negative regulator of HH pathway, Gli3, was increased in the MID1IP1 knockdown HCC cells. The downstream target of Gli, cyclin D, was also downregulated upon MID1IP1 knockdown. In summary, our data suggest that the MID1IP1 plays a significant role in the male predominance of human HCC. MID1IP1 may also play a novel role in the regulation of hedgehog signaling.

#5157

Quantitative proteomic analysis identifying Krt17 plays a critical role in areca nut inducing oral carcinogenesis.

Chang Hsu Chiang, Chih-Ching Wu, Ann-Joy Cheng. _Medical Bioscience, Tau-Yuan, Taiwan_.

Areca nut is known a carcinogen for oral cancer in southeast Asia, however, the molecular mechanism leading to the malignancy is still unclear. To mimic the habit of areca nut chewing, our laboratory has established four oral cancer cell sublines (SAS, OECM1, K2, C9), chronically trained by areca nut extract (ANE). To elucidate the molecular basis of areca nut induced oral carcinogenesis, the differential proteomes between oral cancer cells and the ANE sublines were determined using the technique of isobaric mass tag (iTRAQ) labeling and multidimensional liquid chromatography- mass spectrometry (LC-MS/MS). Over thousand proteins were identified in four sublines, in which 194 proteins were found differential expressions in at least two ANE sublines. Bioinforatmic analysis revealed that these proteins participate in several pathways, with the regulation of epithelial to mesenchymal transition (EMT) most prominent. Fourteen proteins were confirmed differential expression in the ANE sublines, including Krt17. To shed more light on the mechanism of ANE induced carcinogenesis, Krt17 was further investigated. Knockdown Krt17 significantly suppressed ANE-induced cell growth, migration, and cell invasion, along with the modulation of EMT process. Furthermore, in a carcinogen-induced oral cancer mice model by 4NQO/arecoline, an active compound of ANE, Krt17 was found significantly up-regulated in all hyperplasia and carcinoma (p<0.001). In conclusion, we have identified proteome associated with chronic areca nut exposure in oral cancer cells. Krt17 was demonstrated contributing to areca nut induced oral malignancy. This study should attribute to risk assessment, disease prevention or other clinical applications of areca nut-induced oral cancer.

#5158

The change of EZH2 expression in development of colorectal cancer from adenoma.

Mayuko Ohuchi, Yasuo Nakamoto, Ryuma Tokunaga, Kenichi Nakamura, Keisuke Kosumi, Kazuto Harada, Hironobu Shigaki, Junji Kurashige, Masaaki Iwatsuki, Yoshifumi Baba, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. _Kumamoto University, Kumamoto, Japan_.

[Background]Colorectal cancer has been revealed to develop from adenoma through mutation and deletion of various genes such as FAP, KRAS, DCC and p53 in the process called adenoma-carcinoma sequence. Furthermore, proteins of the polycomb repressive complex 2 (PRC2) function as transcriptional repressors by trimethylating histone H3 at lysine 27, and this complex's activity is essential for cell proliferation and differentiation. The histone methyltransferase enhancer zeste homolog 2 (EZH2), a key member of PRC2, is associated with transcriptional repression of tumor suppressor gene. In our previous study, EZH2 expression was reported to increase as pathology worsened in pancreatic IPMN. Thus EZH2 expression was hypothesized to increase in the adenoma-carcinoma sequence as well.[Purpose]The purpose of this study was to investigate the change of EZH2 expression in progressing colorectal cancer. [Materials and Methods]63 patients with colorectal adenoma, 24 patients with carcinoma in adenoma and 14 patients with colorectal carcinoma who underwent surgical or endoscopic resection were enrolled in this study. The normal lesions of the clinical specimens were also examined. We evaluated the association between EZH2 expression, pathology and expression of tumor suppressor gene. Immunohistochemistry of EZH2, Ki-67, p21, p27 and p16 was performed.[Results]The Ki-67 expression increased parallel to the worsening of pathological findings (p=0.009), and EZH2 expression showed a significantly positive association with Ki-67 expression (p=0.02). There was a significant increase of EZH2 expression between normal and colorectal adenoma and carcinoma in adenoma. However, no significance between carcinoma in adenoma and colorectal cancer in EZH2 expression could be identified. Conversely, p21 expression decreased significantly between normal and adenoma lesion; however, p16 and p27 expression showed no major change. [Conclusion]EZH2 expression considerately increased parallel to pathological worsening and p21 expression showed a decrease between normal and colorectal adenoma. Thus EZH2 can contribute to development of colorectal cancer from adenoma via p21 suppression.

#5159

Smad7 knockdown in colon cancer cells activates protein kinase RNA-associated eIF2α pathway thereby leading to cell death.

Veronica De Simone, Gerolamo Bevivino, Silvia Sedda, Roberta Izzo, Massimo Claudio Fantini, Giovanni Monteleone. _University of Rome Tor Vergata, Rome, Italy_.

Background. Up-regulation of Smad7, an inhibitor of TGF-β1, occurs in sporadic colorectal cancer (CRC). Knockdown of Smad7 with a specific antisense oligonucleotide (AS) leads to activation of eIF2α, an attenuator of protein synthesis, and arrest of CRC cells in the S phase of the cell cycle with the downstream effect of inducing cell death.

Aim. To investigate the mechanisms by which Smad7 knockdown activates eIF2α.

Methods. Phosphorylation of eIF2α was evaluated in CRC cell lines (i.e. HCT116 and DLD-1) either untreated or treated with Smad7 sense (S) or AS by Western blotting (WB) and immunofluorescence (IF). Expression of ATF4 and CHOP, two downstream targets of EIF2α, were evaluated by IF and activation of PKR, GCN2 and PERK, up-stream kinases that induce eIF2α phosphorylation, was assessed by WB. Wild type or PKR-deficient CRC cells treated with Smad7 AS were monitored for eIF2α activation and induction of death. Finally, we assessed whether enhanced phosphorylation of eIF2α seen in cells treated with Smad7 AS was also associated with reduced interaction between eIF2α and PP1, a phosphatase that normally dephosphorylates eIF2α.

Results. Smad7 knockdown increased ATF4 and CHOP expression thus confirming previous data showing activation of eIF2α phosphorylation in CRC cells treated with Smad7 AS. Among kinases that induce eIF2α phosphorylation, only PKR was activated by Smad7 knockdown. Consistently, silencing of PKR reduced but did not abolish Smad7 AS-induced eIF2α phosphorylation and cell death, thus suggesting the existence of further mechanisms that control eIF2α phosphorylation in Smad7-deficient cells. Indeed, in CRC cells, Smad7 interacted with PP1 and Smad7 knockdown reduced association of PP1 with eIF2α.

Conclusions

Data show that Smad7 is involved in CRC cell survival and suggest that Smad7 is a valid target for therapeutic intervention in CRC.

#5160

**Establishment of** in vitro **carcinogenic model of high-grade serous ovarian carcinoma using immortalized fallopian tubal secretory epithelial cell.**

Kohei Nakamura,1 Kentaro Nakayama,1 Tohru Kiyono,2 Noriyoshi Ishikawa,1 Masako Ishikawa,1 Hiroshi Katagiri,1 Toshiko Minamoto,1 Tomoka Ishibashi,1 Emi Sato,1 Kaori Sanuki,1 Hitomi Yamashita,1 Kouji Iida,1 Razia Sultana,1 Satoru Kyo1. 1 _Shimane University Faculty of Medicine, Izumo, Japan;_ 2 _National Cancer Center Research Institute, Tsukiji, Japan_.

High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy in the world. Recent studies suggest that fallopian tubal secretory epithelial cells (FTSECs) are candidates for the origin of HGSOC. Several genetic alterations involved in the carcinogenesis have been reported in HGSOC, but their minimal requirement and the specific combinations remained unclear. To clarify these points, we established an in vitro stepwise carcinogenesis model using immortalized FTSECs with overexpressed cyclinD1/cdk4 and hTERT, in which we additionally mimicked selected genetic abnormalities frequently observed in clinical samples. Mutational analysis using clinical samples of HGSOCs identified frequent p53 mutations (96%). Furthermore, RAS/PI3K pathway is important signaling pathway and was deregulated in 45% of HGSOCs. PI3K/AKT pathway is also important pathway in HGSOCs. Thus, we introduced dominant negative form of p53 alone or in combination with oncogenic mutant KRAS allele into immortalized cells, but both failed to exhibit tumorigenic phenotypes. Additional introduction of genetic factors were attempted, and overexpression of c-Myc or phosphorylated Akt (p-Akt) were independently found to confer tumorigenic phenotypes. Importantly, all transformed FTSECs exhibited high-grade serous carcinomas that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Thus, p53/KRAS/c-Myc or p53/KRAS/PI3K-AKT pathways were determined to be minimal required for carcinogenesis. The strength of our study is the use of immortalized FTSECs that have original characteristics of fallopian tube as well as the introduction of genetic mutations clinically observed in human HGSOCs. This experimental model provides a foundation for future studies of early events in carcinogenesis of HGSOCs and the identification of candidate targets for drug development and secreted biomarkers for early detection. It is also likely that a similar model can be broadly applied to studying the normal biology of other epithelial cells where precursor lesions and malignant counterparts are not entirely understood.

#5161

Development of cutaneous basal cell carcinomas from cutaneous actinic keratoses.

W Clark Lambert,1 Claude E. Gagna,2 Muriel W. Lambert1. 1 _New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ;_ 2 _New York Institute of Technology, Old Westbury, NY_.

Actinic keratoses (AKs) are extremely common proliferations of epidermal keratinocytes which show cytological atypia and impaired ability to mature as the cells that comprise them migrate from the basal layer of the epidermis to higher levels of the epidermis. A small percentage of AKs are well known to progress to cutaneous squamous cell carcinomas (SCCs). However, progression of AKs to cutaneous basal cell carcinomas (BCCs) has not been convincingly documented despite several attempts, even though BCCs are significantly more common than SCCs in the general US and European populations. Documented cases have been dismissed as actinic keratoses that have simply occurred superimposed upon basal cell carcinomas due to chance occurrence of two common sun induced skin lesions at the same sites.

In a university based referral dermatopathology service, one of us (WCL) observed 21 cases of AKs progressing to BCCs during a 29 year period (January 1, 1985 to December 31, 2013) of continuous observation in which 115,898 cases of AKs, 10,188 cases of BCCs and 4,809 cases of SCCs were diagnosed. Only cases in which a continuous progression of cells from those showing changes typical of AKs to those showing changes typical of BCCs was observed were included. During the same interval 308 cases of AKs progressing to SCCs were diagnosed. To further test the hypothesis that AKs can progress to BCCs, cases of AKs that progressed to BCCs were further examined to determine those in which neither the AK nor the BCC occupied the entire skin surface (i.e., length of the epidermal basement membrane) present in the specimen. Of the 21 cases, 13 met this criterion. All were shave biopsies of sun damaged skin. For each biopsy, the proportion of the surface covered by the AK, the proportion occupied by the BCC, and the proportion of the surface overlapped by both lesions were measured and the proportion of overlap was compared to the proportion of overlap expected due to chance. Statistical analysis showed that the observed overlap exceeded the expected overlap by 32 per cent with p < 0.05. We conclude that progression of AKs to BCCs, while not as common as progression of AKs to SCCs, does occur in a small proportion of cases.

#5162

Id1 is an oncogenic target of RNA binding protein HuR in gastric cancer.

Shuang Han,1 Xiaoqing Wu,1 Yuxiao Guo,1 Jing Zhang,1 Xuan Gu,1 Rebecca T. Marquez,1 Jeffrey Aube,2 Liang Xu1. 1 _University of Kansas, Lawrence, KS;_ 2 _Department of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC_.

Hu antigen R (HuR), a ubiquitous RNA-binding protein (RBP), has been shown to be overexpressed in gastric cancer. Cytoplasmic HuR is reported to be associated with high stage, distant metastases and unresectability. HuR efficiently regulates cancer-related gene expression by binding to AU- and U-rich sequences (ARE) in the 3' and 5'-untranslated region (UTR) of mRNAs. Id1 (Inhibitor of DNA binding) is a putative oncogene that we previously found was upregulated in gastric cancer and associated with poor prognosis. Id1 mRNA contains several putative HuR ARE sites. Our goal is to determine the impact of HuR on Id1 expression. We first confirmed Id1 is a HuR target by Ribonucleoprotein Immunoprecipitation (RNP-IP) in gastric cancer cells. Then, we found KH-3, a potent and specific inhibitor of HuR, inhibited Id1 mRNA and protein expression. The proliferative and invasive capabilities of the gastric cancer cells were also inhibited by KH-3. We conclude that Id1 is a target of RNA-binding protein HuR in gastric cancer. HuR specific inhibitor, KH-3, may be a promising therapeutic agent in treating HuR/Id1 high gastric cancers.

#5163

Mitochondrial mutations and gene expression analysis in colorectal adenopolyps.

LaShanale M. Wallace, Sharifeh Mehrabi, Xuebiao Yao, Felix Aikhionbare. _Morehouse School of Medicine, Atlanta, GA_.

Colorectal cancer (CRC) is the third most common diagnosed and cause of cancer related deaths in both men and women in the United States. Numerous studies have analyzed mitochondria DNA mutations in CRC and other tumors. Results from these studies have detected high mutation rates which may lead to mitochondrial deregulation and tumor progression. Most CRCs develop from adenopolyps via the adenoma-carcinoma sequence. Therefore, analysis of mitochondrial mutations and gene expression may provide a mechanism for inhibition of this tumoral sequence in individuals with a high risk of developing CRC. In the present study, PCR based sequencing and reverse transcription-quantitative PCR (RT-qPCR) were used to determine if mutations in mitochondrial encoded genes and levels of expression of these genes could influence the progression of the adenoma- carcinoma tumoral sequence. Genes analyzed included MT-RNR1, MT-COI, MT-ATP6, MT- MT-CYB, and mitochondrial ND genes that are involved in the normal metabolism of mitochondria. Measurements were made for 34 tissue sample pairs obtained from various types of colorectal adenomas and corresponding normal surrounding tissues. Additionally, mitochondrial complexes I (NADH: ubiquinone oxidoreductase) and III (CoQH2-cytochrome c reductase) protein was analyzed. There was progressive differential expression of mt-genes and complexes I and III proteins among the colorectal tumor stages relative to their paired normal samples. The level of complexes I and III were higher in tumor tissues relative to normal surrounding tissues. Noticeably, the expression of MT-COI was higher in late stage carcinomas among; all studied transcripts. We detected 54 point mutations in one region ranging from 11871-11877. The frequency of these mutations in all stages was as followed; 71.5% in tubular adenoma, 57% tubulovillous, and 43% in villous and carcinoma. Our results suggest that alterations in mt-gene expression play a role in the transformation of the colorectal tumoral stages.

#5164

Protein kinase C epsilon is a mediator of KRAS-driven lung tumorigenesis.

Rachana Garg,1 Veena Kapoor,1 Martin Abba,2 David M. Feldser,1 Steven M. Albelda,1 Marcelo G. Kazanietz1. 1 _University of Pennsylvania School of Medicine, Philadelphia, PA;_ 2 _Centro de Investigaciones Inmunolo´ gicas Ba´sicas y Aplicadas, La Plata, Argentina_.

Exposure to a number of environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), are the major causative agents of lung cancer, the leading cause of cancer related deaths in the U.S. Oncogenic mutations in KRAS, one of the most common alteration found in non-small cell lung cancer (NSCLC), are induced in high frequency by PAHs and environmental carcinogens. Studies from many laboratories, including ours, established key roles for PKCε, a member of the protein kinase C (PKC) family, in mitogenesis and survival of cancer cells. Notably, PKCε is up-regulated in several epithelial cancers, including human NSCLC, suggesting a role in the development and/or maintenance of the malignant phenotype. We observed that PKCε inhibition/depletion blunt the proliferative, motile, and invasive properties of lung cancer cells with KRAS mutation (PLoS One;7(2):e31714,2012). More importantly, silencing PKCε reduced the ability of human NSCLC cells with KRAS mutation to form tumors in nude mice as well as impaired their metastatic potential (Oncogene 31(20):2593-600, 2011). As Ras-transformed cells have enhanced DAG levels and signaling, we hypothesized that genetic targeting of PKCε reverses lung tumorigenesis driven by oncogenic Ras. To address this, we intercrossed lung-specific mutant Ras mice (KrasLSL-G12D/+) with PKCε knockout mice. Notably, significant inhibition occurred in the formation of lung tumors upon loss of either one or both PKCε gene (PRKCE) alleles, with significant extension of mice lifespan, suggesting a role for PKCε in the initiation of lung tumorigenesis driven by oncogenic mutant KRas. Furthermore, in silico database analysis of KRAS mutated human lung adenocarcinomas revealed a significant association between high PRKCE expression and poor patient outcome. As environmental lung carcinogens (PAHs) induce mutations in Ras, we next intended to test the hypothesis that PKCε is implicated in the action of these carcinogens. Interestingly, alveolar hyperplasia as well as pulmonary adenomas induced by benzo(a)pyrene, a prototypical PAH, were significantly reduced upon loss of one or two PKCε alleles, suggesting that genetic ablation of PKCε impairs chemically-induced lung carcinogenesis. Overall, our results indicate that PKCε is a novel effector of KRAS in lung cancer that may represent a promising target for disease treatment.

#5165

ADAM17: a common effector for RAS and MET-driven transformation of primary keratinocytes.

Christophe Cataisson, Mary Klosterman, Kelly Shibuya, Glenn Merlino, Stuart H. Yuspa. _National Cancer Institute, Bethesda, MD_.

The purpose of the current study is to determine the contribution of the metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) to the transformation driven by activated MET or oncogenic RAS in primary keratinocytes. Transgenic keratinocytes overexpressing HGF (MT-HGF) to activate MET and keratinocytes transduced with an oncogenic RAS share identical phenotypic and biochemical features of transformation and produce squamous tumors in vivo. In both keratinocyte populations, these common features arise from autocrine activation of EGFR through elevated expression and release of EGFR ligands. Amphiregulin (AREG) blockade in MT-HGF keratinocytes decreases MET-mediated EGFR transactivation. Inhibition of EGFR ablates the initiated signature of MT-HGF keratinocytes in vitro and causes regression of tumors from MT-HGF keratinocytes tranplanted in vivo. Deletion or knock-down of ADAM17 decreases EGFR activation in both RAS and MT-HGF keratinocytes and reverses the transformation associated gene signature in RAS keratinocytes. Using AREG release as an indicator of ADAM17 activity, we determined that knock-down of either the SRC kinase or inactive Rhomboid 2 (iRhom2) reduces the ability of keratinocytes to release AREG upon HGF stimulation. Collectively our data suggest that MET-mediated transformation of keratinocytes occurs through EGFR transactivation and that ADAM17 is a necessary effector for RAS and MET-driven transformation.

#5166

Gene expression microarray analysis identifies association of genes in the integrin/fak signaling pathway with claudin-7 in human colon cancer tissue.

Lu Kong, Lei Ding. _Capital Medical University, Beijing, China_.

The role and mechanism of the integrin/focal adhesion kinase (FAK)/ERK signaling pathway in the progression of many malignant tumor types, including colon cancer, have been widely reported. The claudin family of proteins is integral to the structure and function of tight junctions. The role of claudin-7 (Cldn-7, CLDN7) in the regulation of the integrin/FAK/ERK signaling pathway remains poorly understood. Therefore, we investigated differences in gene expression, primarily focusing on CLDN7 and integrin/FAK/ERK signaling pathway genes, in colon cancer and adjacent normal tissues. Quantitative real-time RT-PCR verified the results for mRNA expression, and immunohistochemistry verified the results for protein expression. In silico analysis was used to predict co-regulation between Cldn-7 and integrin/FAK/ERK signaling pathway members. Our results showed marked cancer stage-specific decreases in the protein expression of Cldn-7, Gelsolin, MAPK1 and MAPK3 in colon cancer samples. The observed changes in the expression of all proteins except Cldn-7 were in agreement with the changes in the corresponding mRNA levels. Cldn-7 was predicted not to directly regulate any genes in the integrin/FAK/ERK signaling pathway, but these factors may participate in a common network that regulates cancer progression, in which the MAPK pathway is the central node.

#5167

Expression levels of orexin receptor 1 in different stages of colorectal cancer.

Ana M. Wielandt,1 Cynthia Villarroel,1 Claudia Hurtado,1 Kento Inada,2 Hiroshi Kawachi,2 Daniela Simian,1 Maria T. Vial,1 Marcela Figueroa,1 Magdalena Castro,1 Udo Kronberg,1 Francisco Lopez-Kostner1. 1 _Clinica las Condes, Santiago, Chile;_ 2 _Tokyo medical and dental university, Tokyo, Japan_.

Introduction: Colorectal cancer (CRC) is the fourth leading cause of cancer death in Chile. Currently, treatment is surgical and adjuvance is based on chemotherapy mainly. One of the risk factors of CRC is obesity and metabolic syndrome. In search of factors that induce cell apoptosis, orexins were identified. Orexins are neuropeptides that regulate appetite, inhibit satiety and increase energy expenditure. The altered expression of orexin or its dysregulation has been associated with a wide range of human diseases such as narcolepsy, obesity, drug addiction and cancer. In rats and mice lacking orexin receptor, an increase in body mass index (BMI) was observed. Orexin Receptor 1 (OX1R) is overexpressed in CRC, but is not detected in normal colon tissue. Its activation by Orexin-A promotes apoptosis in cancer cell lines, and can be modulated by diet. Aim: To determine OX1R expression in the adenoma-carcinoma progression of CRC as possible therapeutic target, and its correlation with BMI. Material and Methods: Patients with neoplastic colorectal lesions undergoing surgical or endoscopic treatment between 2014 and 2015 were prospectively enrolled. Tissue samples of 29 patients were divided into 4 groups. Group A: control (n=5), Group B: low-grade adenoma (n=5), Group C: high-grade adenoma (n=7), Group D: adenocarcinoma (n=12). Formalin-fixed-paraffin-embedded samples were used to perform immunohistochemistry (IHC) of OX1R. Fresh tissues from Group D were used to assess protein and mRNA expression of OX1R by Western-blot and quantitative RT-PCR, respectively. Primary cultures were established from fresh tumor tissue of patients with adenocarcinoma. To compare OX1R expression levels we used the t-student test. Results: IHC expression of OX1R was detected both in the luminal membrane of colorectal epithelium and in the cytoplasm. We observed stained cells in 0-1% of the total area in Group A, B and C. In Group D, 7 samples showed staining 0-1%, 2 samples 1-10% and 3 samples 10-20%. High mRNA and protein expression was detected in stage III- IV adenocarcinoma samples compared to stage 0, I and II tumor samples. Normal adyacent tissue of CRC patients does not express OX1R. BMI is inversely associated with OX1R expression. Orexin A was able to induce apoptosis in primary cell cultures from advanced tumors in a dose dependent manner. Conclusions: OX1R is expressed scarcely in early stages of carcinogenesis. Its expression is induced significantly only in the most advanced adenocarcinomas, both at protein and mRNA levels, and is inversely associated with BMI. In primary cultures of patients with CRC, orexin A is able to induce apoptosis in a dose dependent manner. FONDECYT 1140012.

#5168

Mitochondrial 16srRNA variants and complex III expression in serous ovarian cancer.

Shakeria L. Cohen,1 Sharifeh Mehrabi,1 Edward E. Partridge,2 Felix Aikhionbare1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _University of Alabama, Birmingham, Birmingham, AL_.

Patients suffering from serous ovarian cancer (SOC) are typically diagnosed at later stage, drastically decreasing their five-year survival prospect. Diagnostic tools, including the measurement of elevated levels of the CA125 protein released by ovarian tumors, provide valuable information but are inherently limited in specificity in diverse populations. Hence, there remains a need for more advanced diagnostic tests to distinguish the different histopathological subclasses to improve treatment outcome. Reactive oxygen species (ROS), by products of normal energy production within the mitochondria, are among the list of compounds believed to be involved in aging and the pathogenesis of many human diseases. Moreover, elevated ROS levels may cause subtle changes in regions of the mitochondrial genome (mtDNA) inducing oxidative stress and tumor microenvironment. Consequently, the events could be used to establish potential mtDNA markers involved in the progression of SOC as an age related disease. The aim of this study is to determine mitochondrial mutational roles and protein expression level in both precancerous and malignant stages of SOC. In the current study PCR-based sequencing was used to detect mtDNA sequence variants in the 16srRNA gene, known to be highly mutated in SOC. The gene spans 1671 to 3229 bp within the mtDNA and is highly conserved. Thirty-one frozen SOC tissue samples of four histopathological subclasses [cystadenoma, n=7; borderline, n=8; and malignant stages III/IV, n=8 and normal no egg, no surface epithelium as a control, n=8] were examined. Additionally, expression of mitochondrial complex III (Coenzyme Q-cytochrome c reductase) was evaluated using western blot analysis. Forty variants were detected of which three were unreported. G1811A, an unreported variant was detected in 50% of the borderline samples. The unreported variant C2794G was detected in normal, cystadenoma, and borderline with an increasing frequency of 50%, 83% and 100% correspondingly, however did not display in the malignant samples. A3364G an unreported variant was observed in all four categories with a frequency of approximately 70% or higher. Complex III was expressed progressively in normal, borderline and malignant samples. Moreover, the expression of complex III was significantly higher in the malignant samples compared to the normal control. Results from this study could suggest that the genetic instability of 16srRNA region may play a role in ovarian tumorigenicity. Additionally the over expression of complex III in the malignant stages could serve as a target for chemotherapy of serous ovarian disease.

#5169

Functional studies of the prostaglandin E2 receptor EP4 in ovarian cancer.

Joceyln C. Reader, Paul Staats, Olga Goloubeva, Ningbo Jian, Dana M. Roque, Maya E. Matheny, Amy M. Fulton, Gautam G. Rao. _University of Maryland Medical Center, Baltimore, MD_.

Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States. Most cases of ovarian cancer present in late stages leading to recurrent disease which is incurable. Few effective targeted therapies are available to treat ovarian cancer other than non-specific, toxic chemotherapy. Ovarian cancer is often fatal due to innate or acquired chemoresistance. Lipid mediators are linked to many pathological processes including inflammation and cancer. The lipid prostaglandin E2 (PGE2) is synthesized by cyclooxygenases, COX-1 and COX-2, and elevated expression of COX and increased PGE2 levels are functionally linked to the progression of many cancers. PGE2 is exported from the cell where it acts in a paracrine and autocrine manner by activating a family of four G-protein coupled receptors (EP1-4) which are linked to different intracellular signaling pathways. EP1 is coupled to calcium mobilization and PKC; whereas, EP2 and EP4 activate PKA/cAMP, PI3K and ERK pathways. EP3 generally inhibits cAMP levels. COX-1 has been shown to be overexpressed in primary ovarian cancer as well as in many ovarian cancer cell lines. The EP4 receptor is overexpressed and plays a mechanistic role in various malignancies including breast and colon. Due to toxicity concerns surrounding global inhibition of the COX enzymes and, in an effort to identify new targeted therapies for the treatment of ovarian cancer, our objective was to determine the expression as well as the functional role of the PGE2 receptor EP4 in ovarian cancer. We hypothesized that the EP4 receptor is overexpressed in ovarian cancer and that binding of its cognate ligand, PGE2, will drive ovarian cancer progression and that inhibition of the EP4-mediated signaling will lead to inhibition of ovarian cancer growth and metastasis.

In order to test these hypotheses, we analyzed the expression of the EP4 receptor in a human ovarian cancer tissue microarray (TMA) as well as human ovarian cancer cell lines. Immunohistochemical analysis of EP4 on the TMA composed of varying histologies, including serous, endometrioid and clear cell, as well as normal ovarian tissue revealed that EP4 was expressed in 38.7% of ovarian cancer patients; whereas, EP4 was not expressed in the 10 normal ovarian tissue samples. Additionally, in comparison to immortalized human ovarian surface epithelial (HOSE) cells, EP4 is overexpressed in many of the cell lines analyzed, including ES-2, OVCAR-3, COAV-3, SKOV3, OVISE and Kuramochi cells. Treatment of these cell lines with an EP4 antagonist resulted in decreased proliferation and migration compared to vehicle control. Consistent with the pharmacological data, treatment of ovarian cancer cell lines with siRNA directed against the EP4 receptor lead to decreased proliferation and migration. Based on these data, targeting of the PGE2 EP4 receptor should be investigated further for the treatment of ovarian cancer.

#5170

Snail-dependent ESRP1 silencing drives malignant conversion of human pulmonary epithelial cells.

Tonya C. Walser,1 Zhe Jing,1 Linh Tran,1 Ying Lin,1 Li Zhu,1 Sherven Sharma,1 Aik Ooi,1 Brigitte N. Gomperts,1 Jerry W. Shay,2 Jill E. Larsen,2 Boning Gao,2 John D. Minna,2 Micihael C. Fishbein,1 Steven M. Dubinett1. 1 _UCLA David Geffen School of Medicine, Los Angeles, CA;_ 2 _University of Texas Southwestern Medical Center, Dallas, TX_.

The significance of Snail expression in the early pathogenesis of non-small cell lung cancer has not been determined. Here, we report robust Snail expression in premalignant lung lesions relative to histologically normal-appearing pulmonary and lung cancer tissues. Utilizing immortalized human bronchial epithelial cells (HBECs) to isogenically model the contribution of the transcriptional repressor Snail to lung carcinogenesis, we document Snail-dependent anchorage-independent growth of pulmonary epithelial cells in vitro, as well as their transformation, primary tumor growth, and metastatic behavior in vivo. We delineate ESRP1 tumor suppressor silencing as a requirement for the Snail-driven transformation and identify ESRP1 silencing in Snail-expressing pulmonary premalignant lesions in situ. Utilizing this HBEC lung carcinogenesis model, we determined that Snail drives the expansion and malignant conversion of an ALDH+CD44+CD24- subset of pulmonary stem cells. Apoptosis-resistance and expression changes in oncogenes and tumor suppressors were noted, and ESRP1 was identified as a mediator of the malignant conversion of these Snail-primed pulmonary. Collectively, this is the initial report of a Snail-ESRP1-cancer axis that is operative during lung carcinogenesis and the first characterization of potentially targetable mediators of this normal to cancer continuum.

#5171

Interleukin-17 acts through MMP7 to promote prostate cancer.

Qiuyang Zhang, Sen Liu, Keshab Parajuli, Zongbing You. _Tulane University School of Medicine, New Orleans, LA_.

Interleukin-17 (IL-17) is a key cytokine in inflammation, which has been shown to play critical roles in development of prostate cancer, skin cancer, colon cancer, and breast cancer. We have previously demonstrated that IL-17 promotes prostate carcinogenesis, accompanied with increased expression of matrix metalloproteinase 7 (MMP7) in the mouse prostate. The purpose of the present study was to determine whether MMP7 mediates IL-17's action and the underlying mechanisms. Mmp7 knockout (Mmp7-/-) mice were crossbred with phosphatase and tensin homolog (Pten) conditional knockout mice (Ptenpc-/-, prostate-specific through probasin promoter-driven Cre recombinase). The prostate tumor phenotypes were analyzed among Mmp7+/+;Ptenpc-/-, Mmp7+/-;Ptenpc-/-, and Mmp7-/-;Ptenpc-/- mice. MMP7-overexpression or MMP7 knockdown using small interfering RNA (siRNA) was generated in human prostate cancer LNCaP and C4-2B cell lines to explore the molecular mechanisms of MMP7's actions. We found that Mmp7-/-;Ptenpc-/- mice displayed prostates that were smaller than Mmp7+/+;Ptenpc-/- or Mmp7+/-;Ptenpc-/- mice. Mmp7-/-;Ptenpc-/- mice developed a reduced number of invasive prostate adenocarcinomas with lower rate of cellular proliferation and higher rate of apoptosis than Mmp7+/+;Ptenpc-/- or Mmp7+/-;Ptenpc-/- mice. In addition, the invasiveness of prostate tumor cells isolated from Mmp7-/-;Ptenpc-/- mice was significantly reduced compared to those isolated from Mmp7+/+;Ptenpc-/- mice in Matrigel® invasion assays. Expression of the epithelial-to-mesenchymal transition (EMT) markers, such as β-catenin, vimentin, Snail, and Slug, was dramatically reduced in Mmp7-/-;Ptenpc-/- mice compared to Mmp7+/+;Ptenpc-/- or Mmp7+/-;Ptenpc-/- mice. In contrast, expression of the epithelial markers such as E-cadherin and claudin was increased in Mmp7-/-;Ptenpc-/- mice compared to Mmp7+/+;Ptenpc-/- or Mmp7+/-;Ptenpc-/- mice. MMP7 cleaved E-cadherin and disrupted E-cadherin/β-catenin complex to increase cytoplasmic and nuclear β-catenin levels in the prostate cancer cells, thus up-regulating EMT transcription factors. In addition, IL-17 receptor C (Il-17rc) and Pten double knockout (Il-17rc-/-;Ptenpc-/-) mice echoed the reduction of EMT in Mmp7-/-;Ptenpc-/- mice, compared to Il-17rc+/+;Ptenpc-/- mice. Further, IL-17 treatment induced MMP7 expression in LNCaP and C4-2B cells, leading to increased expression of EMT markers. In contrast, MMP7 knockdown inhibited IL-17-induced expression of EMT markers. Taken together, our results demonstrate that MMP7 mediates IL-17's function in promoting prostate carcinogenesis through induction of EMT.

This work was partially funded by National Institutes of Health (R01CA174714 and P20GM103518), Department of Defense (W81XWH-14-1-0050, W81XWH-14-1-0149, W81XWH-14-1-0458, and W81XWH-15-1-0444), and Tulane's Institute of Integrated Engineering for Health and Medicine.

#5172

Forerunner genes contribute to bladder carcinogenesis by altering the basal to luminal urothelial transition program.

Sangkyou Lee, Jolanta Bondaruk, Sooyoung Lee, June G. Lee, Tadeusz Majewski, Woonyoung Choi, Charles Guo, Colin Dinney, Li Zhang, Keith Baggerly, Richard Behringer, David McConkey, Bogdan Czerniak. _UT MD Anderson Cancer Center, Houston, TX_.

Background: The idea that microscopically recognizable dysplasia and carcinoma in situ act as precursor conditions for invasive cancer is the prevailing view of how many common epithelial malignancies develop. However, new evidence suggests that these are not the initiating events of carcinogenesis but represent the evolution of pre-existing occult disease, caused by the field effects of carcinogens and/or chronic inflammation, which shows minimal phenotypic deviation from normal tissue. These effects may form large plaques in the mucosa of the affected organ but the mechanisms driving their developments are unknown.

Design: We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term "forerunner" (FR) genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11) whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. We focused on two model FR genes, LPAR6 and CAB39L, and validated their involvement in the development of mucosal field effects by in vitro mechanistic studies and in vivo models.

Results: We provide evidence that silencing of prototypic FR genes by hypermethylation (LPAR6 and CAB39L) and less frequently by mutations (LPAR6) is associated with the initial expansion of intraurothelial neoplasia and sets the stage for subsequent events of carcinogenesis. We also found that LPAR6 and CAB39L control cell survival and proliferation consistent with the loss of function being contributory to tumorigenesis. We validated their involvement in three large independent cohorts of bladder cancer, which were profiled according to a novel molecular taxonomy and show that they distinctively contribute to the development of luminal (LPAR6) and basal (CAB39L) subtypes. The in vitro studies using cell lines and their ability to form urospheres showed that these genes contribute to carcinogenesis by altering the transition from basal to luminal phenotypes. Consistent with these observations the BBN induced cancers in Lpar6-/- mice were of luminal type while the cancers induced in Lpar6+/+ mice were of basal type.

Conclusion: Loss of prototypic FR genes contributes to the development of mucosal field effects initiating bladder carcinogenesis by altering the basal to luminal urothelial transition program.

#5173

Role of Gli2 in mediating the progression of prostate cancer to CRPC.

Lu Xia, Tai Qin, Luzhe Sun. _UTHSCSA, San Antonio, TX_.

Background: Prostate cancer is a leading cause of cancer-related death in the US men, largely due to the development of castration resistant prostate cancer (CRPC) in the patients treated with androgen deprivation therapy. There is increasing evidence indicating that Hedgehog pathway is involved in the malignancy of prostate cancer, which includes the finding of two prostate tumors with loss-of-function mutations in the SuFu gene, correlation of tumor grade with high expression of Sonic hedgehog, Gli2 and/or hedgehog target genes including Ptch1 and HIP, and increased Gli2 expression in therapy-resistant tumor cells from patients. In addition, treatment of mice with hedgehog inhibitors significantly attenuated the growth of prostate tumor xenografts. Some recent studies also implicated a crosstalk between Hedgehog/Gli signaling and androgen signaling pathways in prostate cancer. However, the role of Gli2 in CRPC progression has not been investigated. Methods: A human Gli2 specific small hairpin RNA (shRNA) expression vector was constructed using Tet-PLKO-puro lentivector, which was delivered into the androgen-dependent human prostate cancer LNCaP cells. RT-PCR, Western Blot and Luciferase Reporter Assay were performed to confirm doxycycline-inducible Gli2 knockdown. The effect of Gli2 knockdown on anchorage-dependent or -independent growth was assessed with MTT and Soft Agar Assays respectively. Cells were cultured in androgen depleted medium or treated with the anti-androgen, Casodex, to mimic androgen deprivation condition. Mouse xenograft models were used to assess the role of Gli2 in the development of CRPC in vivo. Results: Androgen deprivation promoted the expression of Hedgehog signaling components including Gli2 in the LNCaP cells. Gli2 knockdown inhibited anchorage-dependent and -independent growth of LNCaP cells. Casodex complemented the inhibition of Gli2 knockdown on the growth of LNCaP cells. Gli2 knockdown prevented the outgrowth of androgen-independent cells from LNCaP cells cultured in androgen depleted medium. Induced-knockdown of Gli2 by doxycycline inhibited the growth of castration-resistant tumors in vivo in mice. Conclusions: Our study demonstrated the important role of Gli2 in the development of CRPC. Therefore, it may have the potential to serve as a novel therapeutic target for the prevention of CRPC.

#5174

Modeling mesothelioma initiation through disruption of the Hippo pathway revealed involvement of PLCB4 in YAP-driven mesothelioma cell proliferation.

tatsuo kakiuchi. _aichi cancer center, nagoya, Japan_.

Disruption of the Hippo pathway as a result of deletion and/or mutation of the involved genes (e.g., neurofibromin 2 [NF2]) is frequently observed in mesothelioma. The disruption results in reduced phosphorylation of yes-associated protein (YAP), and the non-phosphorylated YAP translocates to the nucleus and regulates gene expression. While the roles of the disrupted Hippo pathway in the maintenance of established tumors have been investigated using mesothelioma cell lines, its involvement in the initiation of mesothelioma remains unclear.

We used immortalized human mesothelial cells to study the transformation process, and found that NF2 knockdown led to transformation of the cells concurrently with reduction in YAP phosphorylation. The cells exhibited enhanced growth in vitro, and formed tumors following transplantation into nude mice. Similar results to those obtained by NF2 knockdown were also obtained using enforced expression of wild-type (wt) or constitutively active (S127A) YAP. Although enforced expression of YAPwt or YAPS127A was insufficient to transform primary (unimmortalized) human mesothelial cells, our findings provide evidence for the crucial roles of activated YAP in the transformation of mesothelial cells.

To identify YAP-regulated genes critical for mesothelial tumorigenesis, we conducted gene expression analysis comparing control- and YAP-transduced immortalized human mesothelial cells. Gene Set Enrichment Analysis (GSEA) using a gene set down-regulated by YAP knock-down in mesothelioma cell lines revealed phospholipase C beta 4 (PLCB4) to be among the top-ranking genes up-regulated by YAP in our experiments. PLCB4 was up-regulated by YAP in immortalized human mesothelial cells, and down-regulated by YAP knock-down in YAP-driven mesothelioma cells. shRNA-mediated silencing of PLCB4 attenuated the growth of YAP-transduced mesothelial cells and Hippo-disrupted, but not -proficient, mesothelioma cell lines.

Our model system thus provides a versatile tool to investigate the mechanisms underlying mesothelioma development. We suggest that PLCB4 may be an attractive drug target for the treatment of malignant mesothelioma. 

### Therapeutic Studies in Patient-derived Xenografts

#5175

Mutational and copy number profiling of cancer-related genes in 26 human tumor xenografts and their correlations with antitumor drug sensitivities.

Takashi Kobunai,1 Kenta Tsunekuni,2 Kazuaki Matsuoka,1 Hiroshi Tsukihara,1 Teiji Takechi1. 1 _Taiho Pharmaceutical Co., Ltd., Tokyo, Japan;_ 2 _Graduate School of Medicine, Osaka University, Osaka, Japan_.

Background: Tumor responses to antitumor drugs are variable, but predicting these responses is important when selecting effective chemotherapy treatments. Our aim was to identify variations or alterations in gene copy number that influence cancer cells' susceptibilities to standard chemotherapeutic agents. Methods: Twenty-six human cancer cell lines representing the five main tumor types were subcutaneously implanted into nude mice and tested for sensitivity to fluorinated pyrimidines (UFT, TS-1, 5'-DFUR, and capecitabine), CDDP, CPT-11, and paclitaxel. The cell lines included lung (AOI, LC-11, Lu-99, LX-1, LC-6, Lu-134, Lu-130), colon (KM12C, KM12C/FU, HCT-15, COL-1, CO-3), pancreas (PAN-3, PAN-4, PAN-12, H-48, MIAPaCa-2, BxPC-3), gastric (SC-2, ST-40, 4-1ST, SC-4) and breast (MC-2, MX-1, MDA-MB-435SHM, MDA-MD-231). Genomic DNA was prepared from frozen tumor tissues. Mutations in 48 genes from the TruSeq Amplicon Cancer Panel were screened using the MiSeq system (Illumina, San Diego, CA). Somatic copy number alterations were analyzed by high-density SNP arrays (Affymetrix, Santa Clara, CA). Results: Of the 225 amplicons (187 non-overlapping regions) in the cancer panel, 86% achieved a minimum average sequencing depth of 1000X and the average coverage across all target regions was 5374X. In 26 tumors, sequencing detected 55 somatic mutations in 18 out of 48 cancer related genes of high prognostic or therapeutic significance, such as TP53, APC, PTEN, and SMAD4. Mutation frequencies across 26 xenografts were 73.1% (TP53), 38.5% (KRAS), 15.4% (APC), 11.5% (SMAD4 and RET), 7.7% (BRAF, GNAS, and PTEN), and 3.8% (CTNNB1, GNAQ, HNF1A, HRAS, IDH1, KIT, NOTCH1, PIK3CA, SMO, and STK11). Tumor xenografts with TP53 mutations were significantly less sensitive to CDDP and CPT-11 than wild-type cell lines (P<0.05). The APC mutation conferred resistance to paclitaxel. Copy number gain was observed at 23.1% (KRAS), 15.1% (EGFR), and 11.5% (JAK2 and CDK2NA). Copy number loss was observed at 30.8% (CDK2NA), 19.2% (SMAD4), and 15.4% (PTEN). Cell lines with more copies of CDK2NA and JAK2 were more sensitive to CDDP, while cell lines with fewer copies of PTEN were more sensitive to CDDP. Similarly, copy number gain of CDH1 conferred resistance to UFT, while copy number gain of KRAS sensitized tumors to 5'-DFUR. The copy number of 48 genes determined by the GeneChip array moderately agreed with those estimated by local GC-content adjusted coverage profiles in the sequencing analysis (average Pearson's correlation coefficient = 0.66, 95%CI = 0.54-0.69). Conclusions: Integrated analysis of mutational profiling and gene copy number may be useful to elucidate candidate genes influencing susceptibility of cancer cells to antitumor drugs.

#5176

Orthotopic syngeneic tumor models for preclinical evaluation of cancer immunotherapy strategies.

Juan Zhang, Sheng Guo, Zhensheng Wang, zhongliang Li, Meng Qiao, Qian Shi. _Crown Biosciences, Taicang, China_.

Background: The recent successes of cancer immunotherapies have stimulated interest in the potential application of these approaches in many solid and hematologic tumors. Syngeneic tumor models have been widely used in cancer immunotherapy assessment. However, most of these experimental models were established as subcutaneous models, while there may be significant difference in immune microenvironment comparing orthotopic vs. subcutaneous tumors. In addition, drug delivery and metabolism are different in orthotopic tumors vs. subcutaneous tumors. To address this unmet need, we set out to validate a panel of orthotopic syngeneic models for anti-cancer therapeutics (mainly immunotherapeutics) evaluation.

Results: A large collection of orthotopic syngeneic models covering breast cancer, liver cancer and various hematological cancers were established in immunocompetent mice. Survival curves were established and the responsiveness to anti-PD1, anti-PDL1 and anti-CTLA4 antibodies were evaluated in the orthotopic models. Orthotopic liver tumors were monitored with ultrasound imaging to obtain longitudinal growth curves. The correlation in drug efficacy between the orthotopic model and its subcutaneous counterpart was analyzed. The role of NK cells in the development of a panel of systemic hematological tumor models was investigated with NK depleting antibodies.

Conclusion: Despite the obvious difficulties, orthotopic syngeneic models represent closer model systems to human diseases and may be a valuable tool to the evaluation of cancer immunotherapies and their combination strategies.

#5177

RNAseq and FACS profiling of syngeneic mouse models treated with immune checkpoint inhibitors enable biomarker discovery and model selection for cancer immunotherapy.

Lan Zhang, Juan Zhang, Sheng Guo, Wubin Qian, Zhensheng Wang, Qian Shi. _Crown Biosciences, Taicang, China_.

Background: Syngeneic tumor models have long been used in cancer research. Recently, the clinical success of anti-CTLA4 and anti-PD1 antibodies resulted in increased interests in using syngeneic models to evaluate cancer immunotherapeutics. Furthermore, as researchers discovered chemo, radio and targeted therapies of cancer may interact or change tumor immune environment, they are looking for suitable models that may evaluate the combination of those therapies. More importantly, in the clinic it is still unknown why some patients respond to certain immunotherapies while others do not. We set out to utilize syngenetic models to address those questions.

Material and methods: Syngeneic cell line models were used to evaluate anti-PD1, PD-L1 and CTLA4 antibodies efficacies. 13 tumors from different syngeneic models were collected before the treatment for RNAseq analysis. Tumors were also collected after treatment for immune cell analysis with FACS for PD response. Biomarkers were analyzed with the RNAseq data to predict treatment response to different immune checkpoint inhibitors.

Results: Crown has established the largest collection of syngeneic models with well characterized immunotherapy data. Those models have a diverse response to anti-PD1, PD-L1 and CTLA4 antibodies, ranging from close to 100% inhibition to inducing tumor growth upon antibody treatment. Mostly recently, we have generated detailed maps of the expressional and mutational profiles of those models. Mutational analysis indicated a number of syngeneic models harbor mutations that may be useful for combination studies of targeted and immuno-therapy. Chemo and targeted therapy in combination with immunotherapy in syngeneic models suggested potential strategies that may be successful in the clinic. Analysis of RNAseq and FACS analysis data indicated markers that may be useful to predict immunotherapy response.

Conclusions: These data will enable selection of models for chemo or targeted therapies in combination with immunotherapy. In addition, predictive biomarkers obtained from the analysis may be useful in understanding patient response in the clinic.

#5178

Determinants of response to temozolomide in an exceptionally sensitive patient derived model.

Lara H. el Touny,1 John Connelly,1 Curtis Hose,1 Anne Monks,1 Mark W. Burkett,1 Erik Harris,1 Rene' M. Delosh,1 Julie Laudeman,1 Chad Ogle,1 Russell Reinhart,1 Michael Selby,1 Thomas Silvers,1 David Evans,1 Dianne Newton,1 Luke Stockwin,1 Melinda Hollingshead,2 Ralph Parchment,1 James H. Doroshow,3 Beverly Teicher,4 Annamaria Rapisarda1. 1 _NCI-Frederick/Leidos Biomed. Research, Inc., Frederick, MD;_ 2 _NCI-Frederick, Frederick, MD;_ 3 _NCI, Bethesda, MD;_ 4 _NCI, Rockville, MD_.

Application of precision medicine to cancer treatment utilizes cutting-edge genomic sequencing techniques to identify specific mutations in tumors that can be matched to targeted therapies designed to treat those abnormalities. To complement NCI-MPACT, an ongoing molecular profiling-based clinical trial (NCT01827384), we used cell lines developed from several patient-derived xenograft (PDX) models to examine response to, and potential biomarkers for, the regimens of the 4-arm MPACT trial: veliparib (VLP)/temozolomide (TMZ), AZD1775/carboplatin, everolimus and trametinib. In vitro sensitivity of the PDX-derived cell lines to clinically achievable concentrations of these MPACT drugs (combinations and single agents) was examined in classic 2D cultures (monolayer on plastic) and 3D cultures (spheroids generated in ultra-low attachment culture plates). Responses in 2D and 3D cultures were similar after 7 days of drug exposure. Moreover, adding VLP (1.7µM or 5µM) in combination with TMZ did not enhance TMZ cytotoxicity. A bladder cancer cell line developed from a PDX model (BLX) showed exceptional sensitivity to TMZ (IC50 ~ 3-5µM) compared to a lung cancer cell line (also produced from a PDX; LUX) which was insensitive (IC50 >40 µM). Loss of MGMT expression in glioma and possibly in colorectal carcinoma is considered a predictive biomarker for response to alkylating agents, such as dacarbazine and TMZ. Indeed, we could not detect MGMT protein expression in BLX, while MGMT was present at high levels in LUX. To elucidate unique determinants of BLX hypersensitivity to TMZ beyond MGMT expression, we examined DNA damage responses elicited in this model. Under both 2D and 3D conditions, exposure to TMZ (13 and 40µM) for various times (4, 8, 12, 24, 48, 72 and 96 hrs) induced γH2AX after 24 hr, while PARP1 cleavage was induced as early as 48 hrs after drug addition indicating the onset of apoptosis. TMZ activated ATR and ATM signaling pathways especially at the later time points, paralleling the pharmacodynamics of PARP cleavage. Moreover, concordant with the sensitivity profiles, signaling activation in 2D and 3D conditions was similar. In contrast, none of these pathways were activated in the TMZ non-responsive LUX model. Cell line models developed from PDXs with intermediate MGMT expression are being evaluated.

A key to success of personalized medicine in oncology will be the identification of genomic determinants that predict which individual cases will show exceptional responses to particular treatments, and our data suggest that PDX cell line models may be valuable for elucidating molecular and genetic characteristics of response to specific drugs and for the identification of predictive biomarkers.

Funded by NCI Contract No. HHSN261200800001E. This research was supported, in part, by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.

#5179

Development of a humanized mouse model for direct evaluation of anti-human PD-L1 antibodies.

Meng Qiao, Juan Zhang, Jian Ding, Rui Zhang, Zhongliang Li, Jia Zheng, Teng Lu, Jinjing Ning, Qian Shi. _Crown Bioscience Inc., Santa Clara, CA_.

Therapies that perturb the binding of programmed death-1 receptor ligand (PD-L1) to its receptor PD-1 achieved unprecedented rates of sustained clinical responses in patients with various types of cancer. Mouse surrogate antibodies were initially evaluated in syngeneic mouse models as the proof of concept for anti-PD-L1 therapy.

However, there's an urgent need to develop appropriate animal model to directly evaluate anti-human PD-L1 antibodies before clinical trial. Here we describe our ongoing efforts in developing a chimeric mouse/human cell line to test human anti PD-L1 antibody. We profiled PD-L1 expression by FACS using anti-mouse PD-L1 antibody and confirmed expression of PD-L1 in a series of murine cancer cell lines. H22, a liver cancer line with moderate expression level of PD-L1 was selected due to its in vivo sensitivity towards anti-PD1 and anti-PD-L1 antibodies. Crispr/cas9 system was employed to knockout murine PD-L1 and replace it with the human counterpart. The targeted knockout had been confirmed by sequencing, the expression of human PD-L1 is illustrated by FACS using antibody that specifically recognize human form and no cross reaction to mouse form. The engineered H22-hPD-L1 cell line was then inoculated subcutaneously to immune-competent BALB/c mice to establish the in vivo model that maintains complete murine immune system and harbor humanized PD-L1. The tumor growth curve of H22-hPD-L1 model and its sensitivity to anti-human PD-L1 antibody have been established.

In summary, our H22-hPD-L1 model may be a valuable tool to evaluate the in vivo activity of anti-human PD-L1 antibody therapies either as a single agent or combinational strategies. The similar engineering may be applied to more murine cell lines (e.g. EMT-6, B16F10 and MBT-2, etc) to provide a spectrum of cell lines with different diseases and genetic makeup for immunotherapies involving anti-hPD-L1 antibodies.

#5180

Bayesian multi-endpoint analysis of syngeneic mouse immunotherapeutic trials.

David Gold,1 Ningning Chen2. 1 _Bristol-Myers Squibb, Princeton, NJ;_ 2 _Purdue University, West Lafayette, IN_.

Recent and notable immunotherapeutic successes for the treatments of melanoma and lung cancer have catalyzed wide-spread development of novel immunotherapies and combinations for a variety of cancers. While promising, responses to immunotherapies are a focal point of present investigations, being as of yet challenging to explain. Amongst the many translational challenges, pre-clinical in bred syngeneic mouse experiments often display heterogeneous tumor responses, whereby a fraction of tumors show resistance, partial, or complete response to the same immuno-intervention(s) implanted in identical in bred mouse strains.

We mathematically modeled heterogeneous tumor responses in syngeneic mouse trials with Bayesian statistical methodology. Our decision criteria for drugs and drug combinations can simultaneously evaluate two or more endpoints. At the heart of our methodology is Bayesian learning with prior information that did not compromise statistical performance; actually, we achieved statistical gains to identify superior immunotherapies, rendering quantifiable efficiencies by design. We illustrate an augmented approach to discovery by Bayesian learning in real syngeneic mouse studies. We propose our powerful multi-endpoint Bayesian statistical criteria to ascertain superior pre-clinical immunotherapeutic activity.

#5181

Dual inhibition of RNA Pol I transcription and PIM kinase as a new therapy to treat prostate cancer.

Richard J. Rebello,1 Eric Kusnadi,2 Don Cameron,2 Analia Lesmana,2 Denis Drygin,3 Ashlee K. Clark,1 Laura Porter,1 Shahneen Sandhu,2 Gail P. Risbridger,1 Richard B. Pearson,2 Ross D. Hannan,4 Luc Furic1. 1 _Monash University, Clayton, Australia;_ 2 _Peter MacCallum Cancer Centre, Melbourne, Australia;_ 3 _Pimera, San Diego, CA;_ 4 _Australian National University, Canberra, Australia_.

Background: The MYC oncogene is commonly over-expressed in prostate cancer (PC). Upregulation of ribosome biogenesis and function is a characteristic feature of MYC-driven tumors. Accordingly, inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically in models of hematological malignancy characterized by elevated MYC expression. Additionally, PIM kinases activate MYC signaling and mRNA translation in PC and cooperate with MYC to accelerate tumorigenesis. Here we investigate the efficacy of a dual approach targeting ribosome biogenesis and function to treat PC by combining CX-5461 with the pan-PIM kinase inhibitor CX-6258 in murine and human models

Methods: The efficacy of CX-5461 and CX-6258, alone and in combination, was tested in PC cell lines, in the Hi-MYC mouse model of PC (n=8-11 per group) and in PC metastatic tissues. Primary cell lines derived from Hi-MYC mice were used to analyze signaling events underpinning therapeutic efficacy. Triplicate experiments were analyzed with ANOVA followed by Dunnett's post hoc test. All statistical tests were two-sided.

Results: CX-5461 reduced anchorage independent growth and induced cell cycle arrest in human PC cell lines and in primary prostatic epithelial cells from Hi-MYC mice (P<0.001). CX-5461 treatment of Hi-MYC mice induced p53 expression and activity and significantly reduced prostate epithelial cell proliferation (P=0.02) and invasion. While CX-6258 showed little effect alone, its combination with CX-5461 further suppressed proliferation (P=0.01), dramatically reduced the incidence of large invasive lesions from 64% to 9% and preserved prostate ductal architecture.

This promising combination strategy prevented the growth of PDX tissue characterized by elevated MYC and resistance to conventional therapy (P=0.04).

Conclusions: Our results demonstrate preclinical efficacy of combination therapies targeting the ribosome at multiple levels and provide a new approach for treatment of PC with high MYC activity.

#5182

**A rapid** in vivo **screen for pancreatic ductal adenocarcinoma therapeutics using the tumor marker Rgs16::GFP.**

Ozhan Ocal,1 Victor Pashkov,1 Rahul K. Kollipara,1 James B. Lorens,2 Galvin H. Swift,1 Rolf A. Brekken,1 Thomas M. Wilkie1. 1 _UT Southwestern Medical Center, Dallas, TX;_ 2 _University of Bergen, Bergen, Norway_.

Pancreatic Ductal Adenocarcinoma (PDA) is the most lethal major cancer in the USA due to lack of early diagnostics and effective treatments. Activating Kras mutations (such as KrasG12D) occur early in tumor progression and are present in about 90% of PDA. Pancreatic Intraepithelial Neoplasms (PanIN) are the most common initial neoplastic lesions and those with activating Kras alleles typically progress to carcinoma in situ and metastasize.

Receptor Tyrosine Kinases (RTK) and G-protein Coupled Receptors (GPCR) can indirectly activate Kras and are therefore potential drug targets. We previously showed that a feedback inhibitor of GPCRs, Regulator of G-protein Signaling 16 (Rgs16), is expressed in pancreatic progenitors during embryonic development. Rgs16 expression continues postnatally in ducts and beta cells during isletogenesis but is absent in normal glycemic adults. On the other hand, Rgs16 expression returns to ducts and islet beta cells after chronic hyperglycemia in mouse models of Type 1 and Type 2 Diabetes mellitus. Our hypothesis is that Rgs16::GFP is induced in these pancreatic cells in response to GPCR agonists released during chronic stress. In an effort to conduct drug screens in primary duct cell culture, we investigated Rgs16::GFP expression in pancreatic neoplasia by crossing it into a mouse model of aggressive PDA, called KIC (KrasG12D; Cdkn2aL/L; Ptf1a::Cre).

In Rgs16::GFP-KIC mice, Rgs16 was expressed at the initiation of PDA in early PanINs, as early as two weeks after birth (P15), and throughout tumor progression. Rgs16::GFP expression increased with tumor mass through one month of age (P29). We used this tumor specific characteristic of Rgs16 expression to set-up a two-week in vivo quantitative assay to test chemotherapeutic drug effectiveness. In a proof-of-principle study, the standard PDA therapeutics gemcitabine and nab-Paclitaxel reduced Rgs16::GFP expression and tumor burden compared to untreated mice at P29. Targeting Axl, an RTK highly expressed in PDA, with two different inhibitors of Axl signaling, BGB324 or warfarin, in combination with gemcitabine and nab-Paclitaxel further reduced PDA initiation and progression compared to standard chemotherapy alone. Survival studies with Rgs16::GFP-KIC mice showed that treatment with gemcitabine and warfarin extended median life span about two weeks while nearly doubling the maximum life span compared to untreated group.

In summary, Rgs16::GFP-KIC mice provide an in vivo model to rapidly identify more effective PDA chemotherapeutics and treatment protocols.

#5183

Efficacy study of APX3330, a Ref-1 redox inhibitor, and Gemcitabine in a mouse pancreatic ductal adenocarcinoma model.

Kyle C. McElyea, Max H. Jacobsen, Max Schmidt, Huiwen Cheng, Mark R. Kelley, George E. Sandusky, Melissa L. Fishel. _Indiana University School of Medicine, Indianapolis, IN_.

High levels of apurinic/apyrimidinic endonuclease/redox factor 1 (APE1/Ref-1 or Ref-1) expression have been reported in numerous cancers such that Ref-1 is an emerging target in a variety of cancer types. Ref-1 is a dual function protein with both DNA repair activity as well as redox activity. Ref-1 is responsible for the repair of baseless sites in DNA caused by alkylation and oxidative DNA damage as well as regulating several transcription factors including HIF-1a, NFkB, AP-1, and STAT3. Using siRNA to knockdown Ref-1 in pancreatic ductal adenocarcinoma (PDAC) cells (MIA-PaCa-2), we quantitated Ref-1 expression following transfection with scrambled control and Ref-1 siRNA using Indica Lab's HALO CytoNuclear software. Knockdown of greater than 85% greatly decreases the proliferation of PDAC cells, and supports Ref-1 as a target in pancreatic cancer.

To assess the efficacy of targeting Ref-1 in vivo, a small molecule Ref-1 redox inhibitor, APX3330, was used in combination therapy with a PDAC standard of care agent, Gemcitabine, in a PDAC xenograft mouse model. In this study, NSG mice were treated with: Gemcitabine (35mg/kg), APX3330 (12.5, 25, and 50mg/kg), and combinations of APX3330 and Gemcitabine (12.5, 25, and 50mg/kg), as well as a vehicle control group.

At termination of the study, tumors were harvested, tissue sections prepared, stained for H&E, and immunostained for Ki67 and CD31. Slides were then imaged via Aperio's ScanScope. Immunostains were quantitated to predict the effectiveness of the combination treatment in a PDAC in vivo model. IHC slides from treated tumors were quantified using Aperio's ImageScope software to determine the percent of cell proliferation and angiogenesis in the various treatment groups.

These preclinical studies, demonstrated additive effects of combining APX3330 with Gemcitabine to reduce pancreatic tumor volumes and cell proliferation. Significantly decreased tumor volumes in the combination treatments of APX3330 with Gemcitabine were found compared to each agent alone. All treatments, single or combination yielded tumor volumes significantly different from the vehicle control.

A statistically significant decrease in cell proliferation determined from Ki67 staining was found amongst the 12.5 and 50 mg/kg doses of APX3330, all three combination groups, and in the Gemcitabine group compared to vehicle control. There was a trend toward, yet not statistically significant, increased anti-angiogenic effects in the 12.5 and 25 mg/kg dose groups of APX3330 alone compared to all other treatment groups. This data continues to demonstrate a single agent response of pancreatic tumor cells in a preclinical model using APX3330, but more importantly, a novel combination effect when APX3330 is combined with Gemcitabine in a xenograft model, both via tumor volume and cell proliferation marker, Ki67 supporting the use of APX3330 in combination with Gemcitabine in PDAC patients.

#5184

MiXeno Humanized Mouse Model as an Efficient Tool to Evaluate in Vivo Activity of Anti-Cancer Immunotherapeutics.

Juan Zhang, Meng Qiao, Jian Ding, Zhensheng Wang, zhongliang Li, Qian Shi. _Crown Biosciences, Taicang, China_.

Background: Fighting cancer by taking advantage of a patient's own immune system is showing considerable success in oncology research. Targeting therapeutic antibodies against tumor-associated antigens or the host immune system has been shown to prolong survival of patients in various tumor types. However, the lack of experimental immunotherapy models is a major obstacle toward better evaluating new immunotherapeutics and testing their exploratory combination strategies. To address this need, we set out to validate a new platform MiXenoTM by implanting human PBMC to immune-deficient mice. The MiXenoTM mice were partially reconstituted with human immune components. In vivo efficacy of several immunotherapeutics were evaluated.

Results: We have reported before that several MiXenoTM models could be used to evaluate anti-human PD-1 or PD-L1 antibodies, where human T cells were well reconstituted in the mouse system. Here one of our MiXenoTM collection: HCC827 model was further optimized and identified as a PD-1/PD-L1 inhibitor highly sensitive model. Involvement of T cells in the tumor microenvironment was observed in HCC827 tumors. Furthermore, the dynamics of NK cell population in blood, tumor and other organs (e.g. spleen and lymph nodes) was established in MiXenoTM models. NK cell function was evaluated as well.

Conclusion: The MiXenoTM model provides an alternative to the full stem cell reconstitution approach, and may allow various types of immunotherapies being evaluated in these partially humanized xenograft models.

#5185

Development of patient-derived xenograft (PDX) models for pancreatic carcinoma as a preclinical platform for drug development.

Jill Ricono, Jayant Thatte, Colleen Scott, Thomas B. Broudy. _Crown Bioscience San Diego, San Diego, CA_.

Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive tumors in humans. A notable feature of PDA is its innate resistance to many chemotherapies. In preclinical studies, Abraxane showed antitumor activity as a single agent and synergistic activity in combination with gemcitabine in murine models of pancreatic cancer. In this study, we have developed and characterized multiple models of patient derived xenograft (PDX) pancreatic models. These models recapitulate major structural and genetic features noted in patient populations. Developed PDX models are from both prior treated and non prior treated patients with varying levels of resistance to Gemcitibine. Here we have investigated efficacy of standard of care agents in these pancreatic models and demonstrate the potential utility of the pancreatic models in drug discovery efforts in oncology for the treatment of pancreatic cancer.

#5186

Using HuTrial™ to aid in predicting a patient population of responders.

Jayant Thatte, Jill Ricono, Colleen Scott, Thomas B. Broudy. _Crown Bioscience San Diego, San Diego, CA_.

One of the biggest challenges in drug discovery is directing drug treatment to the correct population. Crown Bioscience currently has over 1,800 Patient derived xenograft models in >70 indications. These models, HuPrime® human surrogate mouse models, are reflective of the diversity of genetics, tumor heterogeneity and response in patient populations. HuPrime® models are searchable in the HuBase™ database by the expression data, copy number, mutational analysis as well as patient clinical data including diagnosis, pathology and IHC information. The vast number of models available allows one to perform surrogate clinical trials in a Phase II-like trial utilizing one patient sample per tumor bearing animal per treatment group (n=1 studies). In this study we show reproducibility of n=1 studies compared to the traditional preclinical mouse studies with multiple animals with the same tumor being treating with the same test agent. Further we show, the use of one patient sample per treatment allows for the expansion of the study utilizing multiple patient samples thereby reducing cost of preclinical studies. One can leverage the HuPrime® models to discover biomarkers, identify patient responder and non-responder profiles to accelerate drug development and improve chances of success in the clinic.

#5187

PET imaging of trastuzumab emtansine (T-DM1) drug delivery to intracranial patient derived xenograft (PDX) models of breast cancer metastasis.

Carsten Haagen Nielsen,1 Mette K. Nedergaard,1 Lotte K. Kristensen,1 Camilla S. Knudsen,1 Michael J. Wick,2 Kyri Papadopoulos,2 Anthony Tolcher,2 Andreas Kjaer3. 1 _Minerva Imaging, Copenhagen, Denmark;_ 2 _South Texas Accelerated Research Therapeutics (START), San Antonio, TX;_ 3 _Department of Clinical Physiology, Nuclear Medicine & PET and Cluster for Molecular Imaging, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark_.

Background: It is estimated that 10-30% of all breast cancer patients at some point develop brain metastases. Overexpression of the human epidermal growth factor receptor 2 (HER2) is a independent risk factor for development of brain metastases. Up to 37% of patients with HER2-positive metastatic breast cancer develop brain metastases and half of these patients die as a result of failure to control the intracranial disease. A reason for this is the challenge to obtain efficient drug delivery across the blood brain barrier (BBB).

Subcutaneous patient derived xenograft (PDX) models are increasingly used for efficacy studies in drug development. However, when targeting brain tumors or metastases, the major impact of the BBB on drug bioavailability must be taken into consideration. Clinical PET imaging with 64Cu or 89Zr labeled trastuzumab has previously been able to visualize breast cancer brain metastases.

The aim of our study was to investigate if PET imaging with 64Cu-labeled trastuzumab was predictive of the efficacy of trastuzumab emtansine (T-DM1) in a HER2 positive breast cancer PDX model established as an intracranial brain metastases model.

Methods: The intracranial PDX model was established by stereotactic intracranial injection of enzymatically digested ST1339 tumor tissue. At confirmed tumor take, mice were randomized into two arms: control and T-DM1 (10 mg/kg/week x4). Treatment response was monitored by contrast-enhanced T1- and T2-weighted Magnetic Resonance Imaging (MRI) and positron emission tomography (PET) with the amino acid radiotracer O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET). PET/CT imaging with 64Cu-trastuzumab was performed in animals with confirmed intracranial ST1339 tumors prior to treatment with T-DM1 (10 mg/kg/week x4).

Results: T-DM1 treatment with 10 mg/kg/week x4 of mice with intracranial tumors inhibited tumor growth and prolonged survival compared to non-treated animals. A variable response within the treatment group was observed. Forty percent had tumor shrinkage while 60% exhibited tumor growth within the duration of the therapy. The intracranial tumors were clearly visible on the 64Cu-trastuzumab PET images co-registered with T2-weighted MR images for anatomical localization. Interestingly, the intracranial tumor uptake between the animals was rather heterogeneous.

Conclusion: A treatment response to T-DM1 was observed in an intracranial ST1339 HER2 positive breast cancer PDX model. PET imaging with 64Cu-trastuzumab confirmed delivery of trastuzumab to the tumors. Further quantitative image analysis of the intracranial 64Cu-trastuzumab tumor uptake will reveal if the drug delivery measured by 64Cu-trastuzumab PET imaging is predictive of the efficacy of T-DM1 in a HER2 positive PDX breast cancer brain metastases model.

#5188

Combination of radiofrequency ablation and Sunitinib in the treatment of experimental hepatocellular cancer.

Xiaoqiang Qi, Guangfu Li, Dai Liu, Kevin F Staveley-O'Carroll, Eric Kimchi. _Medical University of South Carolina, Charleston, SC_.

Radiofrequency Ablation (RFA) is one of current clinical option in the treatment of hepatocellular cancer (HCC); however, RFA monotherapy is often not curative as a consequence of tumor micro-metastases and recurrence. Thus, induction of systemic response with additive treatment is required. In the present study, we are exploring the combination of RFA and sunitinib, an FDA-approved multiple tyrosine kinase inhibitor, in the treatment of HCC. Using our established strategy a clinically relevant murine model of HCC is prepared which mimics human disease and reflect its typical features. The follow-up magnetic resonance imaging (MRI) is applied to monitor tumor progression until the cumulative tumor volume reaches 2000 mm3. Size-matched tumor-bearing mice are assigned into 3 groups and receive the following treatments: no treatment, RFA alone with our improved machine and optimized parameters, combination of sunitinib and RFA. The therapeutic efficacy is evaluated by measuring tumor volume with MRI every twice weeks. We demonstrate that tumors grow substantially in the mice without treatment. RFA alone effectively destroys tumors, but unable to prevent tumor recurrence and micro-metastasis. In contrast, > 50% size reduction in tumor size is detected in the combined group compared to that in control mice, even no detectable tumors are observed in some treated mice. In conclusion, combination of sunitinib and RFA powerfully suppress tumor growth and could be translate into clinic practice.

#5189

Caloric restriction augments the chemotherapeutic response in a murine triple negative breast cancer model.

Brittany Simone,1 Tu Dan,1 Ajay Palagani,1 Meredith LaRose,1 Jason E. Savage,2 Nicole Simone1. 1 _Thomas Jefferson University Hospitals, Philadelphia, PA;_ 2 _National Cancer Institute, Bethesda, MD_.

Purpose:

Triple negative breast cancers (TNBC) are a highly aggressive subtype of breast cancer, linked with a poor prognosis. TNBC tumors have a higher propensity for metastasis and unfortunately have fewer treatment options due to their lack of estrogen, progesterone or Her-2/neu receptors. We sought to determine if caloric restriction (CR) could augment the response of chemotherapy including docetaxel and cisplatin in a TNBC model and improve survival.

Methods:

An orthotopic mouse model using a highly metastatic, luciferase-tagged TNBC cell line (4T1), was used to generate palpable tumors. Mice were then treated with CR, cisplatin alone, docetaxel alone or a combination of CR and chemotherapy. Three times a week, primary tumors were measured with calipers and in vivo imaging was performed to evaluate the metastatic burden of disease. Molecular evaluation of the tumors was performed, generating a mechanistic hypothesis for increased sensitivity to chemotherapy.

Results:

Combining either docetaxel or cisplatin with CR, significantly decreased the rate of primary tumor growth (p<0.001). Average time to metastases (19 days in ad lib group) was increased by an average of 10.5 +/- 3 days with the addition of either docetaxel or cisplatin, and increased by an additional 6 days in the CR+cisplatin (35 days until metastases, p<0.01). The addition of CR to chemotherapy decreased the overall number and volume of lung metastases, and increased overall survival as compared with those groups receiving chemotherapy alone. Molecularly, CR was noted to augment the effect of chemotherapy by decreasing inflammation that results from IL-1β mediated acute RT toxicity and molecular effectors in the insulin (insulin, IGF-1, IGF-BP3 and IGF-1R) and adipokine signaling pathways (decreased leptin and increased adiponectin).

Conclusions:

CR augments the effect of systemic chemotherapy in an aggressive TNBC mouse model, in part by decreasing the inflammatory response. The novel use of CR in combination with systemic chemotherapy has the potential to enhance clinical outcomes for patients with triple negative breast cancer and should be tested in the clinical setting.

#5190

**Correspondence between clinical patient outcome and** in vivo **carboplatine response of high grade ovarian cancer PDX.**

Stan P. du Manoir,1 Helene Delpech,1 Beatrice Orsetti,2 Jeremy Tessier,1 Pierre-Emmanuel Colombo,3 Charles G. Theillet1. 1 _IRCM-inserm, Montpellier, France;_ 2 _ICM-IRCM, Montpellier, France;_ 3 _ICM, Montpellier, France_.

Survival rate for high grade ovarian cancer (HGOC) remains low and has not significantly evolved for several years. This poor outcome is due to several mechanisms of resistance (initial or acquired) in different context (Homologous recombination deficience, CCNE1 amplification). We developed a collection of High-grade ovarian cancer Patient Derived Xenografts (PDX) with extensive characterization (CGH, transcriptome and exome sequencing, Colombo et al, Oncotarget. 2015 Sep 29;6(29):28327-40). Our objective is to create a panel of HGOC PDX which encompasses main types of platinium sensitivity/resistance in HGOC. We selected 10 PDX models (9 serous histiotypes and one carcinosarcomas) among our collection of 39 HGOC PDXs from two patient extreme platinium response categories (resistant with a progression-free inferior to 6 months and sensible with a progression-free higher than 18 months, some of the sensitives with BRCA1 mutations). These PDXs included PDXs were grafted sub-cutaneously on nude mice and submitted to carboplatin treatment (50mg/kg twice a week, for 4 weeks) and impact on tumor growth was evaluated as well as histological characterization. For the carcinosarcomas, no selection of the epithelial or sarcomatous component upon treatment was observed. Outgrowth of the tumors after end of treatment is currently under assessment.

Remarkably, tumor PDXs response was in accordance with clinical status with complete and partial response for the sensitive PDXs ; slowed growth under treatment or stabilization for resistant PDXs. Outgrowth of the tumor after the end of treatment is currently under assessment.

For each PDXs model, the large majority of the 7-9 treated tumor reacted in identical fashion if classified according to RECIST criteria with occasionally divergent responses reflecting tumor heterogeneity. Genetic and histological characterization of these outliers is ongoing.

This collection is a valuable resource to model carboplatine resistance in a well-defined context and will be the cornerstone of our future studies to conduct preclinical drugs tests, in particular for combination selected in vitro and to evaluate clonal evolution under treatment.

#5191

An orthotopic xenograft renal cell carcinoma Sunitinib resistant murine model.

Sebastian K. Frees, Igor Moskalev, Betty Zhou, Peter Raven, Claudia Chavez-Munoz, Peter Black, Alan I. So. _Vancouver Prostate Centre, Vancouver, British Columbia, Canada_.

Purpose of study

To develop a reproducible and reliable orthopic xenograft mouse model for Sunitinib resistant renal cell carcinoma (RCC).

Introduction

Clear-cell renal cell carcinoma (ccRCC) is the most common malignancy of the kidneys and has been steadily increasing at a rate of 3% yearly over the last decade. Furthermore, 30% of patients with ccRCC have de novo metastatic disease. Until the last decade, immunotherapy was the only option for metastatic ccRCC treatment. Current first-line systemic therapies for metastatic ccRCC all primarily target the angiogenesis pathway via tyrosine kinase inhibition (TKI) and have increased progression free survival dramatically. However, durable responses are rare, drug resistance invariably arises after short-term disease stabilization and metastatic ccRCC remains almost uniformly lethal. There is currently no animal model for TKI resistance in RCC. We developed a reliable and reproducible orthotopic xenograft mouse model of Suntinib resistance in RCC.

Methods

Luciferin expressing-CAKI-1 human renal cancer cells were injected under the renal capsule of athymic nude mice using ultrasound guidance. After initial tumor growth mice were treated with Sunitinib malate at an initial dose of 40mg/kg which was increased sequentially (60 and 80 mg/kg) at each tumor passage (Fig.1). Tumors were monitored twice weekly by ultrasound and bioluminescence (IVIS system). Tumors exceeding 200mm3 by ultrasound or 10^10 photons/sec by IVIS were considered end points. Before passaging, tumors were harvested and immediately dissociated with a Dispase/Collagenase mix. 5x105 cells were injected into the following batch of mice, considering all these steps as one cycle or passage. Tumors were passaged with increasing doses of Sunitinib 5 times.

Results

Tumor take rates were above 95% in each cycle. Initial treatment of Sunitinib at 40mg/kg resulted in a decrease in tumor size via ultrasound and IVIS of about 30%. At around 14 days of treatment tumors started to regrow despite further treatment. In the second cycle tumors did not show a response to a subsequent treatment of 40mg/kg, proving resistance. Even after increasing the dose of Suntinib in the following cycles to 60 and 80 mg/kg respectively, there was no response observed. The fifth cycle (100mg/kg) had to be terminated due to severe side effects in the animals (skin coloring, weight loss), however no decrease in tumor size was observed. We therefore created a Sunitinib resistance model in the range of the maximal tolerated long-term dose in vivo.

Conclusions

This study describes the first orthotopic xenograft renal cell carcinoma Sunitinib resistant mouse model created in situ. The development of Sunitinib resistance in an angiogenic environment establishes a more realistic and translational model, which opens the possibility to study not only the mechanism of Sunitinib resistance but also to find alternative therapies to overcome Sunitinib resistance.

#5192

Utilizing NSCLC PDXs derived from patients on osimertinib (AZD9291) clinical trials to further refine therapeutic strategies.

Sangeetha Palakurthi,1 Man Xu,1 Amanda J. Redig,2 Michael Dills,1 Prafulla Gokhale,1 Jinyun Choi,2 Atsuko Ogino,2 Yanan Kuang,1 Nora Feeney,1 Cloud Paweletz,1 Paul Kirscmeier,1 Jessie English,1 Darren Cross,3 Pasi Jänne2. 1 _Belfer Center for Applied Cancer Science, DFCI, Boston, MA;_ 2 _Dana-Farber Cancer Institute, Boston, MA;_ 3 _AstraZeneca, Oncology IMed, Cambridge, United Kingdom_.

Background: Osimertinib (AZD9291)is a mutant-selective EGFR tyrosine kinase inhibitor (TKI) effective against EGFR activating and the T790M acquired resistance mutations.Osimertinib has been approved by the US FDA for patients with EGFR T790M positive NSCLC with resistance to first line EGFR TKIs. However, as acquired resistance to osimertinib is now emerging, we aimed to develop PDXs from patients with: a) acquired resistance to first-line EGFR TKIs prior to enrolling on osimertinib trials and b) post-osimertinib resistance.

Methods: Pre- and post-osimertinib tumor biopsies were implanted into the flank or sub-renal capsule of NSG mice. Successfully established PDXs were expanded and confirmed by ddPCR and NGS to maintain molecular fidelity to the original patient tumor. The osimertinib efficacy in a subset of PDX models was tested and compared to clinical osimertinib response in the patients from whom the PDXs were derived. Acquired osimertinib resistance models were further treated with a panel of novel targeted agent combinations, matching the potential mechanism of osimertinib resistance.

Results: 46 patients underwent a pre-osimertinib or post-osimertinib biopsy. 26 biopsies were from the AURA trial for patients with acquired resistance to first-line EGFR TKIs; 5 biopsies were from the TATTON trial for patients without a T790M mutation; and 7 biopsies were from patients with acquired resistance to osimertinib. A platform of 16 PDX models have been successfully developed and shown to exhibit diverse mechanisms of TKI resistance as confirmed by NGS. These models include: 6 with EGFR T790M; 4 with EGFR non-T790M resistance to erlotinib; and 4 with acquired resistance to osimertinib. In the drug efficacy studies, PDX sensitivity to osimertinib is shown to be comparable to the corresponding patient's clinical response. In the DFCI-243 model (patient: EGFR T790M+, PR 8.9 months), tumors showed regression during the dosing phase and rapidly regrew upon cessation of osimertinib treatment. In contrast, in the DFCI-217 model (patient EGFR T790M+, PR >24 months), tumors showed sustained regression even after osimertinib treatment cessation. In the DFCI-284, a potential model of primary AZD9291 resistance tumors showed no regressions with osimertinib treatment. PDX models have also been used to refine treatment approaches for acquired resistance to osimertinib. DFCI-306 model established from a patient who developed an acquired BRAF mutation while on osimertinib has been shown to respond to either selumetinib or the selumetinib/osimertinib combination.

Conclusion: We have developed a platform of NSCLC PDXs from patients with acquired resistance to first-line EGFR TKIs and the newly approved third-generation inhibitor osimertinib. These models can be used to refine treatment strategies in patients with acquired resistance to first-line EGFR TKIs with primary or acquired resistance to osimertinib.

#5193

Identification of molecular predictors of differential chemotherapy response using patient-derived xenografts.

Michael T. Lewis, Lacey E. Dobrolecki, Susan G. Hilsenbeck, Lukas M. Simon, Chad A. Shaw. _Baylor College of Medicine Cancer Center, Houston, TX_.

Background: Clinical oncology trials in humans have major limitations, not the least of which is the inability to treat a single tumor with multiple drugs simultaneously to identify the most effective treatment options. In theory, this and other limitations can be overcome using an "animal clinical trial" platform that exploits collections of human cancer patient-derived xenograft (PDX) models.

Clinically, breast cancers are divided into three distinct groups: those that express the estrogen hormone receptor (ER+) (which typically also express the progesterone hormone receptor (PR+)), those with amplified or overexpressed ErbB2 (HER2) oncogene (HER2+), and those that express none of these three markers ("triple negative" breast cancer TNBC).

Unlike ER+ and HER2+ breast cancers, there are currently no targeted therapies against TNBC. Treatment of TNBC entails surgery coupled with radio- or chemotherapy, or both. Chemotherapy options include Taxanes (e.g. Docetaxel, Paclitaxel), Anthracyclin based therapies (Adriamycin/Doxorubicin plus Cytoxan (cyclophosphamide) (AC), and more recently, and platinum-based agents (e.g. Cisplatin, Carboplatin). However, other than BRCA1/2 mutation status correlating with increased efficacy of platinum-based agents, there are currently no clinically useful predictors of differential treatment response among these three commonly used chemotherapeutics.

Hypothesis: We hypothesized that a molecular predictor of differential chemotherapy response could be developed using human breast cancer patient-derived xenografts as the discovery platform.

Materials and Methods: We tested this hypothesis in an "animal clinical trial" using 20 PDX treated with three chemotherapies vs. control. Molecular correlates of response were identified in RNAseq and proteomic data, and are being validated in a separate cohort of PDX.

Results: We show that animal clinical trials are feasible, and can be conducted efficiently, using minimal staff. Further, we extend our previous analysis showing that treatment responses observed clinically are recapitulated in the PDX lines, thereby establishing clinical relevance of observed responses. Finally, we demonstrate that PDX show largely non-overlapping sensitivity to only one of the three agents tested, with corresponding largely non-overlapping molecular signatures delineating those PDX that respond to a given treatment from those that do not. Validation studies in newly developed PDX lines are ongoing.

Conclusions: Together, these data suggest that PDX models should be useful for generating predictors of treatment response. Ultimately, if robust genetic, epigenetic, or molecular predictors of differential treatment response can be identified and applied clinically, it may be possible to avoid ineffective treatments, minimize exposure to cytotoxic agents, and ultimately increase survivorship.

#5194

A Mouse-Human co-clinical trial with patient-derived xenograft (PDX) models demonstrates a predictive signature for dovitinib (TKI258), an FGFR and VEGFR inhibitor,in lung squamous cell carcinomalung cancer (LSCC).

Hye Ryun Kim,1 Han Na Kang,2 Sung Moo Kim,2 Hwan Kim,2 Kyoung-Ho Pyo,2 Myung-Ju Ahn,3 Tae Min Kim,4 Byoung Chul Cho1. 1 _Yonsei Cancer Center, Seoul, Republic of Korea;_ 2 _JE-UK Institute for Cancer Research, Seoul, Republic of Korea;_ 3 _Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea;_ 4 _St. Mary Hospital, Catholic University, Seoul, Republic of Korea_.

Background: We conducted a co-clinical trial with PDX models in conjunction with a phase II clinical trial with dovitinib in patients with fibroblast growth factor receptor 1 (FGFR1) amplified LSCC in order to identify a predictive biomarker for dovitinib, a FGFR inhibitor.

Methods: PDX models were established using tumor samples obtained from LSCC patients enrolled in the trial. We conducted an in vivo efficacy test with dovitinib in PDX models and comprehensive molecular profiling by whole exome sequencing (WES), array comparative genomic hybridization (aCGH), and microarray-based gene expression.

Results: The histological and genomic characteristics of xenograft tumors (F2) resembled case-matched original tumors (F0). Dovitinib treatment resulted in tumor shrinkage in PDXL01, but not in PDXL02-04, which corresponded to responses in LSCC patients enrolled in the clinical trial. The patient, from whom PDXL01 was derived, showed a partial response to dovitinib with progression-free survival of 6 months, whereas the patient, from whom PDXL04 was derived had rapid disease progression within 2 months of treatment. Mutation profiles of F0 and F2 were largely concordant with each other in at least 70% of somatic mutations. The genome-wide copy number alterations were also largely concordant between F0 and F2, suggesting that PDX tumors can successfully represent the genomic features of the tumors from LSCC patients. Notably, mutation in FGFR 1-3 genes was not observed. FGFR1 amplification largely due to arm-level 8p gain was consistently observed, regardless of sensitivity to dovitinib. This finding suggests that FGFR1 amplification may not be a predictor for dovitinib sensitivity. Finally, we compared gene expression between a responder and non-responders. In a Heatmap analysis of the top 50 significantly up- and down-regulated genes, FGFR gene signature including FGF3 and FGF19 was significantly up-regulated in the responder. Gene set enrichment analysis (GSEA) identified that gene sets, such as FGFR ligand binding and activation and SHC-mediated cascade pathway, were significantly enriched in the responder, highlighting FGFR pathway activation as a key molecular determinant for sensitivity to dovitinib. To identify predictive gene signatures from independent datasets, we further performed GSEA in dovitinib-sensitive lung cancer cell lines compared to resistant ones as available in the CCLE (Cancer Cell Line Encyclopedia) database. Among the four functional modules (FGFR signaling, Immune, Cell cycle, and RNA) that we identified, the FGFR signaling module performed well in the prediction of dovitinib sensitivity of PDXL01-04

Conclusions: FGFR gene expression signatures are predictors for response to dovitinib in LSCC.

#5195

Novel patient-derived xenograft and primary tissue culture cells for preclinical study in sarcoma.

Xiaoqing Pan,1 Mami Takahashi,1 Hitoshi Ichikawa,1 Akihiko Yoshida,2 Akira Kawai,2 Toshio Imai,1 Tadashi Kondo1. 1 _National Cancer Center Research Institute, Tokyo, Japan;_ 2 _National Cancer Center Hospital, Tokyo, Japan_.

Sarcomas are a group of malignant tumors displaying a wide diversity of pathological appearances and clinical responses due to the mesenchymal origin. Although certain sarcomas are characterized by the presence of unique fusion genes and mutations, the molecular backgrounds for tumorigenesis, metastasis, and resistance to treatments are remained to be further clarified in sarcomas. To improve clinical outcome, precise diagnosis followed by optimized therapy has long been desired in this field. Recently, clinical trials for application and re-localization of molecular targeting drugs have been conducted, and several drugs have been proven to effective in sarcomas. Preclinical models are indispensable to furthering our understanding of molecular background and facilitate novel therapeutic strategy. However, because of low prevalence the models are quite limited in sarcomas. To this end, we launched a project to establish patient-derived xenografts (PDXs) and primary culture cells of sarcomas. From April 2014 through October 2015, we implanted surgical tumor tissues from 76 sarcoma patients into highly immune-deficient NOG mice, and banked them after three passages. The identities of sarcomas were histologically confirmed by a certified pathologist, and the utility of PDXs were confirmed by re-growing them after storage in liquid nitrogen. 17 PDXs were prepared for preclinical study including myxofibrosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor, liposarcoma, leiomyosarcoma, osteosarcoma and clear cell sarcoma. Tissue cultures were also conducted by utilizing surgical specimens or PDXs. 158 sarcoma tissue samples were subjected to primary culture. Five immortalized cell lines, and 33 primary cultured cell lines with a minimum number of passages were established. The former includes leiomyosarcoma and undifferentiated pleomorphic sarcoma, and the latter includes additional 10 subtypes such as myxofibrosarcoma, malignant peripheral nerve sheath tumor, liposarcoma, osteosarcoma, clear cell sarcoma, etc. Moreover, the molecular backgrounds of the established sarcoma PDXs and tissue cultured cells were evaluated by targeted sequencing using an original cancer gene panel (NCC oncopanel), which can detect targetable mutations and gene fusions. Clinical and pathological observations of the donor patients will be linked to the characters of PDXs and tissue cultures. The applications of these in vivo and in vitro models for preclinical study will be our next challenge.

#5197

Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing.

Agnieszka Wozniak,1 Jasmien Cornillie,1 Yemarshet K. Gebreyohannes,1 Bram Boeckx,2 Haifu Li,1 Thomas Van Looy,1 Giuseppe Floris,3 Jasmien Wellens,1 Lise Vreys,1 Daphne Hompes,4 Marguerite Stas,4 Friedl Sinnaeve,4 Diether Lambrechts,2 Maria Debiec-Rychter,5 Raf Sciot,3 Patrick Schoffski1. 1 _Laboratory of Experimental Oncology, Department of Oncology, KU Leuven and Department of General Medical Oncology, University Hospitals Leuven, Leuven, Belgium;_ 2 _Laboratory of Translational Genetics, Vesalius Research Center, VIB and KU Leuven, Leuven, Belgium;_ 3 _Department of Pathology, KU Leuven and University Hospitals Leuven, Leuven, Belgium;_ 4 _Department of Surgical Oncology, University Hospitals Leuven, Leuven, Belgium;_ 5 _Department of Human Genetics, KU Leuven and University Hospitals Leuven, Leuven, Belgium_.

Background: STS constitutes a rare and very heterogeneous family of mesenchymal tumors. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies.

Methods: A panel of patient-derived xenografts (PDX) was established by subcutaneous implantation of fresh, surgically resected tumor specimens in immunodeficient athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histolopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least two passages. To evaluate the stability of the genomic profile we performed low-coverage whole-genome sequencing on DNA isolated from tumors obtained from at least two passages. In addition RNA-seq was used to better characterize the mutational profile of the ex-mouse tumors.

Results: Until now 139 STS samples from consenting patients treated at the University Hospitals, Leuven, Belgium, have been transplanted. Twenty six well-characterized, stable STS PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Seventeen models, analyzed with low-coverage DNA sequencing, showed a stable genomic profile in at least two different tumor passages. At this point the panel includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (5), dedifferentiated liposarcoma (3), malignant peripheral nerve sheath tumor (3), synovial sarcoma (2), leiomyosarcoma (3) epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1) and undifferentiated high grade sarcoma (2). Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for at least four prospective clinical trials. In addition 22 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an "established model".

Conclusion: Our panel of mesenchymal PDX models is characterized by stable histological and molecular features. These clinically well-annotated models can contribute to reliable preclinical studies for new anticancer treatments for STS. The availability of unique, rare STS xenografts also allows to study the biology of these diseases. The platform is made available to collaborators from academia and industry.

#5198

Combination effect of anti-VEGFR-2 antibody with erlotinib on EGFR mutant non-small cell lung cancer.

Hiroe Kayatani,1 Kadoaki Ohashi,1 Takeshi Imao,2 Kenichiro Kudo,1 Yuka Kato,1 Takashi Ninomiya,1 Toshio Kubo,1 Akiko Sato,1 Ktsuyuki Hotta,1 Mitsune Tanimoto,1 Katsuyuki Kiura1. 1 _Okayama University Hospital, Okayama, Japan;_ 2 _Okayama University, Okayama, Japan_.

Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have excellent effects on non-small cell lung cancer (NSCLC) harboring EGFR mutations. However, EGFR mutant NSCLC inevitably acquires resistance to EGFR-TKIs such as gefitinib, erlotinib, or afatinib. Previously, we found that bevacizumab, an anti-vascular endothelial growth factor-A (VEGF-A) antibody, enhanced the antitumor effects of gefitinib and afatinib in preclinical lung cancer models (Ichihara et al., Caner Res 2009; Ninomiya et al., Mol Cancer Ther 2013). In a phase II trial of OLCSG1001, we demonstrated relatively longer progression-free survival by adding bevacizumab to gefitinib in the treatment of patients harboring an EGFR exon 19 deletion. Seto et al. (Lancet Oncol 2015) also reported that adding bevacizumab to erlotinib in patients harboring EGFR mutations significantly prolonged progression-free survival. A new anti-angiogenic agent, the anti-human VEGF receptor-2 (VEGFR-2) antibody ramucirumab, has also been used clinically (Garon et al., Lancet 2014). We investigated the effect of an anti-VEGFR-2 antibody combined with erlotinib on the growth of lung cancers harboring EGFR mutations.

Materials and Methods: NSCLC cell lines PC-9, which harbors EGFR exon 19 deletion and H3255, which harbors EGFR L858R mutation, were used in this study. Ramucirumab and the anti-murine VEGFR-2 antibody DC101 were kindly provided by Eli Lilly (Indianapolis, IN, USA). BALB/c nu/nu or transgenic mice expressing the delE748-752 mutant version of mouse Egfr driven by the SP-C promoter, which is equivalent to the delE746-A750 mutation of humans (C57BL/6/Egfr15DEL) (Ohashi et al. Cancer Sci 2009), were used for in vivo experiments. For xenograft models, PC-9 or H3255 cells were injected subcutaneously into BALB/c mice. The mice were divided into four treatment groups: vehicle, erlotinib, DC101, or erlotinib combined with DC101.

Results: DC101 alone moderately inhibited the growth of PC-9 or H3255 tumor in the xenograft mouse models or suppressed lung cancers in C57BL/6/Egfr15DEL mice. Combined erlotinib and DC101 therapy inhibited the growth of PC-9 tumor in the xenograft mouse models more markedly than did erlotinib or DC101 alone. There were no differences in toxicity between monotherapy and combination therapy.

Conclusion: Combination therapy with erlotinib and an anti-VEGFR-2 antibody had a stronger antitumor effect than those of the respective monotherapies in lung cancers harboring EGFR mutations in vivo.

#5199

An in vivo reporter to quantitatively and temporally analyze the effects of CDK4/6 inhibitor-based therapies in melanoma.

Jessica L. Teh,1 Timothy Purwin,1 Evan J. Greenawalt,1 Inna Chervoneva,1 Allison Goldberg,1 Michael E. Davies,2 Andrew Aplin1. 1 _Thomas Jefferson University, Philadelphia, PA;_ 2 _MD Anderson University of Texas Houston, TX_.

Aberrant cell cycle progression is a hallmark feature of cancer cells. Cyclin-dependent kinase 4 and 6 (CDK4/6) drive progression through the G1 stage of the cell cycle, at least in part, by inactivating the tumor suppressor, retinoblastoma (RB). CDK4/6 are targetable and the selective CDK4/6 inhibitor, palbociclib (IBRANCE/PD-0332991), was recently FDA approved for the treatment of estrogen receptor-positive, HER2-negative advanced breast cancer. In cutaneous melanoma, driver mutations in NRAS and BRAF signal to promote CDK4/6 activation suggesting that inhibitors such as palbociclib are likely to provide therapeutic benefit most likely in combination with BRAF and/or MEK inhibitors that are FDA-approved. However, the determinants of the response to CDK4/6 inhibitors alone and in combination with other targeted inhibitors are poorly defined. Furthermore, in vivo systems to quantitatively and temporally measure the efficacy of CDK4/6 inhibitors and determine the extent that CDK activity is reactivated during acquired resistance are lacking. Here, we describe the heterogeneous effects of CDK4/6 inhibitors, the expression of anti-apoptotic proteins that associate with response to CDK4/6 and MEK inhibitors, and the development of a luciferase-based reporter system to determine the effects of CDK4/6 inhibitors alone and in combination with MEK inhibitors in melanoma xenografts. These findings are likely to inform on-going and future clinical trials utilizing CDK4/6 inhibitors in cutaneous melanoma.

#5200

Preclinical model of patient-derived tumor xenograft in humanized mice.

Annika Wulf-Goldenberg, Maria Stecklum, Iduna Fichtner, Jens Hoffmann. _EPO GmbH, Berlin, Germany_.

Human patient-derived xenografts (PDX) from different tumor indications transplanted on immunodeficient mice have demonstrated strong predictive power for many drug development programs in cancer research. However, one caveat of PDX models is that they lack a functional immune system, which allows tumor engraftment in the xenogenic host mice.

Immunotherapy has emerged as one of the most promising avenues for cancer therapy. For translating immunotherapies into the clinics preclinical animal models are needed which harbor human immune cell repertoire for rising an immune response.

To overcome these constraints our aim is the development of PDX models on mice with a functional human immune system to improve predictability of drug efficacy and safety.

We reconstituted a human immune system in mice by engrafting human hematopoietic stem cells in immunodeficient mice. We were able to demonstrate that in the mice a full set of human immune cells, including T cells, B cells, NK cells, monocytes and dendritic cells can be analyzed by flow cytometry.

At the time when the human immune system was developed, established patient-derived tumors were transplanted on these reconstituted humanized mice. PDX from different tumor entities were transplanted. The tumors are growing on these humanized mice either similar to non humanized mice or slightly slower. During the observation period no rejections by the human immune cells were evident, although tumor growth was accompanied by an increase of human T cells in the peripheral blood. Ex vivo analysis will be performed demonstrating the cytotoxicity of these T cells and the functionality of the human immune cells.

Humanized mice bearing various PDX tumors were treated with therapeutic checkpoint inhibitors. Ipilimumab that target the CTLA-4 receptor lead to a slight tumor growth delay and an increased percentage of T cells in the blood and in the tumor. Nivolumab that targets the PD-1 receptor showed similar results. The combination of both inhibitors showed a synergistic effect which lead to a greater tumor growth inhibition.

Our humanized mouse models enable appropriate preclinical assessment of immune-based therapeutic anti-tumor strategies especially when combing the humanized mouse with patient-derived tumor xenografts.

#5201

Identification of growth factors that promote the proliferation and drug resistance of human primary acute B lymphoblastic cells.

Peng Li, Zhiwu Jiang. _GIBH, CAS, Guangzhou, China_.

Bone marrow mesenchymal stem cells (BMMSCs) contribute the survival, expansion, and drug resistance of leukemic cells. However, how BMMSCs promote the proliferation and drug resistance of leukemia remains to be poorly understood. Here, we derived a BM-derived stromal cell line that supported unlimited growth of human primary acute B lymphoblastic leukemic cells (B-ALL) by physical contacts with B-ALL cells and secreting growth factors. Nevertheless, its capacity of promoting primary B-ALL proliferation was abolished once the cell line was induced to differentiate into adipocytes. In xenograft models, we demonstrate that the presence of adipocytes suppressed the engraftment of primary B-ALL cells while the decrease of adipocytes enhanced the reconstitution of B-ALL. Consistently, we observed that the increase of numbers and diameters of adipocytes was correlated with remission after chemotherapy treatments in B-ALL patients. By comparing the global transcription profiles between the BM stromal cells and the adipocytes that were derived from the cell line, we identified a combination of cytokines and adherent proteins that were able to support the long-term growth of human primary B-ALL cells and desensitize them to chemotherapy drugs in a stromal-free culture condition. Furthermore, we showed that the PI3K/AKT pathways were involved in chemotherapy-induced resistance because targeting these signaling pathways increased the chemotherapy sensitivity of B-ALL in xenografts. Collectively, our results uncovered the mechanisms of BMMSCs supporting the survival, expansion, and drug resistance of human B-ALL cells.

#5202

Comparison of [124I]-FIAC and [18F]-FAC as potential biomarkers to predict response to gemcitabine therapy in pancreatic cancers.

Kishore K. Gangangari,1 Blesida Punzalan,2 Brendan Huang,2 NagaVaraKishore Pillarsetty2. 1 _The Graduate Center, CUNY / Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Memorial Sloan Kettering Cancer Center, New York, NY_.

Pancreatic cancer (PaCa) is one of the most lethal kinds of cancers because it is virtually symptom free in early stages, metastasizes rapidly and is difficult to resect. Gemcitabine (GEM) as a single agent or in conjunction with radiation is the most commonly used in the management of PaCa. But GEM has shown efficacy only in a small but select group of patients. While the role of key transporters and enzymes has been implicated in sensitizing or conferring resistance to gemcitabine, no clear correlation has been establishe. Currently, there is no a priori way to determine invasively or non-invasively which PaCa patients stand to benefit from GEM therapy. Any biomarker that can act as a prognostic indicator of GEM response will greatly aid in optimizing treatment regimen, reducing unnecessary side effects and subjecting resistant patients to aggressive therapies. Our goal is to develop a PET imaging based biomarker to predict response to GEM therapy in pancreatic cancer patients non-invasively. [18F]-FAC was evaluated as biomarker for GEM response in leukemia models but the short half life (t1/2 = 110 min) combined with background gut activity can complicate image analysis. Therefore our goal was to develop a longer lived analog of GEM that can act as a surrogate marker. Based on modeling studies and ability to label with longer lived isotope (Iodine-124; t1/2 = 4.18 d) we concentrated our efforts on evaluating I-124 labeled 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodocytosine ([124I]-FIAC) as a positron emission tomography (PET) biomarker of GEM response.

[124I]-FIAC was synthesized by iodination of 1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)-cytosine (FAC) with [124I]-NaI using chloramine-T as an oxidant. In vitro phosphorylation efficacy studies were carried out on GEM, FAC and FIAC using recombinant deoxycytidine kinase (dCK) enzyme. In vitro kinetic uptake assay was performed on representattive pancreatic cancer cells lines AsPc-1 and MiaPaCa-2. Subcutaneous tumor xenografts were established in athymic nude mice. [124I]-FIAC was administered intravenously via tail vein and its uptake was studied by PET imaging and sacrificial biodistribution studies at 4, 24, 48 and 72 h. Imaging and biodistribution studies using [18F]-FAC were also performed in tumor bearing mice at 1 and 4 h.

[124I]-FIAC was synthesized in >17% yields with >99% radiochemical purity. The efficiency of phosphorylation was in the following order GEM ~ FAC > FIAC. Cellular uptake assays showed that [124I]-FIAC was taken up preferentially (4X) by AsPc-1 in comparison to MiaPaCa-2.

[124I]-FIAC and [18F]-FAC were synthesized and compared for their efficacy to serve as potential biomarker for gemcitabine response in pancreatic cancer models in vitro and in vivo. Preliminary studies indicated that [124I]-FIAC and [18F]-FAC behave differently in these models with former showing preferential uptake in AsPC-1 cells and xenografts.

#5203

Innovative and predictive models against cancer: an IMODI integrative approach.

Caroline Mignard,1 Laurent Arnould,2 Alain Bruno,3 Loreley Calvet,4 Marc Colombel,5 Jill Corre,6 Olivier Cuvillier,7 Bénédicte Eckel,8 Nico Forraz,9 Alexandra Gonzalez-Jouhanneaud,10 Liliane Goetsch,10 Dominique Guenot,11 Juan Iovanna,12 Mariana Kuras,13 Christophe Lautrette,14 Françoise Le Vacon,15 Bernard Malavaud,16 Philippe Merle,17 Florence Meyer-Losic,18 Françoise Praz,19 Olivier Rosmorduc,20 Jean-Emmanuel Sarry,6 Séverine Tabone,21 Philippe Vaglio,22 Loïc Ysebaert,16 Olivier Duchamp,1 IMODI Consortium. 1 _Oncodesign S.A., Dijon Cedex, France;_ 2 _Centre Georges François Leclerc, Dijon, France;_ 3 _Servier Research Institut, Suresnes, France;_ 4 _Sanofi, Vitry sur Seine, France;_ 5 _INSERM U1033, Lyon, France;_ 6 _INSERM U1037, Toulouse, France;_ 7 _CNRS U5059, Toulouse, France;_ 8 _INSERM U1111, Lyon, France;_ 9 _CTI-BIOTECH, Meyzieu, France;_ 10 _Pierre Fabre Research Institut, Saint Julien en Genevois, France;_ 11 _Strasbourg University, Strasbourg, France;_ 12 _INSERM U1068, Marseille, France;_ 13 _Ariana Pharmaceuticals, Paris, France;_ 14 _OncoMedics, Limoges, France;_ 15 _Biofortis, Saint-Herblain, France;_ 16 _Toulouse Hospital, Toulouse, France;_ 17 _INSERM U1052, Lyon, France;_ 18 _Ipsen Innovation, Les Ulis, France;_ 19 _INSERM U938, Paris, France;_ 20 _Pitie-Salpetriere Hospital, Paris, France;_ 21 _Centre Léon Bérard / Synergie Lyon Cancer, Lyon, France;_ 22 _Modul-Bio, Marseille, France_.

Many reports support that xenografts from patient-derived xenografts (PDXs) in mice or rats well recapitulate the molecular diversity, cellular heterogeneity, and histology seen in patient tumors. However, shortcomings including limited clinical diversity of the PDXs, absence of influence of human microbiome, absence of drug human metabolism and reduced immune system restrain the predictive values of these PDX models. To set-up a holistic integration of these criticisms, we have associated efforts from public hospitals, academic groups, biotechs and private pharmaceutical companies with the financial support of the French Ministry of Industry.

First, to improve the clinical cancer diversity, surgical specimens from patients obtained from 8 different tumor locations (lung, breast, ovary, pancreas, liver, prostate, AML, myelome) are collected to establish large collections of PDXs in mice. In addition, in vitro primary cultures of cells from these samples are conducted to establish a collection of cell lines from the stromal and the tumoral compartments. The established models are evaluated for ex vivo and in vivo sensitivities to relevant anticancer drugs, histological and molecular characteristics. All model characteristics are being compiled in a web-based database for efficient features search and interconnection. These tumor collection and model characterization were performed under specific procedures that are followed by all partners within the consortium, which allows to have harmonized results and high quality material. Second, to improve our knowledge on the role of the human gut microbiota, stools are collected from patients with cancer and from mice bearing PDXs before and after chemotherapeutic treatments allowing a comparison of the microbiota profiles. In order to increase the predictability of the models, we are generating mice with humanized liver showing distinct drug pharmacokinetic profiles as compared with parental mice. Finally, the humanization of the immune system in mice is developed by several approaches including the use of induced pluripotent stem cells from cancer patients.

We will present the first characterized models and will discuss their usefulness and chance to bring benefit to patients via this holistic strategy developed within the IMODI initiative.

#5204

Enhancing susceptibility to PARPi in homologous recombination repair dysfunction (HRD)-associated pancreatic cancer.

Talia Golan, Ella Buzhor, Sharon Halparin, Dikla Atias, Maria Raitses-Gurevich, Chani Stossel, Raanan Berger. _Sheba Medical Center, Tel Hashomer, Israel_.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with limited treatment options. Recent genomic advances have identified a PDAC subtype with homologous recombination repair deficiency (HRD). These patients include BRCA-associated PDAC (5% of sporadic PDAC cases and 10-15% of Jewish Ashkenazi PDAC cases) and a 'BRCAness' phenotype in the absence of BRCA mutation. These patients are of critical importance as they may benefit from a personalized approach towards treatment. Tumors with HRD are highly susceptible to PARP inhibition (PARPi). However, durable anti-tumor responses are limited and accumulating resistance is observed, thus forming the scientific rationale for the development of combination treatments with PARPi. PI3K/AKT signaling has an essential role for cancer initiation, progression, maintenance and homologous recombination (HR) repair steady state preservation. The goal of our study is to test the hypothesis that addition of PI3Ki may enhance the sensitivity to PARPi.

We have developed a unique patient-derived xenograft (PDX) from metastatic lesions (ascites fluid or liver biopsies) thus closely mimicking the clinical scenario. In order to examine feasibility of BRCA-associated PDACs for PI3Ki therapy, we have evaluated PI3K pathway activation by Western blot analysis. Phosphorylated AKT by the protein kinase PDK1 (pAKT 308) was detected in BRCA1-mutated PDX, which supports the addition of PI3Ki to PARPi in our PDAC xenograft model. Therapeutic efficacy of PARP inhibitor (Olaparib) alone and in combination with PI3K inhibitor (BKM120) was evaluated on BRCA-mutated naïve to treatments xenografts. Mice were randomized into 4 groups: a. vehicle control, b. NVP-BKM120 via oral gavage, c. Olaparib I.P. or d. the combination of Olaparib with NVP-BKM120, all once a day, for 5 days a week. Preliminary results have demonstrated significant tumor growth attenuation in the group threated by drug combination (Olaparib and NVP-BKM120), which did not result in measurable toxicity, such as weight loss. NVP-BKM120 alone group demonstrated weight loss of more than 20% of the initial weight in 3 out of 5 mice in this treatment group.

We anticipate that our study will promote an understanding on the role of BRCA and PI3K inhibition in HRD PDAC. The proposed research governs to overcome resistance to DNA damaging agents in BRCAmut and BRCAness PDAC patients. Furthermore, we propose a unique platform to test therapeutic interventions targeting these pathways in a personalized manner.

## EPIDEMIOLOGY:

### Description of Cancer Trends and Next-Generation Sequencing in Epidemiology

#5205

Changes in trends in colorectal cancer incidence rate by anatomic site between 1978 and 2004 in Japan.

Hiroko Nakagawa,1 Hidemi Ito,1 Satoyo Hosono,1 Isao Oze,1 Haruo Mikami,2 Masakazu Hattori,3 Yoshikazu Nishino,4 Hiromi Sugiyama,5 Kayo Nakata,6 Hideo Tanaka1. 1 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 2 _Chiba Cancer Center, Chiba, Japan;_ 3 _Fukui Prefectural Hospital, Fukui, Japan;_ 4 _Kanazawa Medical University, Kanazawa, Japan;_ 5 _Radiation Effects Research Foundation, Hiroshima and Nagasaki, Hiroshima, Japan;_ 6 _Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan_.

Although colorectal cancer (CRC), a major cancer worldwide, has shown a proximal or right-sided shift in subsite distribution in western countries, trends in subsite incidence in Asian countries remain unclear. Here, we evaluated subsite-specific trends in CRC incidence rate between 1978 and 2004 in Japan using large data from 10 population-based cancer registries. The colorectal sites (C18-20) were categorized into three groups, proximal colon (C18.0-18.5), distal colon (C18.6-C18.7) and rectum (C19.9 and C20.9). Trends in age-standardized incidence rates (ASRs) were characterized by Joinpoint regression analysis. A total of 474,651 colorectal cancer cases were analyzed. Overall, ASRs increased remarkably until 1993, with an annual percent change (APC) of 4.9%, and then stabilized thereafter. By subsite, however, ASRs of proximal colon significantly increased, with APCs of 7.1% (1978~1991), 3.8% (1991~96) and 0.9% (1996~2004); distal colon showed an initial significant increase, with an APC of 7.6%, but stabilized from 1991 until the end of observation; and rectal cancer showed an initial significant increase, with APCs of 1.9% (1978~88) and 5.6% (1988~92), but then decreased abruptly in 1992, the year colorectal cancer screening was introduced nationwide, with an APC of -1.0%. Thus, we revealed that changes in incidence trends for the three anatomic sites apparently began to differ in the 1990s, and this is a first report to our knowledge. We also discuss factors that could explain these changes in Japanese. Careful monitoring is necessary to confirm whether these trends are changing in the Japanese population.

#5206

Recent incidence trends of lymphoid malignancies in Hong Kong, 2001-2010.

Bryan A. Bassig,1 Wing-Yan Au,2 Oscar Mang,3 Roger Ngan,3 Lindsay M. Morton,1 Dennis K.M. Ip,4 Wei Hu,1 Tongzhang Zheng,5 Wei Jie Seow,1 Jun Xu,6 Nathaniel Rothman,1 Qing Lan1. 1 _NCI-DCEG, Rockville, MD;_ 2 _Queen Mary Hospital, The University of Hong Kong, Hong Kong;_ 3 _Queen Elizabeth Hospital, Hong Kong;_ 4 _School of Public Health, The University of Hong Kong, Hong Kong;_ 5 _Brown University, RI;_ 6 _The University of Hong Kong, Hong Kong_.

Incidence trends of lymphoid malignancies in the United States (U.S.) have been well-characterized based on analyses of data from the Surveillance, Epidemiology, and End Results Program. These analyses showed marked increases in incidence rates of non-Hodgkin lymphoma (NHL) in the early to mid-1990s, with some evidence that rates have begun to level off in the recent decade. In contrast, there are limited published data on trends in rates of lymphoid malignancies in the Chinese population, particularly for subtypes. Such analyses may provide insight into geographic differences in the descriptive epidemiological patterns of these neoplasms as well as etiological hypotheses. To explore recent trends in incidence rates in HK, yearly age-adjusted incidence rates and annual percent changes (APCs) were calculated using available data over the period 2001-2010 for overall lymphoid malignancies, overall B-cell neoplasms not including Hodgkin lymphoma (HL), overall NK or T-cell tumors, HL, plasma cell neoplasms (PCN), and specific NHL B-cell subtypes that were most common in the HK population over this time period, including diffuse large B-cell lymphoma (DLBCL), marginal zone lymphoma (MZL), and follicular lymphoma (FL). The incidence of overall lymphoid malignancies showed a steady increase in HK over the period 2001-2010 with an APC of +1.4% (95% CI = + 0.4%, + 2.3%). A steady rate of increase was further observed over the ten year period for overall B-cell neoplasms not including HL (APC = +1.6%, 95% CI = + 0.4%, + 2.9%), whereas the incidence of overall T- and NK/T cell neoplasms showed no significant fluctuation over the ten year period (APC = -1.1%, 95% CI = -3.5%, +1.5%). For the most common subtypes diagnosed in HK during the study period, an increasing trend of HL was observed over the years 2004-2010 only (APC = + 10.5%, 95% CI = + 4.9%, + 16.4%) based on the best-fitting model that included one joinpoint, and stable but non-significant rates of increase for DLBCL (APC = + 1.5%, 95% CI = - 0.7%, + 3.6%), PCN (APC = + 1.2%, 95% CI = -0.2%, + 2.6%), and FL (APC = + 2.7%, 95% CI -0.5%, 6.0%) were also observed, all over the period 2001-2010. Conversely, incidence rates of MZL showed no significant fluctuation over the study period (APC = -1.0%, 95% CI = -2.9%, +0.8%). The overall trends observed during even this relatively short time-period suggest that the rate of several lymphoid malignancies in HK may be beginning to shift in the direction of rates that were observed among individuals living in the U.S. Additional analyses of population-based registry data from other regions in East Asia, especially over longer time-periods, are needed to replicate and extend these findings. Further, our results support the need for etiological studies of lymphoid malignancies in this population, particularly as it relates to environmental, occupational and lifestyle risk factors that might influence disease rates and the genetic risk factors that potentially interact with them.

#5207

Comparison of subtype-specific incidence rates of lymphoid malignancies in Hong Kong and the United States.

Nathaniel Rothman,1 Bryan A. Bassig,1 Wing-Yan Au,2 Oscar Mang,3 Roger Ngan,3 Lindsay M. Morton,1 Dennis K.M. Ip,4 Wei Hu,1 Tongzhang Zheng,5 Wei Jie Seow,1 Jun Xu,6 Qing Lan1. 1 _National Cancer Inst., Rockville, MD;_ 2 _Queen Mary Hospital, The University of Hong Kong, Hong Kong;_ 3 _Queen Elizabeth Hospital, Hong Kong;_ 4 _School of Public Health, The University of Hong Kong, Hong Kong;_ 5 _Brown University, RI;_ 6 _Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong_.

Clinical studies of lymphoid malignancies (LMs) have suggested that the descriptive patterns of these neoplasms differ in East Asia compared to Western populations. However, there are very limited available data on population-based, subtype-specific incidence rates of LMs in the East Asian population, particularly in Chinese. Using data from the Hong Kong (HK) Cancer Registry and the United States (U.S.) Surveillance, Epidemiology, and End Results Program, we calculated and compared age-adjusted incidence rates of LM subtypes in HK to those in Whites and Asians living in the U.S. Overall and sex-specific rates were calculated for the period 2001-2010. In order to formally compare the incidence rates between HK and the U.S., standardized rate ratios (SRRs) were calculated comparing the age-adjusted rates of each subtype for U.S. Whites and U.S. Asians in the SEER 13 database to HK. The incidence of most subtypes was low in the HK population, with rates <1 case per 100,000 for all subtypes except for diffuse large B-cell lymphoma (3.26/100,000) and plasma cell neoplasms (1.99/100,000). Age-adjusted incidence rates of all evaluated B-cell subtypes were significantly higher in U.S. Whites compared to HK, with SRRs ranging from 1.6 (Burkitt lymphoma) to 9.1 (chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL)). Rates in U.S. Asians were generally intermediate to those in U.S. Whites and HK, with SRRs for B-cell subtypes ranging from 1.0 (Burkitt) to 2.0 (CLL/SLL) comparing Asians in the U.S. to HK. For T or NK-cell subtypes, rates of extranodal NK/T-cell lymphoma were significantly lower in both U.S. Whites (SRR = 0.2) and U.S. Asians (SRR = 0.5) compared to HK. However, incidence rates of other evaluated T-cell subtypes were comparable or slightly higher in the U.S. population compared to HK. Our findings, which are based on data from population-based disease registries, provide new insight into the subtype-specific patterns of LMs in the Chinese population, and suggest the need for etiological studies of LMs in the East Asian population to elucidate the factors responsible for these differences in the geographic incidence patterns.

#5208

Trends in fatal prostate cancer incidence by race among US men.

Scott P. Kelly,1 Philip S. Rosenberg,1 William F. Anderson,1 Sean D. Cleary,2 Naji Younes,2 Gabriella Andreotti,1 Michael B. Cook1. 1 _National Cancer Institute, Division of Cancer Epidemiology and Genetics, Bethesda, MD;_ 2 _George Washington University, Milken Institute School of Public Health, Department of Epidemiology and Biostatistics, Washington, DC_.

Background: Prostate-specific antigen testing (PSA) has dramatically changed the composition of prostate cancer, making it difficult to assess incidence trends. By defining fatal prostate cancer as the underlying cause of death from disease within 10 years of diagnosis, we have been able to conduct an in-depth analysis of "clinically relevant" prostate cancer incidence trends.

Methods: We extracted incident prostate cancer cases, causes of death and survival among men aged 45-84 years using the US Surveillance, Epidemiology, and End Results (SEER) database (1975-2002). In addition to standard descriptive analyses, the ability to distinguish prostate cancer cases that were ultimately fatal enabled us to fit age-period-cohort models.

Results: Among 51,682 fatal prostate cancer cases, incidence increased 1% per year prior to 1992, declined 15% per year from 1992-1995, and further declined by 5% per year through 2002. Age-specific incidence rates of fatal disease decreased over 2% per year among men aged 55 years and older, yet increased up to 1% per year among younger men. Fatal disease rates were more than 2-fold higher in black men compared with white men, a racial disparity that increased to 3-6 fold among younger men.

Conclusion: In the United States, after widespread use of PSA screening and advances in prostate cancer treatment, fatal prostate cancer rates have substantially declined. Yet, rates of fatal prostate cancer among younger men have remained relatively stable, suggesting the need for additional attention to early-onset prostate cancer that has the potential to result in death. This study also highlights the persistent black-to-white racial disparity in fatal prostate cancer, underscoring the need for a greater understanding of the causes of this difference so that strategies may be implemented to reduce, and ultimately eliminate, racial disparities in prostate cancer.

#5209

Trends in a survival of cancer of the coupus uteri in Japan 1993-2006 (J-CANSIS).

Shusaku Inoue,1 Satoyo Hosono,1 Hidemi Ito,1 Isao Oze,1 Yoshikazu Nishino,2 Masakazu Hattori,3 Akiko Ioka,4 Tomio Nakayama,5 Keitato Matsuo,1 Mika Mizuno,6 Kiyoko Kato,7 Hideo Tanaka,1 Yuri Ito5. 1 _Aichi Cancer Center Research Institute, Nagoya, Japan;_ 2 _Kanazawa Medical University, Kahoku, Japan;_ 3 _Fukui Prefectural Hospital, Fukui, Japan;_ 4 _Hospital, University of the Ryukyus Cancer Centre, Nakagami, Japan;_ 5 _Center for Cancer Control and Statistics, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan;_ 6 _Aichi Cancer Center, Nagoya, Japan;_ 7 _Kyushu University, Fukuoka, Japan_.

The incidence of cancer of the corpus uteri (CC) in Japan is continuously increasing. To explore the trend in a survival of Japanese CC, we analyzed the population-based cancer registry data.

We obtained data on 8,562 CC cases from six prefectural cancer registries. We defined two periods as 1993-2001 (1st period) and 2002-2006 (2nd period). Relative survival (RS) of each period was calculated using cohort analysis, and we compared them using excess mortality model adjusted for age categories and extent of disease. We also estimated RS by age group, extent of disease and histological subtype. For age groups, patients were classified into those aged 15-54, 55-69 and 70-99. For extent of disease, patients were classified into those staged localized, regional and distant group. For histological subgroups, patients were divided into eight histological categories according to 'Cancer Incidence in Five Continents Vol.10' partially modified.

The 5-year RS was 78.1% in the 1st period and 80.1% in the 2nd period. Compared with the RS in the 1st period, RS in the 2nd period was significantly high [Excess Hazard Ratio (EHR): 0.805, 95%Confidence Interval (95%CI): 0.723-0.896, P<0.001]. In the stratified analysis by age group, the 5-year RS of age group 55-69 significantly improved from 74.4% to 79.8% (EHR: 0.751, 95%CI: 0.643-0.877, P<0.001). The 5-year RS in the other age group did not improved dramatically, but the 1-year RS of age group 70 years or older had a trend towards improvement from 81.2% to 83.4% (EHR: 0.777, 95%CI: 0.586-1.029, P=0.079). The stratified analysis by extent of disease indicated that the 5-year RS improved significantly in local disease from 93.0% to 95.2% (EHR: 0.717, 95%CI: 0.554-0.927, P=0.011) and regional disease from 57.3% to 64.2% (EHR: 0.813, 95%CI: 0.692-0.955, P=0.012). Among distant cases, only 1-year RS improved significantly from 46.1% to 57.1% (EHR: 0.715, 95%CI: 0.572-0.895, P=0.003). According to the analysis by histological subgroup, the 5-year RS of endometrioid adenocarcinoma significantly improved from 85.2% in the 1st period to 90.0% in the 2nd period (EHR: 0.700, 95%CI: 0.564-0.870, P=0.001). In the cases of other specified adenocarcinoma and carcinoma, 1-year RS improved significantly from 84.6% in the 1st period to 89.2% in the 2nd period, but 5-year RS did not.

In conclusion, we could reveal the improved prognosis of Japanese CC from the period of 1993-2001 to the period of 2002-2006, using population-based cancer registry data. The improvement of short-term RS was observed among elderly, distant or non-endometriod cases, whereas their long-term prognosis did not change during the study period. Improved prognosis might be attributed to the progress of treatment, especially due to chemotherapy or formulation of treatment guidelines in early 2000's.

#5210

Incidence and mortality trends of prostate cancer in southern Thailand.

Christian Alvarez,1 Shama Virani,2 Rafael Meza,1 Laura Rozek,2 Hutcha Sriplung,3 Alison M. Mondul1. 1 _University of Michigan, Department of Epidemiology, Ann Arbor, MI;_ 2 _University of Michigan, Department of Environmental Health Sciences, Ann Arbor, MI;_ 3 _Prince of Songkhla University, Epidemiology Unit, Hat Yai, Songkhla, Thailand_.

Background: Prostate cancer is one of the most commonly diagnosed cancers in men worldwide. In Asia, the incidence of prostate cancer is expected to increase in the next decades as the population ages and as developing Asian countries undergo the epidemiologic transition. There is a scarcity of studies on the epidemiology of prostate cancer in Thailand. The characterization and projection of the burden of prostate cancer are important to design cost-effective strategies for the prevention and control of this disease. The purpose of this study is to examine incidence and mortality trends of prostate cancer in southern Thailand.

Methods: Incident prostate cancer cases from the Songkhla cancer registry were used to analyze incidence and mortality trends; complete data on incidence was available from 1990-2013 and data on mortality was available from 1990-2010. Characteristics of the cases on sociodemographic factors and tumor characteristics were collected, as was vital status. Joinpoint analysis was used to examine the incidence and mortality trends and calculate the annual percent change (APC). The number of joinpoints, slope of the trends, and their significance was determined by permutation tests. Incidence and mortality rates were standardized to the Segi's world standard population.

Results: Eight hundred fifty-five prostate cancer cases were diagnosed from 1990 to 2013. The median age at diagnosis was 74 and 89.6% of the diagnosed cases were Buddhist, with the remainder being Muslim. The majority (79.8%) of prostate cancers were unstaged. However, among those cases whose tumors were staged, the distribution was as follows: 3.5% stage I, 17.9% stage II, 2.9% stage III, 75.7% stage IV. The proportion of unstaged tumors decreased during the period 2005-2009 compared to earlier periods, and there was an increase in the proportion of stage II tumors for the same period. The overall prostate cancer incidence rates increased significantly at an APC of 5.1% (95%CI: 3.8, 6.4) from 1990 to 2013. Among Buddhist men, the APC was very similar to the overall incidence (APC: 5.2%, 95%CI: 4.0, 6.5), while we observed a very low incidence rate and no evidence of an increasing trend in Muslim men. As of 2010, 72.7% of the cases had died. Similar to incidence rates, prostate cancer mortality rates have significantly increased at and APC of 3.2% (95CI%: 1.7, 4.8) from 1990 to 2010.

Conclusion: This analysis showed that there has been a significant increase in prostate cancer incidence and mortality in Southern Thailand during the period 1990-2013. Next steps include assessing the effects of age, calendar year, and birth-cohort on the incidence and mortality of prostate cancer and projection of the incidence and mortality rates over the next fifteen years in order to fully characterize this cancer and help to design strategies to reduce the future burden of prostate cancer in Thailand.

#5211

Trends in the incidence of retinoblastoma in the United States from 1973-2012.

Arthur G. Fernandes, Benjamin D. Pollock, Aaron E. Hoffman. _Tulane University, New Orleans, LA_.

Background: Retinoblastoma is a rare form of eye cancer that usually develops in early childhood, typically before the age of 5. This study aims to determine the incidence of retinoblastoma in the United States over a 40-year period from 1973 to 2012 using a review of existing databases.

Methods: 882 patients with retinoblastoma (International Classification of Diseases for Oncology (ICD-O-3) codes C69.2 (retina) and C69.9 (eye, NOS)) were derived from the Surveillance, Epidemiology, and End Results (SEER) program database in the United States from 1973 to 2012, using the nine historical SEER registries with the earliest observation collections. Incidence rates were calculated per 1,000,000 children and age-adjusted using the United States Census Bureau data as the standard population. The significance of trend in the incidence rate over time was determined using the Chi-Square test, and 95% CIs were calculated. Descriptive statistics evaluated differences in incidence in terms of age group, gender, race and laterality. Surveillance was evaluated by analysis of diagnosis confirmation and reporting source.

Results: There were a total of 882 cases of retinoblastoma, representing 6.1% of all childhood cancers under age 5 years. 49.1% of cases were males and 96% of all cases were children aged 0-4 years. The mean incidence of retinoblastoma was 12.14 cases per million children aged 0-4 years (95% CI 11.32 to 12.96) and 0.49 cases per million children aged 5-9 years (95% CI 0.36 to 0.65). There was no significant trend in the incidence for children aged 0-4 years old (Xsup2=35.455 p=0.6324) or 5-9 years old (Xsup2=32.247 p=0.7695) for all races/genders. The proportion of bilateral cases (28.9%) versus unilateral cases (71.1%) remained stable over the 40-year period. 85.6% of the cases were confirmed microscopically and hospitals represent 98.8% of the reporting sources.

Conclusions: The incidence rate of retinoblastoma in the United States has remained stable for the last 40 years. The majority of the cases are unilateral, and hospitals are the main reporting source.

#5212

A comparative descriptive study of cutaneous malignant melanoma.

Roxana Moslehi,1 Francis B. Boscoe,2 Nur Zeinomar3. 1 _Department of Epidemiology and Biostatistics, School of Public Health, and Cancer Research Center, University at Albany, State University of New York, Albany, NY;_ 2 _Cancer Registry, New York State Department of Health, Albany, NY;_ 3 _Department of Epidemiology and Biostatistics, School of Public Health, University at Albany, State University of New York and Department of Epidemiology, Mailman School of Public Health, Columbia University, NY_.

Objectives: Incidence of cutaneous malignant melanoma has increased worldwide in the past several decades, particularly in developed countries. Melanoma is believed to be a multifactorial condition, with exposure to ultraviolet radiation (UVR) as a major environmental risk factor. We matched five provinces in Iran with five states in the United States (US) based on measurements of solar UVB exposure and conducted a comparison of population-based incidence rates of melanoma in each province and state.

Methods: Solar UVB exposure was measured using data from NASA's Total Ozone Mapping Spectrometer (TOMS) for the period between January 1, 1997 and December 31, 2000 at a geographic resolution of one degree. UV exposures (in mW/m2) for each state and province were calculated by averaging the values of the grid points within each state and province. Population-based incidence data for five Iranian provinces (Ardabil, Golestan, Mazandaran, Gilan and Kerman) were obtained from their respective cancer registries. Incidence rates among white non-Hispanic individuals in the five US states (Kentucky, Utah, Texas, Oklahoma, and Hawaii) with matching average UVB exposure to the Iranian provinces were obtained from the North American Association of Central Cancer Registries. Furthermore, population-based incidence rates of melanoma for the overall U.S. population were obtained from the Surveillance, Epidemiology, and End Results (SEER) program for the 13 SEER areas (1996-2000). All incidence rates were calculated per 100,000 person-years and were age-adjusted using the 2000 standard populations.

Results: Annual UVB exposure from 1996-2000 ranged from an average of 138.52 in Ardabil to 227.44 in Kerman. The exposures of the corresponding US states ranged from 124.95 in Kentucky to 246.34 in Hawaii during the same period. The overall male and female rates of melanoma were 0.59 and 0.46, respectively, for Iran and 14.9 (95%CI: 14.7-15.2) and 10.5 (95%CI: 10.4-10.7), respectively, for the US. Age-standardized rates (ASR) of melanoma among males for the five Iranian provinces in comparison to their UVR-matched US states were as follows: 0.30 (22.6) for Ardabil (Kentucky), 1.20 (26.6) for Golestan (Utah), 0.73 (18.1) for Mazandaran (Texas), 0.17 (15.8) for Gilan (Oklahoma), and 0.90 (75.3) for Kerman (Hawaii). ASR of melanoma among females were as follows: 0.20 (16.0) for Ardabil (Kentucky), 0.60 (17.5) for Golestan (Utah), 0.47 (10.6) for Mazandaran (Texas), 0.17 (8.9) for Gilan (Oklahoma), and 0.90 (36.5) for Kerman (Hawaii).

Conclusion: The markedly higher incidence rates of melanoma in selected US states compared to Iranian provinces with similar UVR exposure patterns may be due to differences in lifestyle, pigmentation, or genetic profiles, as well as under-reporting in Iran compared to the US. Our findings underscore the need for additional studies to decipher the multitude of extrinsic and intrinsic factors involved in the etiology of this multifactorial condition.

#5213

Remarkably stable sex-ratio in sporadic kidney cancer incidence.

Ghislaine Scelo,1 Estelle Chanudet-van den Brink,1 Peng Li,1 Chanida Vinayanuwattikun,1 David C. Muller2. 1 _International Agency for Res. on Cancer, Lyon, France;_ 2 _Imperial College London, London, United Kingdom_.

Background: Patterns of sex-disparity in cancer incidence can provide clues on risk factors and susceptibility to the disease. While known kidney cancer genetic syndromes follow an autosomal dominant inheritance and equally affect males and females, kidney cancer occurs predominantly outside a syndromic context (97% of cases are sporadic) and more commonly in males than females.

Methods: We used cancer registry data from 10 registries around the world, from countries showing large variations in kidney cancer incidence in 2012 (from 5.2 to 16.7/100,000 person-years) and with at least 20 years of registration. Using age-standardized incidence rate (on the World population), we compared the male-to-female sex-ratio in incidence rates across registries and over time. We also evaluated whether the sex-ratio varied by categories of age at diagnosis. Finally, we compared the kidney cancer-specific mortality after diagnosis in males and females in 2,969 cases using Cox regression model.

Results and interpretation: From 1985 to 2005, the sex-ratio was on average 2.0 and showed little variation across countries: it was 1.7 in Iceland; 1.8 in Australia and Finland; 1.9 in the UK and Norway; 2.0 in Singapore, Estonia, the Czech Republic, and the USA; and 2.1 in Slovenia. The sex-ratios were also remarkably stable in all registries over time, arguing against the role of known risk factors such as tobacco smoking to which the proportion of exposed women has evolved drastically in the last 50 years. In line with the autosomal dominant inheritance, sex-ratio was on average 1.0 in younger ages and progressively increased to stabilize from around 40 years old on. In particular the ratio remained stable at older ages, which suggests that estrogen levels are unlikely to account for the lower risk experienced by women. In contrast to the observed difference in incidence, our survival analysis showed similar survival rates in both sexes after adjustment for age and stage at diagnosis.

Conclusion: Our results suggest that the difference in kidney cancer incidence rates between males and females are not due to varying prevalence of known risk factors or a protection by sex hormones. While the susceptibility to the disease varies, the tumor behavior seems similar in males and females as shown by similar survival rates.

#5214

Stage IV colorectal cancer sub-site and patterns of distant metastases.

Jamaica R. Robinson, Polly A. Newcomb, Sheetal Hardikar, Stacey A. Cohen, Amanda I. Phipps. _Fred Hutchinson Cancer Research Center, Seattle, WA_.

Approximately 20% of individuals diagnosed with colorectal cancer (CRC) in the United States are found to have distant metastases at the time of their diagnosis, indicating an extremely poor prognosis. Although it is well known that primary CRC typically metastasizes to the liver and/or lungs, little is known regarding which factors might explain the anatomic pattern of CRC metastatic spread. Using population-based registry data from the Surveillance, Epidemiology and End Results (SEER) cancer registry of the Seattle-Puget Sound region, we assessed the relationship between primary CRC sub-site and the presence of synchronous metastases in the liver, lungs, and both the liver and lungs. All individuals with stage IV CRC who were diagnosed between 2010-2014 in the 13-county catchment area and who also had complete data on site of metastasis at diagnosis were included in analyses (N=1287). In total, 703 (55%) patients had distant metastases confined to the liver at the time of diagnosis, 78 (6%) had lung-only metastases, 190 (15%) had metastases in both the liver and lungs but not at other sites, and 316 (25%) exhibited different patterns of metastatic spread, including to the bone, brain, and other anatomic sites. Using multiple logistic regression models, we compared individuals with different patterns of metastatic spread at diagnosis with respect to primary CRC site and with separate models comparing cases with liver-only metastases to all other metastatic cases, comparing cases with lung-only metastases to all other metastatic cases, and comparing cases with metastases to both the liver and lungs to all other metastatic CRC cases. All models were adjusted for age at diagnosis and sex, as well as for depth of primary tumor growth within the colorectum (T-stage) and extent of cancer spread to lymph nodes (N-stage). The odds of having a primary CRC tumor in the rectum, versus proximal colon, were significantly lower among patients with liver-only synchronous metastases relative to those with other patterns of metastatic spread at diagnosis [odds ratio (OR): 0.62, 95% confidence interval (CI): 0.46 - 0.82]. Conversely, the odds of having a primary CRC located in the rectum were significantly higher among those with metastatic disease confined to the lungs (OR: 2.48, 95% CI: 1.38 - 4.47) and those with metastases to the liver and lungs (OR: 1.97, 95% CI: 1.29 - 3.01). Our findings suggest that a patient's site of primary CRC may be informative with respect to possible pattern of metastatic spread.

#5215

Parental and birth characteristics and childhood lymphoma in Texas, 1995-2011.

Erin C. Peckham, Michael E. Scheurer, Heather E. Danysh, Austin L. Brown, Joseph Lubega, Philip J. Lupo. _Baylor College of Medicine, Houston, TX_.

Purpose: In the United States, lymphoma represents approximately one-third of all malignancies in those less than 20 years of age. As most cases are of unknown etiology, the identification of risk factors for the prevention of childhood lymphoma is critical. Parental and birth characteristics are often evaluated in studies of childhood cancer to determine the role of inborn variation on disease risk. Thus we sought to evaluate the role of parental and birth characteristics on the risk of childhood lymphoma.

Material and Methods: Cases (n=374) were obtained from the Texas Cancer Registry (TCR) and limited to children born in Texas during or after 1995 and diagnosed with a lymphoma between 1995-2011. Diagnostic information came from the TCR, and case birth characteristic data was obtained from linked Texas birth certificates provided by the Center for Health Statistics. A randomly selected group of 10 controls for each case with subsequent birth characteristic data available was obtained from linked birth certificates. Multinomial logistic regression was used to generate relative-risk ratios (aRRR) and 95% confidence intervals (CI), adjusted for relevant covariates, to evaluate the association between several parental and birth characteristics and lymphoma risk (overall and by subtype).

Results: Most parental and birth characteristics were not associated with risk of childhood lymphoma. However, two factors were associated with lymphoma risk overall and by subtype: maternal race/ethnicity and infant sex. When compared to non-Hispanic white mothers, Hispanic mothers were more likely to have offspring that developed: 1) any lymphoma (aRRR: 1.09; 95% CI: 0.85-1.40) and 2) non-Hodgkin excluding Burkitt lymphoma (aRRR: 1.42; 95% CI: 0.96-2.11). The reverse was seen for non-Hispanic black mothers. There was also a disparity in risk by infant sex. Specifically, female children were at a decreased risk of developing all lymphomas (aRRR: 0.59; 95% CI: 0.47-0.74); Hodgkin lymphoma (aRRR: 0.65; 95% CI: 0.46-0.92); and Burkitt lymphoma (aRRR: 0.18; 95% CI: 0.10-0.33) when compared to male children. Additionally, paternal nativity was suggested to increase overall lymphoma risk (aRRR: 1.29; 95% CI: 0.96-1.73); an increase specifically suggested among those diagnosed with Hodgkin lymphoma (aRRR: 1.46; 95% CI: 0.93-2.32).

Conclusion: In this relatively large population-based study of these factors on the risk of childhood lymphoma, we found little evidence that parental and birth characteristics were strongly associated with disease risk.

#5216

Broadening tumor spectrum in Lynch syndrome: increased incidence for 15 distinct cancer types.

Christina Therkildsen, Steen Ladelund, Lars Joachim Lindberg, Lars Smith-Hansen, Mef Nilbert. _Hvidovre Hospital, Hvidovre, Denmark_.

Introduction: Lynch syndrome is a multi-tumor syndrome predominantly associated with an increased risk of colorectal cancer, but is indeed linked to a yet uncertain number of extra-colonic tumor types.

Methods: In order to determine the risk for extra-colonic tumors in Lynch syndrome, we determined the incidence rates for 21 distinct malignancies in the national Danish Lynch syndrome cohort compared to an age-and sex matched control cohort from the Danish population.

Results: Based on data from 1494 Lynch syndrome mutation carriers, significantly increased risks were demonstrated for 15 cancer types. These included Lynch syndrome-related malignancies, such as cancer of the endometrium, the ovary, the small bowel, the urinary tract and brain tumors as well as tumor types with an undefined role in Lynch syndrome, such as breast cancer, gastric cancer, pancreatic cancer, skin cancer, prostate cancer, lung cancer, kidney cancer, and testicular cancer. Distinctive peak ages were defined with the highest incidence rate ratio at age 30-49 years for ovarian cancer and 50-69 years for endometrial cancer, breast cancer, and brain tumors to continuously increasing incidence rate ratios with increasing age for e.g. urothelial cancer, gastric cancer, pancreatic cancer and skin cancer.

Conclusion: The broad tumor spectrum in Lynch syndrome is of relevance to consider during genetic counseling, whereas the variable peak incidence ages provide a basis for precision surveillance programs that consider the different risks throughout life.

#5217

Cancer target enrichment panels using advanced molecular inversion probes (MIPs) with ability to reduce amplification bias and detect low frequency variants.

Ryan Bannen, Michael Brockman, Mark D'Ascenzo, Keynttisha Jefferson, Dawn Green, Heather Halvensleben, Kurt Heilman, Todd Richmond, Daniel Burgess. _Roche NimbleGen, Madison, WI_.

Next generation sequencing (NGS) of enriched targets is increasingly being used to discover and track mutations in genes implicated in cancer. Amplicon-based target enrichment approaches are frequently used with cancer samples because there is no sample library preparation needed and primer-based approaches are typically efficient with sequencing reads. However, traditional amplicon approaches don't scale up well and may have PCR and coverage uniformity bias. As the number of samples is increased, a fast, reliable, and cost-effective target enrichment method is a critical tool for mutation discovery and tracking.

Existing target enrichment approaches based on molecular inversion probes (MIPs) have presented challenges with probe design and dependability. We have developed HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) using advanced versions of MIPs which enable library-free enrichment, improved scalability, and the ability to remove PCR duplicates. We have also developed companion command line software which trims primer sequences and removes PCR duplicates. This software uses community standard file formats (FASTQ, SAM/BAM) and is intended to be compatible with common "best practice" NGS analysis pipelines.

Here we describe two HEAT-Seq cancer panels which have been optimized to improve coverage uniformity and probe reliability.

The HEAT-Seq Oncology Panel targets both strands for all protein coding bases in 60 cancer-related genes where possible. The target for this design is 245 kb. With 2 million 2x76bp reads for each of 8 replicates, we observe an on-target read rate of >95%. After duplicate removal, the % of target bases with at least 20x coverage is >90%. We observe uniformity (percent of probes >=20% of the target mean) of ~90%.

The HEAT-Seq Ultra Hot-Spot Panel is a mutation-focused design that provides ultra-deep sequencing coverage capability for sequencing of mutation hot-spots in 53 cancer-related genes to detect low frequency variants in heterogeneous samples. The target for this design is 30.5 kb. With 500,000 2x76bp reads for each of 24 replicates, we observe an on-target read rate of ~80%. After duplicate removal, the % of target bases with at least 20x coverage is >95%. We observe uniformity (percent of probes >=20% of the target mean) of ~95%. We also observed detection of validated low frequency variants down to 1%.

In summary, both panels have been shown to capture cancer-related genes and target regions at depths sufficient to identify and track genomic variants.

#5219

Gene expression profiling of prostate tissue identifies biological pathways associated with TMPRSS2:ERG gene fusion.

Ericka M. Ebot, Masis Isikbay, Travis Gerke, Thomas U. Ahearn, Rachel S. Kelly, Svitlana Tyekucheva, Andreas Pettersson, Kathryn L. Penney, Lorelei A. Mucci. _Harvard T.H. Chan School of Public Health, Boston, MA_.

Background: TMPRSS2:ERG, a hormonally regulated gene fusion present in about half of prostate tumors, is the most common somatic event in prostate cancer. There is intriguing evidence to suggest that TMPRSS2:ERG-positive tumors may define a distinct subgroup of prostate cancer. In this study we compared gene expression profiles according to fusion status to identify genes and biological pathways differential expressed by TMPRSS2:ERG.

Methods: The study included men with prostate cancer in the Health Professionals Follow-up Study and the Physicians' Health Study diagnosed between 1982 and 2004 and followed through 2012. Tumor biomarker data was available for archival tumor tissue from 380 cases, 191 of whom also had gene expression data for adjacent normal tissue. Whole genome mRNA expression profiling (20,254 genes) was performed using the Affymetrix 1.0 ST array. TMPRSS2:ERG tumor status was assessed by a genetically validated IHC assay for ERG. To relate expression of individual genes to ERG we used linear regression. Gene Set Enrichment Analysis (GSEA) was used to identify pathways of genes associated with ERG in both tumor and adjacent normal tissue.

Results: Among 380 cases, 186 (49%) of tumors were positive for the gene fusion. 864 genes were significantly associated with ERG status using a false discovery rate (FDR)-corrected p-value threshold of 0.05. Of the 864 differentially expressed genes, 493 were upregulated and 371 were downregulated in ERG-positive tumors compared to ERG-negative tumors. The top ten genes included ERG, WNK2, CACNA1D, HES1, SEPT9, PLA2G7, TDRD1, ANTXR2, SORBS2, and MYO6. No genes reached significance in adjacent normal tissue after correcting for multiple comparisons. GSEA identified nineteen pathways in tumor tissue and six pathways in adjacent normal tissue enriched according to ERG tumor status. A number of the pathways identified were involved in amino acid and fatty acid metabolism.

Conclusions: We identified significant differences in gene expression profiles in prostate tumor tissue according to TMPRSS2:ERG status, providing supportive evidence of this fusion as a unique subtype.

#5220

Evaluation of ACMG guideline classified variants in 180 cancer and incidental non-cancer genes in families with breast/ovarian cancer.

Kara N. Maxwell,1 Steven N. Hart,2 Joseph Vijai,3 Kasmintan A. Schrader,4 Tinu Thomas,3 Bradley Wubbenhorst,1 Vignesh Ravichandran,3 Raymond M. Moore,2 Chunling Hu,2 Lucia Guidugli,2 Brandon Wenz,1 Thomas P. Slavin,5 Susan M. Domchek,1 Mark E. Robson,3 Csilla Szabo,6 Susan L. Neuhausen,5 Jeffrey N. Weitzel,5 Kenneth Offit,3 Fergus J. Couch,2 Katherine L. Nathanson1. 1 _University of Pennsylvania, Philadelphia, PA;_ 2 _Mayo Clinic, Rochester, MN;_ 3 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 4 _University of British Columbia, Vancouver, British Columbia, Canada;_ 5 _City of Hope, Duarte, CA;_ 6 _National Institutes of Health, Bethesda, MD_.

Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG designated reportable cancer and non-cancer genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole exome sequencing in 404 individuals in 257 families and classified 1640 variants from these genes. Potentially clinically actionable (likely-pathogenic/pathogenic, LP/P) versus nonactionable (VUS/likely-benign/benign) calls were 92% and 88% concordant with locus specific databases and Clinvar, respectively. LP/P mutations were identified in 11 of 25 breast cancer susceptibility genes in 27 BRCA1/2 negative families (11%). Evaluation of 84 additional autosomal dominant cancer susceptibility genes identified LP/P mutations in only four additional families (1.7%), suggesting they do not influence risk in this cohort. However, individuals from nine of 257 families (3.5%) had incidental LP/P mutations in 32 non-cancer disease genes, and 7% of individuals were monoallelic carriers of an LP/P mutation in 39 autosomal recessive cancer syndrome genes. Furthermore, 90% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant classification guidelines. In addition, evaluation of extended panels of cancer genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results.

#5221

Linking the molecular profile of colorectal tumors to germline genetic and environmental risk factors.

Syed H. Zaidi,1 Catie Grasso,2 Jasmine Mu,3 Eve Shinbrot,4 Marios Giannakis,5 Charles Connolly,2 Ivan Borozan,1 Hermann Brenner,6 Peter Campbell,7 Andrew Chan,8 Jenny Chang-Claude,6 Mengmeng Du,9 Vincent Ferretti,1 Amy French,10 Charles Fuchs,5 Steven Gallinger,11 Levi Garraway,5 Andrea Gsur,12 Marc Gunter,13 Tabitha Harrison,2 Michael Hoffmeister,6 Li Hsu,2 Wen-Yi Huang,14 Jeroen Huyghe,2 Mathieu Lemire,1 Elaine Mardis,15 John McPherson,1 Polly Newcomb,2 Lincoln Stein,1 Wei Sun,2 Lee Timms,1 Quang Trinh,1 David Wheeler,4 Christina Yung,1 Niha Zubair,2 Shuji Ogino,16 Stephen Thibodeau,10 Ulrike Peters,2 Thomas Hudson1. 1 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 2 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 3 _Broad Institute, Cambridge, MA;_ 4 _Baylor College of Medicine, Houston, TX;_ 5 _Dana Farber Cancer Institute, Boston, MA;_ 6 _German Cancer Research Center (DKFZ), Heidelberg, Germany;_ 7 _American Cancer Society, Atlanta, GA;_ 8 _Massachusetts General Hospital, Boston, MA;_ 9 _Memorial Sloan Kettering, New York, NY;_ 10 _Mayo Clinic, Rochester, MN;_ 11 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 12 _Medical University of Vienna, Vienna, Austria;_ 13 _Imperial College, London, United Kingdom;_ 14 _National Cancer Institute, NIH, DHHS, Bethesda, MD;_ 15 _Washington University School of Medicine, St. Louis, MO;_ 16 _Harvard Medical School, Boston, MA_.

The completion of The Cancer Genome Atlas (TCGA) project for colorectal cancer (CRC) has enabled a new, focused phase of sequencing tumor samples for which genome-wide genetic, epidemiological, clinical and lifestyle data have been collected. By combining somatic mutational profiles with these aforementioned data, we may identify and report effective prevention and treatment approaches for a broader population of individuals. The advent of targeted deep sequencing approaches using DNA obtained from formalin fixed paraffin embedded tissues has made possible the genetic characterization of the large numbers of patients needed to make such an effort relevant.

As a first step, we describe a custom gene panel designed from large-scale studies for targeted deep sequencing, and its application to over 4,200 CR tumors, collected by the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). This study is designed to identify recurrent somatic mutations and copy number alterations (CNAs) for association testing with germline genetic and lifestyle and environmental risk factors for CRC, and thereby identify relevant approaches to impact cancer prevention.

The targeted CRC panel includes 205 genes. Genes were primarily selected as significantly mutated genes identified from the Nurses' Health Study and Health Professional's Follow-up Study (n=700), and TCGA (n=525). We also included 15 genes associated with high penetrance germline mutations and augmented the list to include genes in pathways of somatically altered genes, identified by literature review, from public databases and known to be associated with loss of heterozygosity. For these 205 genes, amplification primers were designed to include all coding regions of transcripts that are listed in the UCSC Genome Browser.

For regions with CNAs, the TCGA dataset was analyzed to include regions with greater than or equal to 2 copy focal gains or losses that were found in more than 4 or 3 tumors, respectively. Candidate genes in regions with significant CNAs from the TCGA CRC publication (Nature 2012) were also included. For CNAs, 6 to 12 amplicons were designed for each of the 56 selected regions (32 gains and 24 losses).

Additional target regions include: 1) 25 microsatellite loci previously used to identify defective DNA mismatch repair and 212 homoploymer repeats. These include microsatellite loci recommended by the NCI Consensus Panel for identifying MSI; 2) amelogenin (for gender); and 3) Fusobacterium to detect a putative CRC-associated pathogen in tumor biopsies.

At the AACR annual meeting, we expect to present results for deep sequencing (~1,000X) of the first 1,000 CR tumors, including any preliminarily identified pathways and subtypes that may provide the basis for association testing with germline genetic and lifestyle and environmental risk factors needed for inferring better approaches to prevention and treatment of CRC.

#5222

Decoding >30 thousand individuals to analyze the most common genetic disorder: Hereditary hemochromatosis (HH).

Mehmet Tahir Aslan,1 Anita Mathew,1 Ferit Akova,1 Raghu Metpally,1 David J. Carey,1 Heinric Williams,1 Marc S. Williams,1 John Overton,2 Aris Baras,2 Adam M. Cook,1 Ryan D. Colonie,1 Nefize Sertac Kip1. 1 _Geisinger Health System, Danville, PA;_ 2 _Regeneron Pharmaceuticals, Tarrytown, NY_.

Background: Hereditary hemochromatosis (HH), one of the most common single gene disorders that alter the body's ability to regulate iron, causes excess iron deposition in liver, heart, pancreas, joints and pituitary gland, with resultant cirrhosis, cancer, diabetes, heart and joint diseases over time. HH can be effectively treated, but if untreated, the increased iron storage can lead to severe organ damage, and death. Therefore, early identification and treatment is imperative. Most severe cases of HH result from a common mutation in the HFE gene (C282Y), but other HFE mutations have also been identified (H63D and S65C), associated with milder disease. Caucasians of northern European descent are at highest risk, and there is ~1 million people in the US with these mutations, and 10% of Caucasians are carriers. Some individuals are asymptomatic due to the low penetrance, and variable genotype-phenotype correlations. Aims: To determine the prevalence of known HFE mutations, identify potential novel variants, and compare the clinical & laboratory findings with HH genotypes for genotype-phenotype correlations. Materials and Methods: Through the collaboration of Geisinger Health System with the Regeneron, 31,058 MyCode biobank participants were evaluated for HFE gene mutations by analyzing exome sequence data from peripheral blood. Results: Compared to controls, significant differences were noted in males with C282Y/C282Y, C282Y/H63D, and H63D/H63D genotypes for ferritin, serum iron, iron binding capacity (IBC), and transferrin saturation (TS), while C282Y and H63D carriers did not show a significant difference for ferritin, but did for other parameters when compared to non-carriers. Slight differences were noted in females when compared to males, with TS also being significant in C282Y/S65C, H63D/S65C, and S65C/WT females, but not ferritin in C282Y/C282Y or IBC in H63D/H63D individuals. Cirrhosis, hepatocellular carcinoma (HCC), and oral cancers were more prevalent in the C282Y/C282Y genotype. HCC, esophagus and urinary cancers were higher in those with H63D/H63D, while, non-melanoma skin cancers were more prevalent in H63D/WT, and uterine and urinary cancers were more prevalent in the S65C/WT genotype. The patients diagnosed with ICD-9 code of HH were significantly lower than the prevalence of these genotypes, indicating a potential issue of underdiagnosis, or misdiagnosis. One short coming of our evaluation is that the results have not been confirmed by assessment of the medical charts. Conclusion: This large scale genomic study allowed us to identify carriers of HH-associated genetic variants, and uncover potential deficiencies in the current screening algorithm. This will enable the implementation of processes to promote precision medicine, minimize misdiagnosis/underdiagnosis, and maximize outcomes in the management of HH patients.

#5223

A novel molecular inversion probe (MIP) system for the streamlined identification of germline and somatic sequence variants in cancer.

Keynttisha Jefferson, Heather Halvensleben, Dawn Green, Ryan Bannen, Michael Brockman, Todd Richmond, Daniel Burgess. _Roche Nimblegen, Madison, WI_.

Characterization of the somatic sequence variations that accrue in cells is critical for understanding the pronounced cellular and clinical heterogeneity observed in cancer. The ability to efficiently detect variations in tumors can help to identify biomarkers which may be relevant to clinical trials, support more accurate prognosis, and help guide more effective choices of therapy. Next-generation sequencing (NGS) has become a valuable tool for discovering somatic mutations in cancers. Here we present an alternative approach to current methodologies for addressing these needs.

HEAT-Seq (High Efficiency Amplification of Targets for Sequencing) is a targeted NGS method based on optimized, multiplexed molecular inversion probes (MIPs). This is a convenient, sensitive and cost-effective target enrichment technology for SNP discovery and SNP validation in cancer-related genes. HEAT-Seq probes target both DNA strands and were designed to facilitate bioinformatic error correction. Molecule identifiers (UIDs) incorporated into the probes tag PCR duplicates and support ultra-sensitive detection of low frequency variants, reduction of false positives, and accurate assessment of molecular complexity free of amplification bias. Sensitivities for allele detection have been measured to below 1%.

The HEAT-Seq workflow from input DNA to sequence-ready sample can be completed in ≤8 hours without any requirement for a separate library preparation step, which saves both time and cost. Additionally, the capture, amplification and sample clean up steps are performed in a single reaction tube. This eliminates the need to transfer samples to subsequent reaction tubes, preserves sample identity, prevents cross contamination, limits sample loss, and makes the HEAT-Seq protocol easy to automate. A comparison of current PCR-based targeted sequencing with the new HEAT-Seq technology indicates significant advantages for HEAT-Seq in the accurate quantification of low frequency cancer variants.

In summary, HEAT-Seq technology offers a rapid, convenient, automatable option to identify genomic DNA variants and enable advances in cancer research.

#5224

Next-generation sequencing of a panel of genes contributing to breast cancer risk.

Nancy A. Uhrhammer,1 Nagi El Saghir,2 Yannick Bidet,3 Manon Delahaye-Sourdeix,3 Flora Ponelle,1 Marie Ollier,1 Sandrine Viala,1 Firas Kreidieh,2 Nathalie Khoueiry-Zgheib,2 Yves-Jean Bignon1. 1 _Centre Jean Perrin, Clermont-Ferrand, France;_ 2 _American University of Beirut Medical Center, Beirut, Lebanon;_ 3 _University of Auvergne, Clermont-Ferrand, France_.

BRCA mutations account for a minority of hereditary breast cancer risk families. Many other genes have been identified, with rare but highly penetrant mutations, or more frequent mutations giving modest breast cancer risk. Next generation sequencing has made it possible to explore a wide sample of genes for their contribution to breast cancer risk. We have sequenced gene panels in two populations of non-BRCA breast cancer families.

First, 47 French families were selected for triple-negative breast cancer in the index case and >1 first degree relative with breast cancer. A panel of 33 genes involved in cancer risk, DNA repair, and/or partners of BRCA1 or BRCA2 was sequenced using DNA capture and GS-Flex sequencing. Second, a Lebanese cohort of 25 non-BRCA cases with Manchester scores >18 was sequenced on a MiSeq after capture of a similar panel of 37 genes. BRCA1 and BRCA2 were included in both panels to confirm previously obtained results. Large rearrangements were detected using MLPA.

Sequence alignment and variant identification was performed using a combination of commercial software and an in-house GATK-based pipeline. Variants were described as neutral or likely neutral if the MAF in online databases exceeded 1%, if in silico analyses suggested neutrality, and if splicing prediction algorithms agreed on the absence of any significant effect.

NGS analysis confirmed the absence of BRCA mutations. Deleterious mutations were observed in three Lebanese cases: two with a deletion in PALB2 and one stop mutation in ATM. Nine additional Lebanese cases and four from the French TNBC group were found to carry variants of unknown significance (VUS) in one or more genes. Most cases were found to have one VUS; two TNBC cases had more than one VUS and one Lebanese case had a VUS and a PALB2 mutation.

The cases with deleterious mutations had Manchester scores of 56, 22 and 19. Scores for cases with VUSs ranged from 18 to 45 (average 23), and those with only neutral or likely neutral variants ranged from 18 to 27 (average 21.8). These differences are not significant, but additional cases are under study and will be presented. No difference was observed between French TNBC families with or without VUSs.

DNA capture of gene panels is a quick and efficient way of analyzing multiple genes involved in hereditary cancer risk. Our panels focused on a limited number of genes involved in DNA repair and/or direct interactions with the BRCA genes: the small number of genes allowed us to analyze a maximum number of patients per experiment on two relatively low-throughput machines. Although the two cohorts are small, they show that the number of variants requiring follow-up in non-BRCA genes is low. The absence of deleterious mutations in genes involved in homologous recombination repair in the TNBC cohort showed that although genes of this pathway are candidates for tumor sensitivity to PARP inhibitors, the candidates we tested may not be frequently constitutionally mutated.

#5225

CDK11B, PTPRN2 and WDPCP were frequently duplicated genes in refractory/relapsed normal karyotype AML patients: Identifying structural variations using whole genome sequencing.

Jeonghwan Youk,1 Sunghoon Cho,2 Daeyoon Kim,3 Youngil Koh,1 Inho Kim,1 Murim Choi,4 Sung-Soo Yoon1. 1 _Seoul National University Hospital, Seoul, Republic of Korea;_ 2 _Pdxen, Biosystem. Inc, Seoul, Republic of Korea;_ 3 _Cancer Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea;_ 4 _Seoul National University College of Medicine, Seoul, Republic of Korea_.

Background

According to the NCCN guideline, the prognosis of acute myeloid leukemia with normal karyotype (NK-AML) is classified based on the mutational status of NPM1, FLT3-ITD or CEBPA. However, patients with NK-AML without the above mutations, who are in intermediate risk, still show various clinical outcomes. Unfortunately, refractory/relapsed patients with intermediate risk NK-AML cannot be predicted before cytotoxic chemotherapy, yet. With the advent of next generation sequencing technology, we tried to reveal prognostic molecular marker in intermediate risk NK-AML, especially focusing on large structural variations (SVs).

Methods

A total of nine patients with intermediate risk NK-AML patients were included. Whole genome sequencing (WGS) was performed using a pair of tumor and germline DNA of the patients. Leukemic blasts were obtained from bone marrow aspiration specimens of each patient at the time of diagnosis. As a germline control, epithelial cells were collected from saliva of each patient at the time of complete remission. Then, genomic DNA was massively sequenced using HiSeq X10 system (Illumina Inc., San Diego, CA, USA). Possible SVs including inversion, duplication and translocation were identified with BreakDancer (Washington University School of Medicine, St. Louis, MO, USA), Pindel (The Wellcome Trust Sanger Institute, Cambridge, UK) and Delly (European Molecular Biology Laboratory, Heidelberg, Germany). Possible SVs were considered when they were recurrently identified from the three pipelines.

Results

This cohort consisted of 7 males and 2 females (median age = 55.6 years old). None of them had secondary AML. Eight patients were relapsed after complete remission and the other patient was refractory to 1st line induction chemotherapy. Through WGS, 25 inversions, 67 duplications and 10 translocations were identified in nine patients. Among them, 4 inversions, 13 duplications and 2 translocations were recurrently mutated. Especially, CDK11B, PTPRN2 and WDPCP were frequently duplicated more than 40% of the patients.

Conclusions

In this study, we performed WGS to investigate SVs of refractory/relapsed NK-AML patients with intermediate risk. Among various mutations, duplications of CDK11B, PTPRN2 and WDPCP were highly prevalent, so they could be a possible prognostic biomarker in NK-AML with intermediate risk. Further functional validation is required in order to clarify their roles.

#5226

Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis.

Kasmintan A. Schrader,1 Carolyn Chu'ng,2 Eric Zhao,2 Hui-li Wong,3 Yaoqing Shen,2 Martin Jones,2 Tom Thomson,4 Howard Lim,3 Sean Young,5 Carol Cremin,6 Robert Holt,2 Peter Eirew,2 Joanna Karasinska,7 Jacquie Schein,2 Yongjun Zhao,2 Andy Mungall,2 Richard Moore,2 Yussanne Ma,2 Alexandra Fok,2 Robyn Roscoe,2 Stephen Yip,8 Gillian Mitchell,6 Aly Karsan,9 Steven Jones,2 David Schaeffer,8 Janessa Laskin,3 Marco Marra,10 Daniel Renouf3. 1 _Department of Medical Genetics, University of British Columbia; Department of Molecular Oncology, BC Cancer Research Centre; Hereditary Cancer Program, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 2 _Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 3 _Department of Medical Oncology, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 4 _Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada;_ 5 _Cancer Genetics Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 6 _Hereditary Cancer Program, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 7 _Pancreas Centre BC, Vancouver, British Columbia, Canada;_ 8 _Department of Pathology & Laboratory Medicine, Vancouver General Hospital, Vancouver, British Columbia, Canada; _9 _Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency; Department of Pathology and Laboratory Medicine, University of British Columbia; Cancer Genetics Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada;_ 10 _Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada_.

Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient's personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient's PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G>A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications.

#5227

Molecular profiling in African American NSCLC patients to identify novel potential driver mutations.

Ann G. Schwartz, Angela S. Wenzlaff, Chrissy M. Lusk, Greg Dyson, Aliccia Bollig-Fischer, Susan Land, Sneh Lata, Michele L. Cote, Gerold Bepler, Shirish M. Gadgeel. _Barbara Ann Karmanos Cancer Institute, Detroit, MI_.

African Americans continue to have poorer 5 year survival after a lung cancer diagnosis than whites for reasons that remain to be fully characterized. This disparity remains even with advances in treatments targeting specific driver mutations that have improved outcomes for some patients. Although the relative frequency of these genetic alterations varies in subsets of individuals defined by sex, histologic subtype, smoking history and race, little is known about the occurrence of these mutations in African Americans. In this study, we characterize the spectrum of known driver mutations in 200 African American NSCLC patients and seek to identify novel somatic mutations in this population. Initially, the population was screened using the Sequenom LungCarta panel of 216 mutations in 24 genes known to contain alterations associated with lung cancer, RET and ROS1 fusion gene expression, and amplification of FGFR1. Whole-exome sequencing is being performed on tumors from those patients with no known somatic mutations. Paired normal and tumor DNA/RNA samples are being genotyped to distinguish germline from somatic mutations in both the screening and sequencing phases. Initial screening has been completed on 130 patients, with the remaining 70 underway. The mean age of the patients enrolled is 61.7 years, 58.5% are female, and 8.5% are never smokers. In profiling the first 130 African American patients, we find that only 28% of patients (N=36) carry a known somatic mutation that is not present in the germline, far lower than the 41% reported in white patients in our previous studies. Of the identified mutations, approximately 23.8% are KRAS and 23.8% are PT53 alterations. The next most frequent somatic alterations are in EGFR (16.7%). The remaining alterations occurred in only 1 or 2 tumors and include EPHA3, ERBB2, FGFR1, MET, NOTCH1, NRAS, NTRK2, PIK3CA, PTEN, RET and STK11. Five percent of patients have tumors with two driver mutations. Half of the tumors carrying 2 driver mutations had concurrent alterations in KRAS and TP53. Patients with known genetic alterations were more likely to be female than patients with no known genetic alterations (p=0.42), but did not differ in age, smoking status, pack-years, family history of lung cancer, history of COPD, stage at diagnosis or histology. Mutations in EGFR were responsible for the differences by sex. Exome sequencing is complete for the first 48 of the 92 patients with no known somatic mutations. Sequences are being aligned using Novoalign and GATK will be used for variant calling; results will be available soon. Genetic profiling of NSCLC in African Americans has the potential to both identify novel mutations, expanding the list of potential targets for tailored treatments, and aid clinical decision making in African American patients leading to improvements in outcomes.

#5228

Estrogen receptor expression is associated with DNA repair capacity in breast cancer.

Jaime L. Matta,1 Luisa Morales,1 Carmen Ortiz,1 Damian Adams,1 Wanda Vargas,1 Patricia Casbas,1 Julie Dutil,1 Miguel Echenique,2 Erick Suarez3. 1 _Ponce School of Medicine, Ponce, PR;_ 2 _Auxilio Mutuo Hospital, San Juan, PR;_ 3 _University of Puerto Rico, Medical Sciences Campus, San Juan, PR_.

Estrogen receptor (ER) status is an established therapeutic and prognostic biomarker for breast cancer (BC). ER signaling has a propensity to increase proliferation, which can lead to mutations when DNA repair is dysregulated. Deficiencies in DNA repair capacity (DRC) is a hallmark of BC. Recent studies have documented interactions between ER and DNA damage response/repair. Moreover, our previous work showed that DRC in BC patients is approximately 50% less than in women without the disease (p<0.001), and a deficiency in the Nucleotide excision repair (NER) pathway figures prominently in the pathobiology of sporadic BC. Estrogen-receptor-positive (ER+) tumors employ complex signaling that engages in crosstalk with multiple pathways through genomic and non-genomic regulation. A greater understanding of these pathways is important for developing improved biomarkers that can better determine treatment choices, risk of recurrence, and cancer progression. In this study, we aimed to determine whether ER signaling influences DRC. DRC, in terms of the NER pathway, was measured in lymphocytes by means of a host cell reactivation assay with a luciferase reporter gene. A multivariate linear regression model (MLRM) was used to estimate the mean of the DRC and analyze the association between ER positivity (% receptor activation) obtained from pathological reports and DRC in 270 BC patients. These were further stratified by HER2 receptor status. An ordinal logistic regression model (OLRM) was used to confirm the relationship between DRC and ER status using the maximum likelihood method for parameters estimation. Our results show an association between ER status and mean DRC values in BC patients who have HER2-negative (HER-) tumors: that is, the mean DRC value in ER-/HER2- tumors was significantly lower as compared with ER-/HER2+ tumors (p<0.05). In contrast, the mean DRC among patients with ER+/HER2+ tumors did not significantly differ from ER-/HER2+ tumors (p>0.05). The data followed similar patterns when DRC was categorized in tertiles. In conclusion, there is an association between DRC levels and ER status, and this association is modified by HER2 receptor status. Supported by grants from the NCI Center to Reduce Health Disparities and NIH-NIGMS MBRS SCORE and RISE Programs grants # S06 GM008239-20, 9SC1CA182846-04 GM082406 and PSM-MCC Partnership grant # 5U54CA163071-04.

#5229

Candidate gene analysis of exome sequencing data in smokers susceptible and resistant to chronic obstructive pulmonary disease.

Yanhong Liu,1 Michael H Cho,2 Dandi Qiao,2 Farrah Kheradmand,1 Spiridon Tsavachidis,1 Georgina Armstrong,1 Michael Scheurer,1 David Wheeler,1 Christopher Amos,3 Edwin Silverman,2 Margaret Spitz1. 1 _Baylor College of Medicine, Houston, TX;_ 2 _Brigham and Women's Hospital, Boston, MA;_ 3 _Dartmouth Medical School, Lebanon, NH_.

Background: Chronic obstructive pulmonary disease (COPD) is a major comorbidity for Lung cancer (LC), the presence of COPD conferring a three- to 10-fold increased risk of LC. Therefore, we employed an extreme-phenotype approach, focused on exome sequencing of 109 susceptibility loci identified in recent genome-wide association studies (GWAS) of LC, COPD, lung function and smoking behavior, to search for COPD disease-causing rare germline mutations.

Methods: We selected two extreme categories of smokers from COPDGene: 1) Resistant long-term smokers with normal lung function defined as post-bronchodilator FEV1 ≥ 80% predicted, FEV1/FVC ≥ 0.7, with smoking histories of 15+ pack-years, considered as resistant to the effects of smoking, n = 318; 2) Susceptible smokers with severe COPD defined as GOLD spirometry grades III-IV (post-bronchodilator FEV1 < 50% predicted and FEV1/FVC < 0.7), with smoking histories of 10+ pack-years, n = 309. We performed whole exome sequencing and analyzed rare (minor allele frequency [MAF] < 0.01 in reference exome databases) variants predicted to be functional in the 109 susceptibility loci previously identified by GWAS. We tested our results using two independent series including the Boston Early-Onset pedigrees sequencing study (n = 350) and the Exome Sequencing Project (ESP)-COPDGene study (n = 400).

Results: Our analysis revealed two genes with multiple rare non-synonymous substitution variants significantly associated with COPD risk. We identified six rare variants in 10q23.33 Myoferlin (MYOF: p.Arg572Gln, p.Phe1400Leu, p.Tyr1163His, p.Glu1339Lys, p.Leu1582Pro, and p.Arg1630Gln) occurring in 12 susceptible smokers with severe COPD, and three rare variants in 15q15.2 Transglutaminase 5 (TGM5: p.Thr42Asn, Pro136Leu, and p.Val202Ile) occurring in nine susceptible smokers with severe COPD, compared with none of these variants identified in resistant smokers. MAF of these mutations were very rare (range from 0 to 0.003) in the 1000 Genomes and ESP databases. All of these mutations were predicted to be protein damaging and deleterious by bioinformatics algorithms (SIFT, PolyPhen2, Mutation Taster, etc.). We also observed three suggestive rare mutations, 6p21.32 ZBTB9 p.Leu43Val, 9q34.2 DBH p.Gly482Arg, and 10q23.33 IFIT3 p.Leu390Arg, each of which were present in three susceptible smokers and none in resistant smokers. However, we were not able to validate these abovementioned candidate variants in the two replication datasets. This could due to potential false positive results or difference of exome sequencing depth and coverage of the target regions from different platforms.

Conclusion: Our results demonstrated potentially disruptive COPD risk-conferring MYOF and TGM5 rare germline mutations that are associated with susceptibility to COPD in one population of smokers, but additional replication of these results in larger populations will be required.

#5230

Large scale whole genome sequencing with imputation into GWAS improves our understanding of the genetic architecture of colorectal cancer.

Jeroen Huyghe,1 Sai Chen,2 Hyun M. Kang,2 Tabitha A. Harrison,1 Sonja I. Berndt,3 Stephane Bézieau,4 Hermann Brenner,5 Graham Casey,6 Andrew T. Chan,7 Jenny Chang-Claude,5 Gallinger J. Steven,8 Stephen B. Gruber,6 Andrea Gsur,9 Michael Hoffmeister,5 Thomas J. Hudson,10 Loic Le Marchand,11 Polly A. Newcomb,1 John D. Potter,1 Conghui Qu,1 Martha L. Slattery,12 Joshua D. Smith,1 Emily White,1 Li Hsu,1 Goncalo R. Abecasis,2 Deborah A. Nickerson,1 Ulrike Peters1. 1 _Fred Hutchinson Cancer Research Center, Seattle, WA;_ 2 _University of Michigan, Ann Arbors, MI;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _CHU Nantes, Nantes, France;_ 5 _German Cancer Research Center, Heidelberg, Germany;_ 6 _University of Southern California, Los Angeles, CA;_ 7 _Massachusetts General Hospital, Boston, MA;_ 8 _Mount Sinai Hospital, Toronto, Ontario, Canada;_ 9 _Medical University of Vienna, Vienna, Australia;_ 10 _Ontario Institute for Cancer Research, Toronto, Ontario, Canada;_ 11 _University of Hawaii Cancer Center, Honolulu, HI;_ 12 _University of Utah Health Sciences Center, Salt Lake City, UT_.

Whole-genome sequencing (WGS) has started a new era in human genetics in which data can be used to more fully understand the role of genetic variation in common complex diseases, including the role of less frequent and rare variants and structural variation. To explore the impact of these variants on colorectal cancer risk we conducted the first large scale WGS study for colorectal cancer (CRC) including 1,961 CRC cases and 981 controls. These WGS data as well as those from the Haplotype Reference Consortium were imputed in 13,104 CRC cases and 15,521 controls with genome-wide association study (GWAS) data that are part of the Colorectal Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Focusing on rare and less frequent variants, insertions and deletions we observed potentially novel variants: a less frequent variant (MAF=0.026) on chromosome 5 located in NREP/STARD4-AS1 (p=value 4E-08); and a novel rare multi-allelic variant (MAF=0.003) on chromosome 9 near KLF9 and TRPM3 (p-value 2E-09; the other allele of this multi-allelic variant had a MAF of 0.0003 and p-value of 0.55). Furthermore, we observed an independent locus close to the known region 8q24 that was located upstream of GSDMC (MAF = 0.16, p-value 5E-08). Within the known region 8q23/EIF3H we identified several low frequency variants with similar MAF (0.0181 to 0.0204) including a 6bp deletion with p-values between 4E-08 and 1E-09 that were independent of the common variant signal in this region. In addition, we identified statistically significant (p<5E-08) deletions, insertions, and an essential splice site within known GWAS loci that present interesting candidates for functional studies. We will follow up these findings in independent samples from the Colorectal Cancer Transdisciplinary Study (CORECT) and CCFR, as well as additional samples currently genotyped in GECCO. In conclusion, next generation sequencing combined with imputation in large GWAS data sets has the potential to identify novel low frequency and rare genetic variants, aid fine-mapping of known CRC susceptibility loci and point to interesting functional candidates.

#5231

Multiple myeloma will become a common cancer in the era of modern therapy.

Philip S. Rosenberg,1 Ana Best,1 William F. Anderson,1 Ola Landgren2. 1 _National Cancer Institute, Bethesda, MD;_ 2 _Myeloma Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY_.

INTRODUCTION: Multiple myeloma (MM) is the second most common hematological malignancy in the United States (US), representing 1.4% of all newly diagnosed cancers. The annual number of new MM patients is expected to increase primarily because the population is ageing. In parallel, several new drugs with unprecedented efficacy have been approved by the FDA. We were motivated to use data from the Surveillance, Epidemiology, and End Results (SEER) Program and statistical models to estimate future numbers of MM survivors in the era of modern therapy.

METHODS: We estimated future MM incidence using age-period-cohort forecasting models. We estimated all-cause mortality rates by years-since-MM-diagnosis, for cohorts of incident cases defined by age-at-diagnosis and year-of-diagnosis; here we used a proportional hazards absolute risk model with age, year, and duration effects specified using linear splines. We calculated current and future prevalence rates based on observed and extrapolated SEER incidence and mortality. We calculated corresponding numbers of MM survivors in the US by multiplying prevalence rates by official observed and projected population counts. We projected life year gains among persons with MM from prevalence forecasts incorporating more and less favorable assumptions about MM prognosis.

RESULTS: We analyzed MM among black and white men and women using 1992 - 2010 SEER data and forecast to 2022. Qualitative patterns were similar in each group. As previously reported, incidence was stable or slightly increasing and highest among black men. However, survival improved in all groups between 1992 through 2010. For example, among white men aged 60-69 years at diagnosis, median survival after MM increased from 2.6 to 5.0 years. If progress continues at the same rate - a conservative assumption in light of recent advances - the projected median will increase to 7.6 years circa 2022. Among black and white men and women ages 40 - 79 years combined, the number of new MM cases is expected to increase by 28%, from 16,000 to 21,000, primarily because of population growth. However, the number of persons living with MM is conservatively expected to increase by 55%, from 76,000 to 119,000, because of improvements in prognosis as well as population growth. The cumulative life year gain between 2011 through 2022 is around 50,000 life years. If recent treatments are as promising as they appear, these estimates will be conservative.

CONCLUSION: Driven by access to modern therapies with unprecedented efficacy, overall survival for patients with MM will continue to improve significantly. Consequently, our forecast is MM will become much more common in the era of modern therapy. The numbers of patients living with MM are expected to increase more quickly than the corresponding numbers of new cases.

#5232

Using an integrated gene-based sequence kernel association test (intSKAT) to identify subtype specific single nucleotide variants in glioma.

Yian Ann Chen, Jamie K. Teer, Zachary J. Thompson, Rebekah L. Baskin, Yonghong O. Zhang, Kate J. Fisher, Zhihua Chen, Alvaro N. Monteiro, Kathleen M. Egan. _Moffitt Cancer Center & Research Institute, Tampa, FL_.

One of the challenges for genome-wide association analyses is that the effect directions and allele frequencies (e.g., rare, common, or combination of them) of true causal variants are unknown. Built on a family of powerful approaches, sequence kernel association test (SKAT), we have devised a gene-based omnibus approach, integrated-SKAT (intSKAT) to perform association tests using next-generation sequence data. This includes a suite of 12 methods: Burden test, SKAT, SKAT-O, SKAT-C (Combined sum test of rare- and common-variant effects), SKAT-A (Adaptive sum test), SKAT-AR, three methods weighted by functional scores, and three rare-variant only methods. This expansive set allows the flexibility to detect the potential combination of different allele frequencies, effect directions, and/or (lack of) functional predictions. Minimum FDR was used to adjust for multiple comparison across methods. We applied intSKAT to investigate sub-type specific susceptibility loci between low-grade glioma (LGG) and glioblastoma (GBM) as proof of principle. We downloaded germline exome sequence data (N = 612) from TCGA, and aligned the sequence reads using the Burrows-Wheeler Aligner (BWA). Insertion/deletion realignment, quality score recalibration, and variant identification were performed with the Genome Analysis ToolKit (GATK). We used 80% SNV call rate for quality control. Following Principal Component Analysis, data for 544 Caucasian subjects were included in analysis. A total of 224K SNVs in 18,053 genes were studied. Ten genes were significantly associated with glioma subtypes (minFDR of 10%). Among these genes, a total of 9 significant SNVs with predicted possible damaging functions were identified in 6 genes, ATP5O, CKAP2L, SORBS1, STK19, VPS13B, and ZCCHC4. Five of the genes are significantly differentially expressed between LGG and GBM (all p<1.02x10-3), and consistently supported by both microarray and RNAseq platforms, with the only exception of ZCCHC4. Three of the significant genes, which would not have been identified using SNV-level univariate analyses, are CALML6, FGF22, and GRIN1. Weighted-SKAT was effective to identify genes with both deleterious and protective variants. Weighted-Burden test was powerful to detect genes with deleterious variants with predicted function. These findings demonstrate the power of our proposed gene-based method.

#5233

Concordance of genomic alterations by next generation sequencing (NGS) in tumor tissue vs. cell-free DNA.

Young Kwang Chae, Andrew A. Davis, Benedito A. Carneiro, Sunandana Chandra, Nisha Mohindra, Aparna Kalyan, Jason Kaplan, Maria Matsangou, Leonidas C. Platanias, Massimo Cristofanilli, Francis J. Giles. _Northwestern University, Chicago, IL_.

Introduction:

Genomic analysis of circulating tumor cell-free DNA (cfDNA) represents a non-invasive method of assessing genomic alterations using peripheral blood. We compared the concordance between cfDNA and tissue biopsies across genomic alterations in 65 genes in this pilot retrospective study.

Methods:

54 consecutive patients with cfDNA NGS testing (Guardant360) were identified. Of these, 28 had tissue DNA NGS testing (Foundation One). 65 genes were common to both assays. Concordance was defined as the absence or presence of the identical genomic sequencing alteration(s) in a single gene. The analysis included non-synonymous DNA mutations, rearrangements, and copy number variants regardless of clone percentage.

Results:

There were 14 lung cancers and 14 other advanced solid tumors. Including all alterations and variants of unknown significance (VUS), the average number of alterations per patient for tissue and cfDNA was 4.82 (SD 3.02) and 2.96 (SD 3.01), respectively. Concordance between the two assays was 91.9%. Among only genes with reported genomic alterations in either assay (n=170), concordance was 12.4%. Concordance was similar when stratifying based on timeframe between biopsies (median time interval between assays; 89 days). 21.5% of alterations found in tissue were detected in cfDNA. 34.9% of alterations found in cfDNA were detected in tissue. Across the 5 most common genes, sensitivity and specificity were 40.0% and 94.2%, respectively (Table 1).

Discussion:

Concordance between tumor tissue and cfDNA NGS was 91.9%. The cfDNA assay had high specificity, but low sensitivity. In addition, more mutations were detected in tissue. Reasons for these findings may include different sequencing techniques, spatial and temporal factors, tumor heterogeneity, and inclusion of subclones.

[AD and YC contributed equally.]

Table 1: Diagnostic Accuracy across 5 Most Common Genes  | |  | |  | |  | |

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

|

Tissue Mutations | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) | Diagnostic Accuracy (%)

cfDNA mutations | (+) | (-) | |  | |

|

TP53 | (+) | 8 | 2 | |  | |

|

|

(-) | 8 | 8 | 50.0 | 80.0 | 80.0 | 50.0 | 57.1

EGFR | (+) | 1 | 2 | |  | |

|

|

(-) | 2 | 23 | 33.3 | 92.0 | 33.3 | 92.0 | 85.7

KRAS | (+) | 4 | 1 | |  | |

|

|

(-) | 2 | 21 | 66.7 | 95.5 | 80.0 | 91.3 | 89.3

APC | (+) | 0 | 1 | |  | |

|

|

(-) | 4 | 23 | 0.0 | 95.8 | 0.0 | 85.2 | 82.1

CDKN2A | (+) | 1 | 0 | |  | |

|

|

(-) | 5 | 22 | 16.7 | 100.0 | 100.0 | 81.5 | 82.1

Total positive | 14 | 6 | |  | |

|

Total negative | 21 | 97 | |  | |

|

Total (positive + negative) | 35 | 103 | 40.0 | 94.2 | 70.0 | 82.2 | 80.4

PPV: positive predictive value

NPV: negative predictive value

#5234

Rare deleterious germline mutations of candidate genes associated with risk of familial esophageal squamous cell carcinoma (ESCC) by targeted sequencing.

Josephine Mun Yee Ko,1 Lu wen Ni,1 Sheyne, Sta Ana Choi,1 Lisa Chan Lei,1 Li Dong Wang,2 Maria Li Lung1. 1 _Univ. of Hong Kong, Pokfulam, Hong Kong;_ 2 _The First Affiliated Hospital of Zhengzhou University, Henan, Henan, China_.

Background and aims:

Esophageal cancer (EC) is a deadly disease with poor survival rates of only ~10%. The EC incidence remains highest in the Shanxi and Henan regions near the Tang Hang Mountain area in Northern China. In endemic esophageal squamous cell carcinoma (ESCC) regions, common familial clustering and historical migration patterns suggest an inherited ESCC genetic susceptibility. We hypothesize that cancer predisposition gene(s) (CPGs) carrying rare deleterious variants play a crucial role in ESCC genetic susceptibility and aim to identify the underlying CPGs of familial ESCC from Henan by applying the targeted sequencing approach.

Methods:

Our discovery set utilized NGS technology to capture a comprehensive gene panel critical for ESCC by targeted re-sequencing of 1271 selected genes (11 Mb) and sequenced for the germline variants in the blood DNA from 222 familial ESCC individuals and 124 Henan non-cancer controls. Familial ESCC was defined as at least two family members developed ESCC. The custom designed panel contains a cancer panel of 578 gene list (4Mb) and a custom list of 693 (7Mb) ESCC-specific genes derived from various approaches including microarray profiling in FH+ ESCC primary tissues, homozygosity-by-descent (HBD), SNP array-based analysis by both GWAS and candidate gene approaches of our own set of MassArray SNP association data. The data from 346 individuals were processed using the analysis pipeline following the GATK guideline. Briefly, raw fastq reads were mapped using the Burrows-Wheeler Aligner (BWA). PCR duplicates are marked using Picard. The raw variants are called by GATK HaplotypeCaller using a joint genotyping of all samples.

Results:

Preliminary data analysis for the 346 samples resulted in the final call set comprised of 52328 rare variants. There are 26486 rare variants present in the 222 FH+ ESCC cases, but absent in the 124 controls. The average sequencing coverage was 88X. Principal component analysis (PCA) and multidimensional scaling (MDS) indicated a homogenous population structure between the case and the control populations. Targeted re-sequencing results of the 1271 selected genes in 222 familial ESCC individuals identified a recurrent missense variant in AIM1L (Absent in Melanoma 1-like) in 14/222 (6.3%) of FH+ ESCC patients but absence in the 124 non-cancer control. Data obtained by a melting curve-based genotyping platform by LC480 and LightSNiP assay for high throughput validation of this recurrent SNP in larger sample cohort are underway and will be presented.

Conclusions:

A SNP in AIM1L may be associated with higher risk for ESCC in Henan and validation in larger cohort is required. The data may provide valuable insight for understanding genetic susceptibility to this deadly cancer.

Acknowledgements:

We acknowledge the Asian Fund for Cancer Research for grant support.

## PREVENTION RESEARCH:

### Chemoprevention Studies

#5235

Dietary administration of δ- and γ-tocopherol inhibits estrogen-mediated mammary tumorigenesis in ACI rats.

Soumyasri Das Gupta, Joseph Wahler, Min Ji Bak, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. _Rutgers University, Piscataway, NJ_.

Tocopherols (T), members of the vitamin E family, occur in four forms designated as α, β, δ and γ. Epidemiological studies suggest that tocopherols could reduce the risk of cancer. α-T is known as the classical vitamin E, being the predominant form of tocopherol found in human blood and tissues. However, clinical studies with α-T to substantiate the role of tocopherols in cancer prevention have provided inconclusive results. Such outcomes indicate that the cancer preventive activity of tocopherols might be attributed to the other forms of tocopherols, but not α-T. γ-T, which is the most abundant tocopherol in vegetable oils, is our major dietary source of tocopherols. Recent studies have demonstrated that δ-T and γ-T have higher anti-cancer activity than α-T in several animal models of cancer. The present study investigated the cancer preventive activity of individual purified forms of tocopherols α-T, δ-T, γ-T as well as a naturally occurring γ-T rich mixture of tocopherols (γ-TmT) in an in vivo model of estrogen receptor (ER)-positive breast cancer, namely estrogen supplemented August-Copenhagen Irish (ACI) rats. ACI rats were subcutaneously implanted with 9 mg of 17β-estradiol (E2) in silastic tubings and administered with diets containing 0.2% α-T, δ-T, γ-T or γ-TmT. Rats were euthanized at 30 weeks of mammary carcinogenesis when the E2 control group exhibited 100% tumor incidence. α-T had no effect on estrogen-induced mammary tumors. However, treatment with 0.2% δ-T, γ-T and γ-TmT reduced the tumor volume by 51% (p<0.05), 60% (p<0.01) and 59% (p<0.01), respectively. Administration of α-T, δ-T and γ-T resulted in a 2.1-, 80- and 62-fold increase of these tocopherols, respectively, in the serum of

ACI rats. Supplementation with the individual tocopherols also increased the levels of their respective short chain metabolite, carboxyethyl hydroxychromans (CEHCs) in the serum. Administration of δ-T and γ-T was associated with 185- and 99-fold increase in their CEHC metabolites, respectively. The high serum levels of δ- and γ-CEHC metabolites could contribute to the enhanced chemopreventive activity of δ-T and γ-T. In conclusion, δ-T, γ-T and γ-TmT could be potential natural agents for the prevention of estrogen-induced mammary tumorigenesis.

(This work was supported by the NIH grant R01 AT007036 and the New Jersey Commission on Cancer Research Postdoctoral Fellowship to Soumyasri Das Gupta).

#5236

Bitter melon juice causes strong efficacy towards pancreatic cancer stem cells by modulating the hedgehog signaling pathway.

Rajesh Agarwal, Deepanshi Dhar, Chapla Agarwal, Gagan Deep. _University of Colorado Denver School of Pharmacy, Aurora, CO_.

According to ACS estimates for year 2015, ~48,960 new cases of pancreatic cancer (PanC) would be diagnosed with 40,560 associated deaths in the US alone. The median life of PanC patients post diagnosis is <6 months and overall 5 year survival is 3-5%. Thus, there is an urgent need for newer strategies to improve the disease outcome in these patients. Recently, considerable efforts have been directed towards natural dietary/non-dietary agents for both prevention/intervention of PanC. One such dietary agent, bitter melon (Momordica charantia) from Cucurbitaceae family, is now being extensively evaluated for its efficacy against various malignancies including PanC. Recently, we have shown significant growth inhibitory effects of bitter melon juice (BMJ) against a panel of PanC cells including Mia PaCa-2 xenograft tumors. Our studies also showed BMJ activity against both gemcitabine (frontline chemotherapeutic in PanC patients) sensitive as well as resistant PanC cell lines. Since the presence of cancer stem cells (CSC) is now recognized as the main cause for initiation, promotion and drug-resistance as well as relapse of most of the cancers, including PanC, there is a possibility that BMJ was affecting the CSC population in the PanC cells. Accordingly, the present study was aimed to determine whether BMJ has the potential to target CSC in PanC. We isolated CSC enriched (CD44+CD24+EpCAMhigh) cell population from the PanC cell lines, MiaPaCa2, PANC1 and AsPC1 and subjected them as well as the unsorted cells to sphere cluster formation assays in the presence or absence (single vs. multiple time treatment) of BMJ (0.25-2% v/v). The % of floating spheroids generated in the sphere cluster assays after 1-2 weeks were determined. The results indicated a dose-dependent effect of BMJ on both number and size (volume) of spheres, with 2% BMJ showing a significant reduction in both number and size of generated spheres in all PanC cell lines; single treatment of BMJ was found to be equally effective as multiple dosing regimens. Since formation of spheroids under specific in vitro conditions is a measure of stemness, our results show that BMJ has the potential to target the self-renewal of CSCs in PanC cell lines. Subsequent studies employing real time RT-PCR and immunofluorescence showed BMJ effect to be mediated in part by a significant down regulation in the expression of CSC marker CD44, CD133, and hedgehog (Hh)-pathway members in PanC cell lines. Further studies found that BMJ also decreases the protein levels of Hh-pathway members SHH and SMO in MiaPaCa2 xenograft tumors. Together, these are significant findings as they clearly show that BMJ interferes with the kinetics of CSC pool expansion via targeting various regulatory signals associated with the survival and multiplication of CSC pool leading to effective anti-cancer efficacy against PanC cells and their resistant phenotypes.

#5237

Protection of tocopherols against estrogen-induced breast cancer via mechanisms targeting cancer stem cells.

Min Ji Bak, Soumyasri Das Gupta, Joseph Wahler, Mao-Jung Lee, Yong Lin, Weichung Joe Shih, Chung S Yang, Nanjoo Suh. _Rutgers University, Piscataway, NJ_.

Estrogens have been implicated to be complete carcinogens through mechanisms involving increased proliferation, oxidative stress and DNA damage. Although the importance of estrogen in breast cancer is well established, the effects of estrogen on breast cancer stem cells are not fully understood. Due to the role of breast cancer stem cells in tumor initiation, maintenance, and progression, targeting cancer stem cells has become important for understanding estrogen-mediated breast carcinogenesis. The vitamin E family, which is consisted of four different forms of tocopherols and tocotrienols, has been shown to exert anti-tumor effects in animal models of various cancers. In the present study, we examined the protective effects of α-, γ-, δ-tocopherol as well as a natural γ-tocopherol rich mixture of tocopherols (γ-TmT) against estrogen-induced expansion of cancer stem-like cells and estrogen-mediated tumor growth. To determine the effects of tocopherols on breast cancer stem cells, the MCF-7 mammosphere cell culture system, which enriches for mammary progenitor cells and putative breast cancer stem cells, was utilized. Treatment of MCF-7 mammosphere with estrogen for six days resulted in an increase in CD44+/CD24- stem-like cell populations as well as the number and size of mammosphere. α-, γ-, δ-Tocopherol, and most significantly γ-TmT, inhibited estrogen-induced mammosphere formation, indicating that tocopherols reverse estrogen-induced expansion of breast cancer stem cells. In MCF-7 xenograft tumor study, MCF-7 cells (5 x 106 cells/mouse) were injected into the mammary fat pad in immunodeficient mice after subcutaneous implantation with 0.72 mg of estrogen pellet. Mice were then administered with diets containing 0.2% α-, γ-, δ-tocopherol and γ-TmT for 5 weeks. Treatment of α-, γ-, δ-tocopherol or γ-TmT reduced the tumor volume by 71% (p<0.05), 55% (p<0.05), 59% (p<0.05) and 42% (p<0.01) as well as tumor weight by 80%, 63% (p<0.05), 61% (p<0.05) and 48% (p<0.05), respectively. Our results suggest that tocopherols significantly repress the expansion of breast cancer stem cell-like population and tumor development. Tocopherols, in particular a naturally occurring tocopherol mixture rich in γ and δ-tocopherols, could thus be effective natural agents for the prevention and treatment of estrogen-induced breast cancer.(This work was supported by NIH R01 AT007036)

#5238

The chemopreventive potential of silver nanoparticles against UVB-induced skin carcinogenesis in mouse model.

Nikhil Tyagi,1 Sumit Arora,1 Sanjeev K. Srivastava,1 James E. Carter,2 Ajay P. Singh,1 Seema Singh1. 1 _USA Mitchell Cancer Institute, Mobile, AL;_ 2 _Department of Pathology, University of South Alabama, Mobile, AL_.

Solar ultraviolet (UV) radiation, particularly its UVB component, is an established cause of human skin carcinogenesis due to its ability to cause DNA damage in skin cells. Although several sunscreen formulations have been developed and commercialized to limit or minimize UVB exposure to skin cells, incidence of skin cancer has continued to increase suggesting their limited or no efficacy in preventing UV-induced skin cancer occurrence. Recently, we revealed the potential of silver nanoparticles (AgNPs; ≤ 50 nm) against UVB radiation-induced DNA damage in human keratinocytes (HaCaT). Here we performed preclinical evaluation of chemopreventive efficacy of AgNPs against UVB-induced skin tumorigenesis in SKH-1 hairless mouse model. The different concentrations (20 and 40 mg/kg) of AgNPs mixed with hydrophilic cream base were uniformly applied topically onto the dorsal area of mouse skin prior to UVB-irradiation to assess chemopreventive efficacy for several weeks. The UVB treatment group showed tumor formation within 13 weeks, and had high incidence (93.3%) rate by the end of the study (29 weeks). Interestingly, the pretreatment of mice with AgNPs significantly increased the latency period (6-9 weeks) of UVB-induced tumor formation. Moreover, overall tumor incidence was found to be significantly decreased (50 and 78%; in 20 and 40 mg/kg respectively) as compared to AgNPs-untreated mice. Tumor multiplicity and average tumor volume/tumor-bearing mouse were also observed to be significantly reduced in AgNPs-treated mice. Additionally, AgNPs treatment alone for 29 weeks did not induce any apparent signs of toxicity and changes in the body weight suggesting the safety of AgNPs. Altogether, these findings suggest that AgNPs are potential chemopreventive agents against UVB radiation-induced skin carcinogenesis and thus opening the ways for human applications.

#5239

Preventive effects of pentoxifylline on hepatic tumorigenesis in a novel mouse model of NASH-related liver cancer.

Yohei Shirakami, Akinori Maruta, Koki Obara, Takayasu Ideta, Tsuneyuki Miyazaki, Hiroyasu Sakai, Takahiro Kochi, Masaya Kubota, Takuji Tanaka, Masahito Shimizu, Mitsuru Seishima. _Gifu University Graduate School of Medicine, Gifu, Japan_.

Overweight and obesity are becoming more prevalent worldwide. As the hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have received attention as major causes of liver disorder. In addition, hepatocellular carcinoma (HCC) associated with NASH becomes an important issue. Although no standard medicinal treatment for NASH is established, pentoxifylline (PTX), a medicine used to improve circulation, is reported to ameliorate histopathological appearance of NASH and is thought to be a candidate therapeutic agent for NASH. In the present study, we investigated effects of PTX on NAFLD/NASH and the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in monosodium glutamate (MSG)-treated mice, a novel mouse model of NASH-related hepatocarcinogenesis, and db/db obese and diabetic mice. Male MSG and db/db mice were administered DEN, and then they received drinking water containing PTX (100 mg/kg/day) throughout the experiment. At sacrifice, drinking water with PTX significantly inhibited the development of hepatic pre-neoplastic lesions compared with respective control groups. MSG mice were thought to be susceptible to liver tumorigenesis. Hepatic triglyceride contents in db/db mice were decreased by PTX administration. The serum levels of total cholesterol, triglyceride, free fatty acid, and alanine aminotransferase were all decreased by PTX treatment, as was the mRNA expression of pro-inflammatory cytokines (TNF-alpha and IL-1beta) the macrophage-inducing chemokine CCL2/MCP-1, and several lipogenic genes (FASN and SREBP1c) in the liver. In vitro studies also revealed that PTX treatment decreased the expression of several genes associated with de novo lipogenesis and CCL2/MCP-1 in hepatoma and hepatic stellate cell lines, respectively. These findings suggest that PTX prevents NAFLD/NASH-related liver tumorigenesis by attenuating chronic hepatic inflammation and decreasing lipogenic gene expression in the liver. PTX might be a promising and efficient agent for treatment of NAFLD/NASH patients, who have a higher risk for developing liver cancer.

#5240

Lacking hepatic retinoid storage in mice suppresses NASH-related hepatocarcinogenesis.

Takayasu Ideta,1 Yohei Shirakami,2 Tsuneyuki Miyazaki,1 Hiroyasu Sakai,1 Takuji Tanaka,3 William S. Blaner,4 Masahito Shimizu1. 1 _Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu, Japan;_ 2 _Informative Clinical Medicine, Gifu University Graduate School of Medicine, Gifu, Japan;_ 3 _Department of Pathological Diagnosis, Gifu Municipal Hospital, Gifu, Japan;_ 4 _Department of Medicine Columbia University, New York, NY_.

Non-alcoholic fatty liver disease (NAFLD), which is strongly associated with metabolic syndrome, has become one of the most common causes of chronic liver disease in developed countries. Some patients with NAFLD develop a more serious form of the disease, non-alcoholic steatohepatitis (NASH), and NASH can develop cirrhosis or even hepatocellular carcinoma (HCC). Although hepatic retinoid (vitamin A) stores are progressively lost during the development of liver diseases, how this affects NAFLD/NASH and NASH-related hepatocarcinogenesis is unknown. In order to investigate this, we used streptozotocin (STZ) and high-fat diet (HFD) to induce NASH and related hepatic tumorigenesis in matched wild-type and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid in the liver. LRAT is the sole enzyme responsible for hepatic retinyl ester synthesis since LRAT KO mice completely lack lipid droplets in hepatic stellate cells. Mice were injected with STZ within 48 hours after birth and then fed HFD from 4 to 16 weeks of age. At the termination of the experiment, liver tumors were observed macroscopically and microscopically in both groups. In LRAT KO mice, the development of hepatic adenoma and HCC was significantly suppressed compared to control group. LRAT KO mice showed decreased expression levels of cyclin D1 and TGF-beta mRNA in the liver. The serum levels of d-ROMs and BAP were measured and the d-ROM/BAP ratio, which indicates oxidative stress, markedly decreased in LRAT KO mice. The fibrosis in the liver of the LRAT KO mice was decreased. In addition, liver fibrosis in LRAT KO mice was suppressed. These findings indicate that LRAT KO mice are less susceptible to STZ and HFD-induced liver tumorigenesis due to regulation of cell cycle and attenuation of oxidative stress.

The amount of hepatic retinoid may affect the progression of NASH and the development of NASH-related HCC.

#5241

Nitric oxide-releasing naproxen prevents muscle invasive bladder cancer.

Venkateshwar Madka,1 Altaf Mohammed,1 Qian Li,1 Yuting Zhang,1 Stanley Lightfoot,1 Xue-Ru Wu,2 Vernon Steele,3 Levy Kopelovich,4 Chinthalapally V. Rao1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _NYU Langone Medical Center, New York, NY;_ 3 _National Cancer Institute, Bethesda, MD;_ 4 _Weill Cornell Medical College, Cornell University, New York, NY_.

Bladder cancer is the second most common and a leading cause of death among genitourinary cancers worldwide. Particularly, untreated muscle invasive bladder cancer has high mortality (>85 % patients) leading to death within 2 years of diagnosis. Preventing this deadly form is highly imperative since the currently available options to patients with invasive disease remained essentially unchanged and no effective drugs have been approved in past two decades. Several non-steroidal anti-inflammatory agents (NSAIDs) have shown promising chemopreventive activity in many cancers. The common adverse events with NSAIDs, especially gastrointestinal (GI) morbidities, including complications in both upper and lower GI tract drives the need for development of safer agents. To overcome this various nitric oxide (NO)-linked NSAIDs have been synthesized. Here we investigated the NO-releasing Naproxen (NO-Naproxen) with proven anti-inflammatory and GI protecting effects for its efficacy in preventing bladder cancer. Transgenic mouse model (UPII-SV40T; n=30/group) that develop muscle invasive urothelial tumors were generated, genotyped and fed modified AIN-76A diet containing NO-naproxen (0, 300 and 600 ppm) starting at 6 weeks of age. At 40 weeks age, control (0 ppm) and experimental diet (300 and 600 ppm) fed mice were euthanized and urinary bladders were analyzed. Control diet fed male and female transgenic mice developed high grade, invasive transitional cell carcinoma (TCC) of bladder resulting in significant increase in bladder weights (140.2±9.8 mg; p<0.0001 and 34.2+0.8 mg; p<0.0001 respectively) compared with wild type mice (27.3±0.8 mg and 14.8±0.53 mg). These tumors had a significant disregulation of proliferation, cell cycle markers and antioxidant enzymes similar to human tumors. NO-Naproxen administered mice had normal body weight gain; and gross tissue analysis and showed no signs of overt toxicities. Treatment of transgenic mice with NO-Naproxen led to significant suppression of bladder weight in both genders (up to 58% in males, p<0.0001; up to 21% in females, p<0.005) compared to control group. While there was no dose-dependent increase in tumor inhibition, mice on NO-Naproxen diet had developed significantly less muscle invasive tumors suggesting inhibitory effect of treatment on disease progression. Urothelial tumor progression to invasive TCC was inhibited in both male (up to 54%; p<0.005) and females mice (up to 85%; p<0.0001) of the experimental diet groups. Molecular analysis of urothelial tumors via real-time PCR, IHC and/or western blotting showed inhibitory effect of NO-Naproxen on proliferation and inflammatory markers (PCNA, Cyclins, COX2, and IL1b) and showed modulation of antioxidant enzymes (CAT, GPx, GST, NQO1, and SOD3). Our results suggest that NO-Naproxen may be a promising agent for preventing urothelial TCC and warrants further investigation. (Supported in part by NCI-CN53300)

#5242

Differential, dose-dependent, effects of curcumin/analogs, on long (L) versus short (S) isoforms of a cancer stem cell marker, DCLK1, in colon and pancreatic cancer cells: biological impact.

Shubhashish Sarkar, Malaney O. Connell, Pomila Singh. _UT Medical Branch, Galveston, TX_.

Curcumin, a dietary agent, has potent anti-inflammatory/anti-cancer activities, but is much less bioavailable, unlike some of its more potent synthetic analogs, such as EF24. In recent years, inhibitory effects of curcumin/analogs on DNMT1 have been described, resulting in de-methylation and re-expression of several genes, including tumor suppressor genes. Double-Cortin Like Kinase 1 (DCLK1) marks colon cancer stem cells, and is required for colon-carcinogenesis in mice. We recently reported a critical role of DCLK1 for maintaining tumorogenic/metastatic potential of human colon cancer (CRC) cells (Kantara et al, Can Res 2014). More recently we reported that the 5'α promoter of hDCLK1-gene is epigenetically silenced in CRCs due to hypermethylation, while a novel short (DCLK1-S) isoform is transcriptionally up-regulated in response to oncogenic pathways, including NFḱB, from an alternate β promoter, located in intron V (O'Connell and Sarkar et al, Sci. Rep 2015). Since curcumin potently inhibits NFḱB pathway, and can potentially de-methylate epigenetically silenced promoters, we examined possible re-expression/loss of expression of L/S isoforms, respectively, in colon/pancreatic cancer cells, in vitro and in vivo, in response to curcumin/EF24, using several molecular approaches and bioassays. Surprisingly, both agents had dose-dependent effects on demethylation. Low concentrations of curcumin (10μM) and EF24 (0.5μM) inhibited DNMT1, resulting in re-expression of DCLK1-L in both colon/pancreatic cancer cells in vitro. However, higher concentrations of curcumin (>20μM) and EF24 (>1.0μM) caused autophagic death of the cells, and re-expression of DCLK1-L was not measured. NFκB activation and DCLK1-S expression, on the other hand, were increasingly decreased in all cell lines in a dose-dependent manner, in response to curcumin/EF24. Low doses were equally effective against DNMT1/NFκB in vivo, resulting in re-expression of DCLK1-L and loss of DCLk1-S, in xenografts of colon/pancreatic cancer cells. However, the high doses used in this study continued to be effective against the two enzymes, suggesting that the high dose used was not enough for causing autophagic death in vivo, and may need to be increased to replicate cell-death effects observed in vitro. Even though DCLK1-L was re-expressed in the xenografts at both does, in vivo, the size and weight of the tumors was reduced in a dose-dependent manner, mimicking the loss of expression of DCLKL1-S. Our results thus show, for the first time, a dose-dependent epigenetic effect of curcumin/EF24 on cancer cells, which could significantly impact treatment decisions. Low to moderate doses of curcumin/EF24 may allow re-expression of normal/silenced genes, such as DCLK1-L, while reducing growth promoting pathways (such as NFκB/DCLK1-S).

#5243

STAT3 inhibition as chemoprevention in a chemically induced mouse model of HNSCC.

Noah D. Peyser,1 Lin Wang,2 Marie Acquafondata,2 Maria Freilino,2 Hua Li,1 Yan Zeng,1 Malabika Sen,2 William E. Gooding,2 Daniel E. Johnson,2 Jennifer R. Grandis1. 1 _University of California San Francisco, San Francisco, CA;_ 2 _University of Pittsburgh, Pittsburgh, PA_.

Background: Head and neck squamous cell carcinoma (HNSCC) is frequently fatal due to carcinogen-induced field cancerization, leading to second primary tumor formation in ~4% of patients per year. Effective chemoprevention could improve survival. Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor and rational therapeutic target in HNSCC. We recently reported that loss of function of PTPRT (protein tyrosine phosphatase receptor type T), a direct STAT3 phosphatase, by frequent somatic mutation or promoter methylation leads to hyper-phosphorylation of STAT3 and may predict sensitivity to STAT3 inhibition using Stattic. In the present study, we sought to evaluate the contribution of PTPRT to chemical carcinogenesis and sensitivity to Stattic-mediated HNSCC chemoprevention in the 4-NQO (4-nitroquinoline 1-oxide) model in vivo using PTPRT wild-type (WT) or knockout (KO) mice.

Methods: PTPRT WT or KO mice received 4-NQO-containing water (100 µg/mL) for 12 weeks. At experiment initiation, a subset of mice received vehicle (PBS) or 50 mg/kg Stattic by oral gavage Q5D. After 12 weeks of 4-NQO administration, mice were randomized to receive vehicle/Stattic for 12 additional weeks. Mice were then sacrificed followed by tongue excision for H&E and IHC analysis. Histologic assessment of dysplasia was scored from 0 to 6 (normal to invasive SCC).

Results: By week 3, we observed unexpected combined toxicity of 4-NQO and Stattic, with 3 mouse deaths. Combined treatment was suspended until the end of the 12-week 4-NQO administration period, and no further toxicity was noted. At the end of the 24-week experiment, we observed a diverse range of neoplasias in the control group, with 7/13 (53.8%) exhibiting SCC of which 4 (30.8%) were invasive. Using a rank transformation statistic, we detected no significant effect of PTPRT status (P = 0.9985) or interaction between PTPRT status and treatment (P = 1.0) with respect to pathology scores. Further analysis revealed a trend toward effective Stattic-mediated chemoprevention (P = 0.0831). Interestingly, the only two mice with normal-appearing oral epithelia were both PTPRT KO and treated with Stattic.

Conclusions: PTPRT status does not appear to predict susceptibility to 4-NQO-mediated carcinogenesis, nor sensitivity to Stattic-mediated chemoprevention in this mouse model of HNSCC initiation. Nevertheless, we observed a trend toward effective chemoprevention in Stattic-treated mice supporting a role for STAT3 in HNSCC carcinogenesis.

#5244

Effects of chronic low dose aspirin treatment in the mouse AOM/DSS model of colon carcinogenesis.

Nadine Rohwer,1 Anja Kuehl,1 Dieter Zopf,2 Fiona M. McDonald,2 Karsten-Heinrich Weylandt1. 1 _Charité-Universitaetsmedizin Berlin, Berlin, Germany;_ 2 _Bayer Pharma AG, Berlin, Germany_.

Although early detection and treatment of colorectal cancer (CRC) have improved in recent years, it remains a significant healthcare problem with high morbidity and mortality. Published data indicate that long-term intake of low dose aspirin reduces the risk of CRC, however the molecular mechanisms underlying this effect are still unclear. The aim of this study was to investigate the efficacy of low dose equivalent aspirin treatment in a mouse colon carcinogenesis model, including evaluation of potential mechanistic contributors.

An initial PK/PD study in healthy mice indicated that aspirin 25 mg/kg/day given via drinking water had a similar PD effect - reduction in plasma thromboxane B2 (TXB2), as an indicator of cyclooxygenase-1 (COX-1) inhibition - as did 5-day low-dose aspirin treatment (100 mg/day) in healthy human volunteers.

Male and female C57BL/6J mice were treated with the carcinogen azoxymethane (AOM) 10 mg/kg once i.p. combined with three 5-day cycles (at 14 day intervals) of dextran sodium sulphate (DSS) 2 % in drinking water. Aspirin treatment was started concomitant with AOM administration and continued for 12 weeks, at which time the data outlined below were obtained.

Aspirin 25 mg/kg/day significantly reduced tumor burden in 3 independent experiments (total tumor number reduced by 43 - 51 %; tumor area per mouse reduced by 59 - 65 %; mean tumor area reduced by 22 - 27 %). Increasing the dose to 50 mg/kg/day did not show better inhibition of tumorigenesis.

Aspirin-induced reduction in tumorigenesis in this model was accompanied by reduction in systemic plasma TXB2 by about 70 %, indicating reduced platelet activation. TXB2 production by colon explants was also reduced, by 30-50 %. Aspirin treatment reduced inflammatory activity (fecal blood; IL-1β and IL-6 production by colon explant cultures; iNOS-positive tumor macrophage infiltration). Immunohistological analysis of tumor tissue showed no reduction in proliferation (BrdU staining) by aspirin but there was a trend towards increased apoptosis (caspase-3 positive cells) and a significant reduction in CD31-positive microvessel density. Aspirin treatment did not result in significant modulation of the Wnt/β-catenin, NF-κB or HIF-1 pathways as assessed by mRNA expression of several of their target genes in tumor tissue.

The results show that aspirin can inhibit tumor development in this established model of colon carcinogenesis, primarily by reducing tumor number with a less marked effect on the size of individual tumors. This effect is associated with COX-1 inhibition of a similar magnitude to that seen with low dose aspirin in humans. The influence of aspirin on intestinal inflammation and platelet activation as well as on tumor cell apoptosis and tumor angiogenesis might all contribute to the beneficial effects seen in this model. Additional studies are planned to further elucidate aspirin's mechanism of action in inhibiting tumor development.

#5245

Sulindac decreases basal-like mammary tumor burden and proinflammatory mediators in obese mice.

Subreen A. Khatib,1 Emily L. Rossi,1 Laura W. Bowers,1 Andrew J. Dannenberg,2 Stephen D. Hursting1. 1 _University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _Weill Cornell Medical College, New York, NY_.

Background: A hallmark of the metabolic dysregulation associated with obesity is a pro-inflammatory environment perpetuated by pro-inflammatory mediators including adipokines and growth factor signaling. We previously showed that inflammation and basal-like breast cancer (BLBC) growth are increased in chronically obese mice and persist following weight normalization.

Purpose: We tested the hypothesis that Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), can reduce chronic obesity-related inflammation and subsequent BLBC growth.

Methods: Mice were administered a control diet (10 kcal % fat) or diet-induced obesity regimen (DIO, 60 kcal % fat). After 15 weeks on diet, DIO mice either continued on DIO diet or were switched to the low fat control diet to induce gradual weight loss, resulting in Formerly Obese (FOb) mice. Half of the mice in all three groups (Control, FOb, DIO, n=17/group) were randomized to receive Sulindac supplementation at 160 ppm in the diet, which remained constant across diets. Twelve weeks after initiating Sulindac supplementation and weight loss in the FOb groups, all mice were orthopically injected with E0771 cells, a model of basal-like breast cancer. Five mice/group were killed, and their tissue collected and stored at a 4-week intermediate time-point after injection, while 12 mice/group continued in a survival study; these mice were killed when tumor size reached 1.2 cm in diameter in any direction.

Results: Sulindac supplementation in DIO mice significantly reduced serum insulin and leptin to levels statistically equivalent to both control and FOb mice. Interestingly, body weight, body fat percentage, and ex vivo visceral white adipose weight were unchanged between all supplemented and non-supplemented counterparts (i.e. DIO vs. DIO+Sulindac). At the 4-week intermediate time point, Sulindac supplementation in DIO mice (but not in control or FOb mice) significantly reduced mean tumor weight relative to their non-supplemented counterparts. In the survival arm of the study, Sulindac significantly increased tumor latency in DIO and FOb groups (but not controls) in comparison to their non-supplemented counterparts.

Conclusions: Sulindac supplementation significantly reduced pro-inflammatory mediators insulin and leptin in DIO mice, and increased tumor latency in DIO and FOb mice. Furthermore, Sulindac supplementation did not modulate body weight or fat depots, suggesting that Sulindac indeed offsets some of the pro-tumorigeneic effects of obesity rather than targeting obesity directly. Ongoing analyses of inflammatory surrogates, including circulating cytokines and prostaglandins, mammary gland crown-like structures and cyclooxygenase-2 levels, will help to determine if Sulindac's effects are mediated through its anti-inflammatory activity.

#5246

Pharmacokinetic evaluation of novel nano-formulations of aspirin, curcumin and sulforaphane for pancreatic cancer chemoprevention.

Arvind Thakkar, Sushma Chenreddy, Zhijun Wang, Jalpa Modi, Jeffrey Wang, Sunil Prabhu. _Western University of Health Sciences, Pomona, CA_.

The objective of this study was to evaluate the pharmacokinetics of orally administered chitosan - coated solid lipid nanoparticles (c-SLN) formulations encapsulating aspirin (ASP) and curcumin (CUR) with free-sulforaphane (SFN) (ACS), in rats. Twenty five rats were divided into 5 groups at random (n = 5), and received three different dosing levels of ACS c-SLN formulations by oral gavage (low: 2 ASP+4.5 CUR+0.16 SFN mg/kg; medium: 20 ASP+45 CUR+1.6 SFN mg/kg; high: 60 ASP+ 135 CUR+ 4.8 SFN mg/kg), and a high dose of unmodified ACS regimen. Blood samples were drawn at 0, 0.5, 1, 2, 6, 24, and 48 h. The concentrations of ASP, CUR and SFN and their major metabolites such as salicylic acid (SA) and tetrahydrocurcumin (THC) in plasma were measured using an LC/MS/MS method. Briefly, the analytes in rat plasma were extracted by a protein precipitation method using acetonitrile containing 0.1% formic acid. SA and ASP were detected in negative ion mode with m/z transition of 136.9 to 93.0 and 179.0 to 137.2 respectively, while CUR, THC, and SFN were detected in positive ion mode with m/z transition of 369.1 to 177.1, 373.1 to 137.2, and 178.1 to 91.1, respectively. The chromatographic separation was carried out using a mobile phase of acetonitrile and formic acid (0.1% v/v) containing 2 mM amino acetate with a flow rate of 0.3 ml/min. The analyses were linear over 0.005 to 2.5, 0.05 to 25, 0.01 to 5, 0.01 to 5, and 0.002 to 1 μg/ml for SA, ASP, CUR, THC, and SFN, respectively. Preliminary results indicated that there was an 18% increase in the area under curve (AUC) of SA (85 mg.hr/L), ASP (3.4 mg.hr/L), and SFN (2.08 mg.hr/L) of c-SLN formulation when compared to the SA (70 mg.hr/L) and ASP (2.8 mg.hr/L) from unmodified high dose ACS combination. No substantial plasma concentrations of CUR and THC were detected due to the extensive metabolism during absorption. The Cmax of SA (11.0 μg/ml), ASP (0.2 μg/ml), and SFN (0.39 μg/ml) were significantly higher indicating the ability of enhancement of drug absorption by the nano-formulation, while the tmax values of SA (2.54 h), ASP (4.5 h), and SFN (1.5h) were longer compared to unmodified SA (1.25 h) and ASP (2.25 h), demonstrating a sustained release of the drug. The elimination t1/2 values were increased in the c-SLN formulation which suggested a slow release of free compounds in the systemic circulation. The results demonstrate that ACS c-SLN could increase drug exposure following oral administration by enhancing the absorption and sustained release of the drugs. Data obtained from this study provides insights to the clinical development of ACS regimen for pancreatic cancer chemoprevention.

#5247

Caffeic acid phenethyl ester (CAPE) reverses aggressive breast cancer in the radiation chimera model.

Coral O. Omene,1 Manan Patel,2 Irineu IllaBochaca,2 Mary Helen Barcellos-Hoff3. 1 _Dept. of Medicine, New York University Langone Medical Center, New York, NY;_ 2 _Dept. of Radiation Oncology, New York University Langone Medical Center, New York, NY;_ 3 _Dept. of Radiation Oncology, New York University Langone Medical Center, University of California, San Francisco, New York, NY_.

CAPE is the major active component of propolis, a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown diverse effects of CAPE in breast cancer. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC) and evaluated this in a radiation chimera mouse model in which the tumor spectrum is shifted to TNBC.

The radiation chimera model consists of surgically clearing the mammary epithelium from the inguinal glands of 3-week old BALB/c mice, the mice were irradiated with 1 Gy or sham irradiated at 10-12 weeks of age, and bilaterally transplanted 3 days later with syngeneic Trp53 null mammary fragments. Mice were placed on a CAPE diet or a control diet at 1 month post transplantation that was maintained for the course of the experiment. Mammary tumor development was monitored by palpation for up to 18 months. The first tumor was resected at 1cm3 and mice monitored for tumor recurrence or second tumor formation. Tumors were immunostained for estrogen receptor (ER) status and ER negative tumors were selected from all the groups for RNA sequencing.

No difference in body weight or tumor incidence was observed between mice on the CAPE versus control diet. Host irradiation significantly accelerated tumor growth rate compared to the control sham irradiated mice. CAPE treatment blocked this effect, but did not affect the control tumor growth rate. Resected tumors in CAPE treated mice recurred at significantly longer intervals (average of 40 days in the control group versus 90 days with CAPE treatment) and much less frequently than tumors from mice on a control diet. Mean expression analysis showed that CAPE induces a distinct gene expression pattern in ER negative tumors from irradiated mice. As previously described, the transcriptional profile of ER negative tumors was enriched in immune response genes in tumors from irradiated mice on a control diet. Interestingly, this difference was abrogated in mice on the CAPE diet.

These findings support the potential use of CAPE to modify the aggressive behavior of TNBC. This may be due to effects on the immune system in which CAPE acts to re-establish anti tumor immunity. Thus, CAPE may be useful in chemoprevention both for women at high risk for TNBC and to prevent or delay TNBC breast cancer recurrence.

#5248

**ED-71, an analogue of Vitamin D** 3 **, blocks the promotion but not the initiation of colorectal tumors in the Apc** +/Min-FCCC **mouse model.**

Wen-Chi L. Chang,1 Harry S. Cooper,1 Esther Kaunga,1 Lisa Vanderveer,1 Jing Peng,1 Suen S. Chen,2 Margie L. Clapper1. 1 _Fox Chase Cancer Center, Philadelphia, PA;_ 2 _National Cancer Institute, Bethesda, MD_.

Results from several studies demonstrate an inverse relationship between circulating levels of Vitamin D and risk of colorectal cancer. Eldecalcitol (1α,25-dihydroxy-2β-(3-hydroxypropyloxy) Vitamin D3; ED-71) is a novel analog of calcitriol, the most active form of Vitamin D3. ED-71 is more potent in stimulating bone remodeling and has been approved in Japan for the treatment of osteoporosis. The goal of this study was to assess the ability of ED-71 to inhibit spontaneous colorectal adenomas in a unique strain of multiple intestinal neoplasia (Apc+/Min-FCCC) mice. Male mice (6 wks of age) were randomized to treatment groups based on colon tumor status (endoscopic confirmation as tumor-free or -bearing) and body weight (bw) and administered: vehicle (MCT), calcitriol (0.25 µg/kg bw), or ED-71 (0.05 or 0.1 µg/kg bw) by gavage. Mice were treated every other day and bws were recorded weekly. After 14 wks of treatment, the small intestines and colons were excised and examined for gross tumors. Colorectal tumors >3 mm in diameter were frozen for gene expression analyses. The remaining tissue was fixed in formalin and processed for histological review. The multiplicity of gross small intestinal tumors in animals treated with ED-71 (both doses) was comparable to that of vehicle-treated controls. In contrast, the multiplicity of small intestinal tumors was elevated 34.6% over that of controls in animals treated with calcitriol (Mean ± SEM: 28.4 ± 2.6 vs. 21.1 ± 2.8, respectively; P = 0.034). The ability of ED-71 to inhibit colon tumorigenesis was evaluated independently in mice with vs. without tumors at treatment initiation. In tumor-bearing mice, neither calcitriol nor ED-71 (both doses) had any significant effect on the multiplicity of colorectal tumors as compared to control mice. However in tumor-free mice, ED-71 (0.1 µg/kg bw) reduced the mean incidence of adenomas (> 4 crypts) by 47% (ED-71 - 45.5%, controls 92.3%; P = 0.02) and the multiplicity by 47.6% (Mean ± SEM: ED-71 - 1.1 ± 0.39, controls - 2.1 ± 0.72; P > 0.05). In contrast, the multiplicity of microadenomas (≤ 4 crypts) was increased in mice treated with ED-71 (0.1 µg/kg bw) as compared to controls (0.9 ± 0.22 vs. 0.5 ± 0.25 P = 0.06, respectively). These findings suggest that ED-71 is effective in preventing the transition of microadenomas to mature adenomas. The lack of an effect of ED-71 on colon tumor development in mice bearing tumors at baseline could be due in part to loss of the Vitamin D receptor in colon adenomas, as confirmed by real-time PCR. Insight into the mechanism by which ED-71 inhibits adenoma development is being gained from analyses of Vitamin D signaling and microRNA expression in treated colonic epithelial cells. These promising data provide support for future studies to determine the potential utility of ED-71 in preventing colorectal cancer in high-risk patients found to be tumor-free during surveillance endoscopy. (Supported by NCI HHSN261201200015I)

#5249

Crocin prevents early lesions of liver cancer:system biology approach.

Amr Amin,1 Alaaeldin A. Hamza,2 Sayel Daoud,3 Kamal Khazanehdari,4 Ala'a Al Hrout,1 Badriya Baig,1 Amphun Chaiboonchoe,5 Thomas E. Adrian,1 Nazar Zaki,1 Kourosh Salehi-Ashtiani5. 1 _UAE Univ., Al-Ain, United Arab Emirates;_ 2 _National Organization of Drug Control and Research, Giza, Egypt;_ 3 _Tawam Hospital, Al-Ain, United Arab Emirates;_ 4 _Molecular Biology and Genetics Laboratory, Dubai, United Arab Emirates;_ 5 _New York University-Abu Dhabi, Abu Dhabi, United Arab Emirates_.

The angiogenesis inhibitor, sorafenib, remains the only available therapy of the poorly diagnosed hepatocellular carcinoma (HCC). Only recently patents of VEGF receptors-3 inhibitors are developed. ; hence,Thus, a novel approach against HCC is essential for a better therapeutic outcome. Saffron and its active constituents were reported to have anti-tumor properties. Objective: The aims of this study were to examine the chemopreventive action of saffron's main biomolecule, crocin, against chemically-induced liver cancer in rats, and to explore the mechanisms by which crocin employs its anti-tumor effects. Method: We investigated the anti-cancer effect of crocin on an experimental carcinogenesis model of liver cancer by studying the anti-oxidant, anti-inflammatory, anti-proliferation, pro-apoptotic activities of crocin in vivo. In addition, we provided a network analysis of differentially expressed genes in tissues of animals pre-treated with crocin in comparison to induced-HCC animals' tissues. To further support our results, in vitro analysis was carried out. We assessed the effects of crocin on HepG2 cells viability by treating them with various concentrations of crocin; in addition, effects of crocin on cell cycle distribution of HepG2 cells were investigated. Results: Findings reported herein demonstrated the anti-proliferative and pro-apoptotic properties of crocin when administrated in induced-HCC model. Crocin exhibited anti-inflammatory properties where NF-kB, among other inflammatory markers, was inhibited. In vitro analysis confirmed crocin's effect in HepG2 by arresting the cell cycle at G2/M phase, inducing apoptosis and down regulating inflammation. Network analysis identified NF-kB as a regulatory hub, and therefore, a candidate therapeutic drug target. Conclusion: Taken together, our findings introduce crocin as a candidate chemopreventive agent against HCC.

#5250

Photopreventive effect and mechanism of AZD4547 and curcumin C3 complex on UVB-induced epidermal hyperplasia.

Alok R. Khandelwal, Rong Xiaohua, Tara Moore-Medlin, Oleksandr Ekshyyan, Abreo Fleurette, Xin Gu, Cherie-Ann O. Nathan. _Louisiana State University-Health Shreveport, Shreveoport, LA_.

Aggressive cutaneous squamous cell carcinoma (cSCC) of the skin is the second most common type of skin cancer in the USA due to high exposure to ultraviolet-B (UVB) radiation. In our previous studies Curcumin C3 complex (C3), a standardized preparation of three curcumonoids, delayed UVB-induced tumor incidence and inhibited multiplicity. Exposure to UVB activates mTOR and FGFR signaling both of which play a key role in skin tumorigenesis. The purpose of this study was to investigate the efficacy of C3 complex to afford protection against UVB-induced hyper proliferation by targeting the mTOR and FGFR signaling pathways. Pre-treatment with C3 complex significantly inhibited UVB-induced FGF-2 induction, FGF-2-induced colony formation, mTORC1 and mTORC2 activation and FGFR2 phosphorylation in promotion sensitive JB6 epithelial cells. Further, FGFR2 was required for UVB-induced mTOR activation suggesting an important role of FGFR2 in UVB-induced mTOR signaling. SKH-1 mice pre-treated with C3 (15mg/kg/b.w) for 2 weeks followed by a single exposure to UVB (50mj/cm2) significantly attenuated UVB-induced mTOR and FGFR activation. To further assess the role of FGFR in UVB-induced hyper proliferation, SKH-1 mice were pre-treated with AZD4547; a selective pan-FGFR kinase inhibitor followed by single exposure to UVB (50mj/cm2). AZD4547 significantly inhibited UVB-induced mTORC1 and mTORC2 activation, epidermal hyperplasia and hyper proliferation. Our studies underscore the importance of FGFR signaling in UVB-induced acute skin changes and the role of FGFR/mTOR pathway in mediating the effects of C3 complex in the pathogenesis of skin cancer.

#5251

Chemoprevention of cadmium induced prostate carcinogenesis.

Suman Suman, Trinath P. Das, Houda Alatassi, Murali K. Ankem, Chendil Damodaran. _University of Louisville, Louisville, KY_.

Cadmium is known carcinogen that causes damages to multiple organs. Cadmium acts as an agonist and activates androgen receptor (AR) in prostate epithelial cells which in turn promotes the carcinogenesis of prostate. Studies have indicated that occupational exposure of cadmium is a risk factor for prostate cancer and hence there is an immediate need to prevent cadmium induced prostate cancer in occupational workers. Various reports have shown that inhibition of AR signaling by AR inhibitors suppressed cadmium induced prostate carcinogenesis. So, the present study focuses to identify dietary agent which can effectively prevent cadmium induced prostate cancer and to dissect the mechanism of action on malignant transformed cells. We have disclosed one such compound, 6-(3, 9-dihydroxy-2-prenylcoumestan (Psoralidin) (Pso) a non-toxic, orally bioavailable compound, which effectively targets only prostate cancer cells and innocuous to healthy cells. Our results have shown cadmium transformed prostate epithelial cells (CTPE) exhibits high proliferative and high colony forming ability in soft agar assays. However, treatment of CTPE cells with Pso resulted in growth inhibition, without causing toxicity to normal prostate epithelial cells (RWPE-1). Finally, in vivo experiments showed that oral administration of Pso (20 mg/kg/BW) effectively suppresses the growth in CTPE xenografts, at physiologically achievable doses without any toxicity. The pathological findings suggest that CTPE tumors exhibits epithelial and mesenchymal transition (EMT) phenotype and infiltrating margins into the surrounding muscle and adipose tissue. However, Psoralidin treated tumors showed a decrease in tumor size with a significant central necrosis (up to 60% of the tumor size). More specifically, margins of Psoralidin treated tumors are less infiltrative and becoming more well circumscribed. We have also noticed that the mesenchymal component is not present and the only viable tumor is the epithelial one after Pso treatment. Altogether, our results confirm that consumption of natural compounds such as Psoralidin may play an important role in the chemoprevention for cadmium induced prostate carcinogenesis.

#5252

Skin cancer prevention by traditional Chinese medicinal formula Si-Wu-Tang and its constituents.

Kevin Huang,1 Mandy Liu,1 Suhui Zhang,2 Steven Yeung,1 Andy Chang,1 Li Qian,1 Payal Chatterjee,1 Rui Li,2 Su Zhou,2 Nan Mei,3 Zhijun Wang,1 Cyrus Parsa,1 Robert Orlando,1 Yun Luo,1 Ying Huang1. 1 _Western University of Health Sciences, Pomona, CA;_ 2 _Shanghai Institute for Food and Drug Control, China;_ 3 _National Center for Toxicological Research, Jefferson, AR_.

Si-Wu-Tang (SWT), comprising the combination of four herbs, Rehmanniae, Angelica, Chuanxiong and Paeoniae, is one of the most popular Chinese medicines for women's diseases. Previously we showed that SWT was able to upregulate genes in the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, suggesting a potential application for cancer chemoprevention. The present study examined the chemopreventive activity of SWT using models of skin carcinogenesis. In JB6 P+ cells, a non-cancerous murine epidermal cell line for studying skin tumor promotion, SWT inhibited epidermal growth factor (EGF)-induced neoplastic transformation. In a 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine skin tumorigenesis model, both topical and oral treatment of SWT inhibited epidermal hyperplasia, proliferating cell nuclear antigen expression, and H-ras mutations induced by DMBA treatment. In addition, SWT exhibited a significant antimutagenic activity against DMBA-induced mutagenicity, determined by the Ames Test using Salmonella typhimurium TA100 in the presence of metabolic activator S9 system. To identify active components in SWT, among nine compounds previously reported in commercial SWT products, in silico molecular docking analysis predicted some as potential Nrf2 activators due to an ability of interfering the forming of Nrf2-Keap1 complex. Three of these compounds, gallic acid, Z-liguistilide and senkyunolide A, were confirmed with highest potency of increasing the antioxidant response element luciferase reporter activity, inducing Nrf2-regulated genes Hmox1, Slc7A11 and Nqo1, and inhibiting EGF-induced JB6 P+ transformation. Further mechanistic studies showed that SWT and the three compounds suppressed EGF-induced activation of the activator protein 1 (AP-1), an essential transcription factor involved in skin carcinogenesis. The antimutagenic activity for the three compounds was also confirmed with the Ames Test. In conclusion, these results provide evidence that SWT and its constituents are able to prevent skin cancer, at least partly, by activating the Nrf2 pathway and blocking the activation of AP-1. Thus, this widely used Chinese medicinal formula may provide a promising option toward preventing skin cancer or may be other types of cancer.

#5253

Oral administration of Withaferin-A effectively suppresses prostate carcinogenesis in PTEN-Knockout mice.

Chendil Damodaran, Suman Suman, Trinath P. Das, Houda Alatassi, Murali K. Ankem. _University of Louisville, Louisville, KY_.

Androgen ablation therapy alone or in combination with radiation therapy is the mainstay for prostate cancer (CaP), which is initially effective in de-bulking the tumor volume, however, eventually these patients will progress to a castration-resistant prostate cancer (CRPC), which requires more aggressive chemotherapies. Treating CRPC with second generation androgen -ablation based therapies like enzalutamide and abiraterone exhibit only a short window of therapeutic benefit resulting in chemoresistance. Hence, there is an immediate need for identification of novel targets to eradicate CRPC effectively. We and others have reported targeting AKT activation could efficiently suppress the growth of CRPC cells; in fact a number of clinical trials have shown some promise that AKT could be an attractive target for CRPC. In fact, clinical studies from our lab have suggested that AKT activation predominantly occurs in Gleason stage specific manner suggesting identification of novel drugs to inhibit AKT activation is imperative. Our earlier finding suggests that Withaferin-A (WA), an herbal molecule effectively inhibits the growth of CRPC cells by downregulating AKT activation and its downstream pro-survival events. Present study investigated whether oral administration of WA may inhibit tumor development in Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4+) (Pten-KO)] which will be a unique and appropriate preclinical model to study the AKT driven prostate tumor. Oral administration of WA for 45 weeks effectively prevented the tumor growth without any significant signs of toxicity to organs in Pten-KO mice. Vehicle and WA (5mg/kg body weight) were orally given up to 45 weeks. Gross pathological studies suggested a significant inhibition of growth and micro metastasis in WA-treated mice as compared to the vehicle treated mice. On microscopic examination of the prostatic tissue, we found that the WA-treated tumors showed more necrosis than the control group and some of the tumors were more differentiated than the controls in the same group. All the organs were completely submitted for histological evaluation. None of the WA-treated mice organs showed any metastatic lesion on the other hand, we found discrete metastasis to lungs in the control tumors. Our ongoing immunohistochemistry analysis may corroborate our in vitro findings that down regulation of AKT and epithelial-to-mesenchymal transition (EMT) markers such as β-catenin, snail, and vimentin in WA treated tumors as compared to the control mice. Overall, these results provide important scientific evidence in support of AKT signaling as a target to inhibit CRPC as well as metastatic CRPC.

#5254

Andrographolide prevents prostate cancer by targeting CXCR3/CXCR7 and regulators of cell cycle.

Hina Mir,1 Neeraj Kapur,1 Rajesh Singh,1 Guru Sonpavde,2 James W. Lillard,1 Shailesh Singh1. 1 _Morehouse School of Medicine, Atlanta, GA;_ 2 _University of Alabama at Birmingham, Birmingham, AL_.

Despite state of the art cancer diagnostics and therapies offered in clinics, prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on phases of cell cycle in LNCaP, C4-2b and PC3 cell lines compared to retinoblastoma protein (RB-/-) lacking DU-145 cells. This agent blocked the G2/M transition in LNCaP, C4-2b and PC3 whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to switching of the activation states of cell cycle regulators in these cell lines. AG induced its effect mainly via cyclin A2 and B1 in LNCaP, C4-2b and PC3 cell lines; whereas, cyclin E related regulation was affected in DU-145 cells. Phosphorylation status of Wee1, a nuclear kinase belonging to the Ser/Thr family and CDC2 was also affected by AG. Intriguingly, AG affected cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggest this agent has potential to be used as a therapeutic and/or preventive modality.

#5255

Lack of chemopreventive effects of dietary P2X7R inhibitors against pancreatic cancer in p48Cre/+-LSL-KrasG12D/+ mice.

Altaf Mohammed,1 Naveena B. Janakiram,1 Venkateshwar Madka,1 Gopal Pathuri,1 Qian Li,1 Rebekah Ritchie,1 Laura Biddick,1 Hannah Kutsche,1 Yuting Zhang,1 Anil Singh,1 Hariprasad Gali,1 Stan Lightfoot,1 Vernon E. Steele,2 Chen S. Suen,2 Chinthalapally V. Rao1. 1 _University of Oklahoma Health Sciences Center, Oklahoma City, OK;_ 2 _National Cancer Institute, Bethesda, MD_.

Pancreatic cancer (PC) still remains a devastating disease that is almost uniformly lethal. Despite advances in the field of molecular genetics in human pancreatic cancers, targeted therapies has not yet translated to an improved overall survival for patients. Chronic inflammation is hallmark of many cancers, including PC. P2X7R is the most potent of the membrane receptors responsible for inflammasome activation and release of inflammatory cytokines. Sustained stimulation of P2X7R drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome in pancreatic tumor progression, we carried transcriptomic analysis of LSL-Kras pancreatic tumors by next generation sequencing. Results showed that the P2X7 receptor (~20-fold); key inflammasome components, IL-1β (~45-fold), caspase-1 (15-fold), IL-18 (~35-fold), IL-33 (~93 folds), TNF-α (~13-fold) and COX-2 (~41-fold) are highly expressed (p<0.05). Analysis of human PC cell lines, human and mouse PC tissues by western immunoblotting, real time (RT)-polymerase chain reaction (PCR), immunohistochemistry and/or immunofluorescence suggest that the P2X7R is a critical contributor to the progression of pancreatic tumor growth. Hence, to target P2X7R, we tested the effects of two P2X7R antagonists A438079 and AZ10606120 at two doses (50 and 100 ppm) on pancreatic intraepithelial neoplasms (PanINs) and their progression to PDAC in p48Cre/+-LSL-KrasG12D/+ mice. Six-week old KrasG12D/+ (24-36/group) mice were fed (AIN-76A) diets containing 0, 50 or 100 ppm of A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, inflammatory and cell cycle makers; PCNA, p21, P2X7R, NLRP, COX-2, IL-33, caspase-3, caspase-1, Cdc25c and p53 expressions by IHC, IHF, Western blotting, and/or RT-PCR methods. Results suggest that control diet fed mice showed 50% incidence of PDAC in male mice. Dietary A4338079 and AZ10606120 showed up to 60% incidence of PDAC. CT-PET imaging analysis showed enhanced tumor growth in treatment groups. Importantly, the carcinoma spread in untreated mice was 24% as compared to 43-53% in treatment groups. Also, marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug treated mice. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of PCNA, P2X7R, NLRP, and IL-33 whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show significant chemopreventive effects in suppression of PC and hence caution is needed while treating high risk individuals for PC with P2X7R inhibitors. Also, it is not clear whether dietary route of administration might be reason for lack of efficacy. {Supported by NCI-CN-N01- 25008-78}.

#5257

Upregulation of glutathione S-transferase P1 by green tea polyphenols: A novel transcriptional target of p53 tumor suppressor gene.

Vijay S. Thakur, Gauri Deb, Mark W. Jackson, Sanjay Gupta. _Case Western Reserve University, Cleveland, OH_.

Green tea polyphenols (GTPs) and its major constituent (-)-epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells. Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. The mechanisms underlying the re-expression of GSTP1 expression by GTP/EGCG in prostate cancer is currently not well understood. Earlier we demonstrated the ability of GTP/EGCG to increase p53 transcriptional activity through suppression of class I histone deacetylases (Carcinogenesis 33(2):377-84, 2012). Here we report that the GSTP1 gene is an unrecognized downstream transcriptional target of the tumor suppressor p53, and that GTP/EGCG has ability to increase GSTP1 expression mediated through p53. Treatment of human prostate cancer LNCaP cells with 5µg/ml GTP and 20µM EGCG, activate p53 through acetylation at the Lys373 and Lys382 residues along with consequent increase in GSTP1 protein expression in time-dependent manner. To study the possible interaction between GSTP1 and p53, LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV) for generation of p53 knockout cells. GTP/EGCG treatment induced p53 activation and acetylation at p53 Lys373 and Lys382 specifically in LNCaPshV cells in time-dependent manner, but not in LNCaPshp53. This increase in p53 acetylation led to activation of GSTP1 in LNCaPshV cells both at protein and message levels. GTP/EGCG-mediated increase in GSTP1 expression is caused by increase binding of p53 to its consensus binding site on the intron 4 of GSTP1 gene as analyzed by electrophoretic mobility shift binding assay. Furthermore, this association was validated by ChIP assay where GTP/EGCG treatment led to an increase in the amount of acetylated p53-Lys373 associated within the intron site of GSTP1, compared to untreated controls. Taken together, our findings indicate the ability of p53 to transcriptionally activate the human GSTP1 gene, which defines a novel mechanism of protection of the genome by green tea polyphenols.

#5258

Inhibition of DNA methyltransferases and histone methyltransferases by plant flavones.

Rajnee Kanwal, Manish Datt, Sanjay Gupta. _Case Western Reserve Univ., Cleveland, OH_.

Methylation of DNA and histone protein is an epigenetic modification mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs) critically involved in methylation and regulation of affinity binding between the histones and DNA backbone. HMTs catalyze the transfer of one, two, or three methyl groups to lysine and arginine residues of histone proteins. The HMTs-mediated increase in histone affinity to DNA causes chromatin condensation, preventing transcription; whereas DNMTs methylate cytosine residues within gene promoters resulting in transcriptional repression. DNMT and HMT activities are reported to be associated with signal transduction, cell growth and death, as well as with the pathogenesis of various human diseases including cancer. Dietary plant flavones can affect epigenetic modifications accumulated over time and have shown health-beneficial effects. However, the epigenetic response on DNMT and HMT elicited by plant flavones has not been elucidated. Through in silico protein-ligand docking studies and molecular studies with plant flavones viz. chrysin, apigenin and luteolin and their effect on DNA and histone methylation was assessed. The ligands were individually docked into the pocket of DNMT and EZH2 using Glide in XP (extra precision) mode (Schrodinger, LLC). Binding of ligands with proteins were evaluated using GlideScore, which is an empirically derived scoring function. Virtual screening approach using the model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Flavones exhibited a high docking rank (Glide score) in the order of chrysin (-5.8), apigenin (-6.4), luteolin (-7.4) compared with the pharmacological inhibitor, 5-Aza-2′-deoxycytidine (-4.2). Notably, all three flavones inhibited EZH2, having a high docking rank compared to the known pharmacological inhibitor, 3-Deazaneplanocin A (DZNep). The docking rank for chrysin was -10.07, apigenin -9.73 and luteolin -11.23, compared to -7.62 for DZNep. Epigenetic studies performed with plant flavones demonstrated reversal of hypermethylation of cytosine bases in the DNA and prevented methylation of cytosine in the GC-rich promoter sequence incubated with MSsI enzyme. Furthermore, decrease protein expression of EZH2 and trimethylation of H3K27 was noted in prostate cancer cells treated with these plant flavones. Taken together, our results suggest that plant flavone can alter DNMT and HMT activities and methylation of DNA and histone proteins that regulate epigenetic modifications providing significant health-effects and prevent various pathological processes involved in the development of cancer.

#5259

Prevention of hepatocarcinogenesis in rats by arabinoxylan rice bran, MGN-3/Biobran.

Nariman K. Badr El-Din,1 Doaa A. Ali,1 Reem Othman,1 Mamdooh Ghoneum2. 1 _Department of Zoology, Faculty of Science, University of Mansoura, Mansoura, Egypt;_ 2 _Department of Otolaryngology, Charles Drew University of Medicine and Science, Los Angeles, CA_.

Objective: Hepatocellular carcinoma is one of the most common cancers in the world and one of the most lethal. MGN-3/Biobran, derived from rice bran hemicelluloses, has been reported to possess a strong anticancer effect against several neoplasms. In the current study, we examined the protective effect of MGN-3/Biobran against N-nitrosodiethylamine (NDEA) and carbon tetrachloride (CCl4)-induced hepatocarcinogenesis in rats and studied the molecular mechanisms underlying its effect.

Materials and Methods: Male albino rats received carcinogen NDEA (200 mg/kg body weight) single dose i.p. plus promoter CCl4 (3 ml/kg b.w.) weekly s.c. for 6 weeks. Another group of rats were treated with MGN-3/Biobran (25mg/Kg b.w.) 5 times/week i.p. for 2 weeks prior to receiving carcinogens and continued for 20 weeks. Tumor incidence, histopathology, body and liver weight, liver marker enzymes, cell cycle progression, cell proliferation and apoptotic-related markers at mRNA and protein expression levels were quantitatively determined.

Results: Rats exposed to carcinogens alone showed loss of liver architecture, and proliferative and neoplastic changes. This group also showed marked decrease in body weight, increase in liver weight, and elevation in the levels of hepatic diagnostic markers: serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyl transpeptidase (gamma GT). In contrast, rats pretreated with MGN-3/ Biobran and subsequently exposed to carcinogens showed significant reduction in liver tumor incidence, marked decrease in the percentage of pre-neoplastic foci in hepatic parenchyma, and inhibition in the development of hepatocellular carcinoma. MGN-3/ Biobran treatment also maintained AST, ALT, ALP, and gamma GT levels close to normal values. In addition, MGN-3/Biobran treatment was associated with marked increase in both the cell cycle sub-G0/G1 population and AnnexinV-binding. The molecular studies at mRNA and protein expression levels in the liver tissues demonstrated that MGN-3/ Biobran reversed carcinogen induced suppression in the level of IkappaB-alpha (IκB-α), downregulated the expression of the nuclear factor kappa-B (NF-κBp65) and Bcl2, upregulated the expression of p53, Bax, caspases-3 and markedly increased Bax/Bcl2 ratio.

Conclusion: This study concluded that MGN-3/ Biobran inhibited hepatocarcinogenesis induced by NDEA and CCI4 via induction of apoptosis and inhibition of cancer cell proliferation. MGN-3/ Biobran may be a promising chemopreventive agent for liver carcinogenesis.

Biobran /MGN-3 was provided by Daiwa Pharmaceutical Co., Ltd. Tokyo, Japan.

#5260

Prevention of oxaliplatin-induced neuropathy by using minocycline as a chemoprotectant: demonstration by imaging and behavioral assessment.

Dawid Schellingerhout, Elizabeth Vichaya, Leo G. Flores, Daniela Ramos, Lucia Le Roux. _MD Anderson Cancer Center, Houston, TX_.

Purpose: To utilize neurography, a novel imaging method based on retrograde transport of a molecular nerve imaging tracer, to assess the protective effect of minocycline on the development of Oxaliplatin-induced neuropathy.

Materials and Methods: Female BALB/c mice received one of four treatments vehicle/vehicle, vehicle/minocycline, Oxaliplatin/vehicle, or Oxaliplatin/minocycline (n=8/group). A 30 mg/kg cumulative dose of Oxaliplatin or dextrose vehicle was given in 10 divided intra-peritoneal doses across 3 weeks using two 5 days cycles. Animals were treated daily with 50 mg/kg minocycline or 0.9% saline vehicle by oral gavage beginning 48 h prior to the first Oxaliplatin treatment. Both imaging and behavioral data were collected at baseline and weekly for 3 weeks. For each imaging session, animals received fluorescently labeled TTc-Alexa790 (15 ug/20 uL) via intramuscular injection into the calf muscles. Fluorescent imaging (Xenogen IVIS 200) was used to image the distribution of TTc over 60 minutes, with ROI measurements taken over the lumbo-thoracic junction of the spine to quantitate fluorescent uptake. Neurobehavioral assessment for mechanical sensitivity was assessed through the use of von Frey nylon filaments to exert calibrated force on the footpads. The 50% hind paw withdrawal threshold was calculated.

Results: Oxaliplatin/vehicle treated animals showed a significant decrease in transport of TTc during the second week of treatment (F (1,12)=39.604, p<0.001), while the TTc transport of the vehicle/vehicle and oxaliplatin/minocycline remained stable across the experiment. The vehicle/minocycline group saw an increase in transport of TTc during the second week of treatments (F (1,12)=42.533, p<0.001). Behavioral data indicated that Oxaliplatin treatment resulted in increased mechanical sensitivity, while minocycline treatment abrogated this effect, such that animals in the Oxaliplatin/vehicle group showed increased sensitivity compared to all other groups. This effect emerged within the first week of treatment and remained throughout the study. A linear correlation between paw withdrawal threshold and TTc transport at week 3 was found, with r = 0.7939, p<0.01, such that subjects with reduced TTc transport also displayed reduced mechanical thresholds.

Conclusion: Oxaliplatin causes a decrease in retrograde axonal transport, and this reduction in transport correlates with neurobehavioral impairment due to neuropathy. We show that this effect can be attenuated by a chemo-protectant, minocycline, and that the protectant effect was apparent with both behavioral and imaging readouts. This suggests that minocycline can prevent the neuropathy induced by Oxaliplatin and that the mechanism of both the pathological effect and its prevention are related to retrograde axonal transport.

#5261

Novel resveratrol analogue, HS-1793, inhibits hypoxia-induced HIF-1 and VEGF expressions and inhibits tumor growth of human breast cancer cells in a nude mouse xenograft model.

Dong Hwan Kim, Min Jeong Kim, Bokyung Sung, Yong Jung Kang, Seong Yeon Hwang, Na Lam Hwang, Nam Deuk Kim. _Pusan National Univ., Busan, Republic of Korea_.

Taking phytochemicals provide various benefits for human health. Among them, resveratrol, from the blueberry, is well known for its beneficial effects for human. However, resveratrol has photosensitive and easily decomposition by digestive enzyme. Thus we synthesized several synthetic analogues of resveratrol with potent activity. One of them, 4-(6-hydroxy-2-naphtyl)-1, 3-benzenediol (HS-1793) treatment showed potent antitumor activity in various human cancer cells. In this study, we investigated the effects of a novel analogue of resveratrol, HS-1793 on the expressions of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in MCF-7 with MDA-MB-231 human breast cancer cells. We found that HS-1793 inhibited hypoxia (1.0% oxygen) induced HIF-1α protein level and its downstream target, vascular endothelial growth factor (VEGF) expression dose-dependently in both cell lines. Decreased HIF-1α by HS-1793 modulated through 26S proteasome pathway. We also found that HS-1793 suppressed tumor growth in tumor xenograft mice model. Treatment with 10 mg/kg of HS-1793 suppressed Ki-67 and CD31 expressions in tumor tissues. Above all, HS-1793 shows more powerful effects than resveratrol. Taken together, these results implied that HS-1793, a novel analogue of resveratrol might be a new potent chemopreventive agent and anticancer therapy in human breast cancer.

#5262

Chemopreventive and angiopreventive activity of a purified polyphenol-rich extract from olive mill wastewaters.

Adriana Albini,1 Antonino Bruno,1 Barbara Bassani,1 Nicoletta Macrì,1 Francesca Caudano,1 Paola Corradino,1 Teresa Rossi,2 Douglas M. Noonan3. 1 _IRCCS MultiMedica, Milano, Italy;_ 2 _IRCCS Arcispedale S. Maria Nuova, Reggio Emilia, Italy;_ 3 _Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy_.

The presence of diverse phenolic molecules in dietary elements, has been shown to prevent the occurrence of a variety of chronic and inflammatory diseases. Olives and extra virgin oil, a basic component of mediterranean diet represent an important source of these poliphenols, including hydroxytyrosol. While the strong antioxidant potential of these molecules has extensively investigated, their anti-angiogenic and chemopreventive activities remain unknown. Here we assessed the anti-angiogenic and chemopreventive activities exerted by an extracts from olive mill wastewaters (OMWWs), termed A009, which represent a waste product from olive oil industry, in vitro and in vivo on endothelial and different tumour cell lines, as compared with hydroxytyrosol alone

The ability of A009 to affect cell proliferation and survival was evaluated on human umbelical endothelial vein cells (HUVECs), six different tumor cell lines, MCF-7 and MDA-MB-231 (breast), HT-29 and HCT-116 (colon) PC-3 and DU-145 (prostate) and the murine CT26 CRC cells by MTT assay, while the induction of apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry. Functional studies evaluated the capacity of OMWWs to interfere with endothelial cell tube formation, migration and invasion by morphogenesis and Boyden chamber assays, respectively. Finally, the inhibition of angiogenesis and tumor cell growth was evaluated in vivo, by the matrigel sponge assay and tumor xenograft.

The A009 extract was able to inhibit both HUVECs and tumor cell lines growth in a dose dependent manner, exerting a stronger inhibitory effect as compared to the pure hydroxytyrosol alone. This effect was directly associated with the induction of apoptosis and ROS on HUVECs. HT-29 anc HCT-116 CRC cell lines exposed to A009 decreased thei ability to release VEGF and IL-8. Functional studies of HUVECs exposed to A009 demostrated impaired migratory and invasive abilities as far as reduced capability to form capillary-lile-structure in a dose dependent manner. Finally, A009s interfered with angiogenesis and CT26 tumor cell growth in vivo.

Our results suggest that the polyphenol enriched extracts from olive oil processing (OMWWs) A009, show promising angio-preventive and chemo-preventive potentials. In particular, our data demonstrate that a pool of specific polyphenols are characterized by stronger anti-angiogenic/anti-tumor properties compared to hydroxytyrosol alone, a well known polyphenol with anti-tumor activity.

#5263

Prostate-specific membrane antigen (PSMA)-targeting nanobioconjugate-encapsulated green tea polyphenol EGCG for prostate cancer prevention and therapy.

Imtiaz A. Siddiqui,1 Dhruba J. Bharali,2 Rahime Jashari,1 Vaqar M. Adhami,1 Shaker A. Mousa,2 Hasan Mukhtar1. 1 _University of Wisconsin-Madison, Madison, WI;_ 2 _Albany College of Pharmacy, Albany, NY_.

Initially we introduced the idea of employing nanotechnology to improve the outcome of natural agents in chemoprevention and coined the term 'Nanochemoprevention'. Recently we reported the efficacy of chitosan nanoparticles encapsulating (-)-epigallocatechin-3-gallate (EGCG) for prostate cancer (PCa) prevention and therapy. Here we employed the concept of targeted uptake of therapeutic nanoparticles, a potentially powerful technology to enhance the efficacy of EGCG for PCa management. We developed chitosan nanobioconjugates encapsulating EGCG and surface functionalized with the A10 2′-fluoropyrimidine RNA aptamers that recognize the extracellular domain of the PSMA, henceforth called chit-EGCG-Apt. Cellular binding and uptake of the fluorescent nanosystem was detected suggesting PSMA specific internalization and accumulation. Further, we observed that chit-EGCG-Apt lead to a PSMA specific increase of anti-proliferative activity in PCa cells, with a better efficacy in PSMA positive cells (LNCaP and C4-2) than in PSMA negative (PC3 and DU145) cells and a decrease in IC50 by at least half the dose of the native agent. Further, we also observed a significant inhibition in the colony formation potency of PCa cells when treated with chit-EGCG-Apt as compared to the native EGCG with the effects more distinct in PSMA positive cells. Next, we established that EGCG in chit-EGCG-Apt retains its mechanistic identity and observed that the treatment resulted in significant (i) shift in Bax/Bcl-2 ratio in favor of apoptosis, (ii) increase in PARP cleavage, (iii) inhibition in caspase 3, 7, 8 and 9, and other key regulators of apoptosis, (iv) modulation of death receptor pathway proteins, and (v) cyclin dependent kinase inhibitors in addition to arrest of the cells in S phase of the cell cycle. Importantly, these effects were only visible at higher doses of the native EGCG. The nanobioconjugate also demonstrated a significant inhibition of invasive potential of several PCa cells and 3-dimensional spheroid BME cell invasion assay. Further, chit-EGCG-Apt treated mice implanted with androgen sensitive CWR22R(nu)1 cells showed significant inhibition in tumor growth and decreased serum PSA levels reciprocating our in vitro findings. In tumor tissues of mice treated with Chit-EGCG-Apt there was significant (i) induction of poly (ADP-ribose) polymerases cleavage, (ii) increase in the protein expression of Bax with concomitant decrease in Bcl-2, (iii) activation of caspases, and (iv) reduction in Ki-67 and proliferating cell nuclear antigen, as compared to native EGCG. We further observed inhibition of angiogenesis, invasion and metastasis markers in these tissues. Translation of these data to appropriate animal model systems could pave way for prostate cancer therapy in humans. 

## BIOINFORMATICS AND SYSTEMS BIOLOGY:

### New Bioinformatic Tools, Databases, Portals, and Data Resources

#5264

The National Cancer Institute's biospecimen research database: A community resource for improving specimen quality.

Kelly B. Engel,1 Sarah R. Greytak,2 Ping Guan,3 Helen Moore3. 1 _Preferred Solutions Group, Arlington, VA;_ 2 _Kelly Government Solutions, Rockville, MD;_ 3 _National Cancer Institute, Bethesda, MD_.

Tissue and blood biospecimens used in cancer research are collected, processed and stored using standard operating procedures (SOPs) that can vary tremendously across institutions. This lack of standardization in biospecimen collection and processing practices across sample sets and institutions can be a contributing source of research variability and can compromise analytical results. In an effort to minimize preanalytical variability and improve the quality of biospecimens used for cancer research, the National Cancer Institute's Biorepositories and Biospecimen Research Branch (BBRB) developed the Biospecimen Research Database (BRD; http://biospecimens.cancer.gov/brd). The BRD is a free and publically accessible online database that aims to (i) improve access to peer-reviewed articles that investigate the impacts that biospecimen collection and handling practices can have on clinical, molecular, and proteomic endpoints, (ii) facilitate transparency between institutions and researchers by promoting SOP sharing and distribution, and (iii) improve the quality of biospecimens through the development of evidence-based procedural guidelines. To date, the BRD houses more than 2,300 articles and 200 SOPs from 30 different participating biobanking institutions. Articles are meticulously categorized and annotated by a team of Ph.D.-level scientists according to the type of biospecimen and technology platform used and the preanalytical factors investigated. Literature contained within the BRD serves as the foundation for internally developed reviewed papers as well as evidence-based procedural guidelines, termed Biospecimen Evidence-Based Practices, which focus on biospecimen collection, preservation, and processing. In addition, more than 90% of the BRD's SOP library was contributed by other government offices, non-profit, private, and international biobanks and institutions. BBRB has invested a substantial and continued level of effort in the establishment of this scientifically accurate and robust database. Ongoing participation by the scientific community in the form of public commenting, article suggestions and SOP submissions is encouraged and will increase both the diversity and value of the BRD.

#5265

Cloud-based somatic analysis platform for populations with paired tumor normal samples.

Melanie Lou, Holly Beale. _Maverix Biomics, San Mateo, CA_.

Maverix Biomics introduces its cloud-based somatic analysis platform, a scientifically sound, end-to-end analysis solution that begins with quality control of fastq reads and leads to somatic variant calling, annotation and interactive analysis and has been validated using paired tumor normal cancer samples from the COSMIC cell line project (CLP). In addition to providing the ability to call somatic variants for cancer populations and features to explore characteristics within each of these populations, another strength of the Maverix Analytic Platform is the ability to compare multiple cancer populations - a limitation in other existing platforms. To support this functionality, our cloud-based platform has been optimized using the biological expectations of COSMIC CLP data and outfitted with interactive features within Maverix's Variant Explorer. Specifically, we maximized the sensitivity and minimized the percentage of unvalidated variants of two COSMIC cancer cell lines - small cell lung carcinoma and renal cell carcinoma. Then interactive analysis of this data using Maverix's Variant Explorer has identified somatic variants and affected genes specific to each cancer population and shared among both cancer populations. Our platform expedites data exploration and the ability to identify similarities and differences among cancer populations which is useful for tumor reclassification and for repurposing targeted therapies based on discovering similar genetic profiles across different types of cancer.

#5266

Fast and accurate fusion transcript detection using the Trinity Cancer Transcriptome Analysis Toolkit.

Brian Haas,1 Timothy Tickle,1 Nathalie Pochet,2 Jing Sun,1 Peggy Hsu,3 Chip Stewart,1 Carrie Ganote,4 Ben Fulton,4 Catherine Wu,3 Aviv Regev1. 1 _Broad Institute, Cambridge, MA;_ 2 _Brigham and Women's Hospital, Cambridge, MA;_ 3 _Dana Farber Cancer Institute, Cambridge, MA;_ 4 _Indiana University, Bloomington, IN_.

Chromosomal rearrangements leading to fusion transcripts represent a class of oncogenic aberrations that are of high interest for understanding cancer biology, treating cancer patients, and as targets for the development of new therapies. Transcriptome sequencing via RNA-Seq coupled with downstream bioinformatics software applications offers an effective method for identifying candidate fusion transcripts, and is more targeted and cost-effective than whole genome sequencing. Although many such fusion detection software tools have recently been made available, they often differ greatly in prediction accuracy, execution times, computational requirements, installation complexity, and in not being readily accessible to non-bioinformatician cancer researchers.

Here we present the Trinity Cancer Transcriptome Analysis Toolkit (CTAT), a newly developed suite of RNA-Seq targeted fusion detection tools leveraging a combination of reference genome read-mapping and de novo transcriptome assembly, coupled to in silico fusion transcript validation, annotation, and visualization. We show that components of our Trinity CTAT fusion detection toolkit, including STAR-Fusion and FusionInspector, yield improved accuracy and run times as compared to alternative leading tools. As a demonstration of the utility of Trinity CTAT, we explore fusion transcript discovery in a large cohort of ~300 chronic lymphocytic leukemia (CLL) patient samples. We identify several novel recurrent fusions that may represent drivers of CLL etiology and provide new avenues to therapies.

In addition to fusion transcript identification, Trinity CTAT aims to provide cancer researchers with easy access to methods for analyzing cancer RNA-Seq, including transcript reconstruction, identification of mutations, expression analysis, and tumor heterogeneity. Trinity CTAT is freely available open source software and readily accessible to all cancer researchers via our public Galaxy web portal: https://galaxy.ncgas-trinity.indiana.edu/ .

#5267

Gene fusion database to create custom panels: Enabling detection of fusion transcripts and gene expression assays.

Efren Ballesteros-Villagrana,1 Jeoffrey Schageman,1 Kelli Bramlett,1 Paul Williams,2 Scott Myrand,2 Guoying Liu,3 Fiona Hyland,3 Seth Sadis2. 1 _Thermo Fisher Scientific, Austin, TX;_ 2 _Thermo Fisher Scientific, Ann Arbor, MI;_ 3 _Thermo Fisher Scientific, San Francisco, CA_.

Gene fusions play an important role in tumorigenesis and are increasingly recognized as important entities for the diagnosis and treatment of hematological malignancies and solid tumors. Fusion events generate a hybrid mRNA transcript comprising sequence from multiple otherwise distinct genes. Oncogenic fusion events often involve tyrosine kinases or transcription factors, leading to aberrant growth signaling, making these events potentially attractive drug targets. For instance, targeted therapies such as known tyrosine kinase inhibitors are currently approved to treat ALK fusion positive Non-Small Cell Lung Carcinoma (NSCLC) patients. Detection of known gene fusion events is an important part of genomic characterization which can inform patient diagnosis. Current methods for fusion detection include chromosome banding analysis (CBA), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). New developments in next-generation sequencing (NGS) enable the efficient and simultaneous assessment of multiple gene fusion targets with high sensitivity. To enable researchers to design their own custom panels and assess a set of gene fusions of interest, we developed a comprehensive RNA gene fusion database. Oncology researchers now have the capability to create custom panels from this comprehensive database which includes over 1,000 well annotated and optimized gene fusion assays and over 20,000 gene expressions assays.

To build this comprehensive gene fusion database, we identified breakpoint information for 1,178 well annotated fusions described in publications and in the COSMIC and NCBI databases. We prepared a target RNA sequence for each breakpoint using transcript sequences from the Ensembl database. We used a proprietary primer designer to generate candidates for each fusion target amenable to the AmpliSeq™ product line requirements. Quality control was performed throughout the design process to identify the best primer set for each target, to avoid primers overlapping common germline SNPs, potential primer/primer or primer/amplicon interactions, or off-target or wild-type amplifications.

With this comprehensive database we provide a complete range of solutions available on ampliseq.com. Making use of the AmpliSeq™ technology, researchers now have the capability to create their own custom fusion panel and place the order within an hour. These custom panels are used with AmpliSeq™ Library reagents and Ion Torrent™ sequencing platforms for targeting next-generation sequencing. The analysis solution is provided through the Ion Reporter™ (IR) software package. Custom fusion panel workflows in IR are used to analyze sequencing data coming from the custom panels, which includes visualization of fusion transcripts and gene expression levels in a heat map feature.

#5268

Seq2C: from sequence to copy number for cancer samples.

Zhongwu Lai, Aleksandra Markovets, Jonathan Dry. _AstraZeneca, Waltham, MA_.

Targeted sequencing is used increasingly in clinics to guide therapeutic decisions by measuring mutations, small indels and/or rearrangements in cancer genes. An ability to use the same platform to detect additional oncogene activation (or tumor suppressor loss) through copy number changes could significantly expand the number of patients able to benefit from targeted therapies. Many currently available tools either requires a matched normal, works only for whole genome or exome sequencing, or don't work for PCR-based targeted sequencing. In addition, no tools available to detect breakpoints within a gene from targeted sequencing. A versatile copy number analysis tool from sequencing is needed to maximize the value of the sequencing routinely applied in clinics.

Here we presented a novel computational tool, Seq2C (Sequencing To Copy Number), which is versatile to handle various situations and reports aberrations at gene level ready for interpretation. Seq2C works at cohort level and does not require a matched 'normal', though it can optionally use one or more normal samples for small (<20) or homogeneous cohorts. Seq2C applies a robust three-step cohort-based normalization to identify and quantify sequencing coverage variability in given regions, which can be sequence or platform dependant. Seq2C works for hybrid or PCR based targeted sequencing, exome and even whole genome sequencing. In targeted sequencing, Seq2C automatically identify and exclude regions that fail to be captured by the assay to prevent false positive calls of deletions, and can accommodate several magnitude differences in PCR efficiency.

Another distinct feature differentiating Seq2C from other currently available tools is that Seq2C identifies breakpoints and detects one or more exon deletion or duplication rearrangements within a gene, which is common in tumor suppressors as a mechanism to lose function. In addition, it can also detect potential fusions in genes such as ALK and ERG, where a copy number change is often accompanied with the fusion and a breakpoint can thus be called by Seq2C. Furthermore, it can predict gender from exome or whole genome sequencing.

We applied Seq2C to exome sequencing of CCLE cell lines, and showed that it produced gene level copy number data highly correlated to those derived from microarrays, the current gold standard. Interestingly, Seq2C identified one or more exon deletions in several common tumor suppressors, such as TP53, PTEN, CDKN2A, NF1, STK11 and RB1, in cell lines with no known aberrations. They were supported by RNA-Seq data. It also correctly called known ERG fusion in prostate cell line NCI-H660 and identified a previously un-reported EML4-ALK fusion in a pancreatic cancer cell line SNU-324, which is confirmed by RNA-Seq data, suggesting EML4-ALK is not limited to lung cancer.

In conclusion, Seq2C is a versatile copy number analysis tool for sequencing and will be useful for cancer research. Seq2C is freely available in GitHub.

#5269

Minor Variant Finder: New software for detecting somatic mutations at low level in Sanger sequencing traces.

Edgar H. Schreiber, Harrison Leong, Stephanie J. Schneider, Marks Jeff, Wallace George, Hardeep Sangha, Yoke Peng Lim, Evelyn Chan, Steve Berosik, Martin Storm, Joel Colburn. _Thermo Fisher Scientific, South San Francisco, CA_.

Introduction: With the rapid adoption of next generation sequencing (NGS) and its use for characterization of mutations in tumor samples, a need has emerged to establish an orthogonal technology for reliable and sensitive detection of somatic mutations which may occur at proportions of 10% or lower compared to the normal allele. Until now, somatic variants with an allelic proportion of 25% or less are often undetected (i.e. not "called") by automated Sanger sequencing software. We have developed Minor Variant Finder software that calls 5% minor variants at 96% sensitivity and 99% specificity in a test data set. The improved variant detection software calls variants de novo, without a priori knowledge of location or presence, and affords all the advantages of Sanger sequencing, of robustness, low error rate, ease of use, human interpretable visual displays of the output data, and low cost per sample and target.

Methods: New algorithms were developed to compare and subtract the baseline noise signals of a control sample from a test sample. Noise minimization and peak detection algorithms enable the detection of peaks representing candidate minor variants which are confirmed in the complementary strand.To test the new algorithms, synthetic mixtures of minor alleles in the range of 2.5%, 5%, 10% and 20% were prepared by combining genomic DNAs containing known mutations. Using an optimized PCR/sequencing protocol to sequence cell line and FFPE derived DNA, 13 different amplicons and seven different genes, including P53, KRAS, BRAF, FLT3, RB1, CDH1, and ERBB2, were sequenced on 3500, 3730, and 3130 Applied Biosystems Genetic Analyzers.

Results: To test the new software, 898,499 total base positions and 3126 total variant positions, spanning variants at 2%, 5%, 10%, and 20%, were interrogated. The software achieved 96% sensitivity and 99.8% specificity for automated detection of 1112 variants present at the 5% level in 274,110 high quality base positions. Sensitivity was 99% for variants at 10%, and 99.7% for variants at 20%. The software displays a noise-minimized trace view to facilitate visual inspection for confirmation or rejection. Further, reference sequences with hg19 chromosomal locations and NGS vcf files can be imported and aligned side by side with the Sanger sequencing data for orthogonal verification. Hyperlinks to dbSNP for known rsSNP variants are provided in the software.

Conclusions: We have developed new Minor Variant Finder software that detects and reports minor variants by Sanger sequencing. We have demonstrated that the new software used in conjunction with standard protocols for fluorescent dye terminator Sanger sequencing enable the identification of de novo somatic mutations to a level of 5% with 96% sensitivity and 99.81% specificity. This technology will also be useful for the confirmation of minor variants identified by NGS.

For Research Use only - Not for use in diagnostic procedures.

#5270

The UCSC Xena system for integrating and visualizing functional genomics.

Mary Goldman, Brian Craft, Jingchun Zhu, Teresa Swatloski, Melissa Cline, David Haussler. _UC Santa Cruz, Santa Cruz, CA_.

UCSC Xena (http://xena.ucsc.edu/) is a bioinformatics tool to visualize functional genomics data from multiple sources simultaneously, including both public and private data. The Xena system consists of a set of federated data hubs and the Xena browser, which integrates across hubs, providing one location to analyze and visualize all data. The lightweight Xena data hubs are straightforward to install on Windows, Mac and Linux operating systems and easily allow hub administrators to authenticate users, ensuring that only authorized users have access to secure data. Loading data into a Xena hub is easy using either our application or command line interface. Hosting public data from major projects, such as TCGA, on public Xena hubs gives users access without having to download these large datasets. The Xena system makes it easy to aggregate across many hubs, allowing users to integrate public datasets and private secure data together or view them separately. This system of the browser and hubs helps researchers combine new or preliminary results from their laptops or internal servers, or even data from a new paper, securely with vetted data from the public sphere.

The largest public Xena hub, based at UCSC, currently hosts an expanding set of searchable data, including 806 public datasets from several large consortiums including TCGA (The Cancer Genome Atlas), ICGC (International Cancer Genome Consortium), Treehouse Childhood Cancer Project, CCLE (Cancer Cell Line Encyclopedia), and more. Xena hubs are flexible enough to handle most data types, including gene, exon, miRNA and protein expression, copy number, DNA methylation and somatic mutation data along with phenotypes, subtype classifications and genomic biomarkers. Dynamic Kaplan-Meier survival analysis helps investigators to assess survival stratification by any information while scatter plots and bar graphs offer new insights into the data. Integration with Galaxy gives users access to a myriad of bioinformatics tools for further analysis and hypothesis testing.

#5271

Optimization and detection of focal somatic copy number variants in whole genome, whole exome and panel sequencing for tumor/normal matched pairs and tumor only analysis.

Jessica Aldrich, Jonathan J. Keats, Austin Christofferson, Winnie S. Liang, John D. Carpten, Lisa Baumbach-Reardon, David W. Craig. _TGen, Phoenix, AZ_.

Often in the clinical setting, tumor samples may be derived from historical tissue or from fresh frozen tissue and it may not be feasible to obtain normal tissue from the individual. Comparing unmatched tissues from different individuals possess a unique challenge of addressing common copy number variations not faced when comparing matched samples. In addition to the challenges of comparing unmatched samples, DNA extracted from samples preserved as formalin-fixed, paraffin-embedded (FFPE) tissue is often highly degraded and when compared to more intact DNA extracted from different source (e.g. blood) can increase the inherent noise in the system. Here we analytically characterize the accuracy and performance of algorithmic approaches for separating germline inherited copy number variation from somatic copy number changes, focusing on both filtering approaches and use of pooled reference samples sequenced under similar conditions. Example approaches include filtering known common copy number variation within 1000 Genomes Phase 3 and Database of Genomic Variants (DGV) Gold Standard. Additionally, we utilized a tumor/reference pool based analysis where a reference pool was constructed by equimolar pooling of multiple individuals. Determination of fold changes between tumor and reference was calculated by determining physical coverage of read pair fragments in 100 bases increments. Next, to address differences in sequencing performance between tumor and reference, the read depth data for each sample is collapsed/averaged into a lower resolution according to user-selected parameters (e.g. distance between points and read depth). Normalized log2 fold-changes between tumor and reference samples are then calculated and an adjustable smoothing window is applied. In addition, we utilize tumor allele frequencies of known heterozygous germline SNPs identified within the normal to both evaluate potential false positives and correct biases. Lastly, a segmentation algorithm is applied to summarize the individual log2 fold-changes into intervals with a constant copy number state. We will present the advantages and limitations of these approaches both when a germline normal is available and when tumor only analysis is necessary.

#5272

Cloud-based informatics enables the design and analysis of massively multiplex custom gene fusion panels for next-generation sequencing on FFPE RNA samples.

Fiona Hyland,1 Rajesh Gottimukkala,1 Efren Ballesteros,2 Heinz Breu,1 Yuandan Lou,1 Scott Myrand,3 Michael Hogan,3 Kelli Bramlett,2 Guoying Liu,1 Seth Sadis3. 1 _Thermo Fisher, South San Francisco, CA;_ 2 _Thermo Fisher, Austin, TX;_ 3 _Thermo Fisher, Ann Arbor, MI_.

Gene fusions, a combination of two genes, comprising their coding and/or regulatory sequences, are caused by structural rearrangements in DNA or in RNA transcripts. Many gene fusions are strong driver mutations in neoplasia, and are important in understanding basic biology, interaction with targeted therapy, and research into risk stratification and outcomes.

Next-generation sequencing enables sensitive, specific and precise detection of particular fusion isoforms for defined gene pairs. Massively multiplex Ampliseq gene fusion assays enable enrichment of fusion transcripts using as little as 10 ng of RNA extracted from FFPE samples. Sequencing on Ion Torrent instruments reveals the full sequence of the gene fusion, for precise definition of the breakpoint and the expressed exons or promoter regions of both genes.

We developed cloud-based software to support the design of a custom Ampliseq gene fusion panel, comprising 1 to 1,000 fusion isoform assays and any gene expression assays for normalization.

We extensively mined the scientific literature on fusions and the COSMIC database to identify over 1000 fusion isoforms. We rigorously curated this data using automated and manual methods, including mapping, confirmation and correction of reported sequence to obtain genomic coordinates, identification of breakpoints, annotation of exon junctions, and selected wet lab testing. We created a database containing over 1000 high quality curated and annotated fusion isoforms, including 70 ALK, 60 RET, 26 ROS1, and 21 NTRK1 fusions. We designed Ampliseq primer pairs for each of these fusions using advanced assay design and pooling algorithms, such that all fusion and gene expression assays can be multiplexed into 1 or 2 compatible pools.

Assays can be selected by gene or gene pair; detailed information about each assay selected includes isoform, genes, exon numbers, and links to COSMIC and to relevant publications.

We developed cloud-based analysis software to analyze the BAM file resulting from amplification and sequencing of custom Ampliseq fusion panels on an Ion Torrent sequencer. This analysis leverages the rich annotation information from the assay design. The reads are mapped to a custom reference sequence tailored to the custom Ampliseq fusion assay, and applying an optimized algorithm to select confidently mapped reads based on read length and overlap with each gene of the gene pair based on the reference and annotated breakpoint. Gene fusions are detected based on the total number of fusion reads and optionally frequency, and on the properties of those reads. Software QC steps for total number of mapped reads, number of reads for gene expression controls, and elimination of cross-talk artifacts result in a highly sensitive and specific detection of fusions, with LOD below 1%. Fusion results for any or all samples can be viewed, annotated, filtered, and visualized, and exported.

#5273

GenePattern Notebooks: an integrative analytical environment for cancer research.

Michael M. Reich,1 Thorin Tabor,2 John T. Liefeld,1 Peter Carr,2 Barbara Hill,2 Marc-Danie Nazaire,2 David Eby,2 Helga Thorvaldsdottir,2 Pablo Tamayo,1 Jill P. Mesirov1. 1 _University of California, San Diego, La Jolla, CA;_ 2 _Broad Institute, Cambridge, MA_.

As the availability of genetic and genomic data and analysis tools from large-scale cancer initiatives continues to increase, the need has become more urgent for a software environment that supports the entire "idea to dissemination" cycle of an integrative cancer genomics analysis. Such a system would need to provide access to a large number of analysis tools without the need for programming, be sufficiently flexible to accommodate the practices of non-programming biologists as well as experienced bioinformaticians, and would provide a way for researchers to encapsulate their work into a single "executable document" that included not only the analytical workflow but also the associated descriptive text, graphics, and supporting research. To address these needs, we have developed GenePattern Notebook, based on the GenePattern environment for integrative genomics and the Jupyter (formerly IPython) Notebook system. GenePattern Notebook unites the phases of in silico research - experiment design, collaborative analysis, and publication - into a single interface. GenePattern Notebook presents a familiar lab-notebook format that allows researchers to build a record of their work by creating "cells" containing text, graphics, or executable analyses. Researchers add, delete, and modify cells as the research evolves, supporting the initial research phases of prototyping and collaborative analysis. When an analysis is ready for publication, the same document that was used in the design and analysis phases becomes a research narrative that interleaves text, graphics, data, and executable analyses, serving as the complete, reproducible, in silico methods section for a publication. GenePattern Notebook also supports programmers and bioinformaticians, providing seamless interoperation between code blocks and GenePattern analyses within a notebook document. We are collaborating with cancer research laboratories to create GenePattern Notebook documents to serve as examples of integrative genomics research that can be enabled through this system. Potential notebook topics include characterization of intratumoral heterogeneity from single cell RNA-Seq data, effective clinical interpretation of comprehensive genomic profiling from whole exome sequencing of a patient's tumor and germ line samples, and identification of master regulators/transcription factors associated with the downstream transcriptional effects associated with the activation of an oncogene. We will make these and other notebooks available in an online GenePattern Notebooks repository, where other researchers may also contribute their notebooks.

#5274

A novel, statistical-based method to determine RNA expression by next-generation sequencing in clinical FFPE samples.

Robert Shoemaker,1 Aaron Berlin,2 Amy Diliberto,1 Heather Ely,1 Marissa Chen,1 Danielle Murphy,1 Jason Christiansen,1 Vince Reuter,2 Abel Licon2. 1 _Ignyta, Inc, San Diego, CA;_ 2 _ArcherDx, Inc, Boulder, CO_.

Introduction: Next-generation sequencing (NGS) has become a critical diagnostic assay to identify pathologic SNVs, CNVs, and gene rearrangements. However, it has not been routinely used to assess expression levels of target genes that could indicate patient populations responsive to therapeutics. This is largely due to the absence of reliable bioinformatics tools for the assessment of expression levels within RNA assays. We have developed a novel within-sample distribution-based method that assesses the relative extremity of expression for individual genes. Our predominate focus has been on assessing the expression of NTRK1, NTRK2, NTRK3, ROS1, and ALK, however this method can be readily applied to other genes and NGS platforms.

Methods: Within the kinase domain, the expression of a targeted region is represented by the number of unique deduplicated reads for NGS studies and normalized probe expression values (based on spike-in controls) for NanoString studies. A Poisson distribution is used to represent primer expression with the parameters estimated via maximum likelihood. The interquartile range (IQR) of the entirety of the sample's read counts is calculated and only those that do not exceed the third quartile bound by more than 150% of the IQR are considered during parameter estimation. A probability is then assigned to each of the primers based on their read counts. A geometric mean of the individual primer probabilities represents the probability value for the entire gene. Expression values are reported as -log10 (p-value) and cutoffs of 6 and 1 were used to call significant expression for NGS and NanoString platforms, respectively.

Results: The NGS-based approach correctly identified significant expression in all gene rearrangement positive cell lines (n=11). In cell lines not harboring gene rearrangements, the NGS and NanoString platforms showed 100% concordance in calling significant expression (n=12). In a gene rearrangement negative FFPE cohort, concordance between NGS and NanoString platforms was 97%-99% for target genes (n=102). ROS1 and ALK were most commonly found to be significantly expressed in FFPE samples (5% and 2%).

Conclusions: We have developed a statistical-based approach to detecting expression levels for RNA-based NGS assays. This can be applied to cohort studies to not only identify clinical samples that may benefit from targeted kinase inhibitor therapeutics but also correlate with predicted outcome of disease.

#5275

An integrated pipeline for TCGA data analysis.

Kaixian Yu,1 Yun Xu,2 Ke Tang,2 Albert Steppi,1 Jun Zhou,3 Zheng Ouyang,3 Jinfeng Zhang1. 1 _Florida State University, Tallahassee, FL;_ 2 _University of Illinois at Chicago, Chicago, IL;_ 3 _Insilicom LLC, Tallahassee, FL_.

The accumulation of publicly available high-throughput experimental data has made analyzing the data a bottleneck in scientific discovery. In this study, we explore a computational, high-throughtput approach for identifying cancer related biological factors using the data available at the Cancer Genome Atlas (TCGA). We developed an integrative genomic analysis (IGA) tool, which performs multiple analysis tasks, including gene/protein level differential expression analysis, gene set level enrichment analysis and network/pathway level comparison. Several data types can be analyzed, including RNA-seq, DNA methylation, miRNA-seq, and proteomics. By varying cancer types, race groups, and data types, we are able to generate a large set of novel findings, which may serve as experimental targets for biologists and biochemists. Interactive reports are generated to facilitate further exploration of the biological significance of findings. With the high-throughput approach and intgrative tools, the bottlenect for us now shifts to publishing these results. We invite researchers in cancer research community to collaborate with us to publish the findings.

#5276

Impact of duplicate removal on low frequency NGS somatic variant calling.

Jeran Stratford,1 Gunjan Hariani,2 Jeff Jasper,2 Chad Brown,2 Wendell Jones,2 Victor J. Weigman2. 1 _Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC;_ 2 _EA Genomics, A Q2 Solutions Company, Morrisville, NC_.

Cancer genomic profiles created by analysis of targeted Next Generation Sequencing (NGS) panels is emerging as a powerful tool for making informed clinical decisions. Of the critical informatics challenges to address, accurate mutation calls and allele frequency estimations after accounting for PCR-mediated artifacts are debated. The process of sample preparation for NGS sequencing involves amplification by PCR. While PCR is relatively error-free, mistakes early in DNA synthesis can be compounded, driving detection of spurious mutations and having an adverse impact on clinical reporting. Previous reports have addressed the utility of detecting and removing PCR duplicate reads in Mendelian applications but have rarely examined its use with targeted NGS panels.

We performed deduplication with 3 widely used tools (Samtools, Samblaster, and Picard) to understand sensitivity to call low frequency alleles and any impact on false positive/negative rates. Furthermore, we evaluated effects of duplicate removal on targeted panels of varying sizes and effects of sample matrices using replicates of 7 verified reference samples with several digitally confirmed alleles with frequencies ranging from 1-5%.

It is not practical to perform deduplication on PCR-enriched panels, therefore, we assessed 3 different hybrid enrichment panels of varying size (387kb, 1.3Mb, and 54Mb). Deduplication by Picard resulted in a greater decrease in the mean depth for the smaller panels (32-59%) compared to Exome (15%), showing that higher molecular diversity lowers duplication rates. Uniformity (percent of ROI with depth within 20% of the mean depth) improved 6-18% after deduplication for the smaller panels, but only 1% for the Exome.

Independent of panel size, about 32% of the total reads were marked as duplicates, reducing the power to call low frequency variants by 18%. Importantly, after added sequencing 95-96% of onco-specific variants were detected post-deduplication with a lower limit of detection of 3% compared to 2.5% pre-removal. For low-quality DNA samples we find no benefit in added sequencing for any panel. We also generally observed higher sensitivity (0% to 10% for SNV and -3% to 3% for indels) post deduplication.

Molecular diversity also varies by sample type. Intact DNA show higher molecular diversity and lower duplication rates than degraded FFPE samples. We profiled mixed-quality FFPE samples (n=85), good-quality fresh frozen samples (n=3), and NA12878 (n=1) on our smallest panel and noted duplication rates of 64±14%, 40±5% and 30% respectively. On average, deduplication cut the number of SNV calls by 17.4%, with the FFPE samples affected the most (2-88%).

From our analysis we recommend performing deduplication during analysis of targeted panels. While we observed the most benefit for smaller panels with low uniformity, improved variant sensitivity was seen regardless of panel size. During experimental design, we advise a worksheet to guide deduplication decisions.

#5277

The cBioPortal for cancer genomics and its application in precision oncology.

Jianjiong Gao,1 James Lindsay,2 Stuart Watt,3 Istemi Bahceci,4 Pieter Lukasse,5 Adam Abeshouse,1 Hsiao-Wei Chen,1 Ino de Bruijn,1 Benjamin Gross,1 Dong Li,1 Ritika Kundra,1 Zachary Heins,1 Jorge Reis-Filho,1 Onur Sumer,1 Yichao Sun,1 Jiaojiao Wang,1 Qingguo Wang,1 Hongxin Zhang,1 Priti Kumari,2 M. Furkan Sahin,4 Sander de Ridder,5 Fedde Schaeffer,5 Kees van Bochove,5 Ugur Dogrusoz,4 Trevor Pugh,3 Chris Sander,2 Ethan Cerami,2 Nikolaus Schultz1. 1 _Memorial Sloan Kettering Cancer Center, New York, NY;_ 2 _Dana Farber Cancer Institute, Boston, MA;_ 3 _Princess Margaret Cancer Center, Toronto, British Columbia, Canada;_ 4 _Bilkent University, Ankara, Turkey;_ 5 _The Hyve, Utrecht, Netherlands_.

The cBioPortal for Cancer Genomics provides intuitive visualization and analysis of complex cancer genomics data. The public site (http://cbioportal.org/) is accessed by more than 1,500 researchers per day, and there are now dozens of local instances of the software that host private data sets at cancer centers around the globe.

We have recently released the software under an open source license, making it free to use and modify by anybody. The software and detailed documentation are available at https://github.com/cBioPortal/cbioportal.

We are now establishing a multi-institutional software development network, which will coordinate and drive the future development of the software and associated data pipelines. This group will focus on four main areas:

1. New analysis and visualization features, including:

a. Improved support for cross-cancer queries and cohort comparisons.

b. Enhanced clinical decision support for precision oncology, including an improved patient view with knowledge base integration, patient timelines and improved tools for visualizing tumor evolution.

2. New data pipelines, including support for new genomic data types and streamlined pipelines for TCGA and the International Cancer Genome Consortium (ICGC).

3. Software architecture and performance improvements.

4. Community engagement: Documentation, user support, and training.

This coordinated effort will help to further establish the cBioPortal as the software of choice in cancer genomics research, both in academia and the pharmaceutical industry. Furthermore, as the sequencing of tumor samples has entered clinical practice, we are expanding the features of the software so that it can be used for precision medicine at cancer centers. In particular, clean, web-accessible, interactive clinical reports integrating multiple sources of genome variation and clinical annotation over time has potential to improve clinical action beyond current text-based molecular reports. By making complex genomic data easily interpretable and linking it to information about drugs and clinical trials, the cBioPortal software has the potential to facilitate the use of genomic data in clinical decision making.

#5278

NGS-SWIFT: A cloud-based variant analysis framework using control-accessed sequencing data from dbGaP/SRA.

Chunlin Xiao, Eugene Yaschenko, Stephen Sherry. _NIH, Bethesda, MD_.

Genetic variation analysis plays an important role in elucidating the causes of various human diseases. The drastically reduced costs of genome sequencing driven by next generation sequence technologies now make it possible to analyze genetic variations with hundreds or thousands of samples simultaneously, but with the cost of ever increasing local storage requirements. The tera- and peta-byte scale footprint for sequence data imposes significant technical challenges for data management and analysis, including the tasks of collection, storage, transfer, sharing, and privacy protection. Currently, each analysis group must download all the relevant sequence data into a local file system before variation analysis is initiated. This heavy-weight transaction not only slows down the pace of the analysis, but also creates financial burdens for researchers due to the cost of hardware and time required to transfer the data over typical academic internet connections. To overcome such limitations and explore the feasibility of analyzing control-accessed sequencing data in cloud environment while maintaining data privacy and security, here we introduce a cloud-based analysis framework that facilitates variation analysis using direct access to the NCBI Sequence Read Archive through NCBI SRA Toolkit, which allows the users to programmatically access data housed within SRA with encryption and decryption capabilities and converts it from the SRA format to the desired format for data analysis. A customized machine image (ngs-swift) with preconfigured tools, including NCBI SRA Toolkit and NGS Software Development Kit, and resources essential for variant analysis has been created for instantiating an EC2 instance or instance cluster on Amazon cloud. Performance of this framework has been evaluated using dbGaP study phs000710.v1.p1 (1000Genome Dataset in dbGaP, http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000710.v1.p1), and compared with that from traditional analysis pipeline, and security handling in cloud environment when dealing with control-accessed sequence data has been addressed. We demonstrate that with this framework, it is cost effective to make variant calls without first transferring the entire set of aligned sequence data into a local storage environment, thereby accelerating variant discovery using control-accessed sequencing data.

#5279

The first ACT: an efficient data tracking and sharing tool for ADC and linker-payload development.

Frank Loganzo,1 Ellie Muszynska,1 Nathan Tumey,2 Megan Tran,3 Tianhong Zhang4. 1 _Pfizer Oncology, Pearl River, NY;_ 2 _Pfizer WWMC, Groton, CT;_ 3 _Pfizer Business Technology, Pearl River, NY;_ 4 _Pfizer Business Technology, Andover, MA_.

Antibody drug conjugates (ADCs) are anti-cancer agents that more selectively deliver potent cytotoxins to tumors via targeted antigens. ADCs are modular biotherapeutics composed of antibody (Ab), linker (L), and payload (P). The LP may be conjugated to different antibody locations, including endogenous cysteine or lysine residues; recent advances in conjugation technology also have enabled site-specific conjugation to engineered cysteine, glutamine, or other sites. Pfizer has utilized a platform approach to identify novel LPs and ADCs, hence large numbers of linkers and payloads have been synthesized and conjugated to many different antibodies at conventional and site-specific locations. This has generated thousands of permutations of antibody, conjugation site, linker, and payload. Moreover, there are large volumes of metadata and analytical data associated with each ADC component as well as with the final conjugated product. Hence, there is an avalanche of data that must be tracked and accessed in order to ensure efficient decision making and communication as ADCs advance through our biological testing funnel. To handle this information challenge, we designed and implemented a new ADC tracking system. First, we created a systematic nomenclature to enable automated assignment of ADC names. ADC names are concatenated in the format "Ab-(site)_L_P". In addition, specific antibody properties are defined within the Ab portion of the name, such as antigen, clone ID, mutation, isotype, etc. Second, we built a Java web-start enabled desktop application known as Antibody Conjugate Tracker (ACT). ACT integrates ADC bioanalytical and structural information into a user-friendly interface. The application recapitulates our synthesis and testing workflow: antibody inventory, LP inventory, project team synthesis requests, ADC registration, and sample physical inventory. ACT enables system-generated ADC name assignment by defined rules as well as registration of every ADC with corporate identifiers to allow biological data reporting. The systematic naming allows for facile uniqueness checking, thus eliminating possible redundancies and minimizing confusion about ADCs that use similar components. Third, we integrated ACT with the corporate registration system and the biological results database, allowing seamless access of in vitro and in vivo screening data for each ADC. By tracking both platform and program ADCs via ACT, we now have a rich database of constructs and biological results that can be used to interrogate structure-activity relationships (SAR) of new linker-payloads. Overall, benefits of the ACT system include more efficient conjugation workflows, inventory tracking, enhanced communication among scientists from synthesis to testing, and streamlined SAR analysis of novel linker-payloads. These efficiencies have ensured accurate tracking and delivery of innovative ADCs into our clinical development pipeline.

#5280

A web-based platform for exploring 'omics and clinical data from healthy breast tissues.

Xingyan Kuang,1 Catalin Marian,1 Cenny Taslim,1 Min-Ae Song,1 Daniel Weng,1 Jo L. Freudenheim,2 Theodore Brasky,1 Kun Huang,3 Kevin Coombes,3 Peter G. Shields1. 1 _OSU Comprehensive Cancer Center, Columbus, OH;_ 2 _Department of Epidemiology and Environmental Health, Buffalo, NY;_ 3 _Department of Biomedical Informatics, Columbus, OH_.

Integrating 'omics, epidemiology and clinical data from healthy patients and their biologic samples is important for identifying the determinants of early carcinogenesis. In a cross-sectional study of 249 women with no history of cancer and who underwent reduction mammoplasty, whole transcriptome (i.e., gene expression) and epigenome (i.e., methylation and microRNA) data were collected from dissected breast tissues. An extensive epidemiologic questionnaire ascertained data on breast cancer risk factors. Subjects with benign lesions considered at risk for future cancer were excluded. In order to manage, analyze, and share these data efficiently, we organized the data into a relational database to support a series of web-based tools for analyzing and visualizing the multidimensional data in an intuitive graphical user interface (GUI). The current platform improves upon existing web resources that integrate 'omics and clinical data by including novel features that allow for greater flexibility of bioinformatics analyses. It also focuses on the 'omics as an outcome, rather, e.g., what epidemiology risk factor or particular gene is associated with downstream effects across the 'omics. Such features include: 1) a plot function that can visualize up to 4 dimension data chosen by a user from the 3 types of genetic profiles and more than 100 clinical and biomedical variables; 2) a differentiation visualization tool based on the 'omics data of 2 groups of samples; and 3) miRNA target gene network generated by the correlations between gene expression and miRNA expression of our patient samples and validated miRNA target gene database. The GUI facilitates the discovery of research scientists by reducing complex biological and clinical data into easily understandable views. Even without the expertise of computational biology, researchers can integrate and analyze the high dimensional data that they are interested conveniently on our user-friendly designed platform.

#5281

Fast and scalable software for comparative variant analysis and visualization of massive next-generation sequencing data.

Riku Katainen, Veli Mäkinen, Lauri A. Aaltonen, Esa Pitkänen. _University of Helsinki, Helsinki, Finland_.

Next-generation sequencing (NGS) techniques produce high quantities of DNA and RNA sequencing data, enabling variation analysis at a whole genome level. Due to the massive availability of NGS data, the processing and analysis now constitute a serious challenge for research in life sciences. This necessitates the development of novel, user-friendly and scalable software tools, which are able to jointly handle large NGS data sets in order to break the bottleneck that has already moved from data generation to data analysis in high-throughput biology.

Here we introduce a highly efficient NGS analysis and visualization software tool (Rikurator), which is designed for researchers who are struggling with NGS data analysis and may not have access to a dedicated bioinformatics team. Rikurator can be applied to a multitude of genomic analysis tasks, including discovery of pathogenic mutations in familial or sporadic sample sets, analysis of structural variants and somatic characterization of cancer genomes. To these ends the software implements interactive variant filtering and annotation methods based on sample comparison, control data (e.g. ExAC), amino acid changes and quality scores, which provide an intuitive view at the studied samples. Shared candidate genes in a set of dozens of individuals can be discovered in a matter of minutes with an ordinary laptop computer. Importantly, the software is able to analyze hundreds of whole-genome sequenced samples using only a modest amount of RAM. The tool also visualizes and integrates genes, variants (VCF file format), sequence reads (BAM) and user defined features (BED/GFF). In addition, Rikurator is not limited to human studies, supporting reference genomes and genome annotations in standard formats. The software supports targeted (e.g. exome), whole-genome, RNA, Oxford Nanopore and PacBio sequencing data.

Rikurator can be run on Windows, Linux and Mac, and it will be freely available after publication.

#5282

A cloud-enabled open source data management platform supporting a federated research and development organization.

Lauren Intagliata,1 Selina Chu,2 Garth McGrath,1 Giuseppe Totaro,2 Daniel Civello,1 Nipurn Doshi,2 Shivika Thapar,2 Michael Livstone,1 Chris Mattmann,2 Paul Ramirez,2 Maureen Cronin1. 1 _Celgene Corporation, San Francisco, CA;_ 2 _Jet Propulsion Laboratory, Pasadena, CA_.

Biopharmaceutical R&D organizations characterize drug candidate target effects and modes of action and create molecular models of target diseases. These data-intensive activities are informed by vast data resources including publicly available data, internally generated data and partnered private data collections. However, rapid evolution in computing, data management tools, analytical and visualization methods, the complexity of data types and the data volumes that must be accommodated present significant technical and logistic hurdles to overcome. It is particularly difficult for a geographically dispersed R&D organization to make data resources easily available to scientists for search, visualization and exploration. Nevertheless, this is required for R&D scientists to gain insight into disease and drug mechanisms and to capture the knowledge needed to sustain the scientific enterprise.

Standardized commercial solutions to R&D data challenges are unattractive since they require significant resource investment in platform configuration, user-training and system maintenance. This strategy necessarily creates delay in adopting newly emerging technologies and provides incentive not to adopt alternatives due to investment in existing systems. In contrast, our solution to R&D data demands was to build a cloud-deployed data platform using state of the art tools developed and maintained by the open source software community at the Apache Software Foundation. Partnering with academic data scientists, we selected the best available tools to fit our specific needs. We integrated them into a platform accessible to our federated R&D scientific community while allowing the system to be freely modified and updated on demand to meet evolving user requirements.

Priorities for our data platform are to ingest, secure and index R&D source data of all types, make these indexed data assets available to computational scientists for analysis and provide faceted search capability based on a comprehensive metadata model. Three products: LabKey server, Apache OODT and ISATools have all been combined into a scientific data management system to provide a unified data resource enhanced by a search platform powered by Apache Solr. The platform supports both internally generated data and data imported from public, contracted or partnered sources. All data are available for interactive exploration by our R&D community, accessed via integrated search, analysis and visualization tools.

Deployment of this system to our R&D organization has been met with enthusiastic adoption. Feedback for improvement or requests for system enhancements and additional capabilities are rapidly addressed in this open source environment, leading to further adoption among the R&D scientists and providing the basis for accessible, stable institutional knowledge collections.

#5283

Shangri-Docs: a browser based tool for document exploration and automatic knowledge extraction from unstructured biomedical text.

Chris Mattmann,1 Lauren Intagliata,2 Selina Chu,1 Garth McGrath,2 Giuseppe Totaro,1 Daniel Civello,2 David Ballard,2 Jeffrey Long,2 Nipurn Doshi,1 Shivika Thapar,1 Michael Livstone,2 Paul Ramirez,1 Maureen Cronin2. 1 _Jet Propulsion Laboratory, Pasadena, CA;_ 2 _Celgene Corporation, San Francisco, CA_.

Biomedical information is available to research and development scientists as unstructured text in the form of scientific manuscripts and reports published in the literature and elsewhere. Scientists focused on specific research programs are burdened with surveying vast numbers of publications and reports to acquire information relevant to their efforts. Employing technology as a research aid provides a mechanism to cope with information overload that characterizes the R&D environment. Text mining can extract knowledge from large corpora of biomedical text and make it available to support scientific research and knowledge collections [1, 2] and intelligent PDF reader tools able to search content and find related articles [3] are available; however, such reader tools are typically desktop applications limited to specific platforms and data sources so they cannot easily support broad based integrated scientific search needs for a dispersed R&D organization with a wide variety of content needs.

Our team has developed a web-browser based document reader with a built-in exploration tool and automatic concept extraction from biomedical text content. This provides R&D scientists with a simple tool to aid finding, reading, and exploring documents relevant to focused research objectives. The tool, Shangri-Docs, combines a document reader with automatic concept extraction and highlighting of relevant terms based on carefully selected ontologies combined with our custom corporate enterprise taxonomy. Shangri-Docs provides the ability to evaluate a wide variety of document formats (e.g. PDF, Word, PPT, text, etc.) and exploits the linked nature of the Web and personal content by performing searches on content from public sites (e.g. Wikipedia, PubMed) and privately cataloged databases simultaneously.

Shangri-Docs incorporates Apache cTAKES (clinical Text Analysis and Knowledge Extraction System) [4] and Unified Medical Language System (UMLS) to automatically identify and highlight terms and concepts, such as specific pathology, disease, drug, and biological terms mentioned in the text. cTAKES was originally designed specifically to extract information from clinical medical records. We have extended cTAKES automatic knowledge extraction process to include the R&D biomedical research domain by improving the ontology guided information extraction process. Shangri-Docs could be adapted to other science fields and further customized across our R&D scientific community via our open source, cloud-based, data management system.

[1] Funk, et.al., BMC Bioinformatics 2014, 15:59 doi:10.1186/1471-2105-15-59

[2] Kang et al., BMC Bioinformatics 2014, 15:64 doi:10.1186/1471-2105-15-64

[3] Utopia Documents, http://utopiadocs.com

[4] Apache cTAKES, http://ctakes.apache.org

#5284

Characterizing the differences of allelic imbalance between tumor and normal tissues by next-generation sequencing.

Yao-Wei Jheng,1 Yu-Chiao Chiu,1 Tzu-Pin Lu,2 Liang-Chuan Lai,3 Mong-Hsun Tsai,4 Tzu-Hung Hsiao,5 Eric Y Chuang6. 1 _Institute of Bioelectronics and Bioinformatics, National Taiwan University, Taipei, Taiwan;_ 2 _Department of Public Health, Institute of Epidemiology and Preventive Medicine, National Taiwan University, Taipei, Taiwan;_ 3 _Graduate Institute of Physiology, National Taiwan University, Taipei, Taiwan;_ 4 _Institute of Biotechnology, National Taiwan University, Taipei, Taiwan;_ 5 _Taichung Veterans General Hospital, Taichung, Taiwan;_ 6 _Bioinformatics and Biostatistics Core Lab, NTU Center of Genomic Medicine, Taipei, Taiwan_.

Genetic variation has proved to contribute to tumor formation. One of the phenomenon caused by genetic variation, allelic imbalance, means different expression abundance between two alleles, recently has become a special landmark on cancer biology, with high prevalence in recent cancer studies. However, the present of allelic imbalance has not been fully understood yet. Taking the advantages of next generation sequencing, allelic imbalance can be identified and quantified through bioinformatics algorithms. Here, we developed a mathematical model to identify with allelic imbalance by concurrently analyzing RNA-Seq and DNA-Seq. First we called heterozygous SNP variants from both RNA-Seq and Exome-Seq data. The allele ratio is counted by the ratio of read depth between two alleles after integrating SNPs into gene level. Next, we identify allelic imbalance genes by differences of RNA and DNA allele ratios between tumor and normal samples. As results, a total of 91143 SNPs were identified in Exome-Seq, and about 3% (2586 out of 91143) SNPs were heterozygous in both tumor and normal tissues. We found that about 118 allelic imbalance genes in tumor tissue are significantly different from those in normal tissue. Furthermore, we investigate the enriched function of these allelic imbalance genes. These result showed that these genes are significantly enriched in DNA repair pathway. We speculated that allelic imbalance in tumor tissue may be involved in the dysregulation of DNA repair. Also other enriched functions were identified, such as z-finger protein pathway.

Our goal is to investigate the landscape of allelic imbalance between tumor sample and normal sample. Our results demonstrate the role of allelic imbalance of dysregulation of tumor. Moreover, our method can be applied to uncover more basic mechanisms on allelic imbalance and tumorigenesis.

#5285

COSMIC: comprehensively exploring oncogenomics.

Simon A. Forbes, Nidhi Bindal, David Beare, Sally Bamford, Charlotte G. Cole, Sari Ward, Kenric Leung, Chai Yin Kok, Mingming Jia, Tisham De, Zbyslaw Sondka, Michael R. Stratton, Peter J. Campbell. _Wellcome Trust Sanger Institute, Cambridge, United Kingdom_.

COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations causing human cancer. Broad and comprehensive in scope, its 75th release (Nov 2015) describes over 3.7 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on almost 200 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy number aberrations, 9 million gene expression variants and almost 8 million differentially methylated CpG's. This information combines a consistent interpretation of the data from the major cancer genome consortia and cancer genome literature, with hand-curation of over 22,000 gene-specific literature publications.

With such a large volume of data, it is increasingly important to indicate which information is most significant. All mutations in COSMIC are now given a functional significance score, calculated using the FATHMM algorithm. In addition, a mutation can also be tagged as low-impact if they are described as a polymorphism in normal human genomes. All this information is available for selection and exploration in the COSMIC website (http://cancer.sanger.ac.uk), and for download via COSMIC Downloads (http://cancer.sanger.ac.uk/download). In addition to this broad database, the Cancer Gene Census (http://cancer.sanger.ac.uk/census) is a project within COSMIC aiming to identify and characterize all genes known to cause cancer, currently describing over 570 genes. This Census is now a priority focus of development, with a dedicated curator explicitly defining the range of genes driving cancer, including primary alleles and mechanisms and the diseases which are induced, with detailed supporting evidence.

In addition to these analytical websites, expert-curated lists and now a GA4GH Beacon, COSMIC also hosts a full Oncology Genome Browser (http://cancer.sanger.ac.uk/genome). This fully-featured system allows the exploration of all cancer somatic mutation data collected in COSMIC alongside genomic annotations including coding genes, ncRNAs, SNPs and regulatory features. All data is vertically integrated, allowing exploration of how these many genetic mechanisms might promote oncogenesis, and how similar activating/inactivating mechanisms correlate. Amongst many interesting examples, there is a clear cluster of structural rearrangements immediately upstream of the BRD4 epigenetic modifier gene, affecting a region of multiple transcription control elements, and a substantial accumulation of abnormally hypermethylated CpG islands in the HOXA gene cluster on chromosome 7 coinciding with a group of HOTAIR and HOTTIP miRNAs. With multiple filters and selections available, these visualizations will increasingly support the exploration of how a variety of mutation mechanisms may act together to cause specific cancer diseases.

#5286

A pipeline for developing a novel, predictive tool to classify variants of uncertain significance (VUS) in Lynch Syndrome.

Sanjeevani Arora, Peter J. Huwe, Rahmat Sikder, Manali Shah, Sanat Deshpande, Michael J. Hall, Roland L. Dunbrack, Erica A. Golemis. _Fox Chase Cancer Center, Philadelphia, PA_.

Lynch syndrome (LS) highly predisposes individuals and their families to an increased risk of colorectal cancers and to cancers of the endometrium, ovary, gastrointestinal tract, pancreas, upper urinary tract, and other tissues. The occurrence of LS has been attributed to germline, heterozygous mutations in four genes in the DNA mismatch repair (MMR) pathway: MLH1, MSH2, MSH6 and PMS2. Mutations in the MLH1 and MSH2 genes account for the majority of detectable mutations in LS, with more infrequent mutations in MSH6 and PMS2. Clinical diagnosis of LS helps direct the management of the disease and risk assessment for future cancers in the family.

A major challenge is the determination of variant pathogenicity in MMR genes, particularly those that code for missense mutations. According the InSiGHT database, there are >5000 unclassified variants of unknown significance (VUS), and this number continues to grow every day due to the increased prevalence of genomic testing. Such VUS present an enormous challenge to effective genetic counseling of LS families. There are numerous predictive methods, such as SIFT and Polyphen-2, for predicting the effects of missense mutations in any gene including LS genes. Further, there is a great deal of promise for gene-specific predictors, such as PON-MMR and CoDP, that are trained on the physical and evolutionary features of known deleterious and benign mutations in the target genes of interest.

Since the number of available mutations for the LS genes with experimental phenotypes is limited, we have developed and applied a pipeline to perform experimental validation of VUS in MMR genes. MMR genes with introduced VUS were assessed in vitro in comparison to wild type genes for the effect of the VUS on steady state protein expression, localization, and functionality in inducing DNA damage checkpoints and influencing cell survival following treatment with a panel of DNA-damaging agents. We then assessed a panel of VUS in MSH2, MLH1 and PMS2 in HEK293 kidney cells and colorectal cancer (CRC) cell lines bearing deletions in the MMR genes being tested.

This mid-throughput pipeline allowed effective validation of the functional consequences of each VUS, generating additional data to enhance the efficacy of gene-specific predictors for the LS genes that we are developing. Such tools may greatly benefit the assessment of MMR gene variants identified through genetic testing and for genetic counseling of LS families.

#5287

Heterogeneity of mutation calling and annotation: a survey of cancer next-generation sequencing initiatives by the Global Alliance for Genomics and Health (GA4GH).

Daniel J. Vis,1 Jeremy Lewin,2 Lillian Siu,2 Rachel Liao,3 Jean Claude Zenklusen,4 Fabien Calvo,5 Edit Szepessy,6 Ana Vivancos,7 Valtteri Wirta,8 Subha Madhavan,9 Keunchil Park,10 Daniel Tan,11 Janessa Laskin,12 Melissa Brammer,13 Emmanuel Dias-Neto,14 Anthony Tolcher,15 Thomas J. Hudson,16 Charles Sawyers,17 Mark Lawler,18 Emile E. Voest1. 1 _Netherlands Cancer Institute, Amsterdam, Netherlands;_ 2 _Princess Margaret Cancer Centre, Toronto, Ontario, Canada;_ 3 _Genomics and Health, Toronto, Ontario, Canada;_ 4 _The Cancer Genome Atlas at Center for Cancer Genomics, National Cancer Institute, Washington, DC;_ 5 _Cancer Core Europe, Gustave Roussy, Villejuif Cedex, France;_ 6 _EORTC, Brussels, Belgium;_ 7 _Vall d'Hebron Institute of Oncology, Barcelona, Spain;_ 8 _Scilifelab, Solna, Sweden;_ 9 _Georgetown University Medical Center, Washington, DC;_ 10 _Samsung Medical Center, Seoul, Republic of Korea;_ 11 _National Cancer Centre Singapore, Signapore, Singapore;_ 12 _BC Cancer Agency, Vancouver, British Columbia, Canada;_ 13 _Genentech/Roche, San Francisco, CA;_ 14 _AC Camargo Cancer Center, Sao Paulo, Brazil;_ 15 _START, San Antonio, TX;_ 16 _OICR, Toronto, Ontario, Canada;_ 17 _MSKCC, New York, NY;_ 18 _Centre for Cancer Research and Cell Biology, Queen's University, Belfast, United Kingdom_.

Although genomic data sharing is widely endorsed, practical barriers exist. For example, annotation and calling of mutations vary significantly with the method used. This poses clear challenges in data harmonization and sharing. In this setting, GA4GH conducted a survey of Cancer Next Generation Sequencing (NGS) initiatives globally to chart the technical implementation of genomic programs.

A total of 59 of 108 invited initiatives responded (response rate=55%) via a web-based survey. In total, 63% of programs share their data, and 10% are partially sharing or planning to share. Most initiatives were North American (33%) or European (28%) based. Of the 59 respondents, 51 responded to queries on technical aspects of their NGS program (Table 1). For diagnostic application, 67% employ a small panel (<50 genes), 55% a medium panel (50-250 genes), 45% a large panel (251-1000 genes); only 22% indicated they (also) use whole exome sequencing (WES). Diagnostic programs tend to favor panels and deep sequencing, while research programs favor WES/WGS. Overall, mutation and copy number calls were stored centrally in a single project database (96% and 92% respectively), with lower rates for raw data (BAM files, 86%) and histological data (75%). Somatic mutations were identified primarily via GATK (57%), Samtools (49%), Varscan (47%), MuTect (40%); all but 7 initiatives used combinations of these tools. Mutational variants were annotated by Cosmic (73%), PolyPhen (66%), and dbSnp (64%). Germline samples were used as control in only 62% of initiatives.

In conclusion, the majority of initiatives use an ensemble of tools for calling and annotating mutational variants. Harmonization efforts on gene panel composition and the standardization of tools (eg, methods, application program interfaces (APIs)) are urgently needed to prevent continued generation of isolated data silos that hamper NGS-enabled advances in precision medicine. | |

|

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

Primary purpose of test | % Diagnostic (n=9) | % Research (n=22) | % Diagnostic & Research (n=20)

Organization | |

|

Centralized testing | 22% | 64% | 30%

Unique sample identifiers | 89% | 77% | 70%

Certification ISO/CLIA/NE | 88% | 45% | 65%

Initiative > 1000 Patients | 33% | 36% | 35%

Sequencing | |

|

Sequencing depth 50-250x | 0% | 55% | 80%

Sequencing depth >250x | 100% | 45% | 20%

Panel sequencing | 89% | 68% | 60%

Whole Exome/Genome Sequencing | 22% | 77% | 65%

Samples | |

|

Fresh frozen (FF) | 11% | 23% | 15%

Formalin Fixed Paraffin Embedded (FFPE) | 44% | 27% | 20%

FFPE or FF | 44% | 50% | 65%

Germline as control | 22% | 68% | 65%

#5288

Dynamic genomic indexing enables accurate somatic variant detection from cancer genome sequencing without sequence alignment limitations.

HoJoon Lee, Jacob J. Parker, John Bell, Hanlee P. Ji. _Stanford University, Stanford, CA_.

Cancer genome sequencing is a widely adopted approach for identifying critical genetic variants and increasingly, for diagnostic application in precision medicine. Accurate analysis of cancer genome sequencing is essential, yet remains challenging - the reliable and accurate identification of somatic variants continues to be a significant issue. One source of issues involves the mapping/alignment of sequences. It relies entirely on the human reference genome, a static data consisting of character strings of chromosome. However, the current reference is derived from few individuals and thus, does not account for the full diversity or complex structure of human genomes. Critical features of cancer genomes are missed or called with errors. As a solution, we developed dynamic genomic indexing (DGI), which identifies somatic variants by using the frequency of short sequences that are 20~200 bases in length (20~200mer) from paired tumor/normal sequencing data.

Any somatic variants will affect the frequency of short sequences. In this study, DIG relies on the presence of neo-20mers, derived by point mutations and insertions/deletions (indels), that are different in frequency between normal and tumor sequence reads. These 20mer are unique sequences that arise from cancer genetic variants. First, the DGI identifies the four groups of differential 20mers that their frequencies are significantly different between tumor and normal; tumor specific (TS), normal specific (NS), tumor dominant (TD), and normal dominant (ND). Second, DIG clusters differential 20mers with neighbor 20mers sharing 19 bp in the middle within each group. Third, clustered tumor specific 20mers are matched to clustered NS or ND 20mers. The number of clustered 20mers represents the number of mutational events and ratio between frequency of TS 20mers and matched ND 20mers implies allelic depth. All calculations are conducted in comparison to matched normal samples. Thus, the reference genome is never required throughout the analysis process. Furthermore, these 20mers can be searched easily in RNA-Seq to see if mutations are expressed.

We evaluated the performance of DGI using simulated whole exome sequencing (WES) data and 24 WES from the Cancer Genome Atlas project. We identified many of reported somatic mutations (>90%) from WES as well as novel mutations. We observed DGI performs as good as other variant callers for substitutions while outperforms for indels. Our results support the principle of non-alignment sequencing analysis. DGI provides simple, but accurate matrices of somatic variants for cancer sequencing data for any population and can be easily scaled to handle population-based studies. This study provides small variant detection (<50bp) as a first step toward in a broader effort to develop non-alignment sequencing analysis for structural variation such as copy number and chromosomal rearrangement.

#5289

Enhance both precision and sensitivity of fusion gene detection by hybrid sequencing.

Jason L. Weirather,1 Tyson A. Clark,2 Elizabeth Tseng,2 Jonas Korlach,2 Kin Fai Au3. 1 _Department of Internal Medicine, University of Iowa, Iowa City, IA;_ 2 _Pacific Biosciences, Menlo Park, CA;_ 3 _Department of Biostatistics, Department of Internal Medicine, University of Iowa, Iowa City, IA_.

Background: New Third Generation Sequencing (TGS) techniques, such as PacBio, can provide very informative insights into the transcriptome, such as expression of fusion genes/fusion transcripts from cancer samples. However, the currently available fusion genes analysis tools are for Second Generation Sequencing (SGS) data, where the short read length and unreliable alignments can lead to uncertain accuracy of fusion gene detections. Hybrid-Seq, which integrates SGS short read data into the analysis of TGS long read data, can complement the strengths of both and thus improve the overall performance and resolution of the output data. It also reduces the required amount of TGS data and thus the sequencing cost. Recently, we developed and reported on a Hybrid-Seq approach, IDP-fusion and the results of the proof-of-concept application to MCF-7 data. We demonstrated that IDP-fusion can identify fusions genes with much higher precision than SGS-based approaches. Although the sensitivity is comparable to the most sensitive SGS-only method, a significant proportion of experimentally verified gold standard fusion genes had yet to be identified by IDP-fusion. It indicated an opportunity to enhance the sensitivity of IDP-fusion while retaining the unparalleled precision of the results.

Method: Here we present an innovative Hybrid-Seq approach which extends IDP-fusion, to filtered fusion gene candidates predicted by SGS short read alignments. The fusion gene candidates are verified by the presence of a TGS long read better aligned to an artificial chromosome created from the fusion candidate than any single genome locus. We applied IDP-fusion to a Hybrid-Seq data from the MCF-7 breast cancer cell line, including Illumina SGS data and a lately TGS data generated by PacBio P5-C3 sequencing chemistry. The new IDP-fusion considered fusion candidates reported by several popular SGS tools (TopHat-Fusion, SOAPfuse, TRUP, FusionMap, and deFuse). We compared performance of our new tool to the original IDP-fusion, and the SGS-only approaches.

Results: The new algorithm of IDP-fusion improved the sensitivity from 33.8% to 54.9%. This is higher than the most sensitive SGS-only tool (deFuse, 38.0%), which is achieved at the cost of a low precision of 13.8%. The improved IDP-fusion retains a precision of 60.9%, which is only down slightly from the original IDP-fusion at 68.6%. This tradeoff is acceptable when considering the overall accuracy described by F-score for IDP-fusion. The F-score has increased from 45.3% to 57.8%, which is also considerably better than the best F-score achieved by SGS-only methods (32.8%).

Conclusions: Fusion candidates identified directly from SGS reads can be screened using alignments of TGS long reads, and supplement fusion candidates detected from long reads. Comparing to SGS-only methods, this Hybrid-Seq approach provides much more sensitive and more accurate reports on the fusion genes.

#5290

ChimericSeq: an easy-to-use program for discovery and analysis of integration events from NGS data.

Patrick Jongeneel,1 Selena Lin,2 Jamin Steffen,1 Surbhi Jain,1 Ying-Hsiu Su,3 Wei Song1. 1 _JBS Science, Doylestown, PA;_ 2 _Drexel University College of Medicine, Philadelphia, PA;_ 3 _Barach S. Blumberg Institute, Doylestown, PA_.

The purpose of this study was to develop a computational method of extracting viral integration events from NGS data in an intuitive way that could accommodate access from users of all disciplines. Viral integration into the host genome is a characteristic of many pathogenic viruses, including the hepatitis B virus (HBV) and human papillomavirus (HPV). Gradual insertion of viral components near proto-oncogenes of the host genome over time can induce uncontrolled cellular proliferation, eventually leading to carcinogenesis. While increased availability of high-throughput next generation sequencing (NGS) has provided tools for researchers to discover these underlying host changes due to viral integrations, there is an emerging need for analytical support of this data. Here we present ChimericSeq, a user-friendly program that can quickly identify viral integration events from NGS data. To fully evaluate this program, we compared the functionality of ChimericSeq to other current viral integration programs. A number of synthetic data sets of HBV sequence fragments integrated into 100bp fragments of random human genomic DNA were created to mimic the nature of chimeric reads of NGS data. ChimericSeq was able to correctly identify reads containing at least 25bp of viral sequence at 100% accuracy. This was a major improvement over the current programs, VirusClip and ViralFusionSeq, which could not detect reads with only 25bp of viral sequence, and even had difficulty correctly identifying viral integration sites containing additional viral sequence. Furthermore, ChimericSeq could detect viral events nearly 10X and 100X faster than these current programs, respectively. Upon testing of NGS data from actual HBV-positive human tissue, we found that ChimericSeq was not only much faster, but was also able to detect more unique viral integration events than current viral integration tools. In conclusion, ChimericSeq expands NGS analytical support to a broader spectrum of the scientific community, being the first program of its kind to offer support in an intuitive graphical user interface (GUI) for commonly used operating platforms, such as Windows and Mac.

#5291

OncoMD: A powerful genomics data analysis and interpretation platform for cancer discovery research.

Ravi Gupta, Kiran V. Paul, Kartik Kumaramangalam, Sam Santhosh, Amitabha Chaudhuri. _MedGenome, San Francisco, CA_.

Cancer cells gain fitness advantage over normal cells by accumulating mutations over time. Next Generation DNA sequencing technologies have made the detection of genetic alterations in tumor genomes affordable. Powerful data analysis and interpretation tools enable extraction of disease-relevant information from diverse genomics data to guide diagnosis and treatment decisions. We have built a robust cancer analytics platform OncoMD that combines tumor mutation profiles, expression signatures, copy number variations, epigenetic alterations and drug sensitivity to create a holistic view of human cancer enabling discovery of new targets for therapy and prognosis. We have analyzed the rich mutational data captured in OncoMD to identify potential T-cell neo-epitopes in different cancers and examined their prevalence in the context of the tumor microenvironment. The presence of T-cell neo-epitopes alone is not sufficient to sensitize tumors to immunotherapy drugs and requires cooperation from the tumor microenvironment. Although mutational burden was a strong predictor of T-cell neo-epitopes in our analysis, other genetic alterations such as overexpression, splicing, gene-fusions and insertions/deletions can produce altered peptides exhibiting high affinity binding to HLA class I and II molecules in cancers with low burden of missense mutations. We determined the number of potential T-cell neo-epitopes after classifying tumors based on their epithelial, stromal and immune cell content using gene expression signatures. We observed that the normalized T-cell neo-epitope burden was strongly anti-correlated with the T-cell content of the tumors, compared to their epithelial or stromal content. Together, our findings suggest that tumors enriched in T-cells may be subjected to immunoediting processes and carry relatively fewer T-cell neo-epitopes to avoid elimination. These tumors are likely to respond to immunotherapy treatments.

#5292

An online tool for biomarker analysis in Gene Expression Omnibus (GEO) datasets.

Jasmine Dumas,1 Michael Gargano,2 Garrett M. Dancik2. 1 _DePaul University, Chicago, IL;_ 2 _Eastern Connecticut State University, Willimantic, CT_.

The Gene Expression Omnibus (GEO) is a public repository of gene expression and sequence-based datasets, that currently includes >42,000 datasets with gene expression profiles obtained by microarray, and >12,000 datasets containing the keyword "cancer". Although GEO has its own analysis tool (GEO2R) for identifying differentially expressed genes, evaluation of a single gene is not straightforward, and GEO2R was not designed to identify prognostic biomarkers based on survival analyses. In this work, we describe an online, easy-to-use tool for biomarker analysis in GEO datasets.

shinyGEO is a web-based tool that allows a user to download the expression and sample data from a GEO dataset, select a gene of interest, and perform a survival or differential expression analysis in order to evaluate gene expression diagnostic or prognostic biomarkers. For both analyses, shinyGEO produces publication-ready graphics and generates R code ensuring that all analyses are reproducible. The tool is developed using shiny, a web application framework for R, a language for statistical computing and graphics.

The web application and source code is available at the following link: http://bioinformatics.easternct.edu/shinyGEO/

#5293

NCI Office of Cancer Genomics: Generating publicly available data and resources for the advancement of precision medicine.

Nadia L. Jaber, Jessica N. Mazerik, Jaime M. Guidry Auvil, Subhashini Jagu, Nicholas Griner, Martin Ferguson, Daniela S. Gerhard. _National Cancer Institute, Bethesda, MD_.

The Office of Cancer Genomics (OCG), within the National Cancer Institute, supports cutting-edge genomics and translational research to advance the molecular understanding of cancer, with the ultimate goal of improving clinical outcomes through precision medicine. OCG-supported cancer research initiatives generate and analyze comprehensive genomics datasets, and translate these data into tools, resources, and clinically-relevant information. OCG currently supports three complimentary initiatives, each uniquely collaborative and innovative.

The Cancer Genome Characterization Initiative (CGCI) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative collect high quality, clinically annotated, tumor and normal matched tissue samples for both discovery and validation cohorts. These initiatives aim to identify therapeutic targets and biomarkers in some malignancies prevalent in HIV-positive individuals and high-risk pediatric cancers. Samples are analyzed by whole genome, whole exome, and transcriptome sequencing and datasets are available to the research community in ways that protect patient privacy.

The Cancer Target Discovery and Development (CTD^2) Network combines computational approaches, high-throughput functional assays, and high content small molecule and genetic screens to accelerate the translation of genomic data into discoveries that will inform patient-specific cancer treatments. The Network develops bioinformatics and analysis tools, generates diverse datasets, and further validates subsets of these data. All resources, datasets (https://ctd2.nci.nih.gov/dataPortal/), and validated results (http://ctd2-dashboard.nci.nih.gov/) are openly available to the research community.

Data, analytical tools, and resources generated by OCG initiatives are made publicly available and can be accessed through the OCG website (https://ocg.cancer.gov/). Investigators can use and analyze the data according to their research interests. In addition, datasets generated by OCG-supported programs can serve as valuable resources for those seeking to validate genomic findings. For this poster presentation we will present the most up-to-date information about what data and resources are available, and how to access them. The use of OCG datasets and resources by the research community will further the collaborative goal of enabling precision medicine in the clinic. 
